BSCI330
2nd law of thermodynamics
All processes in the universe are driven in the direction that increases disorder -Every energy transfer or transformation increases the entropy of the universe.
When do symptoms of Tay Sachs disease start and what are the symptoms?
Around the age of 6 months neurodegeneration in the CNS leads to blindness, deafness, paralysis, cognitive defects, and eventually death by the age of 4 years old >progressive lose of function/pain
What type of linkages are between cellulose molecules?
Beta linkages= producing alternating structures of glucose molecules (one up, one down)= allowing for a linear molecule
what must happen during TIRF?
Molecules must be withing 100-200nm of the cover slip >light does not enter the sample
What is the major sterol used in fungi?
Ergosterol
T/F The nucleus membrane has 2 separate membranes
False it's a single membrane that's folded up/doubled up into a double membrane structure
What is FRET?
Fluorescence Resonance Energy Transfer Uses two fluorescent proteins where the excitation energy of the second matches the emission of the first.
What is FM limited by?
Fluorescence coming from out-of-focus parts of cells >light passes through the cell in both directions and we can get fluorescence from parts of the cells that are not in the focal plain/point of interest
What is NOT useable energy?
HEAT
Higher S=
Higher density (faster settling to bottom)
What are macromolecules in the cell?
DNA, RNA, proteins, and polysaccharides -dry weight 'non water weight'
Waves out of phase=
Light not passing through parts of the cell
Purpose of beam-splitting mirror
Light that shines gets reflected on to the sample and the light that is emitted passes through thekmirror
Difference between light microscpy and confocal microscopy?
Light uses light and confocal uses a laser
Limitation with light microscopy
Limited resolution
Advantage of confocal microscopy?
Limits the detection to the focal plane and takes images from various focal planes ("z-sections"), making a 3-D image -Higher quality
What is a phosphodiester bond?
Links two nucleotides together in a nucleic acid
Different shapes phospholipids can self assemble
Lipid bilayer (packed loosely) or a lipid micelle (spherical structure)
What are the micro-domains in plasma membranes?
Lipid rafts of different compositions
T/F B1-->a1 = a1-->B1
True
T/F Lipids are NOT polyers
True
T/F The nuclear pore allows material to move past both membranes
True
T/F a plasma membrane is a regulated cellular boundary
True
T/F each chromosome is a VERY long strand of DNA
True
T/F eating plants reduces cholesterol
True
T/F humans with progeria have abnormal and strange shapes of cells/nuclei
True
T/F light that comes from other focal planes after hitting the lens will not focus on the pinhole
True
T/F nucleosome is the least condensed of chromatin?
True
T/F nucleotides are amphiphilic molecules
True
T/F phase-contrast and differential interference-contrast can see subcellular structures and make a 3D appearance and depth
True
T/F ribosomes found in mitochondria are more similar to ribosomes in prokaryotes than eukaryotes
True
T/F sterols are not made up of fatty acids
True
T/F the chemical nature of lipids is amphipathic
True
T/F the double membrane of the nucleus is continuous with the endoplasmic reticulum
True
T/F the fluorescent molecules emit a longer wavelength in all directions
True
T/F the hydrocarbon tail can be saturated or unsaturated
True
T/F the hydroxyl group can switch between above or below in linear form
True
T/F the protein MUST be correctly folded before leaving the ER
True
T/F there is membrane asymmetry between the 2 leaflets
True
T/F energy/light is inversely proportionate to wavelength
True long-wavelength can penetrate more deeply into a tissue sample, but uses less energy
T/F plants can survive without the cell wall
True - it's main function is to form structural strength
T/F in the plasma membrane, one region is NOT the same as another region
True = mosaic model
T/F there's a net increase in entropy from cells building order
True even though there's a decrease in entropy in cell
T/F protons are pumped from matrix into inner membrane space in mitochondria
True for the ETC/oxidative metabolism
T/F the cell wall is separate from the plasma membrane of the plant cell
True it is separate and outside of >connected to the plasma membrane
T/F plants wilt when the lose their turgor pressure
True so plants wilt when they are dehydrated and it loses its turgor pressure
T/F phospholipids self-assemble
True, because of the amphiphilic nature they can self-assemble into lipid bilayer membranes
T/F there's no cure for Tay Sachs disease
True, but gene therapy is being studied to correct the mutation. Otherwise, the only option is genetic screening of parents (especially Jews of Easter European descent, French Canadians, and Cajun population in Louisiana)
T/F A fluorescent microscope is a light microscope
True, but it has been modified to take advantage of fluorescent properties
T/F Phospholipids can flip-flop
True, but it rarely occurs
T/F the mitochondria is about the same size as bacteria
True, that's why LM can view it
T/F the mitochondria is the powerhouse of the cell
True, they generate most of the cell's ATP
T/F cellular structures and most animal cells are too small to see with the naked eye
True, they must be magnified to study
T/F heat is the most disorder form of energy
True: it is not confinable or useable
T/F images are in black and white with electron microscopy
True; not in color
What are the layers of the plasma membrane called and what is their structure?
Two leaflets that have an asymmetric distribution of phospholipids -the outer layer is different than the inner = different functions
How is the mitochondria inherited?
ONLY from the mother -not like the nucleus -none of the mitochondria from the sperm survive after fertilization
What type of phospholipids are on the extracellular space side?
Phosphatidylcholine, sphingomyelin, and ganglioside
Which two phosphoglycerides are neutral?
Phosphatidylethanolamine and phosphatidylcholine
What type of phospholipids are on the cytosol side?
Phosphatidylethanolamine and phosphatidylserine
Which phosphoglycerides is charged?
Phosphatidylserine -negative charge
What is the most abundant lipid in the plasma membrane
Phospholipids
What are plasma membranes made up of?
Phospholipids and sterols
Structure of sterols
Rigid ring structure that stiffen portions of the phoshpholipids - essential for the sturctural integrity of the membrane
Structure of a sterol
Rigid steroid ring structure with a non-polar hydrocarbon tail
What does turgor pressure result in?
