Block 1 Structure and Function (SF)
What are gene targeted changes in genome
"gene knockout", taking place of gene already there
Describe the basic molecular structure of cell membranes.
-Cell membranes consist of a phospholipid bilayer that is embedded with proteins, glycoproteins and lipoproteins. -Generally, membrane proteins are classified as either integral or peripheral. -Membranes contain additional lipids such as cholesterol and other hydrophobic molecules. • Although all cell membranes share the same basic structure, the types of associated proteins and the composition of lipids vary greatly in different membranous organelles and in membranous organelles derived from different cell types. • The precise composition of proteins and lipids in a cell membrane account for its corresponding functional properties.
Describe the various phases in the development of the primitive uteroplacental circulation through formation of mature chorionic villi.
-The trophoblast layer has already formed the cytotrophoblast and syncytiotrophoblast; the latter continues to invade the endometrial stroma. -Cells of the embryoblast differentiate into two layers, the epiblast and the hypoblast, also called the bilaminar embryonic disc. -The amniotic and chorionic cavities form. The amniotic cavity forms within the epiblast, while the chorionic cavity forms adjacent to the embryo in extraembryonic tissue (same area as the yolk sac in the above image). Trophoblast --> Cytotrophoblast ---->Syncytiotrophoblast Blastocyst cavity ---> Amnionic cavity ---->Chorionic cavity Embryoblast ----> Epiblast ---->Hypoblast Syncytiotrophoblast cells begin to produce human chorionic gonadotropin (hCG), which maintains the activity of the corpus luteum and forms the basis for early pregnancy tests. Circulating hCG is a signal to the body that a conceptus has implanted. It can be detected in the mothers blood and urine within 1-2 days after implantation.
Describe how selectins contribute to inflammatory cell recruitment to sites of injury.
1) White blood cell in the circulation makes contact with endothelial cells as they bounce through the blood stream. Quiescent endothelial cells do not express cell surface receptors that bind to white blood cells so they don't linger on blood vessel walls. 2) activated endothelium initiate expression of selectins that form a weak adhesion event with white blood cells that tether the white blood cells to the surface of the blood vessel. 3) Further activation of the endothelial cells increases expression of stronger adhesions receptors, integrins. Integrin adhesion signals the white blood cells to bind and to initiate cell migration into the underlying connective tissue.
What are two distinct mechanism by which cell adhesion receptors bind?
1) homophillic binding 2) heterophillic binding
Describe three steps in the maturation of cell adhesion interactions to an adherens junction.
1) membrane protrusions initiate cell-cell contact. small cadherins and catenins cluster 2) actin and cadherin recruitment expands junction. recruitment of more cadherins and catenins 3) actin remodeling and myosin recruitment expands the adherens junction. contractile actin and myosin bundles.
Specify the 4 basic steps involved in cell motility and describe the role of microfilaments and myosin.
1. Examples of Cell Motility a) Migration of embryonic cells during development b) Migration of neutrophils & macrophages to sites of infection c) Migration of fibroblasts during wound healing d) Movement of axons toward synpases e) Migration of cancer cells during metastasis 2. Steps in Cell Motility a) Extension of leading edge toward direction of movement: lamellipodia and filopodia: actin based. b) Attachment of lamellipodia and filopodia to the substratum: integrin based. c) Development of tension and forward movement: actin/myosin based. d) Retraction of trailing edge Role of Microfilaments in Cell Motility • Rapid polymerization of F-actin network at the plus ends generates force that pushes on cell membrane to produce lamellipodia and filopodia Formins are actin-binding proteins that link the plus ends of F-actin to the cytoplasmic surface of the cell membrane. • NM II generates sliding of F-actin in opposite direction toward the minus ends. • Cell moves forward when F-actin polymerization at the plus ends is faster than sliding of F-actin toward the minus ends. Actin polymerization works with actin binding proteins and the actin:myosin motor to drive cell movement.
Describe the protein subunit composition of the 3 main cytoskeletal elements.
1. Microfilaments (F-actin) - 7-9 nm in diameter - Polymers of G-actin: DYNAMIC 2. Intermediate Filaments - 10 nm in diameter - Polymers of heterogeneous proteins: STABLE 3. Microtubules - 25 nm in diameter - Polymers of α and β tubulin subunits: DYNAMIC
Describe the differences between peripheral and integral membrane proteins.
1. Peripheral Membrane Proteins: -Associated with the membrane via protein-protein interactions and/or by weak electrostatic interactions with polar head groups of phospholipids. -These proteins can be released experimentally from the membrane using buffers containing high salt or extreme pH. 2. Integral Membrane Proteins: Associated with the membrane by direct insertion into the phospholipid bilayer. Experimental release of these proteins from the membrane requires solubilization using detergents to disrupt the phospholipid bilayer. There are 3 main types of integral membrane proteins: a) transmembrane b) GPI-anchored c) lipid-anchored.
