Week 3 Flashcards
In terms of microscope scale, why is 200 nm an important number?
Light microscopes cannot resolve particles below this size limit without the aid of computational approaches.
Approximate size of eukaryotic cells.
20 um
Approximate size of prokaryotic cells.
1 um
Where are the lens located on an inverted microscope?
An inverted microscope has the objective lens below the stage and the light source-condenser lens above the sample.
What are inverted microscopes used for?
To look at tissue culture cells in a flask or culture dish.
Resolution
The minimum distance between two distinguishable objects (D).
What does n sin theta represent mathematically?
The numerical aperture of the lens. It is an assessment of how well the lens was made and its ability to focus light.
What does the magnitude of resolution indicate?
You want resolution to be a small number. The bigger the NA of the lens are the smaller the wavelength of light the better your resolution.
Contrast
Allows objects to be seen
Why are cells and subcellular particles usually difficult to see using a microscope with standard brightfield optics?
They typically have very little contrast.
What are the two ways that contrast can be improved?
- Optical tricks
- Stains
Four types of light microscopy
- Brightfield
- DIC
- Phase-contrast
- Dark-field
Which types of light microscopy convert phase shifts in light to differences in brightness?
- Phase contrast
- Nomarski differential interference contrast (DIC)
How does DIC microscopy work?
Light is polarized before it passes through a spectrum and only the light rays that were rotated by the specimen are allowed to form the image plane.
Commonly used fixatives
Glutaraldehyde and formaldehyde
How does glutaraldehyde fix tissues?
The reactive aldehyde ends cross-link proteins together and hold them in place.
Examples of nonspecific stains
Hematoxylin and eosin
What does hematoxylin stain?
It preferentially stains the nucleus of cells.
What does eosin stain?
Eosin stains protein-rich structures.
How are light and fluorescence microscopy different?
A fluorescence microscope has an additional source of light and can add a filter that allows a specific wavelength of light to pass through.
DAPI
A fluorescent molecule that binds to DNA.
What does green fluorescent Ab stain? (in example)
Spindle microtubules
What does red fluorescent Ab stain? (in example)
Centromeres
What is immunofluorescence localization used for?
To localize specific proteins of interest within the cell for imaging.
After cells are fixed, why are detergents added?
This allows antibodies to pass through the plasma membrane and have access to the interior of the cell where they bind to antigens.
How does immunoflurescence localization work?
An antigen is immobilized. A primary antibody is directed against the antigen. Then, marker-coupled antibodies directed against the primary antibody act as a marker.
Co-localization
Antibody stains are in close proximity because their respective proteins are close, causing the colors to mix.
When imaging two different antigens, why are two primary antibodies from different species used?
This ensures no cross-reactivity.
Through which process is GFP (green fluorescent protein) expressed?
GFP is used like an epitope tag–expressed as a gene fusion.
What reacts to form the chromophore of GFP?
Three residues within the beta barrel (Thr, Tyr, Glv) autocatalytically react to form the chromophore.
Why was the discovery of GFP so novel?
The use of GFP allows cells to be stained and observed without killing them during hte fixative process.
Gene fusion process of expressing GFP in drosophila neurons
DNA sequences encoding GFP were cloned into a plasmid behind a promoter element that drives gene expression only in the peripheral neurons of Drosphila embryos. The DNA was injected into a fertilized egg and incorporated into the fly genome. A peripheral set of embryonic neuronal cells now express GFP and can be visualized by fl microscopy. GFP localized throughout the cytosol of these neurons and shows the complex morphology of these cells.
Process by which plant cells express a Talin-GFP fusion protein
DNA encoding GFP is fused in frame with the coding sequences for the cytoskeletal protein talin. The talin-GFP fusion protein was expressed from its own promoter in plant hair cells (spiky-looking structures). The localization of talin (more accurately, talin-GFP)in these cells is visualized by fl microscopy.
What happens when there are objects outside the focal plane of a microscope?
They create a cloud of blurry light that obscures the focal plane.
How does a confocal microscope fix the problem objects outside the focal plane cause?
It concentrates light at the image plane (in the z-dimension). The fluorophores in the plane are preferentially excited. (does not improve resolving power of the microscope)
What does the term confocal mean?
The two pinholes in the microscope are equidistant from the focal plane.