Rigidity and cell expansion during growth
What are black membranes?
Planar lipid bilayers that are formed across a hole in a partition between two aqueous compartments *Phospholipid bilayer spanning a hole in a partition between two aqueous compartments *the edge is not interacting with water
Organelles seen by EM
Plant cell, animal cell, mitochondria, bacterium, virus ribosome, globular protein, small molecule, individual proteins, and atoms
Organelles seen by LM
Plant cell, animal cell, mitochondrium, and bacterium
What is fixation?
Preparing the tissue for observation by cessation of normal life functions in the tissue (killing) and stabilization of the structure of the tissue (preservation).
What is a triglyceride?
a lipid made of one glycerol and three fatty acids -energy storage in animals
Entropy
a measure of the disorder of a system
What does the golgi apparatus consist of?
a series of flattened discs called cisternea arranged in a stack
What is between the "beads"?
a short stretch of linker DNA
What is the golgi apparatus and its function?
Sorting center for proteins and membranes, and is also involved in the modification of proteins -postal service
What is magnification?
Taking a small image and making it larger
What disease are gangliosides associated with:
Tay-Sachs Disease
What are porins?
channel proteins to exchange information between cytosol into mitochondria
Major sterol in animal cell membranes
cholesterol
What makes up chromatin?
chromosome in a complex with a variety of proteins and RNA molecules (DNA, proteins, RNA)
what is vital microscopy?
created in 2-photon microscopy where there is much deeper penetration into a sample without sectioning
What is the practical limit of resolution in LM?
about 0.2um (microns)
What is a nucleosome?
about 150 nucleotides of DNA wound around a core of histone proteins
How much does the ER constitute of membranes?
about 50% in a typical eukaryotic cell
What happens in the trans cisterna?
addition of Gal and NANA
Endo H resistant
addition of different carbohydrates -complex
What is ATP?
adenosine triphosphate - major short term energy carrier in the cell
What did researchers build based off of mtDNA from people around the world?
an evolutionary tree based on human maternal lineage
What type of membrane does the mitochondria have?
double membrane
In a triglyceride, what is the hydrophilic part buried in?
ester linkage, which prevents it from interacting with water, making a triglyceride a completely hydrophobic molecule
How many origins of replication do eukaryotes have vs. prokaryotes?
eukaryotes have many to allow for rapid duplication while prokaryotes have 1
Function of cellulose
fibrils confer tensile strength on all walls
If we increase disorder (entropy) then we decrease?
free energy
if we decrease disorder (entropy) then we increase?
free energy
What direction are linkages made between carbohydrates?
from left to right
Trans face
further from the ER and represents the exit from the golgi apparatus -has secretory vesicles
What does the nucleus contain?
genetic material
What must be removed the protein before transporting out of the ER?
glucose residue
function of N-linked glycosylation?
helps during protein folding in ER, and then can be modified later in the Golgi apparatus to provide additional functions
Endo H sensitive
high mannose carb concentration
What is heterochromatin?
highly condensed, and represents DNA that is resistant to gene expression -contains some specialized structures (telomeres, centromeres, etc.)
Chemical nature of carbohydrate
highly polar and highly soluble in water
What type of membrane barriers are lypids?
hydrophobic
What must cells do to survive?
increase biological order -cells build order from disorder
What makes up the remaining 4% of the cell?
ions and small molecules -inorganic ions (calcium, chloride, sodium) -sugars and precursors -amino acids and precursors -nucleotides and precursors -fatty acids and molecules -other small molecules
What is euchromatin?
less condensed and remains accessible to the RNA transcription machinery
What are gangliosides similar to?
similar to phospholipids, but the phosphate/polar head group is replaced by a carbohydrate
What types of material can diffuse freely through the pores?
small ions and molecules (<5 kilodaltons)
What does the outer membrane of the mitochondria contain?
smooth surface *porins
What happens in the trans golgi network?
sulfation of tyrosines and carbohydrates SORTING
What about proteins smaller than 60 kilodaltons?
that can still transit via passive diffusion, but more slowly
The increase in entropy from the cell building order and releasing heat outweighs what?
the decrease entropy from the increase order in the cell
What is the H1 histone called?
linker histone
What kind of appearance does the ER have?
meshwork appearance
Small subunit of polymers?
monomers (repeating smaller subunit molecules)
components of a nucleotide
nitrogenous base, phosphate, deoxyribose sugar
Saturated bonds
no double bonds
What is needed after the 30nm chromatin from interphase?
more condensation because it's still too long to fit into the nucleus
are ribosomes enclosed in a membrane?
no: they are the only organelle that isn't
pk=
pH at which 1/2 of all molecules of an ionizable substance are charged
What happens in the cis golgi network?
phosphorylation of oligosaccharides on lysosomal proteins
Chemical nature of nucleotide
polar and charged
What if pK is above 7?
they can be ionized under basic conditions
How do scramblase and flippase work together
they can make membranes asymmetric or symmetric
What is one property of all living things
they create and maintain order in a universe that is tending always to greater disorder
What do cells do to the linker DNA when they die?
they cut it up so they die in a coordinated fashion
How do jellyfish have fluorescent properties?
they have the green fluorescent protein (GFP)
when do the ribosomal subunits assemble?
when protein synthesis initiates
What is a dehydration reaction?
when two monomers bond together through the loss of a water molecule
Size of images seen by LM
.75cm to 100nm
Size of images seen with the naked eye?
1cm to about 100um
Size of structure EM can see
1mm to 0.1nm
Bacterial chromosome width vs. Eukaryote
1mm vs. 2um
How many side chains (R) are electrically charged when dissolved in water?
5 -Asp, Glu, Lys, Arg, and His
What percent of the cell is water?
70%
What causes progeria?
