OSSF Exam 1

Identify tumors in nerve cells with

Neurofilaments (Class III)

Mitotic activity: static

No longer capable of division Neurons, cardiac cells

In what cells is the nucleolus more prominent?

Nucleolus is more prominent in cells actively involved in protein synthesis With Romanowsky stains, such as Wright stain, they often stain blue, while the surrounding chromatin is purple.

DNA glycosylases

Only cut the bond between a damaged base and its sugar recognize the alterations and cleave the base from the sugar recognize the alterations and cleave the base from the sugar; specific glycosylases for each type of altered base Part of BER

Chromatin descriptive terms

Open, loose Coarse, coarsely stippled, dense

Regulated exocytosis pathway

Operates only in specialized secretion cells, Secretory vesicles concentrate at plasma membrane but, will not fuse until triggered by an appropriate signal, Contain surface signals and distinct ionic conditions that cause proteins to aggregate operates only in cells that are specialized for secretion

Movement through channels and transporters is dependent on

Osmosis and diffusion

Cells that are normally multinucleate

Osteoclasts Skeletal muscle Inflammatory macrophages

Peroxisome main function

Oxidation of toxic molecules and breakdown of lipids; synthesis of a phospholipid used to make the myelin sheath around nerve cell axons.

Tetrad

Pair of Homologous ChromosomesSynaptonemal Complex - Where Homologous Dyads are Connected

Dispersive model of DNA replication

Parental and daughter DNA are interspersed in both strands following replication each strand of both daughter molecules contains a mixture of old and newly synthesized DNA Each strand of both daughter molecules contain a mixture of old and newly synthesized DNA

Histopathology/Cytology

Perform: Tissue processing (removed surgically as in biopsy and autopsy), Cutting tissue into sections, Staining, and Preparation for microscopic examination by a pathologist Tissue (from biopsy or autopsy) processing, cutting into sections, staining, and preparation for microscopic examination by a pathologist Tissue processing, cutting into sections, staining, and preparation for microscopic examination by a pathologist.

Peroxisomes details

Peroxisomes are membrane-bound,intracellular, spherical organelles that are so named because they contain one or more enzymes that produce hydrogen peroxide, a highly toxic molecule. They contain such high concentrations of the enzymes catalase and urate oxidase that they sometimes stand out in electron micrographs because of the presence of a crystalloid core. A. Catalase uses the generated hydrogen peroxide to oxidize a variety of other substrates - including phenols, formic acid, formaldehyde, and alcohol, detoxifying them. This type of oxidation reaction is particularly important in liver and kidney cells. In addition, if excess hydrogen peroxide accumulates in the cell, catalase converts it to water. B. A major function of the oxidation reactions performed in peroxidase is the breakdown of fatty acid molecules. This is referred to as ß-oxidation, and is a function that also occurs in mitochondria. Beta oxidation shortens the alkyl chains of fatty acids in blocks of 2 carbon atoms at a time, converting the fatty acids to acetyl CoA. This is then exported to the cytosol for reuse in biosynthetic reactions. C. Peroxisomes are also essential for the biosynthesis of a class of phospholipid abundant in the myelin sheath that insulates nerve cell axons. This function explains why peroxisomal disorders can lead to neurological disease.

Phagocytes

Phagocytosis - an activity of specialized cells generally called "phagocytes" • Macrophage (Histiocyte) - tissue • Monocyte - blood • Neutrophil - blood Commonly Phagocytosed Material o Cell debris o RBCs o Carbon particles o Asbestos fibers o Bacteria, yeast

Glycogen PAS

Pink

Multivesicular body

Predominantly when a lysosome fuses with a mitochondria (can also fuse with peroxisome), membranes of the organelles form vesicles within the autophagosome Complex vesicle with invaginating buds and internal vesicles involved in the maturation of early endosomes into late endosomes. • Exosomes released from multivesicular body -Membrane invagination into endosome forms intraluminal vesicles = multivesicular body (MVB) -MVB can fuse with >Lysosome - destruction >Plasma membrane, releasing membrane- bound exosomes. Multivesicular bodies release exosomes Exosomes provide signal to target cells

Identify tumors in mesenchymal cells (connective tissue, muscle) with

Vimentin and Desmin (Class II)

DNA replication

When a cell divides, the DNA must replicate in order for identical copies to be found in every cell

Clinical correlate of of ribosome and protein synthesis

When proteins fold incorrectly, they sometimes form aggregates that damage cells and even whole tissues. Protein folding diseases can be divided into two groups: in the first, excessive quantities of wrongly folded proteins collect in the form of plaques of insoluble protein in the extracellular tissue, which cannot be broken down by enzymes. They are made up of long filaments (fibrils) that are formed from densely packed beta -pleated sheets of identical proteins; their ordered structure gives them crystal-like properties. This is the group of diseases known as amyloidoses, of which Alzheimer's disease is the best-known example. Transmissible spongiform encephalopathies (TSEs), which include mad cow disease (bovine spongiform encephalopathy; BSE), scrapie disease in sheep (and perhaps chronic wasting disease in elk), are special forms of amyloidosis caused by a misfolded, aggregated form of a protein called PrP (for prion protein), a protein normally located on the outer surface of the plasma membrane, most prominently in neurons. The diseases conditions, however, are unique in that the prion disease can spread from one individual to another, providing that the second individual eats a tissue containing the protein aggregate. It is infectious because the abnormally folded prion-form of the protein can convert the properly folded version of the protein in an infected brain into the abnormal conformation. This can form aggregates that spread rapidly from cell to cell, eventually causing death.

Cytokeratin stain

epithelial-derived tumors For epithelial cells (carcinoma)

Adipocytes

fat cells that make up most of the subcutaneous layer

actin filaments (microfilaments)

filamentous structures in the cytoplasm of eukaryotic cells and form part of the cytoskeleton. They are primarily composed of polymers of actin, but in cells are modified by and interact with numerous other proteins. a thin type of protein filament composed of actin proteins that forms part of the cytoskeleton and supports the plasma membrane and plays a key role in cell strength, shape and movement small fibrils that forms a bundles, sheets or networks in the cytoplasm; for structure, support for microvilli, contractility and movement

Formalin

formaldehyde gas dissolved in water at 37% by weight and 40% by volume A dilute solution of formaldehyde used to preserve biological specimens. the aqueous solution of formaldehyde gas; the most commonly used tissue fixative The routine fixative used in pathology is 10% formalin, i.e. a buffered aqueous, solution containing about 3.8% formaldehyde, often stabilized by the addition of a very low volume of methanol.

Blood cells

formed elements erythrocytes, leukocytes, platelets red blood cells, white blood cells, platelets Specialized connective tissue

Marginal chromatin

found along the peripheral ob the nucleus. It is impossible to actually see the nuclear membrane using a light microscope, but you may see a line in this area composed of marginal chromatin.

Karyorrhexis

fragmentation of pyknotic nucleus

Cardiac muscle cells

functional syncytium- electrical synapses, no chemical synapses intercalated disks- connection between cardiac muscle cells, contain gap junctions cardiac conduction system- atria signal is transmitted to ventricles after a delay, since they are separate systems not connected by gap junctions voltage-gated Ca2+ channels- in addition to voltage-gated Na+ channels, these stay open longer for longer depolarization, greater contraction of cardiac muscle, and less tetany (twitches) T tubules- plasma membrane dips into cytoplasm, more surface area for Ca2+ channels, also sarcoplasmic reticulum actin-myosin fibers- contract in response to influx of Ca2+ are maintained by an extensive capillary network. branching chains of cells, uni- or binucleate striations; intercalated discs

Signal recognition particle

functions as an escort that brings the ribosome to a receptor protein built into the ER membrane binds to the signal sequence and the ribosomal subunits and transports the complex to the ER A protein-RNA complex that recognizes a signal peptide as it emerges from the ribosome.

microtubule organizing center (MTOC)

general term for any structure that organizes microtubules in cells Where the - end of the microtubule attaches. Microtubules grow away from the ___ at its + end. The major ____ in animal cells is the centrosome. General term for any structure (e.g., centrosome and basal body) that organizes microtubules in cells

killer lymphocytes

help regulate immune responses by inducing apoptosis in other immune cells that are unwanted or are no longer needed

Most commonly used stain for light microscopy is...

hematoxylin and eosin (H&E). Hematoxylin is alkaline and binds to acidic cellular components such as nucleic acids. Thus cell nuclei are referred to as basophilic and stain blue-purple. Proteins react with the eosin dye and stain pink to orange; they are described as eosinophilic.

H&E stain

hematoxylin and eosin stain Hematoxylin stains DNA and other acidic structures dark blue or purple, and eosin stains other cytoplasmic components and collagen pink Not a strong basic dye so won't see true blue colors, rather dark purple color

Meiosis - Metaphase 1

homologous pairs align on equator; each pair attaches to separate spindle fiber by its kinetochore homologous pairs align independently at the equator Pairs of homologous chromosomes move to the equator of the cell.

Exportins

identify the molecule as being mRNA destined for export out of the nucleus move macromolecules from the nucleus to the cytoplasm mediate transport of the complexes out of the nucleus nuclear export receptor

Enterocyte

intestinal epithelial cell a cell of the intestinal lining Intestinal cells that absorb nutrients.

Prussian blue dye

iron Shows iron (distinguishes from lipofuscin) as blue; used in hemochromatosis

Cytology details

is the microscopic study of individual cells using the light microscope. Cells from a variety of tissues and body fluids can be easily collected and examined for evidence of inflammation, infection, or neoplasia. Cells are aspirated by introducing a needle into a cell mass and applying suction (fine needle aspirate). Alternatively cells can be scraped from their tissue of origin. Cellular material collected by either method is smeared onto a glass slide. Alternatively, the cut surface of a tissue specimen can be gently pressed to the surface of a glass slide to create an "impression smear". Slides are air-dried and then fixed, usually with methanol or ethanol. These preparations are typically stained with a Romanovsky type of stain (see below). Since these preparations require noembedding step, they can beexamined within minutes ofcollection. Cells collected in this wayappear large and details about theirmorphologic features can be seen. However, details about tissue architecture are generally missing. Cellular arrangements (e.g. formation of tubules or acini) and exact location within the tissue cannot be determined. In addition, the supportive stromal elements (various connective tissue cells and extracellular matrix) often fail to exfoliate and will be absent in the aspirated sample. Blood smears are also an example of this type of preparation Blood smears and cytology preparations are stained using a Romanovsky-type of stain, which combines a basic dye (e.g. methylene blue, Azure A or B) and eosin. These stains are known as polychromatic stains because of the variety of colors and hues that can be seen. Methylene blue is positively charged and binds to nucleic acids and ribosomes. Therefore cells with large numbers of ribosomes (i.e. cells actively involved in protein synthesis) will have deep blue cytoplasm (basophilia). Eosin binds to positively charged cytoplasmic structures. Thus red blood cells stain pinkish-red. Nuclei stain a distinctive shade of purple not attributable to either staining component alone. Examples of Romanovsky stains are Wright stain, Giemsa, or DifQuickTM.

Autophagy

lysosomes break down damaged organelles A process in which lysosomes decompose damaged organelles to reuse their organic monomers

Polysome

mRNA molecule simultaneously being translated by many ribosomes all going in the same direction a cluster of ribosomes held together by a strand of messenger RNA that each ribosome is translating. A complex formed when multiple ribosomes are translating the same mRNA into proteins.

RNA transcription

manufacture of RNA from a DNA molecule, occurs in the nucleus Transfers DNA gene base sequence to a complementary base sequence of an mRNA Transcribe DNA into RNA (slightly different language) dsDNA->ssRNA

Regulated secretion

materials are stored in vesicles and discharged in response to a stimulus secretion occurs only in response to a specific stimulus Discharge of materials synthesized in the cell that have been stored in membrane-bound secretory granules in the peripheral regions of the cytoplasm, occurring in response to an appropriate stimulus

Vimentin

mesenchyme Connective tissue Intermediate filaments in connective tissue cells

Duplication of chromosomes in S phase

-Double helix is made of complementary strands of DNA -Double helix is "unzipped" -Complimentary bases align to form two identical double helixes

Basophilia

-blue color resulting from basic dye binding to negative charges >Methylene blue is a strong basic dye >H&E is not a strong basic dye (won't see true blue colors, but rather a dark purple color)

Flippase

(P-type ATPase) moves PE and PS from outer to cytosolic leaflet protein that facilitates the movement of membrane lipids from one leaflet to the other leaflet of a phospholipid bilayer requires ATP to transport lipids from one leaflet to another

Endocytosis

(inward secretory pathway) >Plasma membrane components delivered to endosomes >Components can be recycled or delivered to lysosomes for degradation >Used to import various nutrients or ingest organisms or debris

Exocytosis

(outward secretory pathway) >Delivers new proteins, carbohydrates, and lipids to plasma membrane or the extracellular space

Centrosome composition and function

- 2 centrioles -Matrix containing ring-shaped structures of gamma-tubulin -Gamma-tubulin acts as nucleation site and anchor sites for growth of new microtubules >Minus ends anchored in MTOC >Constant extension and retraction of microtubules -During mitosis, duplicated MTOCs serve as poles for mitotic spindle

Explain how histone modification can increase or decrease transcription including 2 posttranslational modifications.

- Acetylation - increases access to DNA, increases transcription 1. Histone Acetyl Transferase (HAT) 2. Histone Deacetylase (HDAC) - Histone or DNA Methylation - Decreases access to DNA, decreases transcription 1. DNA methyl transferase (DNMT) 2. Demethylase

Heterochromatin

- Most highly condensed chromatin in which genes cannot be expressed >Used to silence genes >Some DNA is permanently in heterochromatin

Examples of checkpoint proteins DNA damage checkpoint

- P53 - Senses DNA damage >Cycle stopped at G1 >Tumor suppressor gene >>Damage to both copies of P53 gene leads to cancer >>50% human cancers exhibit p53 mutations - Forces defective cells to undergo apoptosis

Interphase - S phase

- S phase promoting factor (SPF) is necessary -Duplication (chromosome and centromeres, centrosomes) dyads are formed; cell is diploid and 4n -Cyclin E broken down -Mitotic cyclins (B and A) formed

Factors that affect resolution

- numerical aperture of the objective lens - wavelength of light; shorter wavelength = higher resolution -Wavelength of light >Shorter wavelengths are better >Visible light allows a resolution of about 0.2 um (200 nm) -Numerical aperture of lens (bigger is better) >Ability of lens to gather light and resolve a point at a fixed distance from the lens -Refractive index of mounting media >Slower speed of light = better resolution

Effects of fixation

- solidification of colloid materials - Hardening of tissue ( fortify and stabalize) - optical differentiation - preserves cells and tissue in a life-like form - stainability • Denature & cross-link proteins • Goals - Immobilize components to maintain structural relationships - Prevent autolysis (self-digestion) - Firm up structures to allow sectioning • Incomplete fixation leads to unsatisfactory results.

Chromosome - Dyad mitosis/ meiosis

-Dyad >Consists of chromosome and its replicate attached at centromeres >Dyads are formed during S-phase of the cell cycle -Chromatids (another name for chromosome and its replicate) >Sister >Non-sister -Dyads condense in early prophase of mitosis and meiosis

Protein degradation

- ubiquitin tagging - proteasome degradation 1. Degradation by proteasomes -utilizes ubiquitin tags (ubiquitin-protein ligase recognizes them- destroys the protein- no lysosome is necessary here. Works via proteasome) 2. Degradation by lysosomes 3. Calcium-dependent enzymes Breakdown proteins -In prokaryotes there is a correlation between the amino acids at the N-terminus and the protein stability -Eukaryotes utilize the ubiquitin pathway to target degradation

Interphase - G0

-"Resting phase" >Cell is active -Non dividing -Differentiation may occur -Cann be temporary >Lymphocytes -Can be terminal stage >Neurons, RBC

Some diseases are caused by accumulation of misfolded protein

-A small error in the genetic blueprint to lead to incomplete folding of a protein, which affects its function. -E.g. excessive quantities of wrongly folded proteins collect in the form of plaques of insoluble protein in the extracellular tissue >Alzheimers >Prion disease causing transmissible spongiform encephalopathies (abnormally folded PrP) >>Bovine spongiform encephalopathy (mad cow disease) >>Scrapie disease in sheep >>Chronic wasting disease in elk

Other "special" stains (histochemistry)

-A wide variety of histochemical reactions can be used to highlight generic tissue components not easily identified with H&E -E.g.Fat,iron,copper calcium, carbohydrates, collagen, components of bacterial cell walls

Cells chromosomes - homologous

-All cells contain pairs of homologous chromosomes plus 2 sex chromosomes -One chromosome from each parent • NOT a chromosome and its duplicate (dyad)

Hemosiderin description

-An iron containing pigment derived from the breakdown of RBCs -Usually found in macrophages >Especially in spleen +/- bone marrow >Seen in any tissue after hemorrhage -Appearance >Fixed H&E stain: Yellow-brown chunky pigment >Cytology smear Wright stain: Blue-green -Prussian blue stain can be used to stain the iron turquoise

Cytokinesis also occurs through actin filament contraction

-At end of mitosis (division of nucleus) -Contractile ring forms under plasma membrane >Actin filaments >Myosin II motor proteins -Contraction of filaments pulls plasma membrane inward -Cell is pinched in two -Ring disperses following cell division

Duplication of chromosomes

-Before cells can divide, the chromosomes need to be duplicated -A chromosome and its replicate/duplicate is called a dyad - Both of the homologous chromosomes need to duplicate - A cell containing dyads would be 4n • In the case of humans this would double the diploid number (2 x 46 = 92 total chromosomes)

Decalcification required for mineralized tissue (e.g. bone, tooth)

-Calcium deposits will not section properly -After fixation, strong acid used to dissolve calcium >Tissue incubated in formic acid, nitric acid or HCl (very dense bone) -Takes 30 min to 7 days (hardbone) -Causes damage to morphology and staining properties

Mitosis overview

-Cell division -5 Stages >Early prophase >Late prophase >Metaphase >Anaphase >Telophase

Cells die by two distinct mechanisms

-Cell murder >Mechanical injury Hypoxia >Infectious agents >Toxins -Cell suicide >Cells no longer needed >Irreparably damaged cells >Extrinsic and intrinsic signals >TNFα

Prokaryote and eukaryote commonalities

-Cells are surrounded by a barrier called a cell membrane. -At some point in their lives, cells contain DNA.

2n chromosomes

-Cells with 2 of each chromosome = 2n >Diploid number of chromosomes = every chromosome including the sex chromosomes >>Human has 22 homologous chromosomes, so 44 individual chromosomes + 2 sex chromosomes for a total of 46 chromosomes = diploid number of chromosomes

Interruption of cell cycle

-Check for completion of S phase -DNA damage checkpoints >Gap -> S >During S >Gap 2 after DNA replication -Spindle checkpoints >Spindle attachment to kinetochores >Spindle alignment -Triggers apoptosis if unable to repair

Lipofuscin details

-Comes from oxidative breakdown of mitochondria and lysosomal digestion -Common in cells with high metabolic rate >Liver >Neurons >Muscle -Appearance depends on stain used >H&E: Brown pigment >Wright (Romanowsky) stain: dark green

Cytology: microscopic study of individual cells

-Common technique -Provides very fast results -Aspirate cells with a syringe (fine needle aspirate) and squirt onto slide -Or touch fresh tissue against a slide

Eukaryotes description

-Contain a defined membrane-bound nucleus -Extensive membrane-bound organelles -Include 4 kingdoms: plants, animals, fungi, and protists.

A new type of exocytosis: Extracellular vesicles facilitate cell-cell communication

-Contain bioactive contents that act on neighboring cells or those far away >MicroRNA (non coding) can affect gene expression >Factors involved in inflammation, angiogenesis, tumor metastasis -May serve as biomarkers

Importance of the Golgi apparatus

-Creates heterogeneous mixture of glycoproteins • Cell signaling • Cell-cell communication • Cell protection, etc. -Creates an asymmetrical plasma membrane -Sends vesicles to their final destinations

Light microscopy - you can see basic structures

-Cytoplasm -Nucleus -Nucleolus -Cell borders

A typical cell is made of a nucleus and cytoplasm

-Cytoplasm includes everything other than the nucleus > Organelles > Cytoskeleton > Inclusions > Cytosol

Meiosis I - Prophase I

-Differs from prophase I of mitosis >As chromatin condenses, dyads (homologous chromosomes) align in pairs (form a tetrad) >Dyads are joined at the inner kinetochores forming synaptonemal complexes >Non-sister chromatids can cross- over and attach, forming chiasmata >Genetic Recombination between chromatids can then occur

DNA (replication) repair systems

-Direct chemical reversal of the damage -Excision repair >Base excision repair (BER) >Nucleotide excision repair(NER) -Mismatch repair (MMR)

Karyosomes

-Discrete clumps of chromatin -Irregular in shape -Scattered throughout nucleus

Microfilaments and microtubules

-Dynamic, internal "skeleton" -Three primary types of protein filaments >Actin microfilaments >Intermediate filaments >Microtubules -Differ in size & Function -Also, 100s of cytoskeleton-associated proteins that regulate the distribution & behavior of cytoskeletal proteins.

Intermediate filaments can be used to identify tumor types

-Epithelial cells -> Cytokeratins (Class I) -Mesenchumal cells (connective tissue, muscle) -> Vimentin and Desmin (Class II) -Nerve cells - Neurofilaments (Class III) -Nucleus (all cells) - Lamins (Class IV)

Lipids (fat droplets) description

-Found in >Adipocytes (fat cells) >Steroid hormone producing cells >Some types of glands Extracted during processing so appear as vacuoles

Apoptosis - microscopic appearance

-Fragments of dark nucleus -Few dead cells -Little to no Inflammation

Microtubules play a number of essential roles in the cell

-Functions >Mitotic spindle >Cilia and flagella >Cytostructural support - anchor organelles >Motor proteins move vesicles along microtubular "rail road tracks" -Disruption of microtubules can have disastrous effects on the cell -Some chemotherapy drugs (e.g. vinblastine) suppress microtubule dynamics

Cytoskeleton summary

-Functions >Stabilizes plasma membrane and maintains cell shape (A) >Hold cells together (A; I) >Anchor organelles (A; T) >Movement of vesicles (A; T) >Cell movement (A) >Changes during mitosis >>Segregation of chromosomes (T) >>Pinching cell apart into two new cells (A)

Interphase - G1

-Growth phase -Increase size -Synthesis >RNA >Proteins -Initiated by rising G1 cyclins -S phase cyclins -SPF formation

Interphase - G2

-Growth, protein sythesis -M-phase promoting factor >Mitotic cyclins B and A + Cdk 1 (Cdc2) -Necessary for: >Assembly of mitotic spindle >Breakdown of nuclear envelope >Condensation of chromosomes

General histologic appearance of the nucleus

-H&E and most other commonly used stains will cause the nucleus to appear a dark blue or purple color. -The nuclei in most cells are round to slightly oval. However, some cell types will have different shaped nuclei. >Smooth muscle cells and fibroblasts can have elongated nuclei. In a cytology smear of these cells, the elongated nuclei will be clearly visible. In a section from paraffin embedded tissue, the orientation of the cut will determine whether or not you can appreciate the elongated shape. A cross section of the cell will make the nucleus appear round. >Some inflammatory cells such as neutrophils, eosinophils and basophils have highly lobulated nuclei. >Monocyte nuclei can be round, oval, bean-shaped or slightly lobular. >Some cells, such as skeletal muscle and osteoclasts, are normally multinucleated. Hepatocytes will sometimes binucleated. Multinucleated cells tend to be very active metabolically.

Microtubules are hollow, nonbranching cylinders

-Heterodimer made of alternating globular tubulin molecules -Alpha tubulin at "minus" end -Beta tubulin at "plus" end >Each beta-tubulin globule is bound to GTP -Each microtubule made of 13 parallel protofilaments -Dynamic structures >"Plus" end is growing >"Minus" end is nongrowing

Actin filaments are important for movement of non-muscle cells using ameboid movement

-Important for WBCs -Role in cancer metastasis -Requires polymerization of actin microfilaments to form pseudopodia (false feet)

Cytosol

-Is an amorphous ground matrix that contains solute such as inorganic ions (Na+, K+, Ca 2+), and organic molecules such as carbohydrates, lipids, proteins, and ribonucleic acids (RNAs). The cell controls the concentration of solutes within the matrix, which influences that metabolic activity within the cytoplasmic compartment.

Membrane bound organelles

-Lipid membranes separate different compartments of the cell, many containing mutually incompatible biochemical reactions. -Double membrane >Nucleus >Mitochondria -Single membrane >Rough endoplasmic reticulum >Smooth endoplasmic reticulum >Transport vesicles >Lysosomes >Endosomes >Peroxisomes >Golgi apparatus

Examples of checkpoint proteins spindle checkpoint

-MAD (mitotic arrest deficient) >Bound to kinetochore until at least 1 microtubule attaches >Without attachment, MAD remains and blocks entry into anaphase >Mutations lead to aneuploidy >>A neuploidy found in many cancer cells (abnormal number of chromosomes in a cell)

Actin filaments functions

-Maintains cell shape -Anchors membrane proteins -Can provide either flexible or stable support >3-D network - especially around cell periphery >Core of microvilli -Motility >Scaffold for myosin (motor protein) in muscle cells >Cellular locomotion of other cells >Cytokinesis after completion of mitosis

DNA repair system:

-Many DNA polymerases have proof-reading exonuclease activity -Scans in a 3' to 5' direction; the opposite direction of DNA replication

Actin and myosin II are also responsible for muscle contraction

-Many myosin II molecules organized into thick filaments that sit between actin filaments >Globular heads bind to actin filaments >Ends of actin filaments anchored to plasma membrane -Contraction occurs through pulling opposing active filaments closer to each other

Where is heterochromatin commonly found?