Describe the 3 main types of lipids in cell membranes and explain their corresponding effects on physical properties of membrane fluidity and rigidity.
1. Phosphoglycerides: Phosphatidylcholine (PC) Phosphatidylethanolamine (PE) Phosphatidylserine (PS) Phosphatidylinositol (PI) Phosphatidic Acid (PA) • Esterified long chain Fatty Acids contain an even number of carbons (16, 18 or 20). • Fatty Acid linked to carbon 2 of the triglyceride backbone often is unsaturated. -The phospholipid molecule is amphipathic, that is, it contains a polar head group that is hydrophilic and 2 FA tails that are hydrophobic. -Kinks in unsaturated FA tails increase membrane fluidity by loosening the packing of lipids and causing molecular disorder. 2. Sphingomyelin (Sphingosine-based Phospholipid): LOVES CHOLESTEROL The molecular properties of sphingomyelin in membranes are different from phosphoglycerides: • The polar head of sphingosine has an OH group that increases its capacity to form hydrogen bonds. • The hydrocarbon tails have a greater degree of saturation than phosphoglycerides, thereby increasing packing in the cell membrane. • The saturated hydrocarbon tails of sphingomyelin have a high affinity for cholesterol, thus, these two molecules tend to aggregate in membranes and pack together tightly to increase rigidity of the cell membrane. 3. Cholesterol: • All cell membranes contain some cholesterol. The relative amount of cholesterol in a cell membrane affects its physical properties. • The polar OH group is hydrophilic and is positioned in the outer and inner layers; the non-polar portion of the molecule is buried in the hydrophobic region. • Cholesterol increases packing of FA tails of membrane phosphoglycerides and sphingomyelin to increase membrane rigidity. In addition, lipid rafts are formed by the interactions between sphingomyelin and cholesterol.
Specify the primary components of a typical eukaryotic cell.
1. Plasma Membrane (Plasmalemma) The plasma membrane encloses the cell and separates the cytoplasm from extracellular space. It enables the cell to interact with its environment and to respond to external stimuli. 2. Nucleus a) Chromatin (DNA and Proteins) b) Nuclear Envelope c) Nucleolus 3. Cytoplasm (Protoplasm) a) Membranous Organelles: Specialized membranes with associated proteins form organelles. The internal compartment of a membranous organelle is called the matrix. • Mitochondria • Rough Endoplasmic Reticulum (RER) • Smooth Endoplasmic Reticulum (SER) • Golgi Complex or Apparatus • Early and Late Endosomes, Endocytic vesicles • Lysosomes and Autophagosomes • Peroxisomes • Secretory Vesicles or Granules • Transport or Shuttle Vesicles b) Cytosol: Aqueous Phase • Dissolved ions, CHO, AA, Nucleotides • Cytosolic enzymes and other proteins • Macromolecules, Ribonucleoproteins, Proteasomes • Inclusions: Lipid droplets, Glycogen, Pigments c) Non-membranous Organelles • Free Ribosomes • Centrioles • Cilia and Flagella d) Cytoskeleton • Microfilaments (Actin and Myosin) • Intermediate Filaments • Microtubules
Describe the structure and function of the following membrane specializations: polarity of cell membranes, microvilli, glycocalyx.
1. Polarity of Plasma Membranes: Differences in structure/function of the plasma membrane by segregation of proteins to specific domains of the cell. 2. Microvilli: Protrusions of the plasma membrane on the apical surface to increase the surface area. 3. Glycocalyx: Coat formed by integral membrane glycoproteins on the apical membranes of epithelial cells
Describe the following basic properties of cell membranes: self-assembly.
1. Self-Assembly of Phospholipid Bilayers • The lipid bilayer forms because it is the most thermodynamically stable structure. • Polar head groups of phospholipids form hydrogen bonds with H2O. • Polarity of H2O forces the hydrophobic tails of phospholipids to the interior. • Hydrocarbon tails attract one another by van der Waals forces. a) Micelles: Aggregation of phospholipids-polar head regions in contact with water (solvent) and tail regions packed together to form a hydrophobic core. b) Liposomes: Artificial membrane vesicles-produced by sonication of phospholipids in aqueous medium-spontaneously assemble into a phospholipid bilayer that encapsulates an aqueous core. *Liposomes have clinical utility for delivering to cells either hydrophobic molecules embedded in the phospholipid bilayer or hydrophilic molecules dissolved in the aqueous core (ex. DNA, proteins, drugs).
Specify 3 types of integral membrane proteins and the molecular basis for their insertion into the cell membrane.
