MCB2210 Block 1

Nerve Cells

"Excitable cells" • Nerve cells generate, receive and transmit electrical signals throughout the body • They can be up to a meter or more (!!!!) in length • They grow that long using a specialized type of "motility" in which they extend part of the cell, but never retract the cell body. They just get longer and longer sending axons along pathways through the body.

Three Classes of Membrane Proteins

(1) Integral membrane proteins: tightly associated with the lipid bilayer. - Amino acids interact directly with lipid portion. - Transmembrane proteins span the bilayer one or more times while others associate with only one leaflet - Although typically seen as crossing both leaflets, **amphipathic helices can also position integral membrane proteins in one leaf of the membrane (2) Lipid anchored proteins: covalent addition of a lipid to a protein targets the protein to the membrane. - Fatty acids are added to attach proteins to the inner leaflet - Glycophosphatidylinositol (GPI) added to attach proteins to outer leaflet. - Some lipid anchored proteins can cycle between membrane-bound and soluble forms (3) Peripheral membrane proteins: indirectly attached to the membrane via interactions with other membrane proteins, not lipids

What are the four different structures/conformations of a protein?

(1) Primary: amino acid sequence (2) Secondary: stretches or strands of proteins or peptides with distinct characteristic local structural conformations, dependent on hydrogen bonding. (α-helix and the ß-sheet) (3) Tertiary: the overall three-dimensional shape of an entire protein molecule. (4) Quartenary: how these protein subunits interact with each other and arrange themselves to form a larger aggregate protein complex. - 2 subunits = dimer - 3 subunits = trimer - 4 subunits = tetramer **NOTE: - If the complex is composed of two identical subunits, it is a homodimer. If the two subunits are different polypeptides, it is a heterodimer. - Protein complexes can be stable, or can be triggered by a signal to assemble http://www.particlesciences.com/images/tb/levels-of-protein-structure.jpg

How do we quantify protein-protein interactions? (Kd)

(on rate: reactants --> products, forward reaction) A+B -> AB <- (off rate: products --> reactants, reverse reaction) on rate = [A] x [B] x kon • *HOW FAST MOLECULE WILL COME TOGETHER • The on rate is largely dependent on the concentrations of A and B. • The kon rate constant depends on the rate of diffusion, the size of the molecules, and whether there is a favored orientation required for binding off rate = [AB] x koff • *HOW FAST MOLECULE WILL COME APART • The off rate is largely dependent on the the koff rate constant. **This is what really determines the AFFINITY of the interaction. • The koff rate constant depends on the sum of the forces (ionic bonds, van der waals interactions, hydrophobic interactions) that will hold A and B together.

Transmission Electron Microscope (TEM)

**Considerations • EM must be done in a vacuum for electron gun to work, so you can't have wet samples • Dry tissue does not have enough density to scatter electrons so you have to replace it with something dense, so you must bind heavy metals like uranium, lead or tungsten to membranes and proteins. • Thick, metal stained samples too dense for electrons to penetrate, so you first section fixed material into very thin slices, then stain Procedure • *Fix tissue (glutaraldehyde-proteins; osmium tetroxide-lipids) • Dehydrate with alcohols and embed with plastic • Cut thin slices (sections; 0.02-0.1µm thick)- sample must be thin otherwise electrons don't get through • Stain with uranium, lead etc. NOTE: • What you see is the scattering of electrons by the metal. There is almost no biological material left after this harsh process! • Images appear 2D

What sign is used for outward direction? Inward?

+ = outward direction - = inward

Primary Active Transport (What are the two general types of pumps? What do they do? What is P-type and V-type? What are ABC transporters? What does their name stand for? What process are they important in? What general type of pump do they each of the previous fall under?

- ATP-dependent pumps: ATP used as the direct energy source |> Hydrolyze ATP and use the energy to move one or more molecules across the membrane P-type: Become Phosphorylated by the PO4 from ATP during the transport • Plasma membrane Na+/K+-ATPase, K+/H+ ATPases, Ca++ ATPases V-type: Vesicular H+ ATPases • do not become phosphorylated • pump H+ into membrane compartments • Acidification of endocytic vesicles, lysosomes, golgi (de-acidification of cytoplasm because H+ is taken from there) ABC (ATP Binding Cassette) Transporters • Use ATP for transport but do NOT become phosphorylated • 5% of bacterial genome is this type of gene • Some isoforms = important in cancer (transformed cells hijack these to pump anti-cancer genetic material out = resistant --> multidrug resistance transporters) • 5% of bacterial genome is this type of gene - Light driven pumps: light used as the direct energy source.

Active Transporters (name and describe what happens in each)

- Active transporters can move molecules against a chemical, electrical or electrochemical gradient; they require extra energy input for transport • ATP-dependent pumps • Symporter (a.k.a. co-transporter) carrier proteins • Antiporter (a.k.a. exchanger) carrier proteins

Passive Transport (What is the difference between carrier proteins and protein channels? What are their relative speeds?)

- Carriers have a solute binding site --> slower - Channels do not have a binding site (like a tunnel --> faster

Absorption of glucose by the small intestine requires the activity of three transporters. Which three? What do they do?

- Na+/glucose cotransporter • Secondary active • Accumulates glucose at high concentration inside the epithelial cell - Glucose passive (facilitated) transporter • Passive mediated • Glucose moves down gradient into the blood Na/K ATPase • Primary active transporter • Pumps Na back out and brings K in using ATP energy

Passive Transporters (name and describe what happens in each)

- Passive transporters allow net movement down a chemical (concentration), electrical or electrochemical gradient; they require no energy beyond thermal motion; • Carrier proteins • Ion Channels: typically bi-directional, regulated--can be opened and closed, not a lot of interaction b/w ions and ion channels (b/c transport is so fast

The Kinetics of Transport (rate of transport vs. concentration of transported molecule relation—factors affecting value)

- Simple diffusion and channel-mediated transport not ever peak (i.e. DON'T become saturated) - Facilitated diffusion by carrier proteins CAN become saturated because of binding of solute to carrier

Gradients (Describe. What are two components that are equal? What are these equilibriums called? What are gradients caused by...? Why are they important? Membrane permeability?)

- The ionic composition is not the same inside and out. (AKA ion type isn't the same—not going to have 25% Na+ in every cell. In some, it will be 30%, some it will be 28%, others will be 13%, etc.) - Total cations and anions are equal -electroneutrality. - Total number of particles inside and out is equal - osmolarity is the same. - Cells use transport proteins to generate an internal ionic environment that is different than the external environment and to exploit the resulting gradients to perform critical functions. - ????????????????????????

Kinase

...An enzyme that catalyzes the transfer of phosphate groups from high-energy, phosphate-donating molecules to specific substrates