-Marginal chromatin -Nucleolar-associated -Karyosomes (discrete clumps of chromatin) >Irregular in shape >Scattered throughout nucleus

Peroxisomes

-Membrane bound organelle that may have a crystalline core -Contain enzymes that produce hydrogen peroxide >Peroxidase, urate oxidase >Generated H2O2 used to oxidize & detoxify chemicals such as formaldehyde & alcohol. >Oxidation reactions used to breakdown fatty acids (b-oxidation) - generates acetyl CoA -Catalase converts H2O2 to water -Prominent in hepatocytes (liver cells) & renal tubular cells (kidney) which actively metabolize drugs and toxic compounds

Actin bundles in core of microvilli help them to remain upright

-Microvilli are cell membrane projections that increase surface area >Particularly well developed in cells that absorb things >>Intestine >>Kidney -Microvilli are supported by stable bundles of actin filaments

Mitosis and meiosis overview

-Mitosis - process of cell replication for all cells in the body that divide >Does not include the production of germ cells (sperm and ova) >Parent cells are diploid, 4n >Daughter cells are diploid, 2n -Meiosis - process of cell replication to produce sperm and ova >Meiosis I - first cell division (prophase to telophase) >>Parent cells are diploid, 4n >>Daughter cells are "haploid" (a dyad from one parent), "2n" - Meiosis II - second cell division (daughter cells from meiosis I divide, prophase to telophase) >Parent cells are "haploid" (a dyad from one parent), "2n" • Daughter cells are haploid, 1n

Centrosome

-Mitotic center A structure present in the cytoplasm of animal cells that functions as a microtubule-organizing center and is important during cell division. A centrosome has two centrioles. A structure in animal cells containing centrioles from which the spindle fibers develop. - Paired centrioles - Spindle apparatus (polar microtubules) - Asters - braces for microtubules

P-glycoprotein (P-gp) is a transmembrane pump that transports certain drugs out of cells

-Mutation in multidrug resistance gene (MDR1 gene) found in many herding breeds of dogs: results in a defective p-gp membrane transporter and inability to get drugs out of cells >Certain drugs (e.g. ivermectin used to kill parasites) accumulate in brain causing show neurological symptoms, such as seizures, ataxia, or even death. -Various canine tumors express P-gp: causes resistance to multiple chemotherapy drugs (e.g. vincristine, doxorubicin = multidrug resistance) because the tumor cells pump drugs out of the neoplastic cells >Mast cell tumors >Mammary tumors >Squamous cell carcinomas >Transitional cell carcinomas

Myosin I motor proteins move things along actin filaments

-Myosin I - only 1 head and a short tail >Not contractile >Does not form filaments >Tail is attached to cargo >Single head repetitively binds and releases actin >Moves along actin filaments carrying (e.g. membrane vesicles)

Glucose-Na+ symporters prevent glucose from moving back into the intestinal lumen during fasting

-Na/K transporter in the basolateral membrane expels Na >Creates Na gradient -Glucose-Na symporter on apical surface of intestinal cell, facing lumen. >Na moves down its electrochemical gradient, pulling glucose with it >Cooperative binding - both must be present for transport.

Nuclear appearance in cell death

-Necrosis >Cells swell >Membrane dissolves >Cells burst & contents trigger an inflammatory reaction -Apoptosis >Cells shrink & cytoskeleton collapses >Nuclear chromatin condenses (pyknosis & karyorrhexis) >Nuclear envelope disassembles >Cell forms blebs into fragments (apoptotic bodies) >Macrophages "eat" them

Membrane asymmetry is functionally important

-Negatively charged phosphotidylserine on cytosolic membrane creates a charge difference between two halves of lipid bilayer (membrane potential) -Glycolipids in plasma membrane always face cell exterior forming the protective glycocalyx -Phosphatidylinositol lipid heads used in signaling pathways

Nuclear composition

-Nuclear envelope - double membrane • Inner membrane lined by nuclear lamina • Outer membrane continuous with rER & studded with ribosomes • Nuclear pores filled with nuclear pore complexes -Chromatin -Nucleoplasm -Nucleolus or several nucleoli

Meiosis - Prometaphase I

-Nuclear membrane breaks down -Kinetochores on lateral side of tetrads attach the tetrads to the spindle(tetrads=linked dyads - homologous pairs)

Negatively charged nucleic acids bind basic dyes

-Nuclei appear basophilic (blue) with H&E >Cytoplasm is usually eosinophilic (pink) (mitochondria, smooth ER, proteins) -Nuclei appear purple with Romanovsky stains (e.g. Wright Stain, DifQuik) >Cytoplasm is lightly basophilic or eosinophilic

Some organelles influence the color of the cytoplasm

-Nucleus, ribosomes (nucleic acids) -> Basophilic (blue) -Mitochondria, smooth ER -> Eosinophilic (pink)

Organelles are metabolically active

-Other types of inclusions are metabolically inactive >Fat >Glycogen >Melanin >Hemosiderin >Lipofuscin

Membranes are made up mostly of phospholipids

-Phosphorus atom in head has polar bonds with oxygen atoms (hydrophilic) -Chains of carbon atoms in fatty acid tails are repelled by water - seek to aggregate with other hydrophobic molecules • Result - formation of lipid bilayer • Self-sealing if torn • Other shapes maintained by cellular cytoskeleton

Ploidy versus chromosome number (n)

-Ploidy refers to the genetic contribution to a cell (for each chromosome) >When cells contain homologous chromosomes (one from each parent) they are Diploid - regardless of whether the chromosomes are replicated or not (could be diploid with 2n or 4n). >Cells with chromosomes from only one parent are Haploid - regardless of whether or not the chromosome is replicated (haploid cells could be 1n or 2n - different chromosomes can come from different parents) >N (number of chromosomes) has nothing to do with ploidy >FYI - Tetraploidy is not a normal condition in mammals (plants only)

Melanin

-Produced by melanocytes, but transported into adjacent epithelial cells -Melanin is produced by the oxidation of the amino acid tyrosine, followed by polymerization -Functions >Protection against UV radiation, heat, and chemical damage >Coat and feather coloration >>Feathers with melanin are stronger than those without it >>Possible part of sexual displays >Ink used by many cephalopods

The smooth endoplasmic reticulum is involved in...

-Production & storage of carbohydrates -Detoxification of some drugs (p450 enzymes) -Sequestration of Ca for muscle contraction -Steroid hormone & lipoproteins synthesis -Synthesis of fatty acids & phospholipids >Membrane formation and recycling: major source of lipid for organelles in the endomembrane system

Prokaryotic vs eukaryotic translation

-Prokaryotes - ribosomes recognize a sequence called the Shine-Dalgarno sequence, where they bind to initiate translation -Eukaryotes - ribosomal subunits recognize and bind to the 5' cap -In eukaryotes there are many more protein factors involved (there are 3 Ifs in prokaryotes and at least 10 in eukaryotes) -In eukaryotes, the poly A tail likely increases rate of re- initiation

Stages of meiosis

-Prophase I >(can be divided into early and mid prophase) -Prometaphase I >(can be called late prophase) -Metaphase I -Anaphase I -Telophase I

Meiosis II stages

-Prophase II - Single dyad of each chromosome - "2n", "haploid" -Prometaphase II - like mitosis -Metaphase II - a single dyad of each chromosome lines up on metaphase plate -Anaphase II - each dyad separates and the chromosome and its replicate are pulled to opposite poles (like mitosis) -Telophase - like mitosis -Daughter cells are 1n, haploid

Ribosomes are responsible for protein synthesis

-Proteins that will remain in cytoplasm synthesized by free ribosomes -Proteins destined for export or other organelles: ribosomes bind to rER >Each type of organelle has characteristic membrane & lumen proteins, phospholipids and other specialized molecules related to its function

Clinical notes on organelles

-Ribosomal RNA is the target of several clinically relevant antibiotics, including chloramphenicol and erythromycin -Ribosomal RNA is the target of ricin, a highly toxic agent produced in the season the castor oil plant. This molecule prevents cells from assembling amino acids into proteins, which is an essential process in all living cells. -Genes that encode the rRNA (rDNA) are sequenced to identify an organism's taxonomic group, calculate related groups, and estimate rates of species divergence.

Things that a clinician can control in histology

-Sample collection and submission >Provide pathologist with a clear description of the lesion (location, size, color, consistency) -Use surgical ink to mark mass margins (provide a key with colors and which margin they identify) -Sample cut into pieces that fit on glass slide -Fixation

Catalyzed transbilayer movement

-Scramblase • In sER - creates symmetrical membrane -Flippase/Floppase • In Golgi - creates asymmetrical membrane

The Golgi sends vesicles to their final destinations

-Secretory vesicles (e.g. collagen, hormones) -Components for plasma membrane (e.g. receptors) -Components of intracellular organelles (e.g. lysosomes)

Myosin produces cellular contraction by sliding actin filaments in opposite directions

-Several types of myosin motor proteins >1-2 heads & a tail >Head repetitively binds and releases actin filament in a swinging motion, moving down filament >Hydrolyze ATP in process -Myosin pulls actin filaments towards each other >1 anchored to back of cell >1 anchored further forward in the cell

Chromosome

-Single molecule of DNA -Double helix formation -De-condensed and single chromosome in G0 and G1 >Chromosome needs to be de- condensed for genes to be expressed (see notes) -Chromosomes are condensed during mitosis and meiosis >Visible under a microscope -Chromatin >Mixture of DNA and proteins that form the chromosomes

Smooth muscle histology background

-Smooth muscle has cigar shaped nuclei -Two thick layers of smooth muscle responsible for intestinal peristalsis. >Inner layer wraps like a cuff >Outer layer runs longitudinallydown length of intestine. >Oval nuclei follow direction of muscle fibers

Various cytosolic proteins can stabilize or destabilize the actin filament

-Some bind actin monomers (e.g. profilin) >Prevent binding to filaments >Maintains reserve pool of monomers -Capping protein - stabilizes plus end of filament -Another protein severs the filament

Bundling proteins can provide stronger, stable actin structures

-Some promote nucleation -Some cross link actin into parallel arrays -Some bundle actin filaments at an angle to produce a web like network

Actin filaments and microtubules: both can show spontaneous depolymerization and treadmilling

-Subunits tend to dissociate from minus end and can be reused at plus end (treadmilling) >ATP/GTP molecule added to plus end >Binding causes hydrolysis >ADP/GDP molecule dissociates from minus end >ADP/GDP replaced by ATP/GTP >ATP/GTP molecule added to plus end

New phospholipids are produced in smooth endoplasmic reticulum (ER)

-Synthetic enzymes bound to cytosolic surface of smooth ER -Enzymes randomly move phospholipases from one leaflet of ER membrane to the other - both leaflet are the same -ER membrane pinches off to form vesicles that fuse with other organelle membranes.

Actin cytoskeleton is a potentially vulnerable property of cancer cells

-Targeting this protein might: >Block metastasis of tumor cells >Block cytokinesis of the tumor cells

RNA polymerase in eukaryotes

-The majority of eukaryotes initiate transcription using the TATA box region at approximately -30bp -TATA box consensus sequence is recognized by the RNA polymerase and directs it where to initiate transcription Pol I : large ribosomal RNA Pol II: messenger RNA (mRNA) Pol III: small rRNA, tRNA and other small RNA

RNA polymerase in prokaryotes

-The sigma region of the bacterial RNA polymerase recognizes specific regions of DNA called promoters -At approximately 10 and 35 base pairs from the start site there are similar DNA sequences termed the -10 and -35 sequence

Actin filaments

-Thinnest component of cytoskeleton -Made of globular actin monomers >Each with ATP binding site -Polymerize into a microfilament (6-8 nm) >Two strings of beads twisted together >Monomers oriented in one direction producing polarity >"Plus end" and a "minus" end -Dynamic structures >"Plus" end is growing

Frozen sections avoid some precessing artifacts

-Tissue hardened by rapid freezing & sectioned in cryostat -Rapid results -Eliminates artifacts caused by formalin& alcohol dehydration -Poor morphology

Proteins have variety of functions & are associated with the membrane in different ways

-Transporters (channels, pumps) -Receptors -Enzymes -Linkers between interior & exterior of cell -Anchors of membrane to cytoskeleton or extracellular matrix

Homologous pairs of chromosomes (G0 and G1)

-Two copies of each chromosome (2n) >One maternal >One paternal -Cells that have one copy from each parent are diploid >(Whether or not the chromosomes are replicated - in G0 and G1 they are not replicated)

Histological appearance of the nucleus can vary

-Usually round to oval -Can be lobular or cigar shaped

Immunohistochemistry

-detecting proteins (antigens) in cells or tissues using antibodies tagged with fluorescent labels -localization of antigens or proteins in tissues using labeled antibodies -localizing antigens or proteins in tissues using labeled (colored or fluorescent) antibodies -Antibody against a specific molecule used to label that molecule in a tissue section

Key components of translation

-tRNA -Ribosomes -mRNA

Factors that affect fixation

1) Time until tissue placed in fixative 2) Fixative volume: biopsied tissue should be fixed in at least 10x its volume in 10% buffered formalin and submitted in a leak-proof container. 3) Tissue thickness - Keep it thin!! For optimal fixation, the dimensions of the specimen should be no greater than 1 cm x 1 cm x 1 cm. At times it may be important to submit a larger piece of tissue - for example it may be important to preserve tissue landmarks such as the fact that a tumor is located in the subcutis. In this case, the larger piece of tissue should be "breadloafed", with each incision made at 0.5-1 cm intervals to allow penetration of formalin. 4) Tissue Type: blood, fat, skin, thick connective tissue capsule can take longer to fix!

Organelles involved in protein synthesis

1) ribosomes (on RER) 2) vesicle 3) golgi apparatus 4) vesicle 5) plasma membrane 1. Nucleus 2. Mitochondria 3. ribosomes 4. Endoplasmic Reticulum 5. Golgi Apparatus -Ribosomes & mRNA produced in nucleus Ribosomes translate the mRNA to make proteins >Polyribosomes (cytoplasmic proteins) vs rough endoplasmic reticulum (organelles, secreted) >Protein folding is essential for their function >Misfolded proteins are degraded -Proteins in rER are modified then transported to Golgi apparatus via vesicular transport -Proteins are further modified in Golgi apparatus -Vesicular transport carries them to other organelles, the plasma membrane or secretory vesicles/granules for export

Cell theory

1. All living organisms are composed of one or more cells. 2. The cell is the most basic unit of structure and function in living things. 3. All cells arise from pre-existing, living cells.

Protoplasm includes

1. Cytoplasm - this includes the organelles and the cytosol, an amorphous ground matrix. This cytoplasmic matrix contain solutes such as inorganic ions (Na, K, Ca), and organicmolecules such as metabolites,carbohydrates, lipids, proteins, andribonucleic acids (RNAs). The cell controls the concentration of solutes within the matrix, which influences the metabolic activity within the cytoplasmic compartment. 2. Nucleoplasm - The nucleoplasm is a highly viscous liquid that surrounds the chromosomes and nucleoli and is enveloped by the nuclear membrane or nuclear envelope.

Explain the structure and function of DNA and how it is packaged in chromatin.

1. DNA is the carrier of genetic material. It contains the information about how, when, and where to produce each kind of protein in every cell. 2. DNA is composed of monomers called nucleotides that make specific pairs (A-T; G-C). 3. DNA forms a double helix, which winds around proteins called histones forming nucleosomes which are packaged tightly into chromatin which forms chromosomes.

List 5 enzymes involved in DNA replication and define their specific roles

1. DNA must be precisely copied during DNA replication in preparation for cell division to avoid alterations in protein product. 2. DNA helicase unwinds DNA to allow access to the template strand 3. Topoisomerase I relieves torsional stress caused by unwinding of the DNA 4. DNA primase is a specialized RNA polymerase that forms a short RNA primer that is complementary to the unwound template. It is necessary because 5. DNA polymerase requires a double stranded section to initiate elongation. DNA polymerase uses the template strand to add dNTPs in a 5' to 3' direction. 6. DNA ligase joins the Okazaki fragments on the lagging strand

What advantages does a nucleus give eukaryotic cells over prokaryotic cells? Some of these are:

1. Development of larger genomes-the genome of most mammals is 3 times larger than bacteria. Packaging such a large genome while keeping it functional is one of the chief functions of a nucleus. 2. Spatial separation of transcription and translation-In prokaryotes mRNA in transcribed and translated simultaneously. The separation of these processes in eukaryotes allows mRNA to be translated where the protein is needed. This is very important in constructing complicated highly specialized cells. Separation of transcription and translation also allows the processing of transcripts before they are translated. This is done by RNA splicing and editing. Higher eukaryotes perform very extensive RNA processing which would be hard to achieve in a newly forming transcript that is already being translated. 3. Gene regulation through control of nuclear to cytoplasmic translocation- In eukaryotic cells RNA, transcriptional regulators and RNA processing factors shuttle back and forth between nucleus and cytoplasm. 4. Every cell in an animal's body has the same genetic information but there is different expression of this information in each cell type - this is the reason for cell differentiation.

Changes in nucleosome structure allow access to DNA, so that it can be transcribed into RNA.

1. During interphase, chromosomes are extended as long, thin, tangled threads of DNA nucleus and cannot be easily distinguished in the light microscope (interphase chromosomes). However, interphase chromatin is not uniformly packed. Instead regions of the chromosome that contain genes that are being expressed are generally more extended, while those that contain silent genes are more condensed. Thus, the detailed structure of the interphase chromosome can differ from one cell type to the next, helping to determine which genes are expressed. Most cell types express about 20 to 30% of the genes they contain. a. Heterochromatin, is the most highly condensed form of interphase chromatin. It has a pattern of histone tail modification that causes the DNA to become so highly condensed that it's genes cannot be expressed to produce RNA. (Note that the same condensation occurs in all chromosomes during mitosis). It typically makes up about 10% of an interphase chromosome, and is concentrated around the centromere region, and in the telomeres at the end of the chromosomes. 1) Some DNA is permanently folded into heterochromatin, and most of this DNA does not contain genes. Because heterochromatin is so compact, genes that accidentally become packaged into heterochromatin usually fail to be expressed. This can result in disease. 2) Heterochromatin can also be used to shut down or silence genes. a) For example, condensed chromatin codes important developmental regulatory genes that encode for regulatory proteins early in embryonic development. This code is removed only when each individual gene is needed by the developing organism. b) Another example is the use of heterochromatin to permanently inactivate one of the two X chromosomes in each female cell. This is essential because a double dose of X-chromosome products would be lethal. At random, one or other of the two X chromosomes in each cell becomes highly condensed into heterochromatin early in embryonic development. This is often called a Barr body. Thereafter, the condensed an inactive state of that X-chromosome is inherited in all of the many descendants of those cells. 3) Depending on the level of metabolic activity, the percentage of heterochromatin can vary from cell type to cell type. Some cells, such as mature neutrophils, a type of WBC, are no longer transcribing their DNA and their chromatin is largely in the form of heterochromatin. a. The remainder of the chromatin, scattered throughout the nucleus and not visible with the light microscope, is euchromatin. This is the uncoiled or stretched out, active regions of DNA, in which the genetic material of DNA molecules is being transcribed into mRNA. Consequently, cells with lots of euchromatin are metabolically active and include neurons and liver cells. b. Each type of chromatin structure is established and maintained by different sets of histone tail modifications that attract distinct sets of non-histone proteins. Once heterochromatin is established, it can spread because the histone tail modifications attract a set of heterochromatin-specific proteins which create the same histone tail modifications on adjacent nucleosomes. This change in the chromatin pattern can continue to spread until it encounters a barrier DNA sequence stops the propagation. In this manner extended regions of heterochromatin can be established along the DNA. c. The difference in DNA packing is visible in H & E stained sections and with electron microscopy. 1) Heterochromatin - With light microscopy, it is heterochromatin that binds basic dyes and accounts for the blue to purple color of nuclei (depending on the stain used). Heterochromatin appears densely stained and often clumped. On a cellular level, heterochromatin is most often found in 3 locations. a) Marginal chromatin - found along the peripheral ob the nucleus. It is impossible to actually see the nuclear membrane using a light microscope, but you may see a line in this area composed of marginal chromatin. b) Karyosomes are discrete bodies of heterochromatin, irregular in size and shape, scattered throughout the nucleus. c) Nucleolar-associated heterochromatin usually rims the nucleolus. d) Barr bodies cansometimes appear as adistinct clump ofheterochromatin adjacentto the nuclear membraneor occasionally theyappear to project from thenuclear membrane (e.g. epithelial cells scraped from the inside of the cheek or large motor neurons). In some WBCs the Barr body forms a drumstick-shaped appendage on one of the lobes of the ribbon shaped nucleus. 2) Euchromatin is not actually visible in the light microscope - it is present with the nucleoplasm in the "clear" areas between the darkly stained heterochromatin. e. Disruption of this balance between euchromatin and heterochromatin can have disastrous effects on normal cell functions, and can lead to unrestricted cell growth, resulting in the development of cancer.

Name 2 endogenous and 4 exogenous forms of DNA damage and identify 3 DNA repair systems.

1. Endogenous damage (reactive oxygen species and replication errors) 2. Exogenous damage (radiation, hydrolysis, toxins, viruses) 3. Repair systems: (1) direct chemical reversal (2) Base excision repair (3) Nucleotide excision repair (4) mismatch repair and proof reading

Tissue Processing Steps

1. Fixation 2. Embedding *Dehydration *Clearing 3. Sectioning 4. Mounting 5. Dewaxing

Steps of actin and myosin working together to produce ameboid movement

1. Growth (polymerization) of actin myofilaments in leading edge 2. Myosin on cell membrane pulls actin strand forward - pushing leading membrane forward and contracting cell 3. Depolymerization of actin filaments in back of cell 4. Cytoplasmic streaming 5. Leading edge may be attached to substrate via binding proteins (integrins) or by electrostatic interactions

Describe the ubiquitin system of protein degradation in eukaryotic cells

1. In prokaryotes, stability is correlated with amino acids at the N- terminus 2. Eukaryotes utilize the ubiquitin degradation system a. Ubiquitinylation begins with the ATP-dependent activation of ubiquitin by the ubiquitin-activating enzyme E1 b. Step 2: Ubiquitin is then transferred to E2, or ubiquitin conjugating enzyme c. Step 3: The addition of ubiquitin to the protein substrate is catalyzed by E3 d. This chain of ubiquitins is recognized by a large protein complex called a proteasome which degrades the target protein

Steps of translation

1. Initiation 2. Elongation 3. Termination

Define and describe the three steps of transcription (initiation, elongation, and termination)

1. Initiation - RNA pol recognizes and binds a specific promoter site, separates the DNA strands to expose the template strand and begins synthesis of RNA 2. Elongation - Elongation occurs along the DNA molecule and therefore DNA unwinding must occur. RNA polymerase has "unwindase" activity that opens the DNA helix. Topoisomerases precede and follow the progressing polymerase to prevent super helical complexes. RNA is elongated in the 5'-3' direction, therefore DNA is read in the 3'-5' direction 3. Termination - Rho dependent, rho independent (hairpin loop), and downstream terminator sequence

List and describe 3 post-transcriptional modifications that occur in eukaryotic cells

1. Methylated guanine cap is added to the 5' end of the mRNA a. Ribosomal subunits recognize the 5' cap and use it to initiate translation (in eukaryotes) 2. A poly A tail is added to the 3' end of the mRNA a. We take advantage of this in the lab and can use an oligo dT primer when examining expression levels of mRNA for use in PCR 3. The RNA is spliced to remove introns a. Alternative splicing can provide different protein products as well as a mechanism for differential regulation of the same protein

The basic eukaryotic cell contains the following:

1. Plasma membrane 2. Glycocalyx (components external to the plasma membrane) 3. Cytoplasm (semifluid) 4. Cytoskeleton-microfilamentsand microtubules that suspendorganelles, give shape, and allowmotion. These are often classified as nonmembranous organelles. 5. Presence of characteristic membrane enclosed subcellular organelles, including the nucleus.

Eukaryotic translation

1. Pre-Initiation Complex 2. Initiation 3. Elongation 4. Termination 1. The 40S ribosomal subunit (attached to various initiation factors and tRNAiMet) scans mRNA from the 5' cap. 2: The Kozak sequence is found 3. The 60S ribosomal subunit binds the 40S subunit 4. Elongation factors bring an amino-acylated tRNA molecule into the A site of the ribosome. 5. 28S rRNA activity 6. The 80S ribosome advances toward the 3' end of the mRNA to the next codon 6 -ribosomal subunits recognize and bind to the 5' cap -In eukaryotes there are many more protein factors involved (there are 3 Ifs in prokaryotes and at least 10 in eukaryotes) -In eukaryotes, the poly A tail likely increases rate of re- initiation

Describe the role of the RNA polymerase in transcription.