A point mutations in the protein lamin A, which is normally a component of the nuclear lamina. This mutations prevents lamin A from incorporating in the lamina, reducing structural support for the nuclear membrane -It also prevents the normal organization of chromatin, affecting the ability of cells to divide properly (no replication to replace dying cells)
What is cellulose?
A polymer of glucose that provides tensile strength comparable to steel
T/F flow cytometry is a type of microscope
False
T/F light microscopy can visualize ribosomes
False
Components of amino acid
-amino acid -carboxyl group -side chain (R)
Critical features of TIRF:
-critical angle for total internal reflectance If you hit the cover slip at just the right angle, 100% of the lasers (total internal reflection) is reflected at the boundary of the cover slip and the sample >NO photons enter the sample
What are cristae?
-finger like projections into the interior of mitochondria folds in the inner membrane of mitochondria
How does DNA fit?
-pack into smaller length -nucleus is 3D, so we have a volume -must be regulated fashion
Other functions of golgi apparatus?
-synthesis site for most cellular carbohydrates -Modification of N-linked glycosylation on proteins -Attachment of O-linked glycosylation to proteins
What sizes require using electron microscopy, x-ray detraction, and other specialized techniques
0.2um, 20nm, 2nm, 0.2nm
Mechanism steps of LM that allow the human eye to view the structure
1) Light is passed through a condenser lens 2) The lens focuses on the specimen 3) The light passes through and is refracted by the specimen 4) The light is collected by an objective lens 5) The objective lens focuses the light into the eyepiece 6) The light hits the lens of your eye where it's focused on the retina for you to visualize it
3 covalent modifications of histones
1) Adding acetyl to lysine 2) Adding methyl groups 3) adding a phosphate to serine
The two themes of biology
1) Astonishing variety 2) Astonishing constancy
3 methods for fluorescent stains can be introduced?
1) Chemical dyes 2) Fluorescent molecules attached to antibodies 3) intrinsically fluorescent proteins (green fluorescent protein (GFP) and its variants) expressed by the cell itself
Steps of FM (3)
1) The light source comes in through the side instead of through a condenser lens and passes the first barrier filter: lets only blue light with a wavelength between 450-490nm 2) The light passes through a beam-splitting mirror: reflects light below 510nm but transmits light above 510 3) The light passes through a second barrier filter: cuts out unwanted fluorescent signals, passing the specific green fluorescence emission between 520 and 560nm
2 main functions of nucleotides
1) building blocks of nucleic acids (makes DNA and RNA) 2) short term energy carriers (ATP)
3 functions of carbohydrates
1) energy source 2) structural support 3) binding surface by adding carbs to other molecules
2 compartments of an energetically favorable phospholipid bilayer
1) exterior aqueous compartment 2) interior aqueous compartment -they are separated by a phospholipid bilayer
Functions of the objective lens in FM
1) focuses light and sends it through the beam-splitting mirror 2) serves as a condenser lens to focus light on the object and is absorbed
What is needed for high-level expression of genes in a loop (3)?
1) histone modifying enzymes 2) chromatin remodeling complexes 3) RNA polymerase
8 common features of all cells
1. Cells are highly complex and organized 2. Cells possess a genetic program 3. Capable of producing more of themselves 4. Biochemical factories that constantly acquire and utilize energy 5. Engage in mechanical activities 6. Respond to stimuli 7. Capable of self regulation 8. Evolution first happens at the level of molecules and cells
How many different ways are D-glucose disaccharides formed?
11
How many antifungal approaches are there?
2 - drugs and cream
Cis=
2 carbons coming off double bonds are on the same face of the double bond
What is a trans bond?
2 carbons on opposite sides of face
What is a phospholipid composed of?
2 fatty acids attached to a glycerol
What does 2-photon microscopy use
2 separate long-wave photons of light, instead one of one short-wave photon, to excite the fluorophore
What are lipid bilayers
2-D fluids that allow lateral diffusion of constituents
What is the DNA in the nucleus divided into?
23 pairs of chromosomes
What percent of the cell is composed of macromolecules?
26%
how many faces does the cisternae stack have?
2: the cis face and trans face
Human genome?
2m (46 linear chromosomes)
What size structure can LM see?
2um to 20mm >20mm, 2mm, 0.2mm,20um,2um
Human cell nucleus in diameter?
5um
How many side chains (R) are polar?
6 -Ser, Thr, Cys, Tys, Asn, and Gln
Structure of a sugar molecule
6 Carbon positions
How many side chains (R) are non polar?
9 Gly, Ala, Val, Leu, Ile, Met, Phe, Trp, and Pro
T/F the actual parking structure of the 30nm fiber is known
False because imagining requires manipulation of the DNA
What is every plant cell surrounded by?
A carbohydrate matrix= cell wall
Benefits of using FM specific filters in microscope?
A cell can be illuminated with light of one color, and visualized at the fluorescence wavelength
What is flow cytometry coupled with?
A cell sorter
What do plant cells have that give them their structural strength?
A cell wall (carbohydrate NOT lipid)
What is pectin?
A complex mixture of polysaccharide that provides resistance to compression -Carbohydrate
What type of disease is Tay-Sachs Disease?
A disease of lipid storage >genetic disorder
How is the plasma membrane a fluid?
A fluid is a substance where the components can diffuse past each other and move around -the membrane is a planar fluid where the lipids can move past each other by a diffusion -the lipid rafts are floating on top of the density gradients
What replaces light in electron microscopy?
A focused beam of electrons and a magnetic lens is sued to focus the electrons instead of glass lenses
What is progeria?
A genetic disorder that causes children to age rapidly
One microdomain is?
A lipid raft (increased cholesterol
What causes Tay-Sachs Disease?
A mutation in the enzyme B-hexosaminidase A prevent the cell from properly metabolizing GM2, causing it to accumulate to abnormally high levels in brain neurons
Function of nuclear lamina?
A protein structure that provides structural support
What does confocal microscopy use?