Why is a strong vacuum maintained in the chamber of a transmission electron microscope?
So air molecules won’t interfere with the electron beam.
How must samples be prepared for electron microscopy?
The samples must be fixed and dehydrated, often through osmium tetroxide, which is both a fixative and a stain.
How is scanning electron microscopy different from transmission electron microscopy?
Rather than passing electrons through teh sample, the sample is coated with metal that molds to the contours of the sample. The electrons that bounce off the sample are collected on the side.
What is scanning EM typically used for?
Examining the surfaces of cells or organisms.
How are Protein A conjugates used to detect antibodies?
Protein A is purified from Staphylococcus aureus and binds to the constant (Fc) domain of antibodies from most mammals.
In immuno EM detection of catalase, what is the primary antibody?
Rabbit anti-catalase
In immuno EM detection of catalase, what is the secondary antibody?
Protein A - gold
What is used to tag genes and proteins of interest in living cells
GFP and spectral variants
How does confocal microscopy improve contrast?
By removing out-of-focus light in the z-axis, giving an “apparent” improvement in resolution.
Which type of microscopy has the greatest resolving power?
Electron microscopy (but cells are fixed–no cell dynamics can be observed)
How does transmission EM create 3d structures?
Transmission EM images thin slices of fixed cells (tissues) which can be combined computationally from serial sections to provide high resolution 3D structures.
What is scanning EM used for?
Imaging surface contours of cells, tissues, or organisms.
What type of microscopy can localize proteins?
Immuno-EM
What type of microscopy can determine protein structure?
Cryo-EM
What are the two major parts of a phospholipid?
- Polar head group
- Nonpolar tails
What are the three parts of the polar head group on a phospholipid?
- Choline
- Phosphate
- Glycerol
First step of the biochemical pathway that makes phospholipid
The enzyme fatty acid synthase starts with acetyl CoA (2 carbon building block) and makes fatty acids (fatty acyl-CoA)
Second step of the biochemical pathway that makes phospholipid
An acyltransferase will transfer the fatty acid chains to the sn1 and sn2 positions (1st and 2nd carbons) of glycerol 3-phosphate. This produces phosphatidic acid (PA).
How to produce phosphatidylserine (PS)
The amino acid serine is added to phosphatidic acid (PA)
How to produce phosphatidylethanolamine
Phosphatidylserine (PS) is decarboxylated to produce PE
How to produce phosphatidycholine (PC)
Phosphatidylethanolamine (PE) is methylated three times to produce PC
What is the name of a spingosine-based phospholipid?
Sphingomyelein
Of the four phospholipids mentioned, which one has a negative charge?
Phosphatidylserine (PS)
DAG
Diacylglycerol
PA
Phosphatidic acid
PC
Phosphatidylcholine
Sph
Sphingomyelin
PE
Phosphatidylethanolamine
PS
Phosphatidylserine
PI
Phosphatidylinositol
PIPs (or phosphoinositides)
Phosphatidylinositol phosphates
What produces PIPs?
Phosphatidylinositol (PI) kinases
What does IP3 do?
It opens a calcium channel in the ER membrane, causing an increase in cytosolic calcium concentration.
What does increased calcium and the signaling molecule DAG do?
This activates protein kinase C (PKC), which phosphorylates a number of different proteins to modulate their activity.
What does PI 3-kinase do?
It adds a phosphate to PI4,5-bisphosphate to make PI3,4,5-trisphosphate (PIP3)
What reverses the action of PI3-kinase?
The action of PI3-kinase to produce PIP3 is reversed by a phosphatase called PTEN.
Relation of the phosphatase PTEN to cancer
PTEN is mutated in a large number of different cancers (leads to hyperactive signaling) and is a very important oncogene.
(Cleavage specificity of phospholipases) what does phospholipase D (PLD) generate?
PA + head group (R)
(Cleavage specificity of phospholipases) what does phospholipase A1 and A2 (PLA) generate?
Lysophosphoipid and free fatty acid
(Cleavage specificity of phospholipases) what does phospholipase C (PLC) generate?
DAG and phospho-head group
How does a cis double bond in a fatty acid tail affect the structure?
This puts a kink in the unsaturated chain.