1. RNA polymerase is the primary enzyme responsible for transcription (always in the 5'-3' direction) 2. In prokaryotes, RNA polymerase sigma region recognizes promoter regions in the DNA 3. In eukaryotes, there are 3 RNA polymerases a. Pol I : large ribosomal RNA b. Pol II: messenger RNA (mRNA) c. Pol III: small rRNA, tRNA and other small RNA 4. Pol II often uses the TATA box consensus sequence to initiate transcription 5. Basal expression and regulated expression in eukaryotes are controlled by proximal promoter elements and distal regulatory elements respectively

Exogenous DNA damage

1. Radiation 2. Hydrolysis 3. Toxins 4. Viruses

Endogenous DNA damage

1. Replication errors 2. Attack by reactive oxygen species

List the factors that affect fixation

1. Time until tissue is placed in fixative 2. Fixative volume (minimum of 10 parts formally for 1 part of tissue) 3. Fixation time >At least 24 hours fixation for small tissues >Up to seven days for whole tissues, big masses >Under-fixation is more of a problem than over fixation for most histologic procedures 4. Tissue thickness - keep it thin >Take picture - then ship one or more small pieces of the mass in formalin >Submit entire mass cutting partial slices into it >Bread loafing so that each slice is about 1 cm 5. Tissue type: blood, fat, skin, connective tissue capsule take longer

Late Prophase (Prometaphase)

1. nuclear envelope disintegrates 2. spindle fibers attach to the kinetochore the mitotic spindle begins to capture and organize the chromosomes. nuclear envelope disintegrates, spindle fibers attach to the kinetochore -M-phase promoting factor -Nuclear membrane dissolves -Interaction of microtubules and chromosomes >Kinetochores >Polarity important

Name the RNA components (mRNA, tRNA, ribosomes) involved in translation and describe each components role in the translation process.

1. tRNA - transfer RNA, responsible for transferring amino acids to the ribosome during translation a. Anticodon site - 3 bases that bind to the RNA and pair with the codon of the mRNA. Indicates which amino acid will be added to the peptide chain. b. Amino acyl site - carries a specific amino acid based on the anticodon site. A tRNA molecule can only carry one specific amino acid. 2. Ribosomes - Site of protein synthesis. a. A site - amino acyl binding site - contains the 3 base codon of the mRNA and directs which tRNA will enter next b. P site - peptidyl binding site - contains the growing peptide chain c. E site - exit site - tRNA exits 3. mRNA - The code that dictates the order of amino acids in a protein 4 .Eukaryotic translation involves binding to the 5' cap rather than the shine-dalgarno sequence, is much more complicated, and the poly A tail increases the rate of re-initiation of translation

Nuclear envelope

2 concentric membranes, which are penetrated by nuclear pore complexes. There is a perinuclear cisternal space between the two membranes. The nuclear envelope provides a selectively permeable barrier enclosing chromatin. Bidirectional traffic occurs continuously between the cytosol in the nucleus. A. Although the inner and outer nuclear membranes are continuous, they maintain distinct protein compositions. 1. The inner nuclear membrane is supported by the nuclear lamina, arigid network of filamentous proteins that gives shape and stability to the nuclear membrane and that may serve as scaffolding for chromatin. The nuclear lamina are anchored to the nuclear pore complexes, to integral membrane proteins of the inner nuclear membrane, and to the chromatin. The nuclear lamina is composed of proteins called lamins which are similar to cytoplasmic intermediate filaments, however, unlike cytoplasmic intermediate filaments, nuclear lamins can disassemble during mitoses and reassemble when mitoses ends. 2. The outer nuclear membrane is continuous with the membrane of the rough endoplasmic reticulum (rER). The perinuclear cisternal space, between the inner and outer nuclear membrane, is continuous with the cisternal space of the rER. Like the membrane of the rER, the outer nuclear membrane is studded with ribosomes engaged in protein synthesis. The proteins made on these ribosomes are transportedinto the perinuclear space. B. Nuclear pore complex (NPC): At numerous sites the paired membranes are punctuated by an opening formed through merging of the inner and outer membranes. Although the nuclear pore is relatively large, it is nearly filled with the structures constituting the nuclear pore complex (NPC). 1. Eight protein subunits are arranged in an octagonal framework at the periphery of each pore, forming a cylinder-like structure known as the. The NPC control the bidirectional flow of molecules between the cytosol in the nuclear compartment. Histones, DNA and RNA polymerases, gene regulatory proteins, and RNA-processing proteins are selectively imported into the nuclear compartment from the cytosol, where they are made. tRNAs and mRNAs are synthesized nuclear compartment and then exported to the cytosol. The export process is selective; mRNAs are only exported after they have been properly modified by the RNA-processing reactions in the nucleus. 2. Because of the structural conformation of those subunits, several 9- to 11-nm wide channels are available for simple passive diffusion of ions and small water soluble molecules (< 5000 dalton). 3. Large proteins traverse the NPC much more slowly. Some NPC proteins that line the central pore contain extensive unstructured regions, which are thought to form a disordered tangle that blocks the central opening to the passage of large macromolecules, while leaving small openings to allow the diffusion of smaller molecules. Large molecules are selectively transported via a receptor-mediated transport process. Signal sequences of molecules to be transported through the nuclear pores must be recognized by one of the many receptor sites of the nuclear pore complex. Transport across the nuclear pore complex is frequently an energy-requiring process. a. The bidirectional traffic between the nucleus and the cytoplasm is mediated by a group of target proteins containing nuclear localization signals (NLSs), along with either nuclear import receptors known as importins, or nuclear export receptors (NESs), known as exportins, depending on the direction of transport. These cytosolic proteins bind to special receptor sites in the nuclear pore complex. Exportins transport macromolecules (e.g., RNA) from the nucleus into the cytoplasm, whereas importins transport cargo (e.g., protein subunits of ribosomes) from the cytoplasm into the nucleus. Exportin and importin transport is regulated by a family of GTP-binding proteins known as Ran.

Microtubules shape and diameter

20-25 nm Nonbranching, hollow cylinders

Tissues are built from about ... types of cells

200

Actin shape and diameter

6-8 nm Double stranded, linear array

Intermediate filaments shape and diameter

8-10 nm Rope like fibers

Nuclear appearance in apoptosis

>Cells shrink & cytoskeleton collapses >Nuclear chromatin condenses (pyknosis & karyorrhexis) >Nuclear envelope disassembles >Cell forms blebs into fragments (apoptotic bodies) >Macrophages "eat" them

Nuclear appearance in necrosis

>Cells swell >Membrane dissolves >Cells burst & contents trigger an inflammatory reaction

Transmembrane proteins made in the rER

>Destined for plasma membrane >Destined for membrane of endomembrane organelle >Become embedded in rER membrane

Water soluble proteins made in the rER

>Proteins are modified and folded >Packed into vesicles which are ultimately transported to Golgi (and beyond) >Injected into lumen by ribosome

Symporter

A carrier protein that transports two molecules across the plasma membrane in the same direction. For example, the Na+-glucose cotransporter in intestinal cells is a symporter. moves two substances in the same direction transporter that carries two different ions or small molecules, both in the same direction

Intermediate filaments

A component of the cytoskeleton that includes all filaments intermediate in size between microtubules and microfilaments A component of the cytoskeleton that includes filaments intermediate in size between microtubules and microfilaments. Threadlike proteins in the cell's cytoskeleton that are roughly twice as thick as microfilaments -Structural role -Stable rope-like filaments (8-10 nm) -Functions >Maintain cell shape >Anchor organelles >Cell-cell junctions >Cell-matrix junctions -Exceptions: laming in nucleus disassemble

Cytochrome c

A crucial protein released from mitochondria in the intrinsic pathway of apoptosis is cytochrome c, a water-soluble component of the mitochondrial electron- transport chain. When released into the cytosol, cytochrome c has an entirely different function, binding to a procaspase-activating adapter protein called apoptotic protease activating factor-1 (Apaf1). This causes Apaf1 to form a wheel-like structure called an apoptosome.

Karyotype

A display of the chromosome pairs of a cell arranged by size and shape. - Chromosomes are condensed • Only condensed when cells are dividing in mitosis or meiosis

Lamins

A group of intermediate filament proteins that form a fibrous network, the nuclear lamina, on the inner surface of the nuclear envelope. The type of intermediate filament found inside the nucleus.

Nucleoplasm

A highly viscous liquid that surrounds the chromosomes and nucleoli and is enveloped by the nuclear membrane or nuclear envelope.

Enzyme histochemistry (cytochemistry)

A method for localizing cellular structures using a specific enzymatic activity present in those structures

Both acid filaments and microfilaments grow from...

A nucleation site -Nucleation site (2 monomers bind weakly; 3 form a more stable group >Actin: nucleation often at plasma membrane >Microtubules: nucleation often at microtubule organizing center (MOC) -Binding of subunits causes hydrolysis of ATP (actin) or GTP (microtubules), which decreases strength of binding - dynamic instability

Rho-dependent termination

A protein called Rho-factor recognizes termination signals; a sequence of about 40-60 nucleotides that is C rich and G poor and includes an upstream segment called the rut site. Rho binds the nascent RNA at the rut site facilitation the release of the RNA from the polymerase. in prokaryotes, termination of transcription by an interaction between RNA polymerase and the rho protein at a run of G nucleotides on the DNA template Rho protein recognizes specific DNA sequences and causes a pause in the RNA polymerase

Nuclear lamina

A rigid network of filamentous proteins that gives shape and stability to the nuclear membrane and that may serve as scaffolding for chromatin. The nuclear lamina are anchored to the nuclear pore complexes, to integral membrane proteins of the inner nuclear membrane, and to the chromatin. The nuclear lamina is composed of proteins called lamins which are similar to cytoplasmic intermediate filaments, however, unlike cytoplasmic intermediate filaments, nuclear lamins can disassemble during mitoses and reassemble when mitoses ends.

Signal sequence

A short sequence of amino aids, usually found at the N-terminus of a protein being translated, that directs the ribosome and its associated mRNa to the membranes of the rough ER where trasnlation will be completed. Signal sequences are found on membrane-boudn proteins, secreted proteins, and proteins destined for other organelles. Short tail of amino acids that directs a protein to a specific cellular compartment The sequence within a protein that directs the protein to a particular organelle.

Coccus

A spherical bacterium.

Spirillum

A spiral-shaped bacterium.

Autophagosome

A vesicle containing internal organelles enclosed by fragments of cytoplasmic membranes that fuses with lysosomes. A double-membrane structure enclosing cellular material destined to be degraded; produced by the process of autophagy. a vesicle formed around a worn-out organelle; this vesicle then fuses with a lysosome which digests the organelle.

Formation and movement of transport vesicles

A. Coated vesicles: Most transport vesicles form from specialized coated regions of membranes, and bud off as coated vesicles which have a distinctive cage of proteins covering their cytosolic surface. The coat performs two main functions: 1. It concentrates specific membrane proteins in a specialized patch, which captures appropriate molecules for transport 2. The coat which helps shape the membrane into a vesicle. It does so by deforming the membrane patch to create a bud that enlarges into a vesicle. 3. Before vesicles fuse with a target membrane, they discard their coat, as is required for the two cytosolic membrane surfaces to interact directly and fuse. 4. Note: there is significant variety in coated vesicles and their functions, which allows different types to be used for different transport steps. B. There are 3 well-characterized types of coated vesicles, distinguished by their coat proteins. Each type is used for different transport steps. 1. One type, known as clathrin-coated vesicles mediate transport from the plasma membrane and between the endosomal and Golgi compartments. a. Clathrin is known to assemble into a basketlike convex framework that deforms the membrane to form coated pits on the cytosolic surface of membranes b. Adapter proteins in the vesicle help to trap various transmembrane proteins inside the vesicle, including receptors that capture soluble cargo molecules. There are several types of adapter proteins, each specific for a different set of cargo receptors. c. Cytoplasmic proteins regulate the pinching off of coated vesicles. C. Most vesicles are actively transported by motor proteins that pull the vesicle along cytoskeletal fibers, such as microtubules or actin filaments. D. Vesicle docking - after a transport vesicle buds from a membrane, it must recognize and dock with its specific organelle (or the plasma membrane). This is achieved by the presence of molecular markers on the vesicle's membrane that identify the vesicle according to its origin and cargo. These markers must be recognized by complementary receptors on the target membrane. 1. Identification is provided by Rab proteins, a diverse family of GTPases. Each organelle and each type of transport vesicle carries a unique combination of Rab proteins. 2. Each Rab protein (red structure on vesicle in diagram) is recognized by a corresponding tethering protein (purple structure) on the cytosolic surface of the target membrane. 3. Docking is further assisted by transmembrane proteins called SNAREs (blue and yellow structures). Once a tethering protein has captured a vesicle by binding to its Rab protein, SNAREs on the vesicle (v- SNAREs) interact with complementary SNAREs on the target membrane (t- SNAREs) to firmly hold the vesicle in place. The SNAREs also catalyze fusion of the vesicle membrane with the target membrane. This not only delivers the cargo, but also adds the vesicle's membrane to the membrane of the organelle. E. Exocytosis: In this process, vesicle membranes fuse with the plasma membrane, either dumping the contents into the extracellular space or delivering integral membrane proteins, and new lipids, to the cell surface. 1. Constitutive secretion- a steady stream of vesicles buds from the trans Golgi network and fuses with the plasma membrane in the process of exocytosis. This constitutive exocytosis pathway occurs continuously regardless of environmental factors, and does not require a particular signal sequence like those that direct proteins to endosomes or back to the ER. a. This process supplies the plasma membrane with newly made lipids and proteins. b. It also carries soluble proteins to the cell surface to be released to the outside - a process known as secretion. Some of the secreted proteins remain attached to the cell surface, while others are either incorporated into the extracellular matrix or others diffuse into the extracellular fluid to nourish or signal other cells. . No external signals are needed to initiate this process. Fibroblasts (produce collagen), osteoblasts (form bone) and chondrocytes (form cartilage) are some of the many cells that perform constitutive secretion 2. Regulated secretion (a.k.a. regulated exocytosis pathway) - this process only occurs in cells that are specialized for secretion. a. Each specialized cell produces large quantities of a particular product (e.g. hormone, mucus, digestive enzymes) which are stored within secretory vesicles (sometimes called secretory granules) for later release from the cytosol. These vesicles bud off the trans Golgi network and accumulate near the plasma membrane (often on one side of the cell - perhaps near the organ lumen or near a blood vessel). b. Exocytosis (and secretion of the proteins) only occurs if the cell receives an extracellular signal such as stimulation by a nerve or hormone (e.g. an increase in blood glucose triggers release of insulin from beta cells in the pancreas). Many types of glands (e.g. exocrine pancreas, thyroid gland) use regulated secretion. c. Proteins destined for regulated secretion are sorted and packaged in the trans Golgi network. These proteins have special surface properties that cause them to aggregate with one another under the ionic conditions (acidic pH and high Ca+2) found in this area of the Golgi apparatus. The aggregated proteins are packaged into secretory vesicles which pinch off from the network and await a signal instructing them to fuse with the plasma membrane. The secretory vesicles have unique proteins in their membranes, some of which might serve as receptors for the aggregated proteins - this may, help explain how the aggregates of secretory protein are segregated into secretory vesicles. 1) Aggregation allows high concentrations of secretory proteins to be packaged into secretory vesicles, so that they can release large amounts of the protein promptly when triggered to do so. 2) Initially the immature secretory vesicles resemble dilated trans Golgi cisternae that havepinched off form the Golgistack. However, as they mature they can fuse with one another and their contents become even more concentrated. Hence mature secretory vesicles (secretory granules) appear very dark in an electron micrograph and may appear as eosinophilic granules in a section stained with H&E viewed with a light microscope. 3) By contrast, proteins forconstitutive secretion do notaggregate and are thereforecarried automatically to theplasma membrane by thetransport vesicles. Thesevesicles contain lower concentrations of their proteins. F. Endocytosis: Cells are continually taking up fluid, as well as large and small molecules, through the process of endocytosis. The material ingested is progressively enclosed by a small portion of the plasma membrane, which buds inward and then pinches off to form an intracellular endocytic vesicle. The ingested material, including membrane components, are delivered to endosomes, from which they can be recycled to the plasma membrane or sent to lysosomes for digestion. 1. There are three types of endocytosis: 1) pinocytosis, 2) receptor- mediated endocytosis, and 3) phagocytosis 2. Pinocytosis ("cellular drinking") - the non-specific ingestion of fluid and small protein molecules, along with bits of their plasma membrane, via small vesicles (< 150 nm in diameter). This process is carried out mainly by clathrin-coated pits and vesicles. Extracellular fluid is trapped within the coated pit as it invaginates, and so any substances dissolved in this fluid are internalized and delivered to the endosomes. Pinocytotic vesicles are especially numerous in endothelial cells that line blood vessels. 3. Receptor-mediated endocytosis - this is actually a specialized form of pinocytosis that allows the cell to ingest specific molecules. The macromolecules bind to complementary receptors on the cell surface and both enter the cell as a receptor-macromolecule complex in clathrin- coated vesicles. This is a much more efficient process than routine pinocytosis, allowing uptake of large numbers of a given molecule without a correspondingly large volume of extracellular fluid. Receptor- mediated endocytosis is used to take up many essential metabolites such as iron, and many cell-surface receptors bind extracellular signal molecules that are taken up by this route. 4. The endocytosed materials are sorted in the endosomes. The acidic environment causes many receptors to release their bound cargo. Cargo proteins that remain bound to their receptor share the fate of their receptor. The routes taken by receptors varies depending on the receptor a. Receptors are returned to the plasma membrane, to the same domain they came from. b. Receptors are returned to the plasma membrane, but to a different domain, carrying their bound cargo to this new location. c. Some travel to lysosomes where they are degraded 5. Phagocytosis ("cellular eating") -the ingestion of large particles, such as bacteria, cell debris and other foreign material. In this process large vesicles (>250 nm in diameter) called phagosomes are produced. Phagocytosis is usually performed by specialized cells called phagocytic cells - including macrophages or histiocytes which are members of the mononuclear phagocyte system. Phagocytic cells are important for killing infectious organisms, but they also play a role in scavenging dead and damaged cells and cell debris. a. To be taken up by a macrophage, a particle must bind to the phagocytic cell's membrane and activate one of several receptors. This induces the cell to extend sheet like projections of the plasma membrane, called pseudopods, which engulf the particle and fuse at their tips to form a phagosome. b. The phagosome then fuses with a lysosome (to form a phagolysosome) and the particle is digested by the lysosomal components. 6. Damaged or worn out organelles are surrounded by membrane (perhaps from the endoplasmic reticulum) to form a vacuole called an autophagosome, which will fuse with a primary lysosome and allow re- cycling of components from the organelle. a. FYI - This process, known as autophagy (from Greek autóphagos, meaning "self-devouring") is the natural, conserved degradation lysosome-dependent regulated mechanism of the cell that removes unnecessary or dysfunctional components. It allows the orderly degradation and recycling of cellular components. In disease, autophagy has been seen as an adaptive response to stress, promoting survival of the cell; but in other cases, it appears to promote cell death. Defects in autophagy have been linked to various human diseases, including neurodegeneration and cancer, and interest in modulating autophagy as a potential treatment for these diseases has grown rapidly. 7. Extracellular vesicles are a heterogeneous group of cell-derived membranous structures comprising exosomes and microvesicles, which originate from the endosomal system or which are shed from the plasma membrane, respectively. Even though the generic term extracellular vesicles is currently in use to refer to all these secreted membrane vesicles, they are in fact highly heterogeneous. Extracellular vesicles are now considered as an additional mechanism for intercellular communication, allowing cells to exchange proteins, lipids and genetic material (e.g. non-coding microRNAs which can affect gene expression). Extracellular vesicles will display a set of cell-type-specific proteins that account for their specific fates and functions. They are present in biological fluids and are involved in multiple physiological and pathological processes such as tissue repair, angiogenesis, tumor metastasis, stem cell maintenance, and coagulation. a. Microvesicles are formed by budding of the plasma membrane, b. Exosome arise from intraluminal vesicles (ILVs) formed by the inward budding of endosomal membrane during maturation of multivesicular endosomes (MVEs). MVEs fuse with the plasma membrane to release ILVs that are then called exosomes.

vesicular transport description

A. Every cell must eat, communicate with the world around it, and respond quickly to changes in its environment. To accomplish these tasks, cells continuously adjust the composition of their plasma membrane and rapid response to need. They use an elaborate internal membrane system to add and remove the cell-surface proteins embedded in the membrane, such as receptors, ion channels, and transporters. 1. Exocytosis provides a major outward biosynthetic-secretory pathway that delivers newly synthesized proteins, carbohydrates, and lipids to either the plasma membrane or the extracellular space. In this pathway, molecules can be modified stored until needed. 2. Endocytosis provides an inward pathway (endocytic pathway) in which cells removeplasma membrane components and deliverthem to internal compartments calledendosomes, from where they can berecycled to the same or different regions ofthe plasma membrane or can be delivered tolysosomes for degradation. Cells also useendocytosis to capture import nutrients suchas vitamins, and lipids, cholesterol, and iron.These are taken up together with the macromolecules to which they bind and are then released in endosomes or lysosomes and transported into the cytosol, where they are used in various biosynthetic processes. B. The lumen of each membrane-enclosed compartment along the biosynthetic-secretory and endocytic pathways is topologically equivalent to the lumen of every other compartment. Proteins can travel in the space without having to cross a membrane, being passed from one compartment to another by means of numerous membrane-enclosed transport containers, which can be either spherical or more irregular tubular structures. C. In the system, each transport vesicle must be selective. It must take up only the appropriate molecules and only fuse with the appropriate target membrane. It is the composition of the enclosing membrane, and molecular markers displayed on it cytosolic surface, that serves as guidance cues for incoming traffic to ensure that transport vesicles fuse only with the correct compartment

Bread loafing

Cut sample into slices about 1 cm thick to ensure fixation

Lysosome description

A. Lysosomes are the digestive system of the cell, i.e. the principal site of intracellular digestion. These are membrane bound organelles rich in hydrolytic enzymes synthesized by the rough endoplasmic reticulum (rER), modified by the Golgi apparatus and packed into this membrane bound structure. There are more than 40 different degradative enzymes in lysosomes, including proteases, phosphatases, glycosidases, sulfatases, lipases, and nucleases. These enzymes are collectively known as acid hydrolases because they are optimally active at a very acid pH (~5.0). The lysosome provides a pH of about 4.5-5.0 in its interior. B. The lysosome has a unique surrounding membrane. 1. An H+ ATPase in the lysosomal membrane uses the energy of ATP hydrolysis to pump protons into the lysosome, thereby maintaining the lumen at its acidic pH. In addition to providing a low-pH environment, that is suitable for the action of the acid hydrolases, the H+ gradient also provides a source of energy that drives the transport of small metabolites across the organelle membrane. 2. The lysosomal membrane contains a large number of highly glycosylated membrane proteins, which helps to protect them from the lysosomal proteases in the lumen. 3. Transport proteins in the lysosomal membrane carry the final products of macromolecule digestion - such as amino acids, sugars, and nucleotides - to the cytosol, where the cell can either reuse or excrete them. C. Lysosomes appear morphologically heterogeneous, reflecting the wide variety of digestive functions that acid hydrolases mediate. These organelles are particularly common in phagocytic cells and their enzymes are used to break down (digest) foreign materials that have been brought into the cells. They are essential for the breakdown of intra-and extracellular debris, the destruction of phagocytized microorganisms, and the production of nutrients for the cell. D. Lysosomes are also important for digestion of the cell's own obsolete organelles (autophagy). It is possible in normal cells to see lysosomes containing (and digesting) mitochondria, as well as other organelles. This process starts with the organelle being enclosed by a double membrane of unknown origin, creating an autophagosome, which then fuses with the lysosome. This process is highly regulated and selected cellular components can be somehow marked for lysosomal destruction. E. Lysosomes are too small to see using a light microscope, although you may be able to recognize vacuoles containing cell debris. Even with electron microscopy, it can be difficult to identify a lysosome and distinguish it from other transport vesicles. As with light microscopy, you might be able to recognize them by the presence of intracellular debris (as in the image to the right), remnant organelles, or small, dense, digestion-resistant residues.

Mitochondria description

A. Mitochondria are membrane bound organelles thought to represent aerobic prokaryotes (read: bacteria) that once lived symbiotically within primitive eukaryotic cells. 1. Mitochondria have circular DNA and can undergo division and increase in number independent of cell division. 2. They also have their own ribosomes and a system for protein synthesis. Mitochondria are abundant in cells with high energy requirements (e.g. skeletal muscle) and that need large amounts of ATP. B. Mitochondria produce ATP through a variety of metabolic pathways including oxidative phosphorylation and ß-oxidation of fatty acids. C. Mitochondria have a double membrane. 1. The outer membrane is relatively permeable, containing a pore-forming protein (porin) that allows free passage of small molecules. The outer membrane contains enzymes that convert certain lipids into forms that can be metabolized within the mitochondria. 2. The inner membrane is folded into tubules called cristae, which increase the surface area for chemical reactions. 3. The inner cavity is filled with a matrix thought to contain binding sites for calcium. 4. Aerobic respiration takes place within the matrix and on the inner membrane. The matrix contains most of the enzymes involved in oxidation of fatty acids and the Krebs cycle. The inner membrane contains the cytochromes, the carrier molecules of the electron transport chain, and the enzymes involved in ATP production. Electrons from NADH and succinate pass through the electron transport chain to oxygen, which is reduced to water. This enzymatic series produces a proton gradient across the mitochondrial membrane, producing a thermodynamic state that has the potential to do work.