1. Transmembrane Proteins • Contain alpha-helical domains of 20 to 25 hydrophobic amino acids that span the hydrophobic portion of the membrane, and hydrophilic domains that protrude onto the outer and inner layers. • Transmembrane proteins can span the membrane once (single-pass) or multiple times (multi-pass). Many transmembrane proteins of the plasma membrane are glycoproteins, produced by adding carbohydrate chains to the hydrophilic domains on the cell surface. 2. GPI (Glycosylphosphatidylinositol) Anchored Proteins: -Carboxy terminus of cell surface protein is attached to the outer layer of the plasma membrane by a glycolipid anchor composed of phosphatidylinositol (PI) and a branched oligosaccharide. Examples: Cell surface enzymes, adhesion molecules and complement regulatory proteins 3. Lipid Anchored Proteins: Protein anchored to inner layer of plasma membrane by covalent attachment to either myristate, palmitate or prenyl groups. *Myristoylation: Covalent attachment of N-terminal Gly of membrane protein to C14 fatty acid *Palmitoylation: Covalent attachment of internal Cys in membrane protein to C16 fatty acid *Farnesylation: Covalent attachment of C-terminal Cys of membrane protein to C15 prenyl group Examples: Ras and other membrane-bound cell signaling proteins, Lamins in the inner layer of the nuclear envelope
When is implantation complete?
11-12 days
When are secondary villi visible?
11-12 days when implantation is complete
Specify the primary functions of cell membranes.
2. Primary Functions a) Creation of compartments and permeability barriers b) Transport of ions and molecules in/out of cells and in/out of intracellular compartments c) Site of enzymatic reactions produced by membrane-associated proteins and substrates d) Extracellular and intracellular signal transduction e) Endocytosis and autophagy f) Protein synthesis and secretion g) Site of cell-to-cell and cell-to-substrate adhesion and communication, e.g., interaction of plasma membrane with other cells and components in the extracellular environment
Describe the basic principles underlying fluorescence microscopy.
5. Fluorescence Microscopy • Fluorescence microscopy is used to examine cell structure & function in fixed or living cells. • A specific cellular molecule is labeled by staining with a fluorescent dye. The dye absorbs light at one wavelength and emits the light at a second wavelength. • A fluorescence microscope has an excitation filter to select wavelength of light (e.g. blue) that excites the fluorescent dye. A dichroic mirror deflects the excitation light down to the specimen. • The fluorescent light emitted by the specimen (e.g. green) passes through the dichroic mirror and and a second filter selects the wavelength of light emitted by the dye for visualization.
When is implantation almost complete?
9-10 days, closing plug remains
Describe different pathways by which inner cell masses form twin embryos.
A. Entire zygote may divide, Di/di B. Inner cell mass itself splits into separate amions, di/mo C. Conjoined twins resulting from failure of blastocyst to split, mo/mo
Anchoring junctions
ANCHORING JUNCTIONS: structures associated with the plasma membrane that provide structural anchors in the flimsy lipid bilayer of the plasma membrane allowing force generation between cells or between cells and the extracellular matrix. THEY ARE A BROADER CATEGORY OF JUNCTIONS BECAUSE THEY CAN MEDIATE BOTH CELL TO CELL AND CELL TO ECM INTERACTIONS. -prominent in tissue under mechanical stress such as skeletal/heart muscle and skin
What are anchoring junctions in cell to cell interactions that link to the actin cytoskeleton?
Adherens junctions. Cells are able to connect their actin cytoskeleton through adherens junctions so that force can be transmitted throughout an epithelial sheet.
What are desmosomes?
Anchoring junctions that occur betwen adjacent cells and form button-like rivers at the cell membrane. Like adherens junctions, cadherin family members are the transmembrane components however anchoring proteins connect to intermediate filaments instead of actin cytoskeleton.
Describe the following basic properties of cell membranes: fluid mosaic model
Cell membrane proteins are embedded within the phospholipid bilayer- the hydrophobic domains are buried in the hydrophobic core of the membrane and the hydrophilic domains are exposed on the inner and outer layers of the membrane to the aqueous environment • Membrane proteins can move laterally through the phospholipid bilayer. This experiment proved that proteins are mobile in membranes and redistribute following fusion of human and mouse cells. • Fluorescent antibodies to human and mouse proteins show random distribution in membranes of fused cells.
Identify the cellular contributions to the bilaminar embryonic disc.
Cells of the embryoblast differentiate into two layers, the epiblast and the hypoblast, also called the bilaminar embryonic disc.
Describe the structure and function of cilia and flagella and explain the molecular basis of movement.