A scanning laser and pinhole apertures to limit detection to the focal plane
What is the replication origin and its function?
A site where DNA duplication is initiated
What is required for electron microscopy?
A vacuum (because in air the electron beam is lost), so special techniques are required >The cells must be fixed, and often desiccated, and then sliced into ultra-thin sections
T/F the nuclear pore forms a hole?
False it needs to open/close the membrane for larger molecules; highly regulated
What type of model does the plasma membrane have?
Fluid mosaic model
How wide is a typical animal cell?
About 10-20 um (microns)
Other components of the cell wall
Additional cross-linking polysaccharides and lignin
Why study cells?
All diseases are disturbances at the cellular level -cellular processes form the molecular basis of human health and diseases
What is a cell sorter?
Allows cells to be separated based on fluorescence and scatter properties >purified cells of specific properties
What are the building blocks of proteins?
Amino acids
What can light microscopy image?
Large-scale cellular structures
examples of antifungal drugs
Amphotericin B and nystatin
Limitation to detection?
An item smaller than 0.2um can only be detected if it emits light
What does a confocal microscope create?
An optimal section
Waves in phase=
As the light passes through the cell, different parts of the cell are affected differently
Which are acidic?
Asp and Glu
Which of the 5 electrically charge R groups are ALWAYS ionized at pH 7?
Asp, Glu, Lys, and Arg
Acidic amino acids
Aspartic acid/aspartate Glutamic acid/glutamate
Where does the oligosaccharide attach?
At the -NH2 group of an asparagine
What is the telomere and its function?
At the end of the chromosome and prevents the ends of chromosomes from being mistaken for broken DNA, and allow for proper duplication of the chromosome ends -solves the duplication problem
What are the 11 ways?
B1-->6 B1-->4 B1-->3 B1-->2 B1-->B1 B1-->a1 a1-->a1 a1-->a2 a1-->a3 a1-->a4 a1-->6
What does the inner membrane of the mitochondria contain?
Rich in the special lipid cardiolipin; folded into cristae
What would the nucleosomes look like if stretched out?
Beads on a string
Why can LM resolve/see a typical animal cell?
Because it's diameter is bigger than 0.2um
What do phospholipids assemble into?
Bilayers, which is the basic structure of cellular membranes -due to the amphipathic structures
How are black membranes different than liposomes?
Both sides of the membrane can be modified by adding different proteins -planar not spherical -altering the two aqueous solutions
Peripheral membranes
Bound to the membrane surface
What is a hydrolysis reaction?
Breaking down polymers by adding a water molecule
Structure and function of nucleic acids
Built from nucleotide monomers that are units of information storage, and also short-term energy storage
How do chemical dyes produce color?
By absorbing specific wavelengths of light
What does the intermembrane space of the mitochondria contain?
Cytochrome c (part of ETC) and several factors that regulate programmed cell death
Where does a glycosidic bond ALWAYS have linkage to?
Carbon 1
What type of molecules make up cells?
Carbon-based that are fundamentally the same for all living species >some molecules are specialized and made by some species
What does eosin stain?
Cellular components (pink)
What is considered a carbohydrate in plants?
Cellulose
What is the major component of the cell wall?
Cellulose -they are clustered together to make the cell wall
What do these 3 modifications of histones lead to?
Changes in interaction between histones, leading to either condensation or decondensation of chromatin
What happens when you modify the charges and chemical nature to the histone tails?
Changes the interaction with neighboring histones
What provides contrast and sensitivity for LM?
Chemical strains and fluorescent molecules >increases the power
What are sterols in animal cells?
Cholesterol -Another class of lipids
How are the carbons in the carbohydrate ring numbers?
Clockwise from the oxygen in the ring
What generates order within a cell?
Energy input and this energy from outside the cell was generated by processes that increase entropy
Two features of cells that make them nearly invisible under LM
Colorless and translucent (bag of water)
Features of the subdomains in the nucleus?
Compartmentalized, but not dynamic -they are NOT separated by membranes and can be assembled/disassembled as needed
Structure and function of lipids
Composed of fatty acids that serve as energy storage, and can also assemble into membranes
Function of Pectin
Compressive strength
What type of reaction links together monomers to form polymers?
Condensation reaction
What modifications regulate chromatin structure?
Covalent modifications to the histone tails
Synonymous term with condensation reaction?
Dehydration reaction
Why is the R group important?
Determines if the amino acid is polar or not -theres 20 different side chains
What direction is the world going towards?
Disorder
Unsaturated
Double bonds
Why are some polar head groups more above the surface than others?
Due to varying lengths of tails -corresponds to lipid rafts, so the micro-domains are not uniformly distributed
How many different RNA molecules do Eukaryotes vs. Prokaryotes have ?
E: 4 P: 3
How many proteins are in eukaryotic ribosomes vs. prokaryotes?
E: 80 P: 55
Order of ER to end of golgi apparatus
ER --> cis golgi network --> cis cisterna --> medial cisterna --> trans cisterna --> trans golgi network
Net result of condensing chromatin=
Each DNA molecule has been packaged into a mitotic chromosome that is 10,000 fold shorter than its extended length = fits inside the nucleus
How are cell walls formed between cells?
Each plant cell secretes its own cell wall -Each plant cell wall has contributions from the 2 bordering plant cells Bottom to top Plasma membrane Primary cell wall Middle lamella Another plant cell above
What is needed sense cells are mostly transparent to electrons?
Electron dense materials are used to stain the cells -Cells can also use gold-tagged antibodies to mark specific proteins/structures
1st Law of thermodynamics
Energy cannot be created or destroyed -conservation of energy -amount of energy in a system is constant
What is required for order?
Energy input
What does flow cytometry use to sort the cells?
Expression levels
Technique used when you don't want to only rely on the phases of light
FIXATION
T/F The endoplasmic reticulum has a double membrane
False
T/F The membrane asymmetry is a part of the mosaic nature of the membrane????