Membrane bound organelles overview

A. On average, the membrane-enclosed compartments together occupy nearly half the volume of the cell, and a large amount of intracellular membrane is required to make all. Lipid membranes separate different compartments of the cell, many of which contain mutually incompatible biochemical reactions. For example, protein synthesis and export, which takes place in the rER and Golgi apparatus, must be kept separate from lysosomes which are responsible for digesting old organelles. Microorganisms phagocytosed by cells must be killed and disposed of without damage to normal cellular structures and functions. B. The membrane(s) that surround certain organelles have a structural makeup similar to that of the plasma membrane (lipid bilayer with integral proteins). However, the membrane of each type of organelle will have a different distribution of phospholipids, and each organelle will contain its own characteristic set of enzymes and other specialized molecules related to its function (e.g. catalyze chemical reactions) and serve as organelle- specific surface markers that direct new deliveries of proteins and lipids to the appropriate organelle. To serve the specialized functions of cells, each type of organelle will vary in abundance and may have additional properties that differ from cell type to cell type. C. The spaces enclosed by membranes act as intracellular microcompartments in which substrates, products, and other substances are modified, concentrated, or sequestered (e.g. the membranes of lysosomes protect the rest of the cell from enzymes whose hydrolytic activity might be detrimental). 1. The nucleus and mitochondria have a double membrane; all other membrane-bound organelles have a single membrane. 2. The membranes of some these organelles form structures that may be convoluted (e.g. smooth endoplasmic reticulum) or plicated (e.g. mitochondria). D. Organelle growth and function requires a supply of new lipids to make more membrane and a supply of appropriate proteins. 1. The fate of the synthesized protein depends on the first 5-30 of its amino acids which comprise a sorting signal that directs the protein to the organelle in which it is required. Different sorting signals can direct proteins into the nucleus, mitochondria, peroxisomes, and the ER. Proteins that lack such signals remain as permanent residence in the cytosol. 2. For some organelles, including mitochondria, peroxisomes, and the interior nucleus, most proteins are synthesized in the cytosol and delivered directly to the organelle. Proteins move from the cytosol into the nucleus via the nuclear pores (see chapter on The Nucleus for more details). Fully folded proteins are able to move through these pores 3. For some organelles, that are collectively known as the endomembrane system, including the Golgi apparatus, lysosomes, endosomes, and the inner nuclear membrane, proteins and lipids are delivered indirectly via the ER, which is a major source of lipid and protein synthesis. a. Proteins are ferried by transport vesicles that bud off from one of these organelles and fuse with another. In this process of vesicular transport, vesicles deliver soluble cargo proteins, as well as the proteins and lipids that are part of the vesicle membrane. b. The interiors of these organelles communicate extensively with one another and with the outside of the cell by means of small vesicles that move proteins and other molecules from one membrane-bound compartment to another. E. Many of the membrane-enclosed organelles are held in their relative locations in the cell by attachment to the cytoskeleton, especially to microtubules. Cytoskeletal filaments (actin filaments and microtubules) provide tracks for moving organelles around and for directing the traffic of vesicles between one organelle and another. These movements are driven by motor proteins that use ATP hydrolysis to propel the organelles and vesicles along the filaments (see chapter on Cytoskeleton for more details).

Ribosomes and protein synthesis

A. Ribosomes are important organelles involved in protein synthesis and go hand-in-hand with the rough ER see next section). Ribosomes are responsible for synthesis of both soluble proteins (i.e. in the cytoplasm) and integral membrane proteins. 1. Ribosomes consist of two subunits that are produced in the nucleolus, but which combine inthe cytoplasm around a strand of messenger RNA(mRNA). The smaller subunit has a binding sitefor the mRNA, while the larger subunit will bind transfer RNA (tRNA) which provides the amino acids needed for protein synthesis, and it will also hold the growing peptide chain. B. Ribosomes are extremely tiny and are beyond the limit of resolution of the light microscope. However, when large numbers are present, the RNA will bind basic dyes producing basophilia. This is most notable when tissues are stained with Romanovsky type stains used for staining blood smears and cytology preparations, where large numbers of ribosomes will cause the cytoplasm to stain deep blue (see image, to the right, of an activated lymphocyte stained with Wright stain). - For example, early RBC precursorscontain large numbers of polysomes, forproduction of hemoglobin (and other proteins) (see image of RBC precursors in bone marrow cells stained with Wright stain, a Romanovsky type of stain). C. If a protein being made by the ribosome is destined for another organelle (either its lumen or its membrane) or for export out of the cell, the ribosome will bind to the cytosolic membrane of the rough endoplasmic reticulum (rER; it is the docked ribosomes that cause the ER to appear rough). Ribosomes are not a stable part of this organelle's structure as they are constantly being bound and released from the membrane. As proteins are created, they can either become incorporated into the lipid bilayer of the ER (becoming an integral protein of the membrane) or pass directly into the lumen of the rER. The proteins will move to the Golgi and beyond, via vesicle transport. Proteins in the membrane of the rER will become incorporated into membranes "downstream" (i.e. plasma membrane or organelle membranes). Protein in the lumen of the rER will go into the lumen or an organelle or be excreted out of the cell through exocytosis. D. Proteins destined to remain within the cell are produced by free ribosomes that float around within the cell and don't attach to the rER. As an mRNA molecule is translated, many free ribosomes bindit, forming a polyribosome (polysome). Free ribosomes are important for making proteins such as hemoglobin (RBCs), contractile proteins (actin & myosin in muscle), keratin (skin cells) and neurofilaments (nerve cells). E. Note that free ribosomes and membrane-boundribosomes are identical, differing only in theprotein they are making at any given time. Where the protein ends up is determined by a sorting signal composed of the first 5-30 amino acids on the protein strand. If the protein is destined for an organelle or the plasma membrane, this part of the protein strand will become bound to a translocator protein in the organelle (e.g. rER). F. To be useful to the cell, the newly made polypeptide chain must fold up into its unique 3-dimensional conformation, binding a small molecule cofactors needed for activity, the appropriately modified by protein kinases or other protein-modifying enzymes, and assembled correctly with other protein subunits with which it functions. When a protein folds into a compact structure, it puts most of its hydrophobic residues in an interior core and large numbers of noncovalent interactions form between various parts of the molecule. The information needed for all these changes is contained in the sequence of linked amino acids that the ribosome produces when it translates and mRNA molecule into a peptide chain. 1. For some proteins, folding begins immediately as the protein spins out of the ribosome starting at the N-terminal end, and within a few seconds it forms a compact structures containing most of the final secondary features (e.g. α helices or β sheets) aligned in roughly the right conformation. 2. For other protein domains, the initial folded state (called a molten globule) is the starting point for a relatively slow process in which many side-chains adjustments occur that eventually form the correct tertiary structure. 3. Most proteins do NOT begin to fold during their synthesis. Instead they are met at the ribosome by a special class of proteins called molecular chaperones. There are many different paths that can be taken to convert an unfolded or partially folded protein to its final compact conformation, and for many proteins, without the intervention of chaperone proteins, some of the intermediates formed would form abnormal aggregates and be left as dead ends. a. Many molecular chaperones are called heat shock proteins, because they are synthesized in dramatically increased amounts after a brief exposure of cells to an elevated temperature. This reflects a rapid response to an increase in misfolded proteins, such as those produced by elevated temperatures, and an attempt to help these proteins refold. b. There are several major families of molecular chaperones, with different family members functioning in different organelles. Some hydrolyze ATP, often binding and releasing their protein substrate with each cycle of ATP hydrolysis. Other molecular chaperones form a large barrel shaped structure that acts like an isolation chamber into which misfolded proteins are fed, preventing their aggregation and providing them with a favorable environment in which to attempt to refold. c. Some chaperone proteins are found in the cytosol, helping proteins generated by free ribosomes to fold. d. There are also chaperone proteins found in the lumen of the rER which serve the same function for proteins generated by docked ribosomes (see following section on endoplasmic reticulum). e. The proteolytic machinery of the cell and the chaperones compete with one another to reorganize a misfolded protein. If a newly synthesized protein folds rapidly, at most only a small fraction of the molecules are degraded. In contrast, a slowly folding protein is vulnerable to the proteolytic machinery for a longer time and many more of its molecules are destroyed before the remainder attain the proper folded state. Due to mutations or to errors in transcription, RNA splicing and translation, some proteins never fold properly. It is particularly important that the cell destroy these potentially harmful proteins. The apparatus that destroys the aberrant proteins is the proteasome, an abundant ATP-dependent protease dispersed throughout the cytosol and the nucleus. Incorrectly folded proteins in the cytoplasm are tagged by the protein ubiquitin which allows them to be taken up by the proteasome. Ubiquitin is a small (8.5 kDa) regulatory protein that has been found in almost all tissues of eukaryotic organisms. Ubiquitination affects cellular process by regulating the degradation of proteins (via the proteasome and lysosome), coordinating the cellular localization of proteins, activating and inactivating proteins, and modulating protein-protein interactions f. Incorrectly folded proteins in the lumen of the rER are exported from the ER into the cytosol where they are ubiquinated and then degraded by proteasomes.

Golgi (Golgi Apparatus or Golgi complex) description

A. The Golgi apparatus is usually located near the cell nucleus and it consists of a collection of flattened, membrane enclosed compartments, called cisternae, arranged in parallel and embedded in a network of microtubules. Each Golgi stack consists of 3-20 cisternae which are piled like stacks of pita bread. Like the smooth ER, the membranes that and close the cisternae of the Golgi apparatus lack ribosomes. 1. The number of Golgi stacks per cell varies greatly depending on the cell type; some cells contain one large stack, while others contain hundreds of very small ones. 2. Because it lacks ribosomes, this organelle binds weakly to traditional stains such as H&E and the Romanovsky stains, resulting in a negative Golgi image. 3. Each Golgi stack has two functional faces and is typically organized with a distinct convex and concave side, reflecting its functional polarity. a. The Cis-Face (or "forming face") refers to cisternae that are closest to the ER. Transfer vesicles from the ER carry newly synthesized proteins to Golgi. b. The Trans-Face (or "maturing face") refers to cisternae that are furthest away from the ER. This side of the stack faces the plasma membrane and has a slightly concave appearance. You can often see vesicles budding off of the upper most membrane on this side. 4. The unique architecture of the Golgi apparatus depends on both the microtubule cytoskeleton and cytoplasmic Golgi matrix proteins, which form a scaffold between adjacent cisternae and give the Golgi stack its structural integrity. Some matrix proteins form long filamentous tethers, thought to help retain Golgi transport vesicles close to the organelle. B. Functions: The Golgi apparatus is responsible for post-translational modification, sorting and packaging of proteins and lipids. It modifies the many proteins and lipids that it receives from the ER, and then distributes them to the plasma membrane, lysosomes, and secretory vesicles. 1. Oligosaccharide chains are processed in the Golgi apparatus. In the ER, oligosaccharide intermediates help proteins fold and help transport misguided proteins to the cytosol for degradation. Once these your functions have been fulfilled, the cell can redesign the oligosaccharides for new functions. This happens in the Golgi apparatus, which generates the heterogeneous oligosaccharide structure seen in mature proteins. a. The oligosaccharide processing steps occur in an organized sequence within the Golgi stack, and each cisterna contains an abundance of membrane-bound glycosidases and glycosyl transferases which work with sugar nucleotides to modify lipids and proteins molecules as they pass through the Golgi apparatus. b. Processing occurs in a spatial sequence, with enzymes catalyzing early processing steps concentrated in the cisterna near the cis face of the Golgi stack, while enzymes catalyzing later processing steps are concentrated in the cisterna near the trans face. c. The Golgi apparatus is especially prominent in cells that are specialized for secretion of glycoproteins, such as the goblet cells of the intestinal epithelium, which secrete large amounts of polysaccharide-rich mucus into the gut. 2. Proteoglycans are assembled in the Golgi apparatus. This process involves the polymerization of one or more glycosaminoglycan chains (i.e. long unbranched polymers composed of repeating disaccharide units) onto serines on a core protein. Many proteoglycans are secreted and become components of the extracellular matrix, while others remain anchored to the extracellular face of the plasma membrane. Still others form a major component of slimy materials, such as the mucus that is secreted to form a protective coating on the surface of many epithelia. 3. The sugars Incorporated into glycosaminoglycan's are heavily sulfated in the Golgi apparatus immediately after these polymers are made, thus adding a significant portion of their characteristically large negative charge. C. Once modifications are complete, the protein enters a final saccule in the distal side of the Golgi apparatus. Here proteins are sorted into secretory vesicles destined for the extracellular space (e.g. hormones, collagen), the plasma membrane (e.g. cell surface receptors, adhesion molecules) or intracellular organelles such as lysosomes. 1. Secretory vesicles become increasingly condensed as they migrate through the cytoplasm to form mature secretory granules. Depending on the material in the secretory granule, these may be stored for some time or may migrate quickly to the surface where they are released through a process called exocytosis. 2. The Golgi is prominent in cells such as plasma cells that actively secrete large amounts of protein (immunoglobulin). It is also found in cells that synthesize large amounts of membrane associated-protein such as neurons. D. It is still uncertain how molecules move from one cisterna to another. 1. Vesicular transport model: was initially thought that the Golgi apparatus is a relatively static structure and soluble proteins and membrane move from one cisterna to another through vesicular transport. 2. Cisternal maturation model: an alternate theory is that the Golgi is a dynamic structure in which the cisternae themselves move. Vesicles from the ER fuse with one another to become a cis Golgi network/cisterna. This network that progressively matures to become a medial cisterna, and then finally a trans cisterna. Budding vesicles would move membrane proteins, including the processing enzymes into the newly forming cisterna to me: so a newly formed cis cisterna would receive its normal complement of resident enzymes primarily from the cisterna just ahead of it, and would later pass these enzymes back to the next cis cisterna that forms. 3. These 2 models are not mutually exclusive, and in fact transport may occur by a combination of both mechanisms.

Nucleolus organization

A. The nucleolus is the most obvious structure see in the nucleus of a eukaryotic cells when viewed in the light microscope. It is a non-membrane bound aggregate of macromolecules within the nucleus that can be thought of as a factory in which many different noncoding RNAs are transcribed, processed, and assembled with proteins to form a large variety of ribonucleoprotein complexes. 1. Here ribosomal RNAs are synthesized and combined with proteins to form ribosomes, the cell's protein-synthesizing shades. The nucleolus contains the chromosomes carrying the rRNA genes, precursor rRNAs, mature rRNAs, rRNA-processing enzymes, small nucleolar RNAs (snoRNAs), and small nucleolar ribonucleoprotein complexes (snoRNPs), ribosomal proteins and partly assembled ribosomes. Close association of all these components presumably allows the assembly ribosomes to occur rapidly and smoothly. a. Ribosomes consist of two subunits; a large subunit and a small subunit, made up of 4 types of eukaryotic rRNA and 50 or more proteins. b. Ribosome assembly is a complex process. After further modification, the ribosomal subunits are assembled using some ribosomal proteins imported from the cytoplasm. The partially assembled ribosomal subunits (preribosomes) are exported from the nucleus via nuclear pores, to be fully assembled into mature ribosomes in the cytoplasm. 2. The nucleolus is also the site where other RNAs are produced and other RNA-protein complexes are assembled. Examples follow. a. The genes encoding transfer RNAs (tRNA), which carry amino acids for protein synthesis are clustered in the nucleolus, and tRNAs are processed there. b. Telomerase is believed to be assembled in the nucleolus. Telomerase is a ribonucleoprotein that is an enzyme that adds DNA sequence repeats ("TTAGGG" in all vertebrates) to the 3' end of DNA strands in the telomere regions, which are found at the ends of eukaryote chromosomes. This region of repeated nucleotide called telomeres contains noncoding DNA and hinders the loss of important DNA from chromosome ends. Telomerase is a reverse transcriptase that carries its own RNA molecule, which is used as a template when it elongates telomeres, which are shortened after each replication cycle. B. The size of the nucleolus reflects the number of ribosomes that the cell is producing. It's size therefore varies greatly in different cells and are usually prominent in cells with active protein synthesis (e.g. liver hepatocytes, nerve cells, glandular epithelial cells producing a secretory product). They can change in a single cell, sometimes occupying as much as 25% of the total nuclear volume in cells that are making unusually large amounts of protein. More than one nucleolus can be found in some very metabolically active cells. Multiple nucleoli are common in rapidly dividing malignant tumor cells. 1. In an electron micrograph, the nucleolus appears as a large, irregularly round area of dense chromatin. 2. With H&E staining nucleoli stain a dark purple. 3. With Romanowsky stains(e.g. Wrightstain, DiffQuick),they often stain blue, while the surrounding chromatin is purple.

Mitochondria main function

ATP synthesis by oxidative phosphorylation and the Krebs cycle, oxidation of fatty acids, first stage in synthesis of steroid hormones. Generation of heat in multilocular (brown) fat.

Tumor supressor protein p53

Accumulates in response to DNA damage that cannot be repaired, and activates the transcription of genes that encode proteins that inhibit anti-apoptotic proteins. This triggers the intrinsic pathway, thereby eliminating a potentially dangerous cell that could otherwise become cancerous

A cell or tissue component that binds an acid dye

Acidophilic (acid loving)

Clostridium botulinum neurotoxin causes botulism by...

Acting on key SNARE proteins -Synaptic vesicles containing neurotransmittors are released from neurons and stimulate muscles to contract. -Neurotoxin cleaves one of the SNARE proteins >Prevents fusion of vesicle >Prevents neurotransmitter from stimulating muscle -Result is muscle paralysis -As toxin spreads it can block nerves controlling the respiratory tract and heart, leading to death.

Romanovsky stain

After Marrow Dries on a slide it is stained with this stain. It contains methylene blue and its oxidation products. (Azure and Eosin)

Artifacts result while cutting the section and transferring it to the slide

Air bubbles underneath tissue Dull blade- tears in tissue Wrinkling of tissue Knife chatter

Membranes are a sea of lipid in which float protein and carbohydrate; they separate the cell from its surroundings and compartmentalize the interior of the cells

All cellular membranes (plasma membrane and organelle membranes) have the same basic structure BUT: each organelle has its own sets of lipids and proteins

Protoplasm

All of the cellular contents with the exception of the plasma membrane and glycocalyx

tRNA

An RNA molecule that functions as an interpreter between nucleic acid and protein language by picking up specific amino acids and recognizing the appropriate codons in the mRNA transfer RNA; type of RNA that carries amino acids to the ribosome tRNA

Intrinsic pathway of apoptosis

An amplifying proteolytic cascade triggered in response to cell injury, DNA damage, lack of O2, lack of extracellular survival factors or presence of intracellular developmental signals -Involved in tissue remodeling in embryogenesis. Occurs when a regulating factor is withdrawn from a proliferating cell population (e.g., decreased IL-2 after a completed immunologic reaction --> apoptosis of proliferating effector cells). Also occurs after exposure to injurious stimuli (e.g., radiation, toxins, hypoxia). -Changes in proportions of anti- and pro- apoptotic factors increasing mitochondrial permeability and cytochrome c release. BAX and BAK are proapoptotic proteins; Bcl-2 is antiapoptotic. -Bcl-2 prevents cytochrome c release by binding to and inhibiting Apaf-1. Apaf-1 normally induces the activation of caspases. If Bcl-2 is overexpressed (e.g., follicular lymphoma), then Apaf-1 is overly inhibited, caspase activation and tumorigenesis. mitochondrial leakage of cytochrome c into the cytosol with eventual activation of caspases depends on release of mitochondrial proteins into the cytosol. A crucial protein released from mitochondria in the intrinsic pathway is cytochrome c, a water-soluble component of the mitochondrial electron- transport chain.

Extrinsic pathway of apoptosis

An amplifying proteolytic cascade triggered in response to the binding of extracellular signal molecules to transmembrane death receptors - pathway initiated by engagement of plasma membrane death receptors on a variety of cells binding of TNF to its receptor and eventual activation of caspases The extrinsic pathway cancross talk with the intrinsic pathway, to amplify the cascade in order to kill the cell.

Smooth endoplasmic reticulum

An endomembrane system where lipids are synthesized, calcium levels are regulated, and toxic substances are broken down. Smooth Endoplasmic Reticulum is composed of short, tubules without ribosomes -May be continuous with rER -Appear disorganized; variable shaped lumina

Primase

An enzyme that joins RNA nucleotides to make the primer. An enzyme that joins RNA nucleotides to make the primer using the parental DNA strand as a template. -Synthesizes short RNA sequences called primers -Primers serve as a starting point for DNA synthesis -A type of RNA polymerase

DNA helicase

An enzyme that unwinds the DNA double helix during DNA replication -Separate double-stranded DNA into single strands allowing each strand to be copied -Unwind DNA at positions called origins where synthesis is initiated -Uses ATP for energy to break the hydrogen bonds between the nucleotide base pairs -Forms the replication fork

Magnification

An increase in the apparent size of an object The ability to make an object look larger

Mitochondria

An organelle found in large numbers in most cells, in which the biochemical processes of respiration and energy production occur. Powerhouse of the cell, organelle that is the site of ATP (energy) production Present in all cells except erythrocytes & mature keratinocytes (skin cells) Contribute to cytoplasmic eosinophilia

Endosymbiotic theory

Ancestors of mitochondria and plastids was prokaryotes thatcame to live in a host cell. Mitochondria may have originated as a symbiotic prokaryocyte

Romanovsky stains combine eosin (acidic dye) and methylene blue (strong basic dye)

Examples: Wright stain, Diff-Quik (trade name used loosely to refer to a number of stain brands) • Difference from H&E: -Deeper blues -Nuclear material shows metachromasia (purple color)

Glycogen H&E

Appears as vacuoles

Artifacts

Are unwanted or confusing features in tissue sections that result from accidental damage or poor technique in sample collection, embedding, sectioning, or staining. Artifacts must be recognized to avoid misinterpretation. The most frequent are tissue autolysis due to poor fixation, tissue shrinkage during fixation and dehydration, separation of adjacent structures, and folds or wrinkles that occur during sectioning.

Sectioning

Average section thickness for paraffin wax is 5 μm (μm = microns, or 10-6 m), and sections are cut on a special microtome using metal knives. The sections are laid on glass slides for microscopic viewing.

Prokaryotes

Bacteria and archaea Cells that do not contain nuclei

The inactive X chromosome in females is called a...

Barr bodies are easier to see in smears of cells rather than formalin fixed tissues - drum stick of heterochromatin

Large numbers of ribosomes cause the cytoplasm to look...

Basophilic Increased basophilia most notable with Romanovsky stains.

A cell or tissue component that binds a basic dye

Basophilic (base loving)

Nucleus, ribosomes (nucleic acids)

Basophilic, make cytoplasm look blue

Things that a clinician can control in histology: use surgical ink to mark mass margins

Be sure to provide a key: color at top, bottom, left, and right margins

Why are many molecular chaperones called heat shock proteins?

Because they are synthesized in dramatically increased amounts after a brief exposure of cells to an elevated temperature

Nuclear export receptor

Bind to both the export signal and nuclear pore complex proteins to guide their cargo through the nuclear pore complex to the cytosol. guides mature mRNA through nuclear pore complex into cytosol a complex of proteins that binds to an RNA molecule once it has been correctly spliced and polyadenylated, and facilitates export from the nucleus

Microvesicles

Bleb directly off plasma membrane

Hemosiderin Wright (Romanovsky) stain

Blue-green

Lipofuscin H&E

Brown pigment

CRISPR/Cas9

CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats Cas: CRISPR associated genesEssential in adaptive immunity in select prokaryotes enabling them to respond and eliminate invading genetic material such as viruses

Telomerase

Catalyzes the lengthening of telomeres in germ cells An enzyme that catalyzes the lengthening of telomeres. The enzyme includes a molecule of RNA that serves as a template for new telomere segments. An enzyme that catalyzes the lengthening of telomeres in eukaryotic germ cells.

Cellular organelles

Cell organelles are discrete structureswithin a cell that have specializedfunction. Each organelle has its owncharacteristic set of enzymes and otherspecialized molecules that performmetabolic, synthetic, energy requiringand energy generating functions.

Three main tenants of cell theory

Cell theory states that all living things are composed of cells; cells are the basic units of structure and function in living things, new cells are produced from existing cells.

Death receptor

Cell-surface molecule that triggers apoptosis when bound by an extracellular signal protein Homotrimeric transmembrane proteins which are members of the tumor necrosis factor receptor family. Binding of these receptors by extracellular signal molecules triggers the extrinsic pathway of apoptosis. Specific receptors on the outer surface of cells that will bind to cytokines and initiate the extrinsic pathway of apoptosis.