Cilia and Flagella: Specialized motile projections of cells composed mainly of MTs • Cilia are prominent in epithelial cells lining parts of the respiratory tract and the oviduct • Flagella are vital for motility of spermatozoa (basically longer cilia). __ a) Axoneme: Core structure composed of microtubules in 9+2 array and associated proteins required for structural integrity (nexin) and movement (dynein). b) Basal bodies: The axoneme grows out of the basal body which derives from the centrioles- The minus ends of the MTs are anchored in axoneme doublets which extending from the basal body. c) Molecular Basis of Movement: • Dynein is affixed to A tubules. • Motor heads of dynein move along adjacent B tubules towards the minus end. • Because adjacent doublets are connected by nexins, the forces generated by movement of the dynein heads cause MTs to bend.
Describe how IF are linked to desmosomes and hemidesmosomes.
Desmosomes when need very stable attachment, which is why they utilize IF. Hemidesmosomes very heavy anchoring structures that need stable IF as well.
What allows for cell sorting mechanism in the tissues?
Differential expression and recognition by cell surface adhesion molecules
Explain the concept of dynamic instability of microtubules.
Dynamic Instability of Microtubules: Cycles of rapid growth and shrinkage of MTs that is dependent on the concentration of GTP•Tubulin dimers -- a) High concentration of GTP•Tubulin dimers: Rapid growth of MT occurs as GTP cap is maintained on the plus end. -- b) Low concentration of GTP•Tubulin dimers: Rapid shrinkage of MT occurs because the GTP cap is lost and Tubulin•GDP dimers dissociate from the plus end.
Know the 3 primary types of cell junctions
Elaborate subcellular structure that are visible by electron microscopy. first identified as electron dense structure when viewed by electron microscopy. Each type of junction has specific cellular functions. Each type of junction has its own distinct protein composition. Tight junctions, gap junction, and anchoring junctions are three examples of cell junctions.
What is another way to describe inner cell mass of blastocyst?
Embryoblast or stem cells
What are focal adhesions?
Form between cells and the extracellular matrix. Integrins bind to ECM components and link to actin cytoskeleton via anchoring proteins. Focal adhesions are used for cell movement.
Describe the molecular structure of microtubules including subunit composition, arrangement of protofilaments and diameter.
Found in soluble and filament-bound proteins, similarly to actin Molecular Structure: α-Tubulin and β-Tubulin subunits form dimers that polymerize to produce hollow cylinders approximately 25 nm diameter a) Basic structural unit: α,β-Tubulin heterodimer • α-Tubulin has bound GTP that does not hydrolyze • β-Tubulin has bound GTP that is hydrolyzed to GDP•Pi following assembly into microtubules b) Protofilaments: Heterodimers join end to end to form protofilaments consisting of alternating α & β-Tubulin subunits c) Assembled Microtubule: 13 protofilaments form a cylinder with a hollow core
How does hypoblast form?
From delamination of inner cell mass during implantation
GAP JUNCTIONS
GAP JUNCTIONS mediate cell to cell communication. -gap junctions allow small molecules to pass from one cell to another. -they are composed of proteins called connexins that come together to form channels called connexons. -neighboring cells express connexons on their cell surface, these connexons bind to connexons on adjacent cells to form an intercellular channel.
Anchoring junction types.
GENERAL STRUCTURE OF ANCHORING JUNCTIONS: 1) plasma membrane receptor with an extracellular domain, a membrane spanning domain, and a cytoplasmic tail 2) anchoring proteins that bind to the cytoplasmic tail of the receptors and cytoskeletal elements 3) cytoskeleton TYPES OF ANCHORING JUNCTIONS AND THEIR CYTOSKELETAL ATTACHMENTS: 1. adherens junctions - cell to cell that link to actin cytoskeleton 2. desmosomes - cell to cell that link to intermediate filaments 3. hemidesmosomes - cell to ECM contacts that link to intermediate filaments 4. focal adhesions - cell to ECM contacts that link to the actin cytoskeleton
Phases of Embryonic Development
Growth - increase in size, cell division Morphogenesis - development of form, mass cell movements Differentiation - maturation of physiological processes, establishment of cell fate for function
Describe the basic principles underlying electron microscopy.
Instead of light, an electron microscope uses an electron beam as an energy source to resolve ultrastructure of cells. There are 2 basic types of EM: • transmission (TEM) • scanning (SEM) a) Transmission Electron Microscope -much higher energy with much shorter wavelength -this increases resolution 1.Specimen prep for TEM -specimen must be fixed 2. Staining in TEM -necessary for viewing structure b)Scanning EM • A scanning EM also uses electromagnetic lenses, electrons and depends on a substantial vacuum being maintained in the column. • For SEM, the specimen is coated with a heavy metal during tissue preparation. The electron beam is scanned across the surface of the specimen.The emission of secondary electrons from the metal surface of the specimen are collected by a detector and the signal is amplified. • SEM produces 3-D surface images with high contour.