Omega carbon
The last carbon (methyl group) is called the omega carbon
Omega-3 fatty acid structure
An omega-3 fatty acid has a double bond between the omega-3 (3rd from the last) and omega-4 (4th from the last) carbons.
Arachidonate
An omega-6 fatty acid (a double bond 6 carbons from the end)
NSAIDs
Nonsteroidal anti-inflammatory drugs (Advil and Motrin contain ibuprofen)
Omega-3 long-chain fatty acids are substrates of what molecules?
PLA2 and cyclooxygenase. This interaction is thought to generate anti-inflammatory compounds (or less inflammatory compounds).
What role does cholesterol play in the plasma membrane?
- Provides a better “seal” against water and small molecules.
- Influences membrane fluidity
- Can form microdomains with sphingolipids called rafts
How does a red fluorescent compound change in a model membrane enriched with cholesterol and sphingolipids?
It partitions into membrane regions (lateral phase separation).
Liquid-disordered state
The more fluid regions on a model membrane
Liquid-order domain
Lipid raft domain on a model membrane (gel-like state)
How does the length of fatty acyl chains affect membrane fluidity?
Longer chains produce less fluidity
How does the saturation of fatty acyl chains affect membrane fluidity?
More double bonds produce more fluidity.
Why is the plasma membrane of eukaryotic cells thicker than the ER membrane?
The plasma membrane is enriched in cholesterol and long-chain sphingolipids.
How much of the free cholesterol is found in the plasma membrane?
70%
Free cholesterol
Free means there is no fatty acid esterified to the cholesterol OH group.
If not in the plasma membrane, where else is cholesterol commonly found?
A significant amount of cholesterol can be found stored in lipid droplets covalently attached to a fatty acid (called cholesteryl esters)
Role of flippases in the plasma membrane
Flippases will translocate PS and PE rapidly to the inner leaflet of the plasma membrane, This is an ATP-dependent process driven by a family of P-type ATPases.
Role of floppases in the plasma membrane
Floppases can translocate PC and sphingolipids to the outer leaflet although these enzymes are less specific for the phospholipid species. This is an ATP-dependent process driven by ABC transporters.
What effect does PS exposure on the outer leaflet have?
PS exposure on the outer leaflet occurs during programmed cell death (apoptosis) and serves as a mark of death. Macrophages recognize dying cells exposing PS and eat the cells before they rupture.
What two things determine membrane fluidity?
The length and saturation of the fatty acyl chains.
What shape of lipids is best for forming bilayers? Why?
Cylindrical lipids are good at forming bilayers. Both the shape and amphipathic properties of phospholipid are critical for bilayer formation.
How do lipids move within the plasma membrane?
Lipids diffuse laterally within a leaflet very quickly, but do not flip-flop spontaneously between leaflets.
Definition of the plasma membrane
An asymmetric bilayer structure established by phospholipid flippases.
What is the permeability barrier of a membrane a function of?
It is a function of the lipid composition.
What is the selective permeability of biological membranes to many different ions, sugars, amino acids, nucleotide precursors, etc. a function of?
The specific proteins in the membrane.
Which molecules can always pass through the plasma membrane?
Hydrophobic molecules such as O2, CO2, N2, and steroid hormones.
Which molecules can usually pass through the plasma membrane?
Small uncharged polar molecules such as H2O, urea, and glycerol.
Which molecules can sometimes (though not usually) pass through the plasma membrane?
Large uncharged polar molecules such as glucose and sucrose.
Which molecules can never pass through the plasma membrane?
Ions
Rank the four categories of molecules by decreasing permeability in the lipid bilayer.
- Hydrophobic molecules
- Small uncharged polar molecules
- Large uncharged polar molecules
- Ions
Intracellular (cytosolic) concentration of Na+
5-15 mM
Intracellular (cytosolic) concentration of K+
140 mM
Intracellular (cytosolic) concentration of Ca2+
10^-4 mM
Extracellular concentration of Na+
145 mM
Extracellular concentration of K+
5 mM
Extracellular concentration of Ca2+
1-2 mM
Intracellular pH
7.2
Extracellular pH
7.4
What are the three types of proteins that control the movement of ions and other molecules across membranes?
- ATP-powered pumps
- Ion channels
- Transporters (carriers)
Rank the rate (in molecules/ions per second) of the three types of proteins that move molecules across the membrane.