Interpretation of form in sections of cells: the sampling problem and the plane-of- section problem

Cells and tissues are usually sectioned for light and electron microscopic analysis. This is essential as they are too thick to be observed without sectioning. This means that only a thin slice of the entire cell is observed, and that the apparent contents and shape of the cell will depend on the position in the cell from which the section is taken and on the orientation of the cell with respect to the section

Unfolded protein response

Cellular action triggered by an accumulation of misfolded proteins in the ER This response to an accumulation of unfolded/misfolded ER proteins results in an increased transcription of genes involved in retrotranslocation and cytosolic protein degradation. Molecular program triggered by the accumulation of misfolded proteins in the endoplasmic reticulum. Allows cells to expand the endoplasmic reticulum and produce more of the molecular machinery needed to restore proper protein folding and processing.

Processing artifacts

Changes in cell volume due to fixation and/or dehydration can cause shrinkage artifacts. - E.g. tubular structures shrink within their surrounding connective tissue - E.g. artificial spaces form around cells. Inactivation of most enzymes and some antigenic molecules Ethanol is a fat solvent hence it dissolve fats and lipids Emulsion pickoff, Gelatin buildup, Curtain effect, Chemical fog, Guide-shoe marks, Pi lines, Wet pressure sensitization, and Dichroic stain wash, identify, catalogue, curate

Protein folding is usually facilitated by...

Chaperone proteins (AKA heat shock proteins) -Network of molecular chaperones promote efficient protein folding and prevent aggregation of misfolded proteins >They have several modes of action -These increase in response to heat and attempt to refold denatured proteins -Aberrantly folded proteins can lead to aggregation and possible cellular damage. Chaperones in the ER help proteins properly fold and prevent protein aggregation -Resident chaperone proteins in ER recognize incorrectly folded proteins -Improperly folded proteins are exported from ER and degraded by proteasome in cytosol

Evidence of endosymbiotic theory

Chloroplasts and Mitochondria have different DNA than rest of cell. They have their own membrane. They look like prokaryotes. 1. Mitochondira & chloroplast ribosomes - 70S (around same size as prokaryotes) 2. Mitochondria and chloroplast contain membrane 3. Mitochondria & chloroplast divide by binary fission 4. Genome size (1x10^7-3x10^80 5. Larger cells can engulf 6. Mitochondria & chloroplast have their own DNA 7. Certain antibiotics that affect prokaryotic cells will also inhibit mitochondria 8. Cell size-some mitochondria larger than prokaryotes 9. Protein synthesis & initial amino acids -Have circular DNA - similar to bacteria • >Capable of independent replication >>Can change their shape, location and number to suit the cell's needs -Have ribosomes and ability to synthesize protein >However, most mitochondrial proteins are made by the cell and rapidly imported into mitochondria

Chromatin

Chromatin consists ofnuclear DNA and proteins that bind to the DNA. These proteins can be divided into 2 general classes. 1. Histones: The histones are the major class of DNA-binding proteins involved in maintaining the compacted structure of chromatin. 2. Nonhistone chromosomal proteins: this is a diverse group of proteins that includes the various transcription factors, polymerases, hormone receptors and other nuclear enzymes. In any given cell, there are greater than 1000 different types of non-histone proteins bound to the DNA.

What happens to chromatin during mitosis?

Chromatin is not only tightly condensed but is free - no longer contained within a nuclear envelope Looks like spiders

Chromosomes description

Chromosome number actually has no bearing on the amount of DNA per cell because chromosomes can be big or small DNA molecules store information (we will learn about gene transcription and translation in the next hour)

Some locations of microtubules

Cilia & flagella Basal bodies Centrioles Mitotic spindle

Xylene/propar

Clears alcohol to allow infiltration with paraffin

Prussian blue stain

Commonly used to identify hemosiderin (iron pigment) in tissue Hemosiderin largely comes from breakdown of RBC hemoglobin. - Iron stores - Post-hemorrhage Turns iron blue

Some functions of actin

Component of contractile elements in muscle and motile cells; structural support

Vimentin stain

Connective tissue (Sarcoma tumor marker; mesenchyme)

Nucleus main function

Contains main genome; DNA and RNA synthesis

Nuclear pore complex

Controls exchange of material between nucleus and cytoplasm A large complex of dozens of proteins lining a nuclear pore, defining its shape and regulating transport through the pore. An array of proteins that line pores in the nuclear membrane and control which substances enter and leave the nucleus

Identify tumors in epithelial cells with

Cytokeratins (Class I)

Mitochondria contribute to...

Cytoplasmic eosinophilia

Telomeres

DNA at the tips of chromosomes Repeated DNA sequences at the ends of eukaryotic chromosomes.

Enzymes involved in DNA replication

DNA helicase Topoisomerase I Primase DNA polymerase DNA ligase

DNA organization in nucleus

DNA in Chromosomes is highly compacted. Around 3 linear feet of DNA are crammed into the average, eukaryotic nuclear membrane. To accomplish this, the DNA has to be highly folded and tightly packed. DNA compression is performed by proteins that successively coil and fold the DNA into higher and higher levels of organization. 1. The nucleosome is the most basic level of chromosome packing. The histone core particle forms a protein core around which double- stranded DNA is wound. Each nucleosome core particle is separated from the next by a region of linkerDNA which can vary in length. The result is the appearance of beads on a string, in which the string is DNA and each bead is a nucleosome core particle. The formation of nucleosomes converts a DNA molecule into a chromatin thread about one third of its initial length. 2. Nucleosomes are packed on top of one another generating regular arrays in which DNA is even more highly condensed into a structure known as a chromatin fiber. The tight stacking of nucleosomes is facilitated by nucleosome to nucleosome linkages formed by histone tails. Histone H1, the linker histone, is also thought to pull adjacent nucleosome together into a regular repeating array. 3. During mitosis, the chromatin fiber is folded into a series of loops and these loops are further condensed to produce the interphase chromosomes. Finally, this compact string of loops is thought to undergo at least one more level packing to form the mitotic chromosome. This is the state which duplicated chromosomes can be most easily visualized.

histone modification

DNA may unwrap or be stopped from unwrapping from the histone changes in the structure of histones that make it more or less likely that a segment of DNA will be transcribed adding chemical modifications to proteins called histones that are involved in packaging DNA

the role of histones in transcription

DNA unwinds itself from the histones, allowing for transcription to occur

Lipofuscin Wright (Romanovsky)

Dark green

Melanin Wright (Romanovsky) stain)

Dark green, green-grey

Alcohol

Dehydration makes tissue miscible with paraffin

Some locations of intermediate filaments

Desmosomes; hemidesmosomes Beneath inner nuclear membrane

Secretory granules

Destined for fusion with the plasma membrane Materials that have been brought into the cell and are separated from the cytoplasm by a single membranous boundary Large, densely packed, membrane-bound structure containing highly concentrated secretory materials that are discharged into the extracellular space (secreted) following a stimulatory signal

Lysosomes function

Digest worn out cell parts or unwanted substances Digest worn-out organelles and cell debris; digest material taken up by endocytosis Digestion and recycling -Membrane bound vesicles that hold >40 degradative enzymes >Acid hydrolases with optimal activity at pH ~4.5-5.0 >Proton (H+) pumps maintain a low lumen pH. -Functions >Breakdown of intra-and extracellular debris >Destruction of phagocytized microorganisms >Production of nutrients for the cell >Digestion of the cell's obsolete organelles >H+ gradient that is drives transport of small back to cytoplasm (e.g. amino acids, sugars).

Staining techniques

Direct examination of sample completed in the lab

SNAREs

DockingisfurtherassistedbytransmembraneproteinscalledSNAREs (blue and yellow structures). Once a tethering protein has captured a vesicle by binding to its Rab protein, SNAREs on the vesicle (v- SNAREs) interact with complementary SNAREs on the target membrane (t- SNAREs) to firmly hold the vesicle in place. The SNAREs also catalyze fusion of the vesicle membrane with the target membrane. This not only delivers the cargo, but also adds the vesicle's membrane to the membrane of the organelle.

Bacteria

Domain of unicellular prokaryotes that have cell walls containing peptidoglycan (microbiology) single-celled or noncellular spherical or spiral or rod-shaped organisms lacking chlorophyll that reproduce by fission single-celled organisms that lack a nucleus; prokaryotes -Single closed compartment surrounded by plasma membrane -Lacks a defined nucleus -Simple internal organization

Toluidine Blue Dye

Dye stains cells differentially depending on nuclear configuration Used during biopsy to define margins of lesions and to aid in discover of secondary lesions Disadvantage: It detects cellular changes in inflammatory cells Binds to different cellular components. Color produced is based on what it binds to. (CHEEK CELLS) -Differentially stains cells, depending on their nuclear configuration -Used during biopsy to define margins of lesions and to aid in discover of secondary lesions -Disadvantage: detects cellular changes in inflammatory cells

Characteristics of microtubules

Dynamic instability & treadmilling; stable structures also possible

Movement along microtubules is mediated by...

Dynein and kinesin motor proteins -Two globular ATP-binding motor heads and a tail (stalks) -Move only in one direction along microtubule >Dyneins move toward nucleus >>Cause cilia to bend by sliding microtubules past each other >Kinesins move away from nucleus

Meiosis - Anaphase I

Homologous chromosomes separate Segregation - Homologous pairs separate to opposite poles. Homologous chromosomes separate, but sister chromosomes stay together Chiasmata separate Migrations of dyads to opposite poles

Plane of section also affects appearance

How tissue is trimmed, how it is placed in the cassette and angle of cut, will impact how we see the tissue on the slide

Clinical correlate apoptosis

Dysregulation of apoptosis leads to a variety of pathologies, including cancer, autoimmune disease, and neurodegenerative disorders. Mutations that inactivate the genes that encode the Fas ligand on killer lymphocytes, or the death receptor on others target cells, can prevent the normal death of some lymphocytes, causing the cells to accumulate in excess of numbers in the spleen and lymph nodes. In many cases this leads to autoimmune disease, in which the lymphocytes react against the individual's own tissues. Decreased apoptosis is also an important contributor to many tumors, as cancer cells often regulate the apoptotic program abnormally. The gene encoding the tumor suppressor protein 53 is mutated in 50% of human cancers that it no longer promotes apoptosis or cell-cycle arrest in response to DNA damage. The lack of P 53 function enables cancer cells to survive and proliferate even when their DNA is damaged; in this way the cells accumulate more mutations, some of which make the cancer more malignant. Many anticancer drugs induce apoptosis (and cell cycle arrest), by a 53- dependent mechanism. Therefore the loss of P53 function makes the cancer cells less sensitive to these drugs.

Mitochondria, smooth ER

Eiosinophilic makes the cytoplasm look pink

Transmission electron microscopy

Electrons penetrate an ultrathin section of tissue to strike a photographic plate • Accelerated electrons as source of illumination • Stained areas of the sample absorb or scatter electrons - Electrons interact with heavy metal atoms, that deflect electrons (dark areas) - Electrons pass through unstained areas (light areas) • Allowsthestudyof inner structure and contours of objects (tissues, cells, viruses) • Requiresverythin sections, tissue stained with heavy metals

Rough and smooth endoplasmic reticulum

Endoplasmic reticulum (ER) is a large, membranous organelle consisting of a net-like labyrinth of branching tubules andflattened sacs, or cisternae, which extendthrough the cytosol. The tubules and sacs interconnect, and their membrane is continuous with the outer nuclear membrane.Thus, the ER and nuclear membranes form a continuous sheet, enclosing a single internal space called the ER lumen or ER cisternal space, which often occupies more than 10% of the total cell volume. The ER has a centralrole in lipid and protein biosynthesis. It is thesite of production of all the transmembrane proteins and lipids for most of the cell's organelles, including the ER itself, the Golgi apparatus, lysosomes, endosomes, secretory vesicles, and the plasma membrane. It also makes most of the lipids for the mitochondrial and peroxisome membranes. While the various functions of the ER are essential to every cell, the relative importance varies greatly between individual cell types and to meet different functional demands, distinct regions of the ER become highly specialized. A. Smooth endoplasmic reticulum (sER) is structurally similar to rough endoplasmic reticulum (see next section), but is not associated with ribosomes. 1. This portion of the ER is composed of membrane bound tubules or saccules, that are short and anastomosing - in a section cut for electron microscopy they will usually appear disorganized, with no real obvious organizational pattern, compared to rER, which appears as parallel arrays of tubules. As the section passes through these twisting and branching saccules, their lumens may appear round, oval or much more irregular in shape. They are not associated with ribosomes, so lack the rough surface seen with rER. 2. Smooth ER Functions: most cells contain relatively small amounts of smooth ER and many cells contain what might be called transitional ER because it has membranes that are partly smooth and partly rough. However, depending on their function, some cell types will contain much larger amounts of smooth ER. a. This organelle is important in the production and storage of carbohydrates (e.g. glycogen), b. The smooth ER contains enzymes that make cholesterol and modify it to produce steroid hormones. Hence it is prominent in the cells within endocrine organs that produce this type of hormone (e.g. adrenal cortex and testosterone secreting cells of mammalian testis). c. Hepatocytes (liver cells) contain large amounts of smooth ER, in part due to its importance in the synthesis of lipoprotein particles (e.g. LDLs, HDLs, VLDLs) which carry lipids via the bloodstream to other parts of the body. d. The membrane of the sER contains enzymes that catalyze a series of reactions to detoxify both lipid-soluble drugs, noxious products such as pesticides, and various harmful compounds produced by metabolism. Some of these detoxification reactions are carried out by the cytochrome p450 family of enzymes. These enzymes carry out reactions in which water-insoluble drugs or metabolites, which would otherwise accumulate to toxic levels in cell membranes, are rendered sufficiently water-soluble to leave the cell and be excreted in urine. Again, these reactions take place, in large part, in the sER of the liver. Chronic exposure to certain toxic substances can lead to increased amounts of sER. e. A crucial function of sER is to sequester Ca2+ from cytosol. The release of Ca2+into the cytosol, from the sER, and itssubsequent reuptake, occurs in manyrapid responses to extracellular signals.A calcium pump transports Ca2+ from thecytosol into the ER lumen. A high concentration of Ca2+-binding proteins in the ER facilitates Ca2+ storage. Muscle cells have abundant modified smooth ER, known as sarcoplasmic reticulum. The release of Ca2+ from the sarcoplasmic reticulum, and its subsequent reuptake, triggers myofibril contraction and relaxation, respectively, during a round of muscle contraction. f. Since sER lacks ribosomal RNA, it binds to acidic dyes, and increased amount of sER results in an eosinophilic appearance of the liver cell cytoplasm, rather than basophilia produced by large amounts of rER. B. Rough endoplasmic reticulum (rER) 1. In an electron micrograph, the rough ER has flattened saccules arranged in parallel arrays. The saccules are studded with protein manufacturing ribosomes giving this structure a "rough" appearance when viewed with transmission electron microscopy. Ribosomes are not a stable part of this organelle's structure as they are constantly being bound and released from the membrane. 2. Cells with large amounts of rER (e.g.the activated lymphocyte shown, stained with Wright stain) have a grey to blue-staining (basophilic) cytoplasm especially when stained with Romanovsky stains (e.g. Wright stain, Giemsa, DiffQuick) and to a lesser extent when stained with H&E. 3. The rER is involved in production of protein. Ribosomes that are synthesizingprotein are directly attached to the ERmembrane, enabling one end of the protein tobe translocated into the ER while the rest ofthe polypeptide chain is being assembled. (In contrast, import into mitochondria, nuclei, and peroxisomes is a post-translational process.) The ER, therefore, serves as an entry point for proteins destined for other organelles, as well as for the ER itself. Proteins destined for the Golgi apparatus, endosomes, and lysosomes, as well as proteins destined for the cell surface, all first enter the ER from the cytosol. All these proteins are initially directed to the ER by an ER signal sequence. Once inside the ER lumen, or embedded in the ER membrane, individual proteins will not re-enter the cytosol during their onward journey. They will instead be ferried by transport vesicles from organelle to organelle within the endomembrane system, or to the plasma membrane. a. Some of the proteins entering the ER are water soluble proteins that are completely translocated across the ER membrane and released into the ER cisterna (lumen). These proteins are destined for either secretion (by release at the cell surface) or for the lumen of an organelle of the endomembrane system. b. Other proteins are destined to be transmembrane proteins. These are only partly translocated across the ER membrane and become embedded in it. They can remain there, as an integral protein of the ER membrane, or they can become part of the membrane of one of the other membrane bound organelles or the plasma membrane. Vesicles budding off from the rER will contain these proteins in their membranes. When these vesicles fuse with their target, the integral proteins will diffuse into the organelle membrane or plasma membrane. 4. A ribosome only binds to the RER once a specific protein-nucleic acid complex forms in the cytosol. This special complex forms when a free ribosome begins translating the mRNA of a protein destined for processing in the ER. a. The signal sequence (those first 5-30 amino acids) is recognized, as it emerges from the ribosome, and bound by a signal recognition particle (SRP) present in the cytosol. Binding of the SRP to a ribosome halts protein synthesis by that ribosome as soon as the signal peptide has emerged from the ribosome. This presumably gives the ribosome enough time to bind to the ER membrane before completion of the polypeptide chain. This is particularly important for hydrolases that might wreak havoc on the cell if released into the cytosol. Also, this pause ensures that large portions of the protein that could fold into a compact structure are not made before reaching the tranlocator in the ER membrane. b. The SRP then binds to an SRP receptor, embedded in the ER. Once bound, the SRP is released and the receptor passes the ribosome to a protein translocator (sometimes referred to as a translocon) in the ER membrane. Translation and protein synthesis recommences, with the nascent protein forming into the RER lumen and/or membrane. The SRP and SRP receptor might be considered molecular matchmakers, uniting ribosomes that are synthesizing proteins with an ER signal sequence and available translocation channels in the ER membrane. 5. Not all of the proteins made by ER-bound ribosomes are released into the lumen. Some remain embedded in the ER membrane as transmembrane proteins. During translocation some parts of the polypeptide chain must be translocated completely across the lipid bilayer, whereas other parts remain fixed in the membrane. 6. Most proteins that enter the ER are chemically modified there. Proteins destined to remain in the ER contain an ER retention signal. Some of these proteins function as catalysts that help the many proteins that are translocated into the ER to fold and assembled correctly. a. One resident protein is responsible for oxidation of free sulfhydryl groups (SH) on cysteines to form disulfide bonds (S-S). Disulfide bonds help to stabilize the structure of proteins that will encounter degradative enzymes and changes in pH outside the cell - either after they have been secreted or after they are incorporated into the plasma membrane. b. Most of the proteins that enter the ER lumen or ER membrane are converted to glycoproteins in the ER by the covalent attachment of short branched oligosaccharide side chains composed of multiple sugars. 1) This process of glycosylation is carried out by glycosylating enzymes present in the ER, but not in the cytosol. Very few proteins in the cytosol are glycosylated, and those that are have only a single sugar attached to them. 2) Rather than adding sugars one-by-one, a preformed, branched oligosaccharide containing a total of 14 sugars is attached en bloc to all proteins to carry the appropriate site for glycosylation. 3) A special lipid molecule called dolichol holds the precursor oligosaccharide in the ER membrane. The oligosaccharide is then transferred to the amino group of an asparagine side chain on the protein. 4) Although the oligosaccharides are initially similar.There is a remarkable diversity of oligosaccharides on mature glycoproteins due to extensive modification of the original precursor structure. Modification of carbohydrates on glycoproteins takes place in the Golgi apparatus. 5) The oligosaccharides on proteins can protect a protein from degradation, hold it in the ER until it is properly folded, or serve as a transport signal for packaging the protein into appropriate transport vesicles. When displayed on the cell surface, oligosaccharides form part of the cells outer carbohydrate layer or glycocalyx and can function in the recognition of one cell by another. c. While in the ER cisternae, the glycolipid (FYI) glycosylphosphatidyl- inositol anchor may be added to some proteins destined for the plasma membrane d. Protein folding and assembly of proteins into multisubunit complexes is another important modification that can take place in the ER. 1) This is handled by some resident molecular chaperone proteins. These proteins recognize incorrectly folded proteins. 2) Proteins that failed to fold correctly, and dimeric or multimeric proteins that do not assemble properly, are actively retained in the ER by binding to chaperone proteins that reside there. The chaperones hold these proteins in the ER until proper folding or assembly occurs. Chaperones prevent misfolded proteins from aggregating, and steer proteins along a path toward proper folding. Improperly folded proteins are exported from the ER back into the cytosol, where they are degraded. 3) (FYI) This quality control system can become overwhelmed and when that happens, this folded proteins accumulate in the ER. If the buildup is large enough, it triggers a complex program called the unfolded protein response (UPR). This program prompts the cell to produce more ER, including more chaperones, proteins involved in retrotranslocation (i.e. export of improperly folded proteins back into the cytosol) and other related proteins that help to increase protein folding. This response allows the cell to adjust the size and function of its ER according to the load of proteins entering the pathway. However in some cases even an expanded ER cannot cope and the UPR directs the cell to self-destruct by undergoing apoptosis. Such a situation may occur adult-onset diabetes, where tissues gradually become resistant to the effects of insulin and the pancreas produces more and more insulin. As the unfolded proinsulin, an insulin precursor, accumulates, the UPR can trigger cell death. 6. The sER membrane synthesizes nearly all the major classes of lipids, including both phospholipids and cholesterol, required for the production of new cell membranes. The enzymes involved in this process are in the cytosolic leaflet of the ER membrane, facing the cytosol, where all the required metabolites are found. Trans-bilayer movement of new lipids to the opposite leaflet of the membrane is mediated by a phospholipid translocator called a scramblase. Specific phospholipids can be moved into the leaflet facing the cytosol by a special phospholipid translocators referred to as flippases and floppases.

Most endocytosed materials are sorted in...

Endosomes -Most receptors are returned to the plasma membrane -Some materials are carried to lysosomes where they are degraded

RNA polymerase

Enzyme that links together the growing chain of ribonucleotides during transcription. enzyme that links together the growing chain of RNA nucleotides during transcription using a DNA strand as a template Enzyme similar to DNA polymerase that binds to DNA and separates the DNA strands during transcription -Enzyme that catalyzes the reaction of rNTP polymerization -Always synthesized in the 5' -> 3' direction

New phospholipids and the Golgi apparatus

Enzymes in Golgi apparatus creates distinct "inside" & "outside" faces

Proteins are usually... in staining

Eosiniphilic -> pink

Ran

Exportin and importin transport is regulated by a family of GTP-binding proteins known as Ran.

actin, intermediate filaments, microtubules

Filaments in the cell's cytoskeleton responsible for cell shape and movement

Fixation

Fixative solutions denature, cross-link, and precipitate proteins in tissue samples, stabilizing protein-protein and protein-nucleic acid interactions. Structural relationships between cells and tissues are preserved. Once cells and tissue are removed from the body, for example in a biopsy, cell death and degeneration (autolysis) would degrade cells and result in destruction of the cellular relationships. The ideal aim would be instantaneous immobilization of structural components as in life. Fixation also immobilizes fats and carbohydrates, reduces enzymatic and immunological reactivity, and kills microorganisms present in tissues. Fixation stabilizes the structure and helps provide support so that very thin slices can be cut for microscopic viewing. The routine fixative used in pathology is 10% formalin, i.e. a buffered aqueous, solution containing about 3.8% formaldehyde, often stabilized by the addition of a very low volume of methanol.

Histologic sections

Fixed tissue, embedded, sectioned and stainedwith various dyes We will use the equivalent of a light microscope

Prussian blue

For hemosiderin granules Ferric ferrocyanide iron

The nucleolus is...

Found inside the nucleus and produces ribosomes Responsible for the synthesis of ribosomes -Contains >RNA >DNA with genes responsible for: >>Production and processing of rRNA >>Production of tRNA >>RNA-protein complexes for spliceosome (pre-mRNA splicing) >Ribosome subunits export from nucleus and assemble in cytoplasm

Silver stain

Fungi, Legionella, H. pylori used to visualize: fungi, Legionella a stain used for visualizing proteins

Periodic Acid Schiff

Fungi, amebae Stain for glycogen and mucoproteins Magenta: glycogen and carbohydrate-rich molecules

Exocytosis occurs through...

Fusion of vesicle's membrane with the cell's membrane -Delivery of integral membrane proteins to cell surface -Secretion of proteins >Constitutive secretion >Regulated secretion - triggered by ligand binding to receptor

Asymmetrical distribution of transporters in epithelial cells is essential for transport of solutes

Glucose uniporters located in basolateral membrane near blood vessels As cytosol glucose levels rise, facilitated diffusion moves it out of the cell into the blood stream

Glycosylation of proteins in the ER

Glycosylation of proteins in ER is "one size fits all" -Glycosylate proteins by adding a preformed branched oligosaccharide >Protect protein from degradation >Hold protein in ER until folded >Transport signal for packaging into vesicles

Lipofuscin

Granular pigment with no known function a pigment that occurs as clumps of yellowish brown granules in the cytoplasm yellow brown indigestible pigment in old cells stuck in the cytoplasm

Mitotic chromosome

Highly condensed duplicated chromosome with the two new chromosomes still held together at the centromere as sister chromatids •Duplicated chromosome •2 sister chromatids •narrow at centromeres •contain identical copies of original DNA Highly condensed duplicated chromosome in which the two new chromosomes (also called sister chromatids) are still held together at the centromere. The structure chromosomes adopt during mitosis.