False
T/F chromatin is homogeneous during interphase
False
Other "cool" Fluorescence Microscopy techniques
FRET (fluorscence resonance energy transfer) and TIRF (total internal reflection fluorescence) 2-photon microscopy
Fatty acids -->
Fats, lipids, membranes (phospholipids)
What are phosphoglycerides?
Fatty acids based around glycerine
What is a sphingolipid?
Fatty acids based around sphingosine rather than glycerine
What movements do the tails make?
Flexion (bending) and rotation
Example of studying lipid membrane properties?
Fluorescent molecules in lipid membranes to study the motion of individual lipid molecules >notice if we have micro-domains in these structures based on the diffusion of different molecules
Emitted fluorescent light from in-focus point =
Focused at pinhole and reaches detector
Lipid micelle
Forms a spherical structure -helps dissolve dirt, grease, and makeup on the face
What is another class of lipids?
Gangliosides
Why is it advantageous for the polar heads to associate on the outsides of the membrane?
Gives water more room to make H+ bonds on the outside, which increases entropy and decreases energy -it's a spontaneous structure formation
What are fatty acids usually linked to?
Glycerol (alcohol linkage)
Components of a triglyceride?
Glycerol and 3 fatty acids
Where are proteins transferred to after ER?
Golgi apparatus
Benefits of electron microscopy
High resolution than LM
Function of glucosyl transferase
Helps with correct folding of protein
What is a commonly used dye combination?
Hematoxylin and eosin because they have different chemical properties that stain different parts of the cell
What two types of chromatin exist in the interphase portion?
Heterochromatin and euchromatin
Which R group switches between neutral and positively charged at pH 7?
His
Basic amino acids
Histidine Lysine Arginine
What reaction breaks apart polymers to form monomers?
Hydrolysis
Is a triglyceride hydrophilic or hydrophobic?
Hydrophobic even though glycerol is hydrophilic and the fatty acids are amphiphilic
Where does all lipid synthesis occur?
In the cytoplasmic face of the ER lipid bilayer (smooth ER)
What is transmission electron microscopy (TEM)?
Imaging internal structures with high resolution
Example of TIRF
Imaging of myosin-GFP molecules attached to actin filaments
What is scanning electron microscopy (SEM)?
Imaging the outside surface (staining, but no sectioning) >3D images
How do cells not break the 2nd law of thermodynamics?
In the course of order-generating reactions, the cell converts part of the energy into heat. the heat is released into the cell's environment, thereby disordering it and thus also increasing entropy overall
Where are ribosomes found?
In the cytoplasm (free ribosomes) and within mitochondria (and chloroplasts) -along the ER
Where are gangliosides found and what is their function?
In the plasma membrane (external surface) and is involved in cell-cell communication and neuronal plasticity (learning, etc.)
Benefits of the inner membrane being folded into cristae?
Increased surface area
Which are basic?
Lys, Arg, and His
Function of membrane proteins
Molecular transport, signal transduction, anchorage to the cytoskeleton (internal structure), interaction with other cells/extracellular matrix
What does the nuclear pore look like in the membrane?
It folds around the membrane and it's larger than 50nm across
Chemical features of carbohydrate
It is constantly going between a linear chain and ring in solution and several OH groups
Why is a laser used?
It provides a light of a single wavelength (don't need a barrier)
Limitations to using chemical dyes?
It requires fixation, which kills and preserves cells
What is so special about histidine
It switches between charged and uncharged around neutral so it can be used to do acid/base chemistry
What is flow cytometry?
It uses fluorescent labels to measure levels of specific biomolecules and ions
Why is a mutation in the enzyme B-hexosaminidase A bad?
It's supposed to degrade GM2, but it doesn't so there is extra GM2, which is toxic *we only want a small amount of GM2 on the cell surface
What type of diffusion do phospholipids move by?
Lateral diffusion where they move past each other
Advantages of FM?
Localize fluorescent molecules in cell more specifically than chemical dyes >*Allows detection of very small numbers of fluorescent molecules, since they are viewed against a dark background >structure will emit light, allowing for visualization to detect them
What is amphiphilic?
Loves both polar and non polar
Histone core components
MAJOR structural proteins -two copies each of 4 different histones (H2A, H2B, H3, and H4) -each core of histones is 8 polypeptides
Structure and function of proteins
Made up of amino acids and perform most cellular functions
Purpose of polymers
Major molecules that provide structure and function to a cell
Function of overall endoplasmic reticulum
Major storage site (lumen) for Calcium ions, which is important for cellular signaling
How do scientists study the property if lipid bilayers?
Make liposomes (artificial bilayers)
Effects of adding a phosphate group to a histone
Makes serine phosphorylation, which makes serine (originally neutral) now a negative charge, disrupting interactions between histones
Effects of adding methyl groups to histone
Mask the charges (hide it), disrupting the interaction
Example of antifungal creams
Miconazole and lamisil
What is the smallest organelle LM can see?
Mitochondria, which are about 0.5um (microns) in diameter
Who is the common ancestor of mtDNA called?
Mitochondrial Eve
How much better is resolution in electron microscopy than light microscopy?
More than 200x so we can see much smaller/closer together structures
Function of vesicular tubules?
Move clusters from rough ER to the cis Golgi network
what are z-sections?
Moving the lens up and down
What is an oligosaccharide composed of (3)
N-acetylglucosamine glucose mannose
What is the attachment site called?
N-linked glycosylation
What is O-linked different than N-linked?
N-linked is pre-assembled with oligosaccharide and transferred to the protein -O linked needs the addition of sugar molecules one, by one (serine and threonine)
Modification of N-linked glycosylation on proteins in golgi
N-linked oligosaccharides are trimmed and further processed by addition of other sugars to generate a wide range of complex oligosaccharides and high mannose oligosaccharides
What does GFP expressed in specific neurons in fruit fly embryos highlight?