- Ion channels (fastest)
- Transporters
- ATP-powered pumps (slowest)
Three types of transporters
- Uniporter
- Symporter
- Antiporter
Movement of a uniporter
Moves molecules down their gradient via passive transport.
Movement of a symporter
Moves one molecule down its gradient and one molecule up its gradient in the same direction. Active transport
Movement of an antiporter
Moves one molecule down its gradient and one molecule up its gradient in opposite directions. Active transport.
How does passive transport occur?
Passive transport occurs by facilitated diffusion down a concentration gradient, electrical gradient, or both (electrochemical gradient).
How does active transport work?
Active transport couples an input of energy to the uphill movement of substrate molecules against the electrochemical gradient.
Possible inputs of energy for active transport
Sunlight, ATP, electron transport, or an electrochemical gradient.
For uncharged molecules moving passively across the membrane, what is the sole force driving them?
The concentration gradient across the membrane.
What establishes the electrochemical gradient in the cell?
The Na+/K+ ATPase and ion channels in the membrane.
Rank the magnitudes of ∆G of the three ways charged molecules move across a membrane.
- Electrochemical gradient with membrane potential negative inside (most favorable)
- Electrochemical gradient with no membrane potential
- Electrochemical gradient with membrane potential positive inside (least favorable)
Common substrates for carrier proteins (passive transport)
Ions, sugars, amino acids, and nucleotides.
Four types of ATP-powered pumps
- P-type
- V-type
- F-type
- ABC
F-type ATPases
The ATP synthases found in mitochondria and chloroplasts. Move protons down their gradient to drive the synthesis of ATP (essentially run in reverse of the other ATP pumps)
V-type ATPase
The V-type ATPases hydrolyze ATP and pump protons into the lumen of the lysosome (plant vacuole), endosomes, and late Golgi compartments.
Which two types of ATPases are rotary?
V-type and F-type
ABC transporters
Stands for ATP-Binding Cassette. ABC transporters are a large and diverse family of ATP-powered pumps that typically pump small hydrophobic (or amphipathic) compounds out of cells.
In relation to ATPases in the membrane, what becomes overexpressed in tumor cells and what effect does this have?
MDR1 and MDR2 become overexpressed in tumor cells that become multidrug resistant (gene amplification and selection by drugs that kill tumor cells) and are able to pump a variety of drugs out of cells.
Bile
Bile is a detergent-like set of molecules made from cholesterol and phosphatidylcholine that is secreted by certain liver cells into bile ducts that deliver bile to the small intestine. The bile helps us digest our food.
CFTR
incidence of about 1 in 3000 people of European descent. CFTR is unusual in that it is a chloride channel gated by ATP binding and hydrolysis.
What transports bile and cholesterol?
Floppases
What is the terminal phosphate from ATP transferred to in a P-type ATP pump?
The terminal phosphate from ATP is transferred to an aspartic acid residue in the pump. This phosphorylated intermediate is unusual for ATPases and is the basis of the name P-type.
Basis of the function of the Na+/K+ ATPase
Na+/K+ ATPase uses energy of ATP hydrolysis to pump Na+ out and K+ into cells - against their concentration gradients.
In the Na+/K+ ATP pump, what is the hydrolysis of one ATP coupled to?
The export of 3 Na+ and import of 2 K+ ions against both [ ] gradients.
What process consumes ~ 25-50% of the ATP in mammalian cells?
The pumping mechanism of the Na+/K+ ATP pump.
Why is the Na+/K+ ATP pump considered electrogenic?
This pump is electrogenic because there is a net movement of 1 positive charge out of the cell and establishes electrochemical gradients that drive many other transport processes.
Why does the Na+/K+ ATP pump have a major effect on cell volume?
Through osmotic effect of pumping out more solute than is pumped in.
What are the three ways cells store potential energy?
- Chemical bonds (fats, sugars, ATP, NADH, etc.)
- Electrochemical gradients (Na+, K+, Ca2+, and H+ in particular)
- Very transiently in high energy protein conformations (proteins can act as springs).
Post-Albers cycle
The catalytic cycle for P-type ATPases.
ABC transporters are primarily responsible for transporting what three things?
Drugs, cholesterol, and bile
Which type of hormones can move across the plasma membrane without a transporter?
Sex hormones