PAS staining

Identifies sugars (glycogen, mucopolysaccharides, etc.)

Cytokeratin

Immunohistochemical stain for Epithelial cells commonly used immunohistochemical marker of epithelial-derived tissues. epithelial cells Intermediate filaments in epithelial cells

Eosin

Imparts pink to red color to basic components like the cytoplasm and extracellular products

Post transcriptional modification

In eukaryotes, mRNA must be modified before it is ready for transport to the cytoplasm for translation into proteins 1. The 5' end of the RNA becomes capped (a methylated guanine residue is added to the end of the RNA transcript 2. The 3' end is modified by the addition of about 200 adenines by the poly-A polymerase 3. The RNA must undergo splicing to remove the introns

Notes on topoisomerase type I in prokaryotes vs eukaryotes

In prokaryotes, topo I can only remove negative supercoils In eukaryotes, topo I can remove positive or negative supercoils

Early prophase

In this subphase of cell division, chromosomes become visible, centrosomes separate and migrate toward opposite poles and mitotic spindles and asters form Centrioles move to opposite ends of the cell Centrosomes have duplicated. Chromatin is condensing into chromosomes, and the nuclear envelope is fragmenting. -M phase promoting factor -Chromatin condensation >Condensin, ATP -Formation of mitotic spindle >Fibers from centrosome >Each spindle fiber, bundle of microtubules >Aster - "brace"

Cytoplasm

Includes the organelles and the cytosol, an amorphous ground matrix. This cytoplasmic matrix contain solutes such as inorganic ions (Na, K, Ca), and organicmolecules such as metabolites,carbohydrates, lipids, proteins, andribonucleic acids (RNAs). The cell controls the concentration of solutes within the matrix, which influences the metabolic activity within the cytoplasmic compartment.

To aid in determining completeness of gross excision...

India ink or other marking dyes can be applied with a cotton-tipped applicator to tumor margins. Different colored inks can be useful for orienting the tissue and distinguishing between left and right margins. These work best when applied to fresh, rather than fixed tissue. If submitting a smaller, representative, section of a larger tissue, - it is important to provide detailed information about the gross appearance of the original tissue and where the section was taken. We love to get photos of the lesion, with info on its location on the animal. That information can be very helpful for the pathologist).

Cellular inclusions

Inert, not metabolically active -Common inclusions >Melanin >Lipofuscin >Hemosiderin (a hemoglobin breakdown product) >Glycogen >Lipid -things that are being stored/held inside the cell -EX: melanin - accumulates in cells and darkens skin -EX: mucus - stored in the cell and secreted later when needed storage compartments, whose contents are not produced by the cell materials produced by and found or stored within the cells like hemoglobin, melanin, glycogen, lipids, mucus, etc.

Lysosome main function

Intracellular degradation of worn out organelles as well as macromolecules and particles taken into the cell by endocytosis/ phagocytosis

Phagosome

Intracellular vesicle containing material taken up by phagocytosis. vesicle formed as a result of phagocytosis Intracellular vesicle containing material taken up by phagocytosis.

Membrane proteins (and lipids) float in the fluid lipid bilayer and diffuse within the membrane. Why/when might this be important?

It is important that membrane proteins (and lipids) float in the fluid lipid bilayer and diffuse within the membrane because • It ensure even distribution of molecules between daughter cells after mitosis • Lipids and proteins can easily diffuse from their original membrane insertion sites • It enables interactions between membrane proteins

Identify tumors in the nucleus of all cells with

Lamins (Class IV)

More heterochromatin...

Less active cell

Routine samples are evaluated with a light microscope

Light source located below - beam focused on and passes through specimen Light enters objective lens and image is magnified (e.g. 2x, 4x, 10x, 40x, 100x) Image further magnified by the ocular lens (10x)

Scramblase

Non-specific lipid translocator which flips phospholipids between membrane leaflets, resulting in an equal distribution between monolayers. catalyzes transfer of random phospholipids from one monolayer to another moves lipids in either direction, toward equilibrium

Phagocyte in tissue

Macrophage (Histiocyte)

Periodic acid-Schiff (PAS)

Magenta: glycogen and carbohydrate-rich molecules Stains fungi (except Actinomycetes) magenta against a light pink or green background. Stains glycogen cells magenta. May use hematoxylin as a counterstain s used to stain carbohydrates such as mucus or glycogen within cells, the carbohydrate-rich glycocalyx at cell surfaces and the polysaccharides in basement membrane. These components stain a magenta or purplish-red color.

Artifacts and processing errors

Many different causes for artifacts in tissue section Artifacts resulting from antemortem procedures - Suture material - Bone fragments - Plant, bacteria, or hair contaminants - Thermal dehydration (i.e. cautery)

vesicular transport model

Materials are transported between the Golgi cisternae via membrane vesicles that bud from one compartment in the Golgi and fuse with another compartment Model of transport through the Golgi apparatus in which vesicles transport proteins between static cisternae Cargo is shuttled from the CGN toward the TGN in vesicles. The actual stacks remain relatively unchanged.

Constitutive secretion

Materials are transported in secretory vesicles and discharged in a continual manner performed by most cells: functions to maintain the extracellular matrix and plasma membrane discharge of materials synthesized in the cell into the extracellular space in a continual manner

Some functions of intermediate filaments

Mechanical strength and resistance to shearing forces

Lysosomes structure

Membrane sac of digestive enzymes membranous sacs containing acid hydrolases -Membrane bound vesicles that hold >40 degradative enzymes >Acid hydrolases with optimal activity at pH ~4.5-5.0 >Proton (H+) pumps maintain a low lumen pH.

Vimentin IF

Mesenchyme, Connective tissue Sarcoma

What type of cells tend to have more euchromatin?

Metabolically active cells tend to have more euchromatin than quiescent cells

Intermediate filaments polarity

Nonpolar

Resolution details

Microscope resolution is the most important determinant of how well a microscope will perform. The resolution of an optical microscope is defined as the shortest distance between 2 points on a specimen that can still be distinguished as separate entities. If the 2 points are closer together, they will blur into a single point. Resolution depends on a number of parameters, and these determine how well 2 point can be seen as distinct entities. These include the following factors: Wavelength of light being used toilluminate the specimen. Longerwavelengths of light offer less resolutionthan short wavelength illumination.Near-ultraviolet light has the shortestusable wavelength and offers thegreatest resolution. Following near-ultraviolet in descending order ofwavelength are red, orange, yellow,green, blue and violet. The range innanometers of the wavelength of thevisible light is from 380nm to 750nm.The wavelength of visible light limitsresolution to that of relatively largecytoplasmic organelles such as thenucleus, the Golgi apparatus or certainvacuoles and inclusions such asphagocytosed debris, hemosiderin ormelanin granules, but the wavelength ofan electron beam (electron microscopy)is much shorter (0.006 nm), allowing much better resolution, down to the level of macromolecules. Numerical aperture of the objective lens in the light microscope: This number indicates the ability of the lens to gather light and resolve a point at a fixed distance from the lens. The smallest point that can be resolved by an objective is in proportion to the wavelength of the light being gathered, divided by the numerical aperture number. Consequently, a higher number corresponds to a greater ability of a lens to define a distinct point in the view field. Refractive index of the media between the specimen and the microscope objective lens: Resolution can be improved by increasing the refractive index between the objective lens and the specimen. The refractive index is merely a ratio expression of the relative speed of light passing through a substance as a proportion of the speed of light in a vacuum. As the refractive index increases, the speed of the light passing through a medium is slowed. As light slows down the wavelength gets shorter and yields better resolution. Refractive index of air is 1.0, of water 1.33, and oil 1.40. Thus the oil immersion lens will allow better resolution. Examples of resolution: 1. Human eye: 0.1 mm 2. Light microscope using air as media (10x or 40x lens): 0.3 um 3. Light microscope using immersion oil (100x lens): 0.2 um (200 nm) 4. Scanning electron microscope: 0.8-5.0 nm 5. Transmission electron microscope: 0.3 nm Magnification is the ability to make an object look larger -- it does not improve clarity. Magnification also utilizes lenses, but if the resolving power is poor, increasing magnification only magnifies a blurry specimen.

Cytology

Microscopic study of individual cells -Common technique -Provides very fast results -Aspirate cells with a syringe (fine needle aspirate) and squirt onto slide -Or touch fresh tissue against a slide

Cilia and flagella are composed of...

Microtubules 9 bonded pairs of microtubules arranged around 2 unbonded pairs. -9 pairs of circularly arranged microtubule doublets -2 central microtubules (9+2) -Linker proteins -Arise from basal body >9 triplets, no central microtubules (9+0)

Dynamic instability of microtubules

Microtubules come from the centrosomes - link to chromosomes - then pull chromosomes back to a corner of the cell - in preparation for cell division Stable microtubules have a GPT cap; follows the rate of polymerziation - goes away if polymerization stops Once GTP cap is gone; microtubule is unstable and will depolymerize allows microtubules to perform their functions. GTP promotes monemer tubulin elongation, so when GTP is present growth is promoted Over time however GTP will hydrolize to GDP and GDP promotes disassembly So the two are in equilibrium, the rate of GTP to GDP is roughly constant Which ever one is more present at a given time creates a favoring effect to whatever they promote

Some locations of actin

Microvilli & terminal web; beneath plasma membrane; muscle contractile elements

Microtubules polarity

Minus end is nongrowing and embedded in MTOC; Plus end is growing end

Actin polarity

Minus end is slow-growing; :Plus end is fast growing

Golgi apparatus main function

Modification, sorting and packaging of proteins and lipids for either secretion (out of the cell) or delivery to the plasma membrane or other organelles

Phagocyte in blood

Monocyte and neutrophil

Vesicular transport

Moves materials between organelles & the plasma membrane -Vesicles bud off from one organelle and fuse with another (or the plasma membrane) contributing >Proteins from rER (modified in Golgi) >Lipids from sER -Things in lumen remain in lumen or pass into the extracellular space -Vesicles move from place to place by motor proteins that pull them along the cytoskeletal proteins

Apoptosome

Multiprotein complex that consists of cytochrome c molecules and Apaf-1 and helps to initiate apoptosis by activating procaspase 9 into caspase 9. A complex formed by the aggregation of cytochrome c and molecules of a protein called Apaf-1, that cleaves and activates the initiator caspase, caspase 9, thus triggering the caspase activation cascade in the intrinsic pathway of apoptosis. a large quaternary protein structure formed in the process of apoptosis. Its formation is triggered by the release of cytochrome c from the mitochondria in response to an internal (intrinsic) or external (extrinsic) cell death stimulus.

Membrane properties

Must be impermeable - to keep regions separate Allow communication (proteins) Expand and change shape (lipid and protein mobility) Import/export specific ions/molecules (channels, pumps) Interact with and recognize the outside world and other cells (receptors, carbohydrates) Protect the cell from the elements (carbohydrates)

Some functions of microtubules

Network for movement of organelles; Movement of cilia & chromosomes during mitosis

Five differences between eukaryotes and prokaryotes

Prokaryotes consist of a single closed compartment surrounded by the plasma membrane and lack a defined nucleus. Eukaryotes are more complex, have a defined membrane- bound nucleus, and are larger.

Rough ER is an important site of...

Protein synthesis -"Interconnected membrane- lined, flattened cisternae (pita pockets) arranged in parallel arrays -Rough ER appears stippled due to docked ribosomes -Ribosomes constantly bound & released

rER main function

Protein synthesis for distribution to most organelles and to the plasma membrane

Ribosome main function

Protein synthesis, both soluble and integral membrane proteins

What happens to proteins that fail to fold properly?

Proteins that the fail to fold properly are ubiquinated and then destroyed by a proteasome, an ATP-dependent protease The proteolytic machinery of the cell and the chaperones compete with one another

Electron microscopy

Provides information about the details of neuronal structure Microscopy using a microscope that employs a beam of electrons to illuminate the specimen. As electrons have a much smaller wavelength than light they produce images with higher resolutions than light microscopes. Uses electrons instead of light, the shorter wavelength of electrons gives greater resolution

Initiation of transcription

RNA polymerase attaches to the promoter region on the DNA and begins to unzip the DNA into two strands. Attachment of RNA polymerase to the promoter RNA polymerase binds to a promoter start (start!) promoter, transcription factors and start point RNA polymerase recognizes and binds a specific promoter site RNA polymerase separates the DNA strands to expose the template and create the transcription bubble Initiation is complete when the first two ribonucleotides of an RNA chain are linked by a phosphodiester bond

Termination of transcription

RNA polymerase reaches a special sequence of nucleotides that serves as a termination point. In eukaryotes, the termination region contains the DNA sequence AAAAAA. The third, and last, phase of transcription in which the mRNA transcript is released when RNA polymerase reaches the terminator sequence RNA polymerase reaches a terminator sequence (Stop codon) and detaches from the template Stop signal causes RNA polymerase to dissociate and the RNA transcript is complete Certain factors cause the RNA polymerase to dissociate - Rho dependent, rho independent (prokaryotes) - poly A signal (eukaryotes) Pre mRNA is released (but must still be processed)

Elongation in transcription

RNA polymerase traverses the template strand and uses base pairing complementarity with the DNA template to create an RNA copy RNA nucleotides are added to the chain RNA polymerase unzips the DNA and assembles RNA nucleotides using one strand of DNA as a template. DNA is unwound, the template strand is read in the 3'-5' direction and RNA is synthesized in the 5' to 3' direction by RNA polymerase Elongation occurs along the DNA molecule and therefore DNA unwinding must occur Transcription bubble is approximately 20 base pairs per polymerase molecule RNA polymerase has "unwindase" activity that opens the DNA helix Topoisomerases precede and follow the progressing polymerase to prevent super helical complexes RNA is elongated in the 5'-3' direction, therefore DNA is read in the 3'-5' direction

Glycogen Carmin stain

Red

Endomembrane system

Regulates protein traffic and performs metabolic functions in the cell The collection of membranes inside and around a eukaryotic cell, related either through direct physical contact or by the transfer of membranous vesicles. A network of membranes inside and around a eukaryotic cell, related either through direct physical contact or by the transfer of membranous vesicles.

Passive transport

Requires NO energy, Movement of molecules from high to low concentration, Moves with the concentration gradient the movement of substances across a cell membrane without the use of energy by the cell • Passive transport (a.k.a. facilitated diffusion) >Channel or Transporter >Requires no expenditure of energy >Allow solutes to flow down electrochemical gradient >Transports only substrates with a relatively specific molecular configuration >Availability of carriers limits the process

Mechanically gated ion channels

Respond to membrane distortion Found in sensory receptors that respond to touch, pressure, or vibration found in sensory neurons and open in response to physical forces such as pressure or stretch respond to mechanical vibration or pressure open when mechanical force applied to the channel.

Vesicular transport is facilitated by

SNARE proteins -Vesicle docks with the appropriate organelle (e.g. lysosome) through molecular markers on vesicle membrane and SNARE proteins >SNARE on vesicle (red line)interacts with with SNARE on target (blue squiggles) >Holds vesicle in place -SNARE proteins also catalyze vesicle fusion with membrane of target organelle or plasma membrane

Defects in membrane transporters can cause diseases such as congenital cystinuria (FYI)

Selective defect in the membrane transport protein that moves cystine from the urine back into the bloodstream. Leads to accumulation of cystine (and cystine crystals) in the urine, (upper right image) Over time this can result in bladder stones composed of cystine (lower right image). FYI

What regulates the shape and number of actin filaments in the cell?

Shape and number of filaments is regulated by the nucleus - which can increase or decrease the number of regulatory proteins

Resolution

Shortest distance between 2 points on a specimen that can still be distinguished as separate entities.

Mitochondria functions

Site of ATP synthesis; powerhouse of the cell 1. Production of Cell Energy 2. Cell Signaling 3. Cellular Differentiation 4. Cell Cycle Growth & Regulation Production of cell energy, cell signaling, cellular differentiation, and cell cycle and growth regulation -Production of ATP >Aerobic (oxidative) phosphorylation >Krebs cycle >Fatty acid oxidation >Amino acid oxidation -Steroid hormone synthesis -Initiate apoptosis (programmed cell death)

Rough Endoplasmic Reticulum (RER)

Site of synthesis of secretory (exported) proteins and of N-linked oligosaccharide addition to many proteins. processes and transports proteins made at attached ribosomes; synthesizes phospholipids the region of the endoplasmic reticulum that is studded with ribosomes and engages in protein modification rER well developed in secretory cells Embedded ribosomes (RNA) cause blue tinge to cytoplasm (especially with Romanovsky stains)

Mitotic activity: continuously dividing

Skin, intestinal epithelial cells, and bone marrow

Endosome main function

Sorting of endocytosed material, with recycling of some molecules back to the plasma membrane

Cytology: use squash preparation to smear material

Squirt material on glass slide and squash with a second slide, pulling one past the other Air-dry the smears

Toluidine blue stain

Stains acid mucopolysaccharides reddish purple (metachromatic) More complicated than alcian blue and used less often Metachromatically stains mast cells Stains mast cell granules metachromatically (dye blue but granules purple) Used to identify mast cells and connective tissue. Mast cells are found in the connective tissue and their cytoplasm contains granules (metachromatic) composed of heparin and histamine. Toluidine blue should stain mast cells red-purple (metachromatic staining) and the background blue (orthochromatic staining).

PAS stain

Stains glycogen (PASs the sugar) Dx of Whipples dx (tropheryma whipplei)

Glycogen

Storage form of glucose

Intermediate filaments characteristics

Strong, stable structures

Organelles

Structures specialized to perform distinct processes within a cell. A tiny cell structure that carries out a specific function within the cell Organelles include mitochondria, endoplasmic reticulum, Golgi apparatus, etc.

sER main function

Synthesis of lipids and lipoproteins, phospholipids, and steroids, carbohydrate metabolism, detoxification of drugs and toxic molecules such as alcohol; sequester Ca2+ from the cytosol; modified form of smooth ER (a.k.a. sarcoplasmic reticulum) regulates calcium ion concentration in muscle cells

Factors that affect fixation

Temperature, Size, Volume ratio, Time, Choice of fixative, Penetration, Tissue storage, pH, Osmolality

Immunohistochemistry (IHC)

Test to detect antigens (e.g., proteins) in cells of tissue by exploiting the principle of antibodies binding to specific antigens. Can detect a protein in tissue: -An antibody binds to the protein -Chemical treatments make the antibody visible -Reveals cells that have a common protein a technique in which labeled antibodies are used to visualize the histological distribution of specific proteins Is the localization of a specific antigen by the use of antibodies. The tissue slice is incubated first with an antibody that is specific for the tissue antigen in question, then with a second antibody specific for an epitope on the first antibody. This second antibody is tagged to an enzyme which will react with a substrate and produce a colored chemical, which precipitates in place, thus "staining" the tissue. Since the antibody that reacts with antigen in the tissue section is itself an antigen for the second antibody the resultant staining of cellular components will be highly specific to the intracellular or extracellular location of the antigen in question. This process is best done on lightly fixed tissue or frozen sections in which the antigen has been minimally denatured.

DNA methylation

The addition of methyl groups (—CH3) to bases of DNA after DNA synthesis; may serve as a long-term control of gene expression. The addition of methyl groups to bases of DNA after DNA synthesis; may serve as a long-term control of gene expression.

Rab proteins

The availability of SNAREs for membrane fusion are regulated by what? Family of monomeric GTPases which associate with specific membranes and function in the specificity of vesicle docking A family of small GTP-binding proteins present on the surfaces of transport vesicles and organelles that serves as a molecular marker to help ensure that transport vesicles fuse only with the correct membrane.

DNA

The carrier of genetic information Composed of monomers called nucleotides (bases): Adenine, thymine, guanine, and cytosine

semiconservative model

The double-stranded DNA contains one parental and one daughter strand following replication the two strands of the parental molecule separate, and each functions as a template for synthesis of a new, complementary strand Type of DNA replication in which the replicated double helix consists of one old strand, derived from the old molecule, and one newly made strand.

Cell cycle

The genes that make up the genetic code are arranged in chromosomes. These chromosomes must be able to replicate, and the replicated copies must be separated and reliably partitioned into daughter cells of each cell division. This process occurs through an ordered series of stages, collectively known as the cellcycle. More information will provided later in the course about the process of mitosis. For now understand that the cell cycle can be divided into 2 broad phases; 1) interphase, when chromosomes are duplicated in 2) mitosis, when chromosomes are distributed to the 2 daughter nuclei

The Golgi generates...

The heterogeneous oligosaccharide structure seen in mature proteins -Glycoproteins are essential for: >Cell signaling >Cell-cell communication >Cell protection, etc. >Part of glycocalyx on outer membrane

Histones

The histones are the major class of DNA-binding proteins involved in maintaining the compacted structure of chromatin.

Nucleus

The nucleus is a membrane bound compartment within the cell and contains genetic information in addition to the necessary machinery for DNA replication and RNA transcription. In reality the largest cellular organelle!

Toluidine blue

The pH indicator of the DNase test is: Which mast cells stains stains granules purple and is also a stain rarely used to stain mucin red-purple? Differentiates wbcs from renal tubular cells

RNA splicing

The removal of noncoding portions (introns) of the RNA molecule after initial synthesis. The removal of introns and joining of exons in eukaryotic RNA, forming an mRNA molecule with a continuous coding sequence; occurs before mRNA leaves the nucleus. Process by which the introns are removed from RNA transcripts and the remaining exons are joined together.

Hemosiderin fixed H&E

Yellow-brown chunky pigment

Can cells increase the amount of euchromatin if needed?

Yes

Histology (from Greek root for tissue)

The study of the microscopic anatomy of cells and tissues (the examination of a thin slice, or section, of tissue under a microscope). The object of this Histology course is to help you understand the microanatomy of cells, tissues, and organs and to correlate structure with function.

Pyknotic

The term used to describe a nucleus that has died. The chromatin has become densely compacted, so no pattern is visible. Composed entirely of heterochromatin

receptor-mediated endocytosis

The uptake of specific molecules based on a cell's receptor proteins binding of ligands to receptors triggers vesicle formation The movement of specific molecules into a cell by the inward budding of membranous vesicles containing proteins with receptor sites specific to the molecules being taken in; enables a cell to acquire bulk quantities of specific substances.

Receptor-mediated endocytosis

The uptake of specific molecules based on a cell's receptor proteins binding of ligands to receptors triggers vesicle formation The movement of specific molecules into a cell by the inward budding of membranous vesicles containing proteins with receptor sites specific to the molecules being taken in; enables a cell to acquire bulk quantities of specific substances. entry of specific molecules into endosomes

Membrane potential

The voltage across a cell's plasma membrane.

Control of the cell cycle

There are checkpoints after G1, S and G2 which the cell monitors for errors. If errors are found and the cell can't fix them, the cell will undergo "apoptosis" or cell death.

Actin characteristics

Thin, flexible filamentsDynamic instability & treadmilling; more stable structures also possible

Electron microscopy details

This includes both transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Both techniques required samples to be "stained" or mixed with a particular element in order to produce images. Both methods use an electron microscope and electron beams (rather than the photons in visible light) to produce highly magnified images with a high resolution that allow one to study the sub-microscopic architecture (ultrastructure) of tissues - structures that can't be seen using light microscopy. TEM creates high-resolution images of the two-dimensional arrangement of molecular structures in plastic-embedded tissue sections. Glass or diamond knives are used to cut these ultrathin sections. More details about how tissues are processed for TEM an be found in the following section. SEM offers high-resolution, three-dimensional, surface views of both natural and fractured tissue surfaces. The diagram below shows the limits of resolution available with a light microscope as compared to an electron microscope. 1. The micrometer or micron (μm) is the most convenient unit for measuring the dimensions of tissues, cells and cellular components at the light microscopic level. Study the subdivisions of one millimeter into micrometers. The micrometer (μm) is too large a unit to conveniently measure ultrastructure with the transmission electron microscope. The Angstrom (Å) unit or, preferably, the nanometer (nm), which is 10 Å, is used instead. 2. Transmission Electron Microscopy (TEM): TEM is based on transmitted electrons. In TEM, electrons are pointed directly toward the sample. When electrons interact with heavy metal atoms, either naturally resident in the tissue or within the stain chemically bound to the tissue, they deflect the electrons, no signal is detected, and such a region registers as "black". When electrons pass through the sample, those parts are illuminated in the image and that region is registered by the signal detector as "white". TEM delivers a two-dimensional picture that illustrates what is inside the cell or in the environment around it. The resulting image is black and white because the image was produced by electrons, rather than with visible light. a. Tissues for TEM must be fixed, embedded and sectioned much like those for light microscopy, but with some differences. Glutaraldehye is used as the primary fixative because, unlike formalin, it does not cause tissue shrinking. Samples (tissue slices) must be <3 mm3 for good morphology in electron microscopy. The fixed tissue is cut into 2-mm2 tissue blocks and post fixed in 1% osmium tetraoxide. It is then stained en block with 5% aqueous uranyl acetate. Paraffin wax will not support the extremely thin sections that must be made to allow an electron beam to penetrate for electron microscopy. Tissues for ultrastructural electron microscopy study are dehydrated in graded ethanol series and then embedded in resin, a liquid plastic solution that polymerizes, when heated, to a very hard block. To preview the tissue and find interesting sections of the sample, a thin section (1 μm) is cut and stained with toluidine blue (remember that for light microscopy we are usually looking at sections that are 3-5 μm in thickness). Areas of interest are then thin sectioned. Ultrathin sections (50-70 nm) for electron microscopy are cut on an ultramicrotome fitted with magnifying binocular eyepieces to view the tiny sections. They are cut with extremely sharp diamond knives and picked up on copper grids. The ultrathin sections are stained with uranyl acetate and lead citrate. Contrast in ultrathin sections is provided by the differential scattering of electrons. Electrons are scattered most strongly by atoms with high atomic number. For this reason cellular components are stained with heavy metal compounds such as osmium tetroxide, uranyl acetate and, sometimes, lead citrate. The heavy metal atoms interact with the electron beam, creating black and white images that reflect cellular architecture. 1) Osmium tetroxide embeds a heavy metal directly into cell membranes, binding to phospholipid head regions, thus creating contrast with the neighboring protoplasm (cytoplasm). Osmium tetroxide also cross-links macromolecules and functions as a fixative, stabilizing many proteins without destroying structural features. 2) Uranyl acetate is made from depleted uranium and the uranium, binds strongly to the phosphate groups of DNA. Notice that for light microscopy, staining is done on tissues after they have been fixed, dehydrated, embedded and sectioned. For electron microscopy, the staining is done immediately after tissue fixation, before they have been dehydrated or embedded. Materials that are often removed during the dehydration process (e.g. glycogen and lipid) remain present in these samples. The samples largely have to be viewed in vacuum, as the molecules that make up air would scatter the electrons.