Specify the main types of intermediate filament (IF) proteins.
Intermediate filaments are composed of a heterogeneous group of proteins that are expressed in different cell types. The main function of intermediate filaments is to provide mechanical support and structural integrity to the cell. These filaments are not dynamic. I. Acidic Keratins - Epithelial Cells II. Neutral/Basic Keratins Epithelial Cells III. Vimentin- Many Cell Types Desmin-Muscle Cells and others Glial Fibrillary Acidic protein (GFAP) - Glial Cells Peripherin - Peripheral Neurons IV. Neurofilament Proteins (NFL, NFM & NFH) - Neurons V. Nuclear Lamins - All Nucleated Cells VI. Nestin - Stem Cells
Describe how inhibition of implantation works
Large doses of progestin compounds; "morning-after pills", levonorgestrel; Trade names: Plan B One Step®, Next Choice®, Mirena® Others include Loestrin® (Microgestin) • disrupts balance between estrogen and progesterone; thus, uterus is 'not ready'and implantation does not occur. • delay ovulation • inhibit ovulation • prevents fertilization by altering transport of sperm/ova
Describe how cell membranes compartmentalize eukaryotic cells.
MEMBRANES ESSENTIALLY FORMED BY THE HYDROPHOBIC EFFECT • Eukaryotic cells have an extensive array of cell membranes that organize the cytoplasm into internal compartments such as the nucleus and membranous organelles. • Cell membranes function as a lipid phase or medium that consists of a phospholipid bilayer and associated proteins, glycoproteins and lipoproteins. • Phospholipids have a polar head group that constitute the hydrophilic regions of the membrane and fatty acid tails that form an interior hydrophobic region. • The precise composition of proteins and lipids in a cell membrane determines its corresponding functional properties. Cell membranes create subcellular domains and facilitate interactions between components in the cytoplasm
Differentiate between membranous and non-membranous organelles.
MEMBRANOUS: Specialized membranes with associated proteins form organelles. The internal compartment of a membranous organelle is called the matrix. NON-MEMBRANOUS: free ribosomes, centrioles, cilia and flagella
Explain how microtubules are assembled and disassembled.
MTs are dynamic structures that can undergo rapid assembly and disassembly Note that most of the MT is composed of Tubulin•GDP dimers a) Tubulin heterodimers bound to GTP are added onto the plus (growing) end of the MT b) A short time after polymerization, GTP bound to β-Tubulin is hydrolyzed to GDP•Pi c) β-Tubulin•GDP is less stable and dissociates from either end of the MT d) Growth of microtubules occurs when GTP•Tubulin dimers are added to the plus end of the MT at faster rate than hydrolysis of GTP to GDP•Pi e) Growing MTs maintain a GTP•Tubulin "cap" on the plus end; Shrinking MTs have lost GTP•Tubulin cap- GDP•Tubulin is exposed on the plus end
Describe how lipid rafts are formed and their functional significance
Membrane "microdomains" enriched in specific proteins and lipids. Rafts are dynamic and move freely within the lipid bilayer- can join to form larger rafts or break down into smaller rafts. a) Lipid Composition: Highly enriched in cholesterol and sphingolipids (sphingomyelin). Cholesterol holds raft together by interacting with saturated FA chains of sphingolipids. Lipid rafts are more ordered and tightly packed than surrounding region of bilayer, thereby creating a partitioning effect. b) Protein Composition: Enriched in specific types of membrane proteins such as GPI-anchored proteins, alpha-subunits of G proteins and protein kinases. Rafts serve as a platform for organizing membrane proteins into distinct functional compartments.
Mesenchyme vs mesoderm
Mesenchyme is mobile and can come from any of the germ tissues Mesoderm is one of the three germ tissues
Explain how microfilaments are assembled and disassembled continuously.