Neural connections in live animal
What is the charge of an amino acid in its ionized form?
Neutral because the ionized form of the carboxyl and amine group is lost, which causes it to lose polarity
Unsaturated bonds
One cis double bond
Is 'S' linear with MW?
No
Saturated=
No double bonds
Is condensing of DNA permanent?
No, it must be able to recondense
Do cells violate the 2nd law of thermodynamics?
No: cells must increase entropy in the universe MORE so than the decrease in entropy within the cell
What type of reactions decrease entropy?
Non spontaneous
What supports the nuclear membrane?
Nuclear lamina
Nucleotides -->
Nucleic acids
What is the best understood subdomain of the nucleus and what is its function?
Nucleolus and it's the site of ribosome assembly
What is the problem with liposomes?
Once they are formed, there is no access to the interior so only the exterior can be modified
What is the difference between the two hydrocarbon tails of a phospholipid
One is saturated and one is unsaturated
How many different types of cells are there in the human body?
Over 200
What is an optimal section?
Only viewing a plane of the cell with much better resolution
Emitted light from out-of-focus point =
Out of focus at pinhole and is largely excluded from detector
What are cellulose microfibrils interwoven with?
Pectin
How to solve the problem of cells being translucent?
Phase-contrast and differential-interference-contrast (DIC, aka Nomarski optics)
What is the major sterol used in plants?
Phytosterols
What are cells bounded by?
Plasma membrane composed of mainly phospholipids
Structure of hydrocarbon chains
Polar head: carboxylic acid group Hydro-phobic tail: hydrocarbon chain
Sugars (monosaccharides) -->
Polysaccharides
What is a disease of the nuclear structure?
Progeria
What are ribosomes and their function?
Protein-synthesizing organelles in all cells
Amino acids -->
Proteins
Which reactions are not energetically favorable?
Reactions that increase the availability of useable energy (decrease entropy)
What is desiccation?
Removing moisture
Limitations to EM
Requires special preservation and staining techniques >Lose the possibility to see color images
What is tensile strength?
Resistant to stretching
What is the Endoplasmic Reticulum divided into?
Rough and smooth ER
Differences between SEM and TEM
SEM= external surfaces and 3D images TEM= internal cellular structures with high resolution
When and Who developed high quality light microscopes
Schleiden and Schwann in the 19th century, developing the Cell Theory
Most energetically favorable structure of phospholipid bilayer
Sealed compartment formed by phospholipid bilayer -the hydrocarbon tails never have access to water =spontaneous even =completely nature effect
Importance of the structure of chromatin?
Serves to condense the linear DNA molecules to fit within the nucleus
What structures are in each chromosome that is required for proper duplication?
Several specialized sequences: replication origin, centromere, and telomere (heterochromatin: don't have genes)
Function of Smooth ER?
Site of lipid synthesis, including sterols, and detoxification of lipid-soluble compounds
Function of Rough ER?
Site of synthesis of transmembrane and secreted proteins (plus proteins found in lumens of organelles along the secretory pathway: golgi)
What does the 'S' mean in ribosomes?
Size designation -how quickly components settle to the bottom during centrifugation
Cell cycle analysis using DNA-staining dye (propidium iodide) in flow cytometry
Size is increasing and the amount is increasing >amount of propidium ioidide is proportionate to amount of DNA
What are Liposomes?
Spherical vesicles that assemble spontaneously from phospholipids in water. They can vary in size depending on the conditions used (concentration of lipid, specific phospholipid used, etc.)
What type of reactions increase entropy?
Spontaneous
What two things are cell walls resistant to?
Stretching and compression
Structure and function of carbohydrates
Sugar monomers that serve as energy storage and structural support for cells
Effects of adding acetyl to lysine
Takes away the positive charge, making it neutral -disrupts neighboring interactions, making the histones closer or repealing
T/F chromatin is a highly dynamic structure
TURE, it changes to regulate gene expression, DNA replication, and cell division >open/close chromatin's structure to regulate gene expression when completing DNA replication -switching between dividing to nondividing
Difference between the ER lipid bilayer and plasma membrane lipid bilayer?
The ER is symmetric while the plasma membrane is asymmetric
B1 OH on sugar A and alpha4 OH on sugar B?
The OH is above the plane on Carbon 1 for sugar A and below the plane on Carbon 4 for sugar 2
What is detection?
The ability to actually see something
What is resolution?
The ability to distinguish 2 objects that are close to each other
When disorder increase what happens to energy?
The amount of useable/available energy tends to decrease
Purpose of fluorescence microscopy?
The fluorescent molecules absorb light at one wavelength and emit light of a longer wavelength
Process of flow cytometry
The laser is going to excite the fluorescence labeled molecules in the cell and the cells emit the light, which is collected by the machine and the cells are separated based of data and scatter
What does the different shape of the saturated and unsaturated tails allow for?
The lipids aren't packed tightly allowing for fluidity
How is the plasma membrane a "mosaic"?
The membrane is made up of tiny pieces (microdomains), so within each microdomain the concentration of particular lipids may be increased or decreased than the surrounding regions -Each region is made up of a different amount of components/differing lipids **One region is NOT the same as another region
What is the Nuclear lamina made of?
The meshwork of proteins called lamins (A,B, and C) -what you would be left with if you dissolved away the nuclear membrane
Why does flip-flopping rarely occur?
The polar head group would have to push through the tails to get to the other side and that requires a lot of energy to do so, making it not spontaneous
What part of the phospholipid and sterol are associated with eachother
The polar heads
How do phospholipids self-assemble?
The polar heads groups associate with water on the "outsides" of the membrane and the hydrocarbon tails associate with each other on the "inside of the membrane
What is turgor pressure?
The structural rigidity of the plant cell wall allows the generation of a large internal pressure
What is the detection surface of the eye?
The retina
Importance of the sterol structure
The ring structure can't rotate without braking and it helps orient sterols in a plasma membrane
What is the centromere and its function?