Virtual microscopy

This is a digital procedure to capture the light microscopic representation of glass slides, often at multiple magnifications. This relies on the digital stitching together of tiles of images along 2-dimensional planes to allow the remote viewing of slides that can be zoomed in/out. Computerized optical scanning of a prepared slide or specimen, usually using modified bright-field or fluorescence microscopes; 2D Typically used for study of brightfield microscopic preparations, involves the conversion of a stained tissue preparation to high- resolution digital images and permits study of tissues using a computer or other digital device, without an actual stained slide or a microscope.

Nonhistone chromosomal proteins

This is a diverse group of proteins that includes the various transcription factors, polymerases, hormone receptors and other nuclear enzymes. In any given cell, there are greater than 1000 different types of non-histone proteins bound to the DNA.

For conventional observation by light microscopy...

Tissue and organ specimens must be thin enough to allow transmission of the illuminating radiation (photons). This is accomplished by cutting thin sections of the specimen (typically 5 microns thick; for reference, a canine erythrocyte is 7 microns in diameter - the human erythrocyte is 8 microns). A specimen that is to be sliced thinly must first be hardened so that it can be cut cleanly. The typical histology samples in this course have gone through a series of steps in which they have been chemically fixed, dehydrated, embedded in paraffin, thinly sliced and stained, usually with H&E.

Frozen sections

Tissue can be frozen and then sectioned in slices nearly as thin as those with paraffin wax support. Use of frozen tissue that has been very lightly fixed allows preservation of immunological reactivity and is also a much quicker process than dehydration and embedding in wax or plastic. Frozen sections can be prepared within minutes and used for rapid diagnosis of tissue removed during surgery before the procedure is completed. Frozen sections (6-8 um) are cut in a cryostat, or microtome in a cold chamber

Necrosis

Tissue death •Mechanical Injury •Hypoxia •Infectious Agents •Toxins

Staining

Tissue has little inherent contrast, appearing featureless unless the sections are stained with various dyes. Forlight microscopy, staining with cationic(basic) and anionic (acid) dyes is the principal way of creating the color contrasts necessary for structural analysis >A cell or tissue component that binds a basic dye is termed basophilic ("base loving"). Conversely, a component that binds an acid dye is acidophilic. These designations are important distinctions drawn to characterize cellular and tissue components, although they do not conform to "conventional" definitions of an acid and a base. 1) A basic dye carries a net positive charge and reacts with a negatively charged tissue group (e.g. the phosphate groups of nucleic acids). 2) An acidic dye carries a net negative charge and reacts with a positively charged tissue group (e.g. basic proteins) in cells and tissues, particularly with the ionized amino groups of proteins. 3) Metachromasia: In a tissue section, certain basic dyes will react with tissue components that shift their normal color from blue to red or purple. This color shift is called metachromasia. This is caused by polyanions within the tissue (e.g. heparin). When these tissues are stained with a concentrated basic dye solution (e.g. toluidine blue, methylene blue), the dye molecules are close enough to form dimeric and polymeric aggregates. The absorption properties of these aggregations differ from those of the individual nonaggregated dyes molecules. Cell and tissue structures that have high concentrations of ionized sulfate and phosphate groups - such as cartilage or heparin-containing granules of mast cells will exhibit metachromasia and toluidine blue and methylene blue will appear purple to red when it stains these components. >The most commonly used stain for light microscopy is hematoxylin and eosin (H&E). Hematoxylin is alkaline and binds to acidic cellular components such as nucleic acids. Thus cell nuclei are referred to as basophilic and stain blue-purple. Proteins react with the eosin dye and stain pink to orange; they are described as eosinophilic. >Many stains for light microscopy are aqueous and require that the paraffin wax is removed from the section. >Special stains are used to highlight tissue components, such as fat, iron, copper, calcium, carbohydrates, collagen, components ofbacterial cell walls. Note - that these stains will not identify very specific proteins (e.g. specific hormones such as cortisol or insulin) or specific infectious agents (e.g rabies virus). The list below includes some of the more commonly used special stains, but see following table and the e- Lab guide on special stains for more examples and more details. 1) Periodic Acid-Schiff (PAS) is used to stain carbohydrates such as mucus or glycogen within cells, the carbohydrate-rich glycocalyx at cell surfaces and the polysaccharides in basement membrane. These components stain a magenta or purplish-red color. 2) Trichrome is a three-color stain used especially to highlight collagen from cellular components (there are several different other types of trichrome stain recipes, all used to highlight collagen). Collagen appears blue or blue-green, and thus is easily distinguished from muscle and cell cytoplasm, which turns red, and nuclei, which turn brown or black. 3) Silver stains are used to identify fungi (black) in tissue. Silver stains can also be used to stain type III collagen (reticular fibers) in connective tissue and neuron processes (axons and dendrites). 4) Acid fast stain highlights Mycobacterium tuberculosis and related bacteria, by staining them bright red or fuscia (when the usual carbol-fuchsin stain is used). Acid fast will also stain certain protozoa (e.g. Cryptosporidium) and some bacteria (e.g. Nocardia). 5) Oil Red O is used to stain lipids. 6) Orcein stain & Weigert's elastic stain are two stains used to highlight elastic fibers (red-brown or blue-black, respectively)

Control of cell numbers and cell size

Tissue homeostasis is maintained by a subtle balance between cell death and cell proliferation. The number of cells in an organisms is tightly regulated, through both the control of cell division, as well as by controlling the rate of cell death. Any changes in one of these can form the basis for disease. A. Cell Cycle: All cells arise from a cycle of cell growth and division. That process is carried out through an orderly sequence of events inwhich the cell duplicates its contents and then divides into two daughter cells. In this process the cell duplicates the DNA in chromosomes, in order to maintain a normal size, the cell will typically also duplicate its other macromolecules and organelles. This material is then segregated into genetically identical daughter cells. The cell cycle is tightly regulated by a complex network of regulatory proteins, which will be discussed in a subsequent lecture. B. The duration of the cell cycle, and the rate of mitosis, varies greatly from cell type to cell type. Some cells types remain in G0. 1. Mitotic activity varies from tissue to tissue and they can be classified as: a. Static - generally not capable of division (i.e. neurons, cardiac muscle) b. Stable - undergoing periodic division to maintain normal function (i.e. liver, CT cells) c. Continuously dividing - always dividing (i.e. epithelial cells & bone marrow). 2. If the cell cycle is disrupted and mitosis is allowed to proceed at an inappropriate rate, neoplasia can result. C. Mitotic chromosomes, present during the mitosis phase of the cell cycle, can be seen in a histologic section or cytologic smear. They will appear as much darker chromatin than seen in surrounding cells, and the nuclear envelope will be missing. The chromosomes will sometimes be arranged in a straight line along the equator of the cell. In other cells, they will be less organized and have been described as looking like a spider, as chromosomes extend away from the mitotic spindle. D. Programmed cell death: If cells are no longer needed, they can activate an intracellular desk program known as apoptosis (pronounced: apo-toe'-sis; note that the second "p" of the word is silent). This comes from a Greek word meaning "falling off" as leaves fall from a tree. Apoptosis is essential during early development, when body parts are developing. For example, developing paws start out as spade-like structures, until the cells in between the developing toes die off. In adult tissues, cell death usually exactly balances cell division, unless the tissue is growing or shrinking. In a healthy adult, billions of cells die in the bone marrow and intestine every hour, while new cells are constantly being produced. In addition, virus-infected animal cells will frequently kill themselves to prevent the virus from replicating and infecting other cells. 1. Cells dying by apoptosis undergo characteristic morphologic changes. They shrink and condensed, the cytoskeleton collapses, the nuclear envelope disassembles, and thenuclear chromatin condenses (becomes pyknotic; i.e. composed entirely of heterochromatin) and breaks up into fragments (process referred to karyorrhexis; cells would be described as karyorrhectic). Unlike mitotic figures, pyknotic nuclei and the fragments produced by karyorrhexis, are round. The cell surface often blebs and if the cell as large, it breaks up into smaller membrane-enclosed fragments called apoptotic bodies. The surface of the cell or apoptotic bodies becomes chemically altered, so that a neighboring macrophage (a specialized phagocytic cell) rapidly engulfs them, before they can still their contents. In this way, the cell dies neatly and is rapidly cleared away, without causing a damaging inflammatory response. Because the cells are being eaten (phagocytized) and digested quickly, there usually few dead cells to be seen in a given area, even when large numbers of cells have died by apoptosis. By contrast, cells that die accidentally, in response to an acute insult such as trauma, a lack of blood supply, hypothermia, excessive and you, low pH, microbial pathogens or ionizing radiation, you so by a process called cell necrosis. Cells that die by this process undergo swelling, the cell membrane dissolves and the cells burst, spilling their contents over neighboring cells, triggering an inflammatory response. Typically, all the cells in the area of insult are affected, such that large numbers of dead cells will be seen in that area. In contrast to necrosis which is always associated with disease, apoptosis is often a completely normal process that occurs in all living organisms. 2. Apoptosis is caused by an intracellular proteolytic cascade mediated by caspases. Caspases are proteases that have a cysteine at their active site and cleave their target proteins at specific aspartic acids (hence "c" for cysteine and"asp" for aspartic acid).Caspases are produced in the cell as inactive precursors, or procaspases, which are typically activated by proteolytic cleavage. Once activated, caspases cleave and thereby activate other procaspases, resting in an amplifying proteolytic cascade. 3. While not all caspases mediate apoptosis, at least 7 of the 14 known mammalian caspases irreversibly commit a cell to die. They apoptotic caspases are generally divided into two classes: the initiator caspases and the effector (or executioner) caspases. a. Initiator caspases operate at the start of the proteolytic cascade. When initiator procaspases are activated they cleave and activate downstream executioner procaspases. b. The activated executioner caspases then cleave and activate other executioner procaspases, as well a specific target proteins in the cell. Among the many target proteins cleaved by executioner caspases are the 1) Nuclear lamins 2) a protein that normally holds an endonuclease (DNA- degrading enzyme) in an inactive form - its cleavage frees the endonuclease to cut up the DNA in the cell nucleus 3) Cytoskeleton components 4) Cell-cell adhesion proteins that attach cells to their neighbors - the cleavage of these proteins helps the apoptotic cells to round up and detach from its neighbors making it easier for a healthy neighboring cell to engulf it; or, in the case of an epithelial cell, for the neighbors extrude the apoptotic cell from the sheet of tightly cohesive cells. 4. Apoptosis can also be initiated through a variety of external and internal cell stimuli. Keep in mind that healthy cells continuously make the procaspases and other proteins required for apoptosis. Thus, the apoptosis mechanism is always in place and all that is needed is a trigger to activate it. The two best understood signaling pathways that can lead to apoptosis in mammalian cells are called the extrinsic pathway and the intrinsic pathway. Each uses its own initiator procaspases and activation complex. a. The extrinsic pathway is initiated by the binding of an extracellular death ligand to a cell-surface death receptor. The ligand- receptor complex recruits further cytosolic factors to form a death- inducing signaling complex. Formation of this complex leads to activation of the initiator caspase, which then cleaves inactivates the effector caspase. 1) The extrinsic pathway cancross talk with the intrinsic pathway, to amplify the cascade in order to kill the cell. 2) A subset of white blood cells known as killer lymphocytes, use this mechanism to induce apoptosis of a target cell. The killer lymphocyte has a death ligand (FAS) on its membrane. When the lymphocyte contacts it target cell, this ligand binds to the death receptor on the target cell membrane, triggering the apoptotic cascade. 3) Many cells produce inhibitory proteins that act to restrain the extrinsic pathway. Some produce cell surface decoys receptors, while others produce intracellular blocking proteins which resemble an initiator procaspase, but lack the proteolytic domain. b. Cells can also activate their apoptosis program from inside the cell, usually in response to injury or other stresses, such as DNA damage or lack of oxygen, nutrients, or extracellular survival signals. Many animal cells require continuous signaling from other cells to avoid apoptosis. In this way, the number of cells is automatically adjusted, during both development and in adulthood, so that is appropriate. 1) This intrinsic pathway depends on release of mitochondrial proteins into the cytosol. A crucial protein released from mitochondria in the intrinsic pathway is cytochrome c, a water-soluble component of the mitochondrial electron- transport chain. 2) When released into the cytosol, cytochrome c has an entirely different function, binding to a procaspase-activating adapter protein called apoptotic protease activating factor-1 (Apaf1). This causes Apaf1 to form a wheel-like structure called an apoptosome. The Apaf1 proteins in the apoptosome recruit initiator procaspase proteins (procaspase 9), which are activated by proximity in the apoptosome. The activated caspase-9 then activates downstream executioner procaspases to induce apoptosis. 3) The intrinsic pathway of apoptosis is tightly regulated to ensure that cells kill himself only when it is appropriate. The balance between pro-apoptotic and anti-apoptotic factors largely determines whether a mammalian cell lives or dies by the intrinsic pathway of apoptosis. a) Survival factors, secreted by other cells, usually bind to sell-surface receptors, which activate intracellular signaling pathways that suppress apoptotic program. Cells deprived of survival factors kill themselves by producing and activating pro apoptotic factors only. b) The tumor suppressor protein p53 accumulates in response to DNA damage that cannot be repaired, and activates the transcription of genes that encode proteins that inhibit anti-apoptotic proteins. This triggers the intrinsic pathway, thereby eliminating a potentially dangerous cell that could otherwise become cancerous

Tissue biopsy

Tissue sample for microscopic examination. - Removal of piece of tissue/ organ at surgery, or necropsy - Fixed in 10% buffered formalin, processed, embedded in paraffin - Result: section of tissue, stained

Actin bundles are anchored...

To a terminal web of actin in cell cortex at base of microvilli

Golgi apparatus is responsible for

Translational modification and sorting Processing and packaging of proteins -Stack of flattened saccules , near nucleus, without ribosomes >Usually curved - shaped dependent on cytoskeletal element and Golgi matrix proteins -Proteins are modified as they move through the saccules becoming glycosylated, phosphorylated, sulfated, etc.

Glycocalyx

a capsule made up of a fuzzy coat of sticky sugars a bacterial capsule that is made of a fuzzy coat of sticky sugars The external surface of a plasma membrane that is important for cell-to-cell communication

DNA repair

a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome The removal and replacement of damaged DNA by the correct sequence Collective term for the enzymatic processes that correct deleterious changes affecting the continuity or sequence of a DNA molecule. -Direct reversal -Base excision repair - Nucleotide excision repair -Mismatch repair -Double strand break repair >Homologous recombination >Nonhomologous end joining

Death ligand

a cytokine in the membrane of cytotoxic T cells that binds to a Fas receptor, starting a signal cascade leading to apoptosis Soluble or cell surface protein that activates death receptors. a cytokine in the membrane of Tc cells that binds to a Fas receptor, starting a signal cascade leading to apoptosis

Euchromatin

Uncoiled chromatin with active DNA -Most cell types express about 20 to 30% of their genes. are discrete bodies of heterochromatin, irregular in size and shape, scattered throughout the nucleus.

Special stains

Used to distinguish parts of microorganisms Capsule stain Endospore stain Flagella stain Capsule stain Endospore stain Flagella stain Used to distinguish parts of cells Capsule stain Endospore stain Flagella stain are used to highlight tissue components, such as fat, iron, copper, calcium, carbohydrates, collagen, components ofbacterial cell walls. Note - that these stains will not identify very specific proteins (e.g. specific hormones such as cortisol or insulin) or specific infectious agents (e.g rabies virus). The list below includes some of the more commonly used special stains, but see following table and the e- Lab guide on special stains for more examples and more details. 1) Periodic Acid-Schiff (PAS) is used to stain carbohydrates such as mucus or glycogen within cells, the carbohydrate-rich glycocalyx at cell surfaces and the polysaccharides in basement membrane. These components stain a magenta or purplish-red color. 2) Trichrome is a three-color stain used especially to highlight collagen from cellular components (there are several different other types of trichrome stain recipes, all used to highlight collagen). Collagen appears blue or blue-green, and thus is easily distinguished from muscle and cell cytoplasm, which turns red, and nuclei, which turn brown or black. 3) Silver stains are used to identify fungi (black) in tissue. Silver stains can also be used to stain type III collagen (reticular fibers) in connective tissue and neuron processes (axons and dendrites). 4) Acid fast stain highlights Mycobacterium tuberculosis and related bacteria, by staining them bright red or fuscia (when the usual carbol-fuchsin stain is used). Acid fast will also stain certain protozoa (e.g. Cryptosporidium) and some bacteria (e.g. Nocardia). 5) Oil Red O is used to stain lipids. 6) Orcein stain & Weigert's elastic stain are two stains used to highlight elastic fibers (red-brown or blue-black, respectively)

Silver stains

Used to identify fungi (black) in tissue. Silver stains can also be used to stain type III collagen (reticular fibers) in connective tissue and neuron processes (axons and dendrites).

Red oil O

Used to stain lipids

Scanning electron microscopy (SEM)

Used to visualize the surface of tissues. Surface of tissue sputter coated with metal (e.g. gold) Electrons bounce off surface & are collected to form 3D image

Scanning electron microscopy

Uses an electron beam and backscatter of electrons to study the surface topography of cells and tissues. Tissue for SEM is fixed, dehydrated and then the outer surface of the tissue is sputter coated with a metal such as gold. The electron beam is then directed toward the coated tissue and electrons bounce off the tissue surface. The microscope collects and counts the scattered electrons, and forms a 3-D image from the reflected electrons.

Nucleolar-associated heterochromatin

Usually rims the nucleus

What does vesicular transport allow?

Vesicular Transport allows movement of materials in & out of cell -Exocytosis (outward secretory pathway) >Delivers new proteins, carbohydrates, and lipids to plasma membrane or the extracellular space -Endocytosis (inward secretory pathway) >Plasma membrane components delivered to endosomes >Components can be recycled or delivered to lysosomes for degradation >Used to import various nutrients or ingest organisms or debris

Scanning Electron Microscope (SEM)

a microscope which excites electrons on the surface of the cell, and shows the specimen's topography A microscope that uses an electron beam to study the surface architecture of a cell or other specimen. A microscope that uses an electron beam to scan the surface of a sample, coated with metal atoms, to study details of its topography.

Secretion

a process by which substances are produced and discharged from a cell, gland, or organ for a particular function in the organism or for excretion.

Na+/K+ transporter

a protein found in the membrane of all cells that extrudes sodium ions from and transports potassium ions into the cell a type of ATPase transporter in the plasma membrane of most cells that is responsible for accumulating intracellular K+ and extruding intracellular Na+. Also known as the Na+ pump. Active transport Antiporter

Transport vesicles

a small membranous sac in a eukaryotic cell's cytoplasm carrying molecules produced by the cell A tiny membranous sac in a cell's cytoplasm carrying molecules produced by the cell. Vesicles in transit from one part of the cell to another

Neuron

a specialized cell transmitting nerve impulses; a nerve cell. a nerve cell; the basic building block of the nervous system

Methylene blue

a staining dye/indicator that interacts with nucleic acid molecules and proteins, turning them to a very dark blue color Strong basic dye

Eosinophil

a white blood cell containing granules that are readily stained by eosin. a granular leukocyte, named for the rose-colored stain of its granules, that increases in allergic and some infectious reactions white blood cell containing granules that stain red; associated with allergic reactions

Histone acetylation

acetyl groups are attached to positively charged lysines in histone tails the attachment of acetyl groups (-COCH3) to certain amino acids of histone proteins, the chromatin becomes less compact, and the DNA is accessible for transcription The attachment of acetyl groups to certain amino acids of histone proteins.

Primary active transport

active transport that moves ions or small molecules across a membrane and may create a difference in charge across that membrane Active transport in which ATP is hydrolyzed, yielding the energy required to transport an ion or molecule against its concentration gradient. Active transport that relies directly on the hydrolysis of ATP.

acidophilia or eosinophilia

affinity for eosin which is an acid stain (yellowish-pink) Pink color resulting from acid dye binding to positive charges

scanning electron microscope

an electron microscope that generates a three-dimensional image. a microscope that produces an enlarged, three-dimensional image of an object by using a beam of electrons rather than light An electron microscope used to study the fine details of cell surfaces

Acidic dyes

anionic, negatively charged chromophore are negatively charged and work best at low pH Acid fuschsin (red) Aniline blue (blue) Eosin (red) Orange G (orange)

Acinar cell

basic secretory cell of salivary glands secretes digestive enzymes in pancreas A cell from the pancreas. It produces enzymes that are used in digestion.

Autolysis

breakdown of cells by their own enzymatic action the spontaneous breakdown of cells as they self-digest self-destruction of cells; decomposition of all tissues by enzymes of their own formation without microbial assistance Destruction of cells or tissues by their own enzymes

Acid fast stain

carbolfuchsin used to stain Mycobacterium species A staining procedure for identifying bacteria that have a waxy cell wall. a differential stain used to identify bacteria that are not decolorized by acid-alcohol

Basic dyes

cationic, positively charged chromophore Methylene green (green) Methylene bue (blue) Pyronin G (red) Toluidine blue (blue)

Lysosomes

cell organelle filled with enzymes needed to break down certain materials in the cell Uses chemicals to break down food and worn out cell parts An organelle containing digestive enzymes Lysosomes are the "digestive system" and "recycling plant" of the cell -Membrane bound vesicles that hold >40 degradative enzymes >Acid hydrolases with optimal activity at pH ~4.5-5.0 >Proton (H+) pumps maintain a low lumen pH. -Functions >Breakdown of intra-and extracellular debris >Destruction of phagocytized microorganisms >Production of nutrients for the cell >Digestion of the cell's obsolete organelles >H+ gradient that is drives transport of small back to cytoplasm (e.g. amino acids, sugars).

Constitutive exocytosis

constant stream of transport vesicles off the trans Golgi that delivers newly made lipids and proteins to the plasma membrane

Gene expression

conversion of the information encoded in a gene first into messenger RNA and then to a protein The process by which information encoded in DNA directs the synthesis of proteins or, in some cases, RNAs that are not translated into proteins and instead function as RNAs. process by which a gene produces its product and the product carries out its function The combined process of transcription of a gene into mRNA, the processing of that mRNA, and its translation into protein Required for: - Adaptation - Tissue specific differentiation - Development

Large amounts of sea can cause...

cytoplasmic eosinophilia (pink)

Translation

decoding of a mRNA message into a polypeptide chain (genetics) the process whereby genetic information coded in messenger RNA directs the formation of a specific protein at a ribosome in the cytoplasm Process by which mRNA is decoded and a protein is produced

Sorting signal

directs the protein to the organelle in which it is required. Proteins that lack such signals remain as permanent residents in the cytosol A short amino acid sequence in a protein that directs the protein to its correct location; also known as a traffic signal. directs the protein to the organelle in which it is required

Cytokinesis

division of the cytoplasm to form two separate daughter cells Division of the cytoplasm during cell division

Cisternal maturation model

each cistern "matures" as it moves from the cis face to the trans face Recent model for the Golgi apparatus that says cisternae of the Golgi progress from the cis to trans face, dynamically modifying their contents. These also means some vesicles transport proteins back to the Golgi to recycle enzymes. the cisternae of the golgi actually progress forward from the cis to the trans face, carrying and modifying their cargo as they move

Orcein stain and Weigert's

elastic stain are two stains used to highlight elastic fibers (red-brown or blue-black, respectively)

Sources of DNA damage

endogenous (replication errors, attach by reactive oxygen species) and exogenous (radiation, hydrolysis, toxins, viruses)

DNA ligase

enzyme that chemically links DNA fragments together an enzyme that eventually joins the sugar-phosphate backbones of the Okazaki fragments A linking enzyme essential for DNA replication; catalyzes the covalent bonding of the 3' end of a new DNA fragment to the 5' end of a growing chain. Responsible for joining the Okazaki fragments Joins DNA strands together by catalyzing the formation of a phosphodiester bond Completes short-patch DNA synthesis occurring in DNA repair process

Histopathology

microscopic examination of tissues for signs of disease

Secondary active transport

movement of material that is due to the electrochemical gradient established by primary active transport use pre-existing gradient to drive transport of solute Form of active transport which does not use ATP as an energy source; rather, transport is coupled to ion diffusion down a concentration gradient established by primary active transport.