Molecular Structure: G-actin monomers polymerize to form F-actin **G-actin monomers must be bound to ATP for polymerization; ATP is hydrolyzed to ADP•Pi after polymerization into F-actin INTRINSIC CAPACITY TO SELF POLYMERIZE Assembly and Disassembly: Microfilaments are dynamic- assembly and disassembly occur continuously in the cell a) Polarity of F-actin: G-actin•ATP is added to plus ends of F-actin at a much faster rate than the minus ends because ADP•actin dissociates more readily than ATP•actin • Plus (+) end = faster growing end • Minus (-) end = slower growing end b) Treadmilling: Results from the difference in critical concentrations of G-actin required for polymerization at the plus end versus the minus end - At intermediate G-actin concentrations, loss from the minus end is balanced by addition at the plus end
Describe the molecular structure and corresponding cellular functions of myosin I and Non-muscle myosin II
Myosins are a superfamily of motor proteins present in all cell types that interact with F-actin to function in cellular processes that require generation of force or translocation of molecules and organelles. Myosin molecules can: 1) carry cargo by "walking" along microfilaments 2) generate tension on microfilaments 3) propel the sliding of microfilaments for cellular contraction and movement CONVENTIONAL MYOSIN/MYOSIN II a) Myosin Heavy Chains (MHC) - MHCs contain the following 3 domains: • Motor (head) - globular domain, conserved sequence containing ATPase activity • Neck (lever) - domain contains sequences that bind to MLCs or calmodulin • Tail (rod) - a-helical domain, most variable in sequence and length - diverse functions b) Myosin Light Chains (MLC)- Bind to MHCs and regulate myosin function: Ca2+ binding proteins regulated by intracellular Ca2+ levels • Essential Light Chains (ELC) • Regulatory Light Chains (RLC) Regulation of Myosin II Contraction: NM II and SM II a) Phosphorylation of RLC by Myosin Light Chain Kinase (MLCK) b) Effects of RLC Phosphorylation on Myosin Activity • Increases ATPase activity in MHC motor domain • Enables assembly of bipolar filaments via interactions in the MHC tail domains NONCONVENTIONAL MYOSINS: Non-conventional myosins are generally thought to be involved in movement of proteins and organelles along actin microfilaments as opposed to force generation. An example is Myosin I: a) Myosin I: Smallest class- consists of single Myosin Heavy Chain, Myosin Light Chain = calmodulin. • Motor domain - binds to microfilaments • Tail domain - binds to proteins and organelles b) Functions of Myosin I: • Myosin I moves along microfilament toward plus end carrying cargo linked to the tail domain • Myosin I has an important function in microvilli where it forms lateral links or "struts" between bundles of microfilaments and the cell membrane
Explain how subunits are assembled into intermediate filaments.
Protein subunits of each specific type of IF proteins assemble into higher order structure. • All IF proteins have a central α-helical (rod) domain that is critical for assembly of coiled-coils. • Anti-parallel alignment of IF protein dimers produces tetramers. • Tetramers align end to end to form protofilaments. • Intermediate filaments consists of 8 protofilaments wound into a ropelike structure
What are transgenic mice?
Putting in gene into genome randomly, not changing endogenous genes.
Bilaminar embryonic disc
Still present at end of implantation
Which tissue is site of synthesis and release of hCG?
Syncytiotrophoblast start releasing hCG as they start invading endometrium tissue basis for pregnancy tests
Tight Junctions
TIGHT JUNCTIONS or occluding junctions contribute to the barrier function of epithelial cell layers, which separate fluids on either side with different chemical compositions. Claudins and Occludins are transmembrane proteins that compost tight junctions. Claudin/occludin on one epithelial cell binds to that on neighboring cells to form a tight seal Example: Gut Epithelia - glucose actively transported from the lumen of the gut through NA+ driven symports
Describe the structural and functional differences of tight junctions, adherens junctions, and gap junctions
TIGHT JUNCTIONS: seals neighboring cells together in an epithelial sheet to prevent leakage of molecules between them ADHERENS JUNCTIONS: joins an actin bundle in one cell to similar bundle in neighboring cells GAP JUNCTIONS: allow passage of small water-soluble ions and molecules
How does tension play a role in cell adhesion?
Tension created by cell attachment unfolds cytoplasmic proteins bound to cell surface receptors (such as alpha-catenin), exposing sites on alpha catenin that interact with other cytoplasmic proteins and cytoskeletal elements.
What are adherens junctions?
They allow cells to use the cytoskeleton to drive morphogenic processes critical for development.
When doe primary chorionic villi develop?
They are evident at 9 days and extraembryonic mesoderm is appearing
What are integrin receptors?
They bind cells to the extracellular matrix. Heterodimers composed of one alpha subunit and one beta subunit. Integrins can be in an inactive state on the cell surface and become activates (able to bind to ECM ligands) by intracellular signaling molecules. They are cell receptors used in both hemidesmosomes and focal adhesions.
How to hemidesmosomes use integrin receptors?
They use integrin receptors to bind extracellular matrix and connect to intermediate filaments. Hemidesmosomes are critically important in anchoring the epidermis to the dermis. They are not used for cell movement. These structures are holding cells in place. *In the skin, epidermal epithelial cells bind to basement membrane to anchor the epidermis to underlying dermal connective tissue.