The site of attachment to the mitotic spindle, which allows one copy of each duplicated chromosome to be pulled into each daughter cell during mitosis
What is involved during further condensation?
The structure is not well understood, but it likely involve extensive looping and interactions with many non-histone proteins, including the nuclear lamina
Energy concept behind self-assembly of phospholipid membranes
The tails don't want to associate with each other, the water molecules are more energetically favorable if they associate with polar head groups instead of hydrocarbon tails
What MUST happen during FRET?
The two proteins must be very close together, exciting protein 1 will allow protein 2 to fluoresce w/o emitting a photon >must be very close together >determines if the 2 molecules are physically in contact with each other
What makes visualizing individual cells challenging?
Their size and transparency
Why isn't there a bond to C5?
There's no OH group
Why are cells not isolated systems?
They are able to exchange energy with their environment
Where are microdomains found?
They are stiffer and less dense so they are floating
How do antifungal drugs work?
They specifically bind to ergosterol and form ion pores, allowing ions to leak out of the cell >changes the osmotic pressure natural of the cell, so it can't properly regulate osmolarity --> death
What is TIRF?
Total Internal reflection fluorescence Uses laser light that is reflected off the cover slip where the molecules close to the cover slip can be excited by the EM field, providing a very narrow band of fluorescence -high resolution image of single molecules
Benefits of cutting tissue samples into thin sections?
Tissues are too large to be visualized by most microscopy techniques so sections allow for particular structures to be seen
Where is the 3rd OH attached in a phospholipid?
To a phosphate, which is attached to a polar head group=phosphoglycerides
Why does flow cytometry need a computer?
To collect fluorescence data (color, intensity) as well as scatter
What is the function of B-hexosaminidase A?
To degrade GM2
What are black membranes used for?
To study the motion of individual lipids molecules
How can we alter the exterior surface of liposomes?
Transferring them to a different environment, which alters the exterior environment and creates a plasma membrane
What type of pressure do cell walls have?
Turgor pressure
What two positions can the OH group be when attached to each carbon in the ring?
UP or below the plane of the ring
How do you see a tiny bag of mostly water?
Use staining with chemical dyes to reveal details of cells
What do Phase-contrast and differential-interference-contrast do?
Use the change in phase of light waves as they pass through a cell: Visualization of live, unmanipulated cells, but with limited details
What is flow cytometry?
Using fluorescence to analyze live cells in real-time in an aqueous stream as they pass through a laser -NOT microscopic
Goal of Phase-contrast and differential-interference-contrast
Visualize the live cell with unlimited detail >determine how the cell itself affects the phase of light
Function of Lignin
Waterproof polymer reinforcing secondary cell walls
When are gold particles used in electron microscopy?
When antibodies are conjugated with gold particles allowing localization of specific molecules within cell by TEM >different sizes represent different antibodies binding to different protein inside the cell (instead of color)
Transmembrane protein/region
When integral membranes span both leaflets or may be anchored via attachment of lipid groups that insert into one leaflet -part is on the outer surface and the other is on the inner surface
What do you add pectin to?
When making jam or jelly because it helps form a jell structure in an aqueous solution
What is O-linked glycosylation?
When some proteins also get carbohydrates added to the hydroxyl group of serine or theronine
Measuring changes in the protein location over time in SYTO13 (nucleus) and NFAT2
When stimulated, the DNA and NFAT overlap but do not overlap in unstimulated cells >the antibodies recognize the NFAT and the material moves from the cytoplasm into the nucleus
What stage is interphase?
When the cells are not actively dividing
How does turgor pressure work?
When the intracellular environment has an excess of solutes water will flow into the cell via osmosis, and the pressure allows the cell wall to expand and not burst
What is a condensation reaction?
When two molecules bond through the loss of a water molecule. -the large molecule is becoming dehydrated
Limitation to magnification?
When you magnify an image, it may turn out blurry
What can fixation reveal?
Where lipids and DNA are and pH level
How is the fluorescent specimen illuminated?
With a focused point of light from a pinhole
Model Organisms (examples)
Yeast -minimal eukaryote Drosophila - fruit fly Mouse
Can cells build order?
Yes (reducing entropy), but must expend energy to do so
Are ribosomes protein?
Yes, they are large complexes of protein plus RNA
What shape is a trans bond?
Zig zag
What is lignin?
a complex organic polymer deposited in the cell walls of many plants, making them rigid and woody. (waterproof) -complex alcohol
What is chromatin?
a complex with histones and other proteins -it is a dynamic structure, which can change depending on the needs of the cell
What is the nucleus bounded by?
a double membrane (two lipid bilayers)
Function of euchromatin
allow for gene expression
DOWN=
alpha
Synonymous term with amphipathic
amphiphilic
What can ergosterol be used for in animal cells?
antifungal drug
Rough ER
associated with many ribosomes, giving it a rough appearance in microscope images -flattened structure
IS the plasma membrane asymmetric or symmetric?
asymmetric
Flippase promotes
asymmetry
What position is the OH in glucose?
below the plane of the ring
Goal of H1
bends the DNA so the histone chords can approach each other, forming a tighter structure
What shape is a cis-double bond?
bent
UP=
beta
Function of the linker histone H1
binds to the outside of each nucleosome and also helps to condense the chromatin by flexing the DNA
Amphipathic meaning
both polar and non polar
How are most rough ER-synthesized proteins modified?
by adding a common oligosaccharide
Function of flippase?
catalyses flipping of specific phospholipids to cytoplasmic membranes
Function of scramblase?
catalyzes flipping of phospholipid molecules and transports new lipids in a proportional manner to the lumen face
What makes up the golgi stack?
cis cisterna, medial cisterna, and trans cisterna
Function of glucosidase
cleaves off glucose that allows the protein to be released from calnexin, so it can fold properly
Cis face
closest to the ER and receives vesicles containing ER-synthesized proteins -has golgi vesicles
What is considered fluorescence data?