Floppase

moves phospholipids from cytosolic to outer leaflet (ABC transporter) moves phospholipids from cytosolic to outer leaflet

Skeletal muscle cells

muscle fibers Are very long Develop through fusion of mesodermal cells (myoblasts) Become very large Contain hundreds of nuclei cells that move organs and body parts

Metachromasia

n a tissue section, certain basic dyes will react with tissue components that shift their normal color from blue to red or purple. This color shift is called... . This is caused by polyanions within the tissue (e.g. heparin). When these tissues are stained with a concentrated basic dye solution (e.g. toluidine blue, methylene blue), the dye molecules are close enough to form dimeric and polymeric aggregates. The absorption properties of these aggregations differ from those of the individual nonaggregated dyes molecules. Cell and tissue structures that have high concentrations of ionized sulfate and phosphate groups - such as cartilage or heparin-containing granules of mast cells will exhibit metachromasia and toluidine blue and methylene blue will appear purple to red when it stains these components.

Dehydration and embedding

n order to embed the fixed tissue in either paraffin (light microscopy) or plastics (light or electron microscopy), the water must be removed from the specimen since these embedding media are not miscible with water. This is accomplished by a gradual replacement of the tissue water with an organic solvent, usually ethanol, and then the replacement of the ethanol with an additional solvent in which the paraffin or plastic is soluble. Paraffin wax is the most commonly used embedding material. Samples are immersed in melted paraffin, which infiltrates into the cells and then solidifies to a hard block at room temperature. Thus most tissue samples prepared for microscopic study have been submerged in a series of increasing concentrations of alcohol to remove all water around and within cells. This is followed by an organic solvent miscible with paraffin. Since body tissues are approximately 70% water, this process can distort tissue structure, and any predominantly aqueous layer in the tissue is removed completely.

Phagocytic cells

neutrophils and macrophages neutrophils, macrophages, dendritic cells cells that engulf, ingest, and destroy foreign bodies or toxins

Commonly phagocytosed material

o Cell debris o RBCs o Carbon particles o Asbestos fibers o Bacteria, yeast

Voltage gated ion channels

open in response to changes in membrane potential A specialized ion channel that opens or closes in response to changes in membrane potential Channels that open or close in response to a change in the membrane potential. open when a certain membrane potential has been reached.

Ligand gated ion channels

open in the presence of a specific binding substance, usually a hormone or neurotransmitter membrane ion channels operated by the binding of specific molecules to channel proteins Ion channels that respond to chemical signals rather than to the changes in membrane potential generated by ionic gradients. The term covers a large group of neurotransmitter receptors that combine receptor and ion channel functions into a single molecule. open when a specific ligand binds to the channel

Initiator caspases

operate at the start of the proteolytic cascade. When initiator procaspases are activated they cleave and activate downstream executioner procaspases.

Eukaryotes

organisms made up of one or more cells that have a nucleus and membrane-bound organelles Cells that enclose their DNA in nuclei Cells that contain nuclei Eukaryotic cells are divided into functionally distinct, membrane-enclosed compartments

Conservative model of DNA replication

parental double helix remains intact, both strands of daughter helices are newly synthesized two parental strands reassociate after acting as templates for new strands thus restoring the parental double helix Both parental strands stay together after DNA replication

Biosynthetic-secretory pathway

pathway of a protein getting processed synthesis, modification and transport of proteins and lipids through the endomembrane system for ultimate release from secretory cells Pathway which includes the delivery of newly synthesized proteins and lipids from the ER to the Golgi apparatus, delivery from the Golgi apparatus to lysosomes or the plasma membrane, and the release of secretory products into the extracellular space

Phagosome (from phagocytosis) fuses with lysosome to form...

phagolysosome or secondary lysosome • Large particles may be visible using light microscope

Metaphase

phase of mitosis in which the chromosomes line up across the center of the cell second phase of mitosis, during which the chromosomes line up across the center of the cell Chromosomes line up in the middle of the cell -Dyads line up at equator -Also called metaphase plate or equatorial plate -Dyads are attached to the microtubules of the spindle via kinetochores >Kinetochores are located on both sides of the centromere of the dyad

Telophase

phase of mitosis in which the distinct individual chromosomes begin to spread out into a tangle of chromatin the final phase of cell division, between anaphase and interphase, in which the chromatids or chromosomes move to opposite ends of the cell and two nuclei are formed. After the chromosome seperates, the cell seals off, Final Phase of Mitosis. -Nuclear membrane formation -Chromosomes (chromatids) de-condense -Cytokinesis >Actin ring contracts

DNA polymerase

principle enzyme involved in DNA replication An enzyme that catalyzes the formation of the DNA molecule. Enzyme involved in DNA replication that joins individual nucleotides to produce a DNA molecule Responsible for forming new copies of DNA by adding nucleic acids in a sequence based on the template strand Cannot initiate synthesis of new strands, can only elongate existing DNA or RNA

Pinocytosis

process by which a cell takes in liquid from the surrounding environment A type of endocytosis in which the cell ingests extracellular fluid and its dissolved solutes. nonspecific ingestion of fluids & small proteins into endosome

Phagocytosis

process in which extensions of cytoplasm surround and engulf large particles and take them into the cell A type of endocytosis in which a cell engulfs large particles or whole cells ingestion of large particles into phagosome >Sometimes is receptor mediated

Apoptosis

programmed cell death • Cells no longer needed • Irreparably damaged cells • Extrinsic & Intrinsic Signals • TNFα

SRP receptor

protein on the cytosolic surface of the ER that allows its bound ribosome complex to interact with the ER A protein on the membrane of the endoplasmic reticulum that binds the signal recognition particle (SRP). embedded in the ER membrane, recognizes the SRP

Hemosiderin

protein-containing storage form of iron found in the bone marrow, liver, and spleen pigment released from hemoglobin process an iron-storage protein primarily made in times of iron overload

Protein translocator

proteins embedded in the membrane that help move cargo across the dual membranes Moves proteins through membranes on the mitochondria and ER. Proteins containing the mitochondrial signal sequence bind to the mitochondrial import receptor, which targets the protein to this structure on the outer membrane of the mitochondria. The protein is unfolded and threaded through one of these in both of the mitochondrial membranes. Once inside, the mitochondrial signal sequence is cleaved off and the protein is refolded. this allows for protein to enter the ER and has a cylinder shape. It can open and connect to the hydrophobic lipid bilayer, and close again to continue the process.

Acid fast

stain highlights Mycobacterium tuberculosis and related bacteria, by staining them bright red or fuscia (when the usual carbol-fuchsin stain is used). Acid fast will also stain certain protozoa (e.g. Cryptosporidium) and some bacteria (e.g. Nocardia).

Enzyme histochemistry

providing substrate and cofactor to reveal the presence of specific enzymes in tissue Frozen sections are commonly used for: A method for localizing cellular structures using a specific enzymatic activity present in those structures is the demonstration of enzymes and their location within tissues. It is performed by allowing a colored enzyme reaction product to precipitate and bind to tissue proteins in the immediate vicinity of the reacting enzymes, thus localizing and roughly quantifying enzyme activity. Since native enzymes are needed for this technique, it is usual to employ frozen tissue sections of sufficient thickness (e.g., 15 microns) to overcome the minimal enzyme activity threshold for the enzymatic starting reaction. Enzyme histochemistry constitutes a link between biochemistry and morphology and is useful in the diagnosis of metabolic imbalances.

pancreatic acinar cells

pyramidal (shaped like pyramids or slices of a pie) in shape and together form glands called acini that are spherical clusters. Their secretions are secreted into the lumen of the spherical acinus. produce pancreatic juice

rER vs Golgi glycoproteins

rER - glycoproteins all have the same type of sugar Golgi - heterogeneous mixture of glycoproteins

Topoisomerase I

relaxes negative supercoils Making single-stranded DNA breaks to relieve supercoiling at origin -Removes negative supercoils without leaving nicks in the DNA molecule -Cuts one strand while simultaneously generating a covalent phosphodiester bond between the released 5' phosphate on the DNA and a tyrosine residue in the enzyme (does not require ATP) -The free 3' end of the DNA is held non-covalently by the enzyme -The DNA strand that has not been cleaved is passed through the break and thus one negative supercoil is removed

AP endonuclease

removes the damaged sequence from DNA removes the damaged sequence during base excision repair Recognizes apurinic/apyrimidinic site (abasic site) and cleaves behind it to base excise repair removes the damaged sequence from DNA Recognizes sites with a missing base; cleaves sugar-phosphate backbone Part of BER

Deoxyribose phosphodiesterase

removes the sugar-phosphate lacking the base in base-excision repair removes the sugar-phosphate lacking the base removes deoxyribose-phosphate to make 3' OH Removes the sugar-phosphate lacking the base Part of BER

baciullus

rod shaped

nuclear localization signal (NLS)

sequence of amino acids in a protein that is recognized by a transport receptor leading to translocation of the protein from the cytoplasm to the nucleus Part of a protein required for its transport from the cytoplasm to the nucleus. A short amino acid sequence that marks a protein for delivery to the nucleus.

ER retention signal

short amino acid sequence on a protein that keeps it in the ER Four amino acid sequence at the C-terminal of a protein which prevents it from being translocation from the ER to other organelles.

Uniporter

single molecule or ion transporter that carries one specific ion or molecule A carrier protein that transports a single molecule across the plasma membrane.

Smooth muscle cells

single, fusiform, uninucleate; no striations Small and tapered Can divide and regenerate long and slender for contraction

Epithelial cells

skin cells that cover the external body surface and line the internal surfaces of organs skin cells that cover the outside of the body and line the internal surfaces of organs

Molecular chaperones

speed folding of the protein help proteins fold correctly in cells A protein that helps other proteins fold or refold from a partially denatured state

Mitochondria characteristics

spherlike or rod or filamentous bounded by double membrane -outer membrane smooth -inner membrane: contains enzymes of respiratory chain has cristae - Contains DNA, RNA, and proteins - Site of catabolism - Site of ATP production - Double membrane bound - Located in cytoplasm of cell - Main function: cellular respiration - Has its own 70s ribosomes - Used to be prokaryotic - DNA is circular -Round, cigar shaped• --Double membrane - creates 4 "compartments" >Outer membrane is relatively permeable >>Signaling sequence allows large proteins to cross membrane >Intermembrane space >Inner membrane folded into tubules called cristae >The inner cavity is filled with a matrix

Tethering protein

stabilises polymerase attach to Rab proteins on vesicles to help them enter the desired membrane Filamentous transmembrane protein involved in the docking of transport vesicles to target membranes.

Meiosis - Telophase I

• Nuclear envelope reforms• Dyads decondense • Cytokinesis >Daughter cells enter prophase II without going through S phase of cell cycle

Hematoxylin

stain that stains acidic structures blue. Areas of the cell that contain lots of DNA and RNA, such as the nucleus and rough ER, will stain dark blue. -commonly used dye -shows nucleic acids (DNA,RNA) Basic dye Imparts blue color to acidic components like DNA & RNA and some cellular secretions (i.e. mucin)

PAS dye

stains carbs magenta

Trichrome stain

stains collagen blue stain commonly used for fecal specimens

Downstream terminator sequence

stops transcription Termination in eukaryotes is less well understood

Inclusions

stored glycogen granules, crystals, pigments, and so on Chemical substances such as stored nutrients or cell products Include things like fat droplets and glycogen

Polyribosome

string of ribosomes simultaneously synthesizing same protein A group of several ribosomes attached to, and translating, the same messenger RNA molecule. string of ribosomes simultaneously translating regions of the same mRNA strand during protein synthesis

Nuclear pores

structures in the nuclear envelope that allow passage of certain materials between the cell nucleus and the cytoplasm holes in the nuclear envelope that allow materials to pass in and out of the nucleus

endocytic pathway

takes materials from outside the cell and brings them into the cell for digestion, degradation, or incorporated into endosome Responsible for the ingestion and degradation of extracellular molecules, moves materials from the plasma membrane, through endosomes, to lysosomes. Route for moving materials from outside the cell (and from the membrane surface of the cell) to compartments, such as endosomes and lysosomes, located within the cell interior

Histone methylation

the addition of methyl groups to histones usually reversibly represses DNA transcription, but can activate it in some cases. the condensing of chromatin structure (heterochromatin), prevents transcription

executioner caspases

the enzymes that hydrolyze diverse biological molecules The activated executioner caspases then cleave and activate other executioner procaspases, as well a specific target proteins in the cell. Among the many target proteins cleaved by executioner caspases are the 1) Nuclear lamins 2) a protein that normally holds an endonuclease (DNA- degrading enzyme) in an inactive form - its cleavage frees the endonuclease to cut up the DNA in the cell nucleus 3) Cytoskeleton components 4) Cell-cell adhesion proteins that attach cells to their neighbors - the cleavage of these proteins helps the apoptotic cells to round up and detach from its neighbors making it easier for a healthy neighboring cell to engulf it; or, in the case of an epithelial cell, for the neighbors extrude the apoptotic cell from the sheet of tightly cohesive cells.

Active transport

the movement of materials through a cell membrane using energy the movement of ions or molecules across a cell membrane into a region of higher concentration, assisted by enzymes and requiring energy. Energy-requiring process that moves material across a cell membrane against a concentration difference • Analyte is moved against its own electrochemical gradient. >Primary active transport: ATP is used to drive the transport involved ("pumps") >>Allows cell to regulate the concentration of solute within its cytoplasm >>May result in an imbalance of solute across a membrane (i.e. creates an electrical differences across a membrane that can be used to do work) >>>Na+/K+ pump >>>H+ pump• Ca2+ pump • Secondary active transport: Electrochemical gradient of one analyte (e.g. sodium) is used to drive the movement of another analyte (e.g. glucose).

Centromere

the region of the chromosome that holds the two sister chromatids together during mitosis Region of a chromosome where the two sister chromatids attach Area where the chromatids of a chromosome are attached Part of the chromosome -Area of adhesion of sister chromatids -Kinetochores (kinetochore microtubules)

Anaphase

the stage of meiotic or mitotic cell division in which the chromosomes move away from one another to opposite poles of the spindle. the third phase of mitosis, during which the chromosome pairs separate and move toward opposite poles Phase of mitosis in which the chromosomes separate and move to opposite ends of the cell -Separation of chromosome from its replicate (chromatids) -Anaphase Promoting Complex >Controls separation >Controls spindle- Inactivates MPF >Activates G1 cyclin

Histology

the study of the microscopic structure of tissues

Channels

the visual and auditory means by which a message is transmitted from sender to receiver means of conveying and receiving messages regulate water flow and solutes through membrane -Fast rate of transport: multiple water molecules or ions move through simultaneously at a rapid rate -A few types are large and permissive• Gap junctions - communication between cells -Most are narrow and highly selective -Many other channel proteins are usually closed - open only in response to specific signals (gated channels) >Exception: K- leak channel proteins: generally open and are critical to generating the normal, resting membrane electric potential

gram positive vs gram negative bacteria

there is an extra outer membrane in gram-negative that stains red Gram positive to take a positive stain and associated with respiratory and soft tissue infections Gram negative to obtain a negative stain and associated with genitourinary or gastrointestinal tract infections Gram-positive: have simple cell walls with a thick layer of peptidoglycan. Gram-negative: more complex cell walls, less peptidoglycan, which is located between 2 membranes. More resistant to antibiotics.

Exosomes

tiny vesicle that removes debris and transports material Small visicles (<120 nm in diameter) which allow the transfer of membrane proteins and other materials to nearby cells

Functions of melanin

to protect the DNA of keratinocytes from mutations induced by UV radiation, decrease the synthesis of vitamin D in response to UV radiation Provides protection against solar damage Important for sexual displays

Antiporter

transporter that carries two ions or small molecules in different directions transporter that carries two ions or small molecules in different directions A carrier protein that transports two molecules acrss the plasma membrane in opposite directions.

Mitotic activity: stable

undergo periodic division to maintain normal function liver, fibroblasts

Immunofluorescence

uses fluorescently tagged molecules, makes use of the specificity in binding of antibodies, AND would require a special UV microscope. a method of tagging antibodies with a fluorescent dye to detect or localize antigen-antibody method of tagging antibodies with a luminating dye to detect antigen-antibody complexes combinations Is localization of specific proteins by allowing reaction with an antibody tagged with a fluorescent chemical that can be visualized in a special fluorescence microscope.

Where is the smooth endoplasmic reticulum very prominent?

very prominent in the liver, muscle, and steroid hormone producing cells

Trichrome staining

was used as the name of a staining method (Mallory's trichrome) which differentially coloured erythrocytes orange, muscle red and collagen blue. It now has become a general term to describe any histological staining method which uses two or more acid dyes in conjunction with a "polyacid" to differentially stain two basic materials in contrasting colours. employ two or more acid dyes. Normally it would be expected that acid dyes would all stain the same basic proteins, but by applying them sequentially the staining pattern may be manipulated by removing dye from less intensely stained components with a "polyacid" is a three-color stain used especially to highlight collagen from cellular components (there are several different other types of trichrome stain recipes, all used to highlight collagen). Collagen appears blue or blue-green, and thus is easily distinguished from muscle and cell cytoplasm, which turns red, and nuclei, which turn brown or black.

Death inducing signaling complex

what does the death receptor DISC stand for Formation of this complex leads to activation of the initiator caspase, which then cleaves inactivates the effector caspase.

Receptor mediated transport

when substances bind to specialized molecules on the cell surface before being engulfed involves membrane surface receptors to internalize the substance type of active transport where substances bind to specialized molecules on the outer cell surface before being engulfed.

Processing artifacts occur

while the film is in the automatic processor and include pi lines, guide shoe marks, and chemical fog.

The nuclear pore complex (NPC) controls bidirectional flow of molecules

• 30 different proteins form octagonal framework • Proteins in central pore form disordered tangle that blocks passage of large molecules >Several aqueous channels allow passive diffusion of small water-soluble molecules >Larger molecules required receptor-mediated active transport by receptors: importins and exportins >>Energy provided by GTP hydrolysis

Golgi apparatus does not stain well with H&E and Romanovsky stains

• Appears as a pale, perinuclear zone • Prominent in secretory cells

Membranes provide a selectively permeable barrier

• Bidirectional movement of molecules >Passive diffusion of small water-soluble molecules >Import histones, DNA & RNA polymerases, gene regulatory proteins, RNA-processing proteins >Export tRNA, mRNA, ribosome subunits

IHC for intermediate filaments used to determine tumor type

• Cytokeratin: - Intermediate filaments in epithelial cells • Vimentin: - Intermediate filaments in connective tissue cells

Rho independent termination

• DNA sequences transcribed into RNA hairpins that stall RNAP • Stretch of Us following hairpin cause RNAP to pause, hairpin forms, RNA falls off hairpin structure formed by inverted repeats, followed by a string of uracils Formation of a hairpin loop that stalls the RNA polymerase and transcription terminates

An ion's electrochemical gradient is a combination of concentration gradient and electrical gradient

• Each solute has a concentration gradient • Charged particles also affected by both concentration and electrical gradient >The difference in voltage across a membrane is called membrane potential >Interior of cell more negative than outside >>Negatively charged proteins >>Negative charged phospholipids >>Slow leak K out of cells

During interphase chromatin is not uniformly packed

• Heterochromatin - most highly condensed chromatin in which genes cannot be expressed >Used to silence genes >Some DNA is permanently in heterochromatin • Euchromatin - uncoiled chromatin with active DNA • Most cell types express about 20 to 30% of their genes. • Proportions vary between cell types

Membranes behave like a two dimensional fluid

• Individual phospholipids are not fixed -they move around withinthe bilayer, much like the movement of molecules in liquids >Can spin, flex & move laterally >Can only move in 2 dimensions. >Almost never flip to other membrane leaflet

Stages of transcription

• Initiation-promoter, transcription factors and start point • Elongation-DNAisunwound,the template strand is read in the 3'-5' direction and RNA is synthesized in the 5' to 3' direction by RNA polymerase • Termination-Stopsignalcauses RNA polymerase to dissociate and the RNA transcript is complete

The nucleus is the control center of the cell

• Membrane bound organelle that contains genetic material • Advantages >Larger genome >Spatial separation of transcription & translation >Controls expression of genes

Membrane fluidity is dependent on lipid composition

• Membranes with more unsaturated hydrocarbon tails are more fluid >Double bonds between carbon atoms creates kink >Hydrocarbon tails can't pack as tightly together as straight saturated hydrocarbon tails • Addition of cholesterol stiffens the bilayer, making it less flexible and less permeable to water soluble molecules >Cholesterol is short and rigid >Fills spaces between neighboring phospholipids • Protects against lipid crystallization at low temperatures

Two types of Extracellular Vesicles

• Microvesicles bleb directly off plasma membrane • Exosomes released from multivesicular body -Membrane invagination into endosome forms intraluminal vesicles = multivesicular body (MVB) -MVB can fuse with >Lysosome - destruction >Plasma membrane, releasing membrane- bound exosomes.

Histology stains

• Most histology slides are stained with hematoxylin and eosin - Hematoxylin: imparts blue color to acidic components like DNA & RNA and some cellular secretions (i.e. mucin) - Eosin: imparts pink to red color to basic components like the cytoplasm and extracellular products

Three types of endocytosis

• Phagocytosis - ingestion of large particles into phagosome • Sometimes is receptor mediated • Pinocytosis - nonspecific ingestion of fluids & small proteins into endosome • Receptor mediated endocytosis - entry of specific molecules into endosomes

Things that a clinician can control in histology: sample collection and submission

• Provide pathologist with clear description of lesion - Location - Size, color, consistency • We love pictures!

Several types of proteins are made in the rER

• Ribosomes are released after synthesizing protein Protein translocator • Water soluble proteins - injected into lumen by ribosome >Proteins are modified and folded >Packed into vesicles which are ultimately transported to Golgi (and beyond) • Transmembrane proteins - become embedded in rER membrane >Destined for plasma membrane >Destined for membrane of endomembrane organelle

Cytology: fast but nonspecific information

• Romanovsky stain • Improved cellular detail • Easier to find organisms • No architecture - No plane of section issues - Less specific diagnosis

EM provides better magnification and resolution

• Shorter wavelength than visible light - Better resolution - Image (black & white) shown on fluorescent screen Magnification as much as 10,000,000x Visualize cellular organelles & macromolecules as small as 1 nm

Meiosis II

• Similar to mitosis • No duplication (no S stage) of genetic material prior to meiosis II, therefore, resulting daughter cells are • Stages - Prophase II (start "2n" "haploid") - Prometaphase II - Metaphase II - Anaphase II - Telophase II

Membrane transport often depends on specialized membrane transport proteins

• Small nonpolar molecules: rapidly cross membrane • Metabolites of cholesterol (e.g. steroids) readily cross membranes • Uncharged, polar molecules slowly diffuse across >Small molecules more rapidly than large ones >Aquaporin channels facilitate more rapid movement of H2O across membranes via osmosis • Membranes are impermeable to charged molecules (ions)

Proteins function in their properly folded state

• Some proteins properly fold as the protein spins out of the ribosome

Coupled pumps link the uphill transport of one solute to the downhill transport of another

• Symporter: moves 2 or more solutes in the same direction across the membrane • Antiporter: moves 2 or more solutes in opposite directions across the membrane • Uniporter: moves 1 solute in one direction (not coupled)

Meiosis - Metaphase I

• Tetrads (homologous dyads) line up on metaphase plate (random) • Start of separation of synaptonemal complex • Still attached at chiasmata - Chiasmata - potential crossover of non-sister chromatids - genetic recombination (occurs during tetrad formation at prophase I)

For functional reasons, it may be important to limit diffusion of lipids and protein throughout the entire cell membrane

• There are several ways for cells to limit the diffusion of membrane proteins (and lipids) and localize them to one region of the cell >Connection to another cell by (occluding) junction >Connection to another cellby gap (communicating) junction >Connection to cytoskeletal components Connection to extracellular matrix

There are several mechanisms for moving solutes across membranes

• Transport can be passive or active (requires energy) • Two general types of membrane transport proteins >Transporters: Bind molecule and undergo transformational change that transfers solute across membrane >Channels: Form aqueous pores that allow solutes to quickly cross membrane >Each protein transports a class of molecule (e.g. ions, sugars, or amino acids) - can be very selective • Endocytosis or Exocytosis - involves membrane folding.

Gated channels can be opened by several different types of signals

• Voltage-gated ion channels open when a certain membrane potential has been reached. • Ligand-gated ion channels open when a specific ligand binds to the channel • Mechanically-gated ion channels open when mechanical force applied to the channel.