Describe the procedure involved in indirect immunohistochemistry
This method utilizes a labeled antibody (IgG) to detect a specific antigen in cells of a tissue section. In most cases, the antibody is labeled with either a fluorescent compound, an enzyme such as peroxidase or alkaline phosphatase, or electron-dense gold particles (TEM). The method is referred to as immunocytochemistry when the antibody is used to detect antigens in cultured cells, cell suspensions or in a smear. DIRECT: The label is complexed with an antibody that binds directly to a specific antigen in the cells. INDIRECT: Two different antibodies are used. Primary antibody (not labeled) binds directly to a specific antigen in cells. Secondary antibody (labeled) binds specifically to an epitope in the primary antibody. The indirect method has 2 advantages: • The signal is amplified because more than one secondary antibody molecule can bind to each primary antibody. • The same secondary antibody can be used in tandem with many different primary antibodies.
Describe how actin binding proteins alter structure and function of microfilaments.
To harness and direct the energy of actin polymerization, the cell has a large, heterogeneous group of proteins that regulate structure and function of F-actin in all cell types - Some examples are listed in the Table below: Formin: F-actin nucleation & polymerization Arp 2/3 complex F-actin branching Fimbrin; α-Actinin; Villin: F-actin cross-linking (bundles) Filamin F-actin cross-linking (networks) Gelsolin; ADP/cofilin: F-actin severing & depolymerization Cap Z Capping of F-actin plus ends α-Catenin; Spectrin; Vinculin; Talin: Linkages to other cytoskeletal proteins
What are cadherins?
Transmembrane receptor proteins in cell to cell junctions (Adherens junctions and desmosomes). Ca+ - dependedent interaction strengthens cadherin association.
Why are identification of specific IFs in tumor pathology useful?
Tumor Cell Markers: Specific antibodies are used for immunohistochemistry -looking for specific IF expression in cells. • Keratins - Carcinomas (epithelial cell derived) • Vimentin - Sarcomas (connective tissue and bone derived) • GFAP - Glial cell tumors
Describe alterations in structure and function of microtubules produced by microtubule associated proteins (MAPs).
a) Assembly - Binding to Tubulin dimers to increase polymerization rate b) Stabilization - Capping of plus ends to prevent disassembly of MTs c) Cross-linking - Spacing of MTs or linking to membranes and cytoskeletal components d) Tracking - Binding to plus end and directing MTs toward specific cellular locations e) Destabilization - Binding to tubulin dimers and preventing MT assembly f) Severing - Destabilizing MTs by generating plus ends without GTP cap and minus ends without γ-Tubulin cap
Explain how contraction of non-muscle myosin is regulated and specify 3 contractile assemblies that can form via interactions with microfilaments.
a) Stress Fibers in Focal Adhesions are Used for Cell Migration: NM II interacts with microfilaments of stress fibers to generate tensionenables The cell to "pull" on cell membrane and to counteract outward forces on the cell membrane generated by the ECM b) Adherens Junctions allows for contraction of sheets of cells: Adherens junctions form a continuous "belt" of microfilaments around a layer of epithelial cells - NM II interacts with these microfilaments to generate a contractile belt that is mechanically linked to the cell membrane and ECM via Vinculin, α and β Catenin and Cadherin. c) Contractile Ring A contractile ring containing microfilaments and NM II forms during telophase of mitosisthe microfilaments are connected to the cell membrane which causes constriction and ultimately cleavage into 2 cells
main function GAP JUNCTION
allows the passage of small water-soluble ions and molecules
main function hemidesmosome
anchors intermediate filaments in a cell to the basal lamina
Specify the main components of the cytosol.
b) Cytosol: Aqueous Phase • Dissolved ions, CHO, AA, Nucleotides • Cytosolic enzymes and other proteins • Macromolecules, Ribonucleoproteins, Proteasomes • Inclusions: Lipid droplets, Glycogen, Pigments
What is cell adhesion?
cell adhesion precedes the formation of cell junctions. cells need to first establish whether they would like to form a cell junction by sensing cell surface signals through cell adhesion. **Cell adhesion does not necessarily lead to formation of cell junction
What does the trophoblast form during implantation?
cytotrophoblast and syncytiotrophoblast (will invade into the endometrium)
How doe spontaneous abortions happen?
due to abnormal development/defective cleavage of the zygote due to chromosomal abnormalities. -This natural screening process of embryos results in a significantly decreased incidence of congenitally malformed infants **most likely during first two weeks of development
Where does the embryo attach to the uterus?
embryonic pole, part of blastocyst w inner cell mass
What are hemidesmosomes?
form between cells and the extracellular matrix and involved integrin receptors. Intermediate filaments connect to theses structures as well.
What is gastrulation?
formation of germ layers endoderm, ectoderm, mesoderm -the process by which the bilaminar embryonic disc is converted into a trilaminar embryonic disc, the beginning of morphogenesis -it begins with the formation of the primitive streak and results in the generation of the three germ layers.