color and intensity (how much there is)
What is the endoplasmic reticulum and its function?
complex membranous organelle that is responsible for modification and maturation of integral membrane and secreted proteins, and for lipid synthesis
What is the result from this human maternal lineage?
convergence on a single woman in Africa 140-200,000 years ago, who is the ancestor of all current living humans
O-linked is equivalent to
custom made clothing
What does the matrix of the mitochondria contain?
cytosol of mitochondia -contains mtDNA, ribosomes, and other components for expression of mitochondrial genome
What does the Golgi apparatus do?
directs proteins (rough ER) and lipids (smooth ER) produced by the ER to their final destination, transported by vesicles
What is the inner membrane the site of?
electron transport chain
What is free energy?
energy available to do work (usable)
Where is the ER found?
extends throughout the cytoplasm and it's inside the cytoplasm
What does hematoxylin stain?
extracellular matrix (purple) - acidic
Structure of a phospholipid
hydrophilic head attached to glycerol and hydrophobic tail with two fatty acids
How much pressure can the cell wall resist to swelling?
hydrostatic pressure of 10 or more atmosphere -allow cells to become extremely rigid
What is the endosymbiont hypothesis?
hyp. of how mitochondria evolved from independent organisms to become part of cells
When is energy useable?
if it is confinable -in chemical bonds or in the form of potential energy
In what direction does turgor pressure work?
in all directions horizontally and vertically
How is the DNA formed?
in chromatin
How do the antifungal creams work
inhibit ergosterol synthesis, by blocking fungal cell growth -so the fungus can't make new cell membrane
Integral proteins
inserted into the membrane interior
What is important in forming the 30nm fiber?
interactions between the N-terminal tails of the histones in neighboring nucleosomes -allow for higher order structure
What does the chromatin look like during interphase?
it is condensed into a fiber that is about 30nm in diameter (not in bead form)
What is linker DNA?
it ranges from a few nucleotides to about 80 nucleotides, such that the nucleosome structure repeats on average every 200 nucelotides or so
Smooth ER
lacks attached ribosomes -tube with extensions( connects rough ER together)
What are ribosomes composed of?
large and small subunits
What about larger material (>60 kd)?
macromolecules and protein complexes (such as ribosomes), can traverse the nuclear pores by active transport
What is the mitochondria and its function?
major energy production organelles, and are responsible for oxidative metabolism of carbohydrates, lipids, and amino acids
What is the most highly condensed form of chromatin?
mitotic chromosomes -during mitosis
What regulates chromatin structure?
modification of histones, which results in gene expression
What does 2-photon microscopy result in?
much deeper penetration into a sample without sectioning AKA vital microscopy -each photon has lower energy we only see fluorescence where the photons intersect
Difference between eukaryotic and prokaryotic ribosomes?
much larger and extra RNA/proteins
What type of DNA is present in mitochondria?
multiple copies of a small (about 16kb;37 genes) of circular DNA
How does the nucleus communicate with the cytoplasm?
nuclear pores -allow for exchanging information
What is the basic unit of chromatin?
nucleosome
What is the ER continuous with?
nucleus membrane
Double membrane structural components of the mitochondria
outer membrane, inner membrane, intermembrane space, and matrix
What is the matrix the site of?
oxidative metabolism
What is the mitochondria the site of?
oxidative metabolism of carbohydrates, amino acids, and lipids
Which reactions are energetically favorable?
reactions that decrease the availability of useable energy (increase entropy)
What do the sterol rigid ring structures do under different temperatures
reduce fluidity under warm temperatures and prevent solidity at colder temperatures
What is considered scatter?
reflecting size, shape, and internal architecture
Regulating chromatin structure=
regulating gene expression
Function of proteins and glycoproteins in cell wall
remodeling and help defend against pathogens
What happens in the cis cisterna?
removal of Man
What happens in the medial cisterna?
removal of man and addition of GicNAc
Where are the ribosomal subunits assembled?
separately in the nucleolus (exported in the cytosol) and then they come together to initiate protein synthesis
Why are these conversions never 100% efficient? (1st Law)
some energy is always lost/converted as heat, which is NOT confinable or useable energy
N-linked is equivalent to
standard clothing shelf
IS the ER membrane asymmetric or symmetric?
symmetric
Scramblase promotes?
symmetry
what are ribosomes often associated with?
the ER (where transmembrane proteins are synthesized)
Why is the ER continuous with the nuclear membrane?
the ER lumen merges with the nuclear intermembrane space
Why is it bad to metabolize a lot of amino acids?
the amine group on the amino group gets released as ammonia, which is toxic
What is the charge of the inner face and outer face of the membrane?
the inner face is negatively charged and the outer membrane is neutral, so the voltage across the membrane affects the movement of ions and interactions
what are nucleoporins?
the nuclear pore complex consisting of over 30 different proteins that span the double membrane
What if pK is below 7?
then they can be ionized under acidic conditions
Function of heterochromatin?
to silence gene expression -ex: brain cells are shut off in the liver cells
T/F amino acids can be metabolized for energy
true
T/F the golgi apparatus is a discontinuous organelle
true
T/F the lamina is not a histone protein
true
T/F the nucleolus is smaller if there is no active ribosome assembly
true
T/F lysine acetylation and methylation are competing reactions
true -you can't do both at the same time and they have slightly different effects
T/F with the lower levels of packing, the fine details are unknown
true, but can be postulated/assumed
T/F there are many opportunities for modifications to the histone tails
true, each histone tail has multiple residues that can be regulated
T/F ATP is an energy currency
true; every exchange is standard
How many times is the DNA wrapped around the histone cords?
twice
Chemical features of amino acid
uniform chemical structure with directionality (amino terminus - carboxyl terminus) and side (R) group variability
Chemical features of nucleotide
uniform chemical structure with some side group variability (nitrogenous base component)