What is hCG?
human chorionic gonadotrophin produced by synctiotrohpoblast which stimulates progesterone by corpus luteum hCG can be assayed in the blood day 8 or urine day 10 low hCG - spontaneous abortion or ectopic pregnancy high hCG - multiple pregnancies, hydatidiform mole, gestational trophoblastic neoplasia
What is hydatidiform mole?
hydatid = greek hydatidos or drop of water complete: -development of trophoblast, no embryoblast -pregnancy without embryo -placental villi become swollen and vesicular -trophoblastic tissue secretes abnormally high levels of hCG -typically abort early in pregnancy -diploid but contain only paternal chromosomes (two sperm or diploid sperm, femal pronucleus is lost or absent) partial: -usually some evidence of embryonic development -spontaneous abortion usually occurs second trimester -triploid with double dose of paternal chromosomes (two sperm or diploid sperm)
main function ADHERENT JUNCTION
joins an actin bundle in one cell to similar bundle in a neighboring cell
main function DESMOSOME
joins intermediate filaments in one cell to those in a neighbor
Main function TIGHT JUNCTIONS
seals neighboring cells together in an epithelial sheet to prevent leakage of molecules between them
What does the inner cell mass form?
the embryo
What is another way to describe outer cells of blastocyst?
trophoblasts, extraembryonic very important in implantation.
Describe ectopic pregnancy
tubal, cervical, ovarian, abdominal delay or prevention of transport of cleaving zygote 1.mucosal adhesions 2.blockages caused by abdomino-pelvic cavity infections
What happens to zygote after fertilization?
undergoes successive cleavages while traveling through uterine tube
Most common site of ectopic pregnancy
uterine tube
When does fertilization begin?
w contact of secondary oocyte membrane and finishes with the intermingling of maternal and paternal chromosomes
Describe the basic principles underlying light microscopy.
• For LM, cells and tissues are fixed, sectioned and stained. Visible light is focused on the specimen by the condenser and the light beam that passes through it is collected by the objective lens. • The optimal resolving power of LM is approximately 0.2 µm. This permits good images of a cell at 1000-1500X magnification. Objects that are smaller than 0.2 µm cannot be resolved by LM.
Describe the molecular structure of kinesins and dyneins and their corresponding roles in intracellular transport.
• Intracellular transport of macromolecules, organelles and vesicles • Two main types of microtubule-dependent ATPases carry cargo along MTs: a) Kinesins: Carry cargo outward towards the plus end of MTs (kick out) b) Dyneins: Carry cargo inward towards the minus end of MTs (drag in) • Globular head domains contain ATPase and "walks along MT • Tail domains carry cargo using sequence-specific binding to proteins in membranes and organelles
Describe the following basic properties of cell membranes: fluidity
• Lipids can rotate and move laterally in the membrane. • Fluidity of membranes affected by lipid composition, specifically the length of FA chains and the degree of saturation of FA chains. • Cholesterol decreases membrane fluidity and increases membrane rigidity *The nonpolar part of cholesterol molecule is embedded in the hydrophobic region of the bilayer; the FA chains become more tightly packed and their range of motion is reduced because FA chains can wrap around the cholesterol molecule. *The OH group of cholesterol molecule is positioned as a polar head group that forms hydrogen bonds with polar head groups of phospholipids.
Specify the main components of the cytoskeleton.
• Microfilaments (Actin and Myosin) • Intermediate Filaments • Microtubules
Define asymmetry of cell membranes and explain the function of flippases.
• Phospholipids are distributed unevenly in the bilayer by a family of ATP-dependent translocases referred to as Flippases. • The outer layer of the plasma membrane contains mainly PC and SM; the inner layer contains more PS, PE and PI. • Cholesterol is distributed evenly in the bilayer
Describe the following basic properties of cell membranes: selective permeability.
• Small uncharged molecules (gases) diffuse freely. • Small uncharged polar molecules (H2O, ethanol) can diffuse. • Large uncharged polar molecules (glucose) are not permeable. • Ions (Na+, K+, Ca2+, Cl-) & charged polar molecules (amino acids) are not permeable. • Transmembrane proteins such as glucose transporters and ion channels are required to facilitate transport of these molecules across membranes.
Define resolving power and explain its importance in microscopy.
• The key determinant in obtaining a sharp, detailed image with a microscope is its resolving power, that is, the smallest distance between two objects that allows them to be seen as separate objects. • Magnification is independent of resolving power. Magnification of an image has utility only when accompanied by high resolution. • If an object is magnified beyond the resolving power of the microscope, a fuzzy image will be produced without an increase in structural detail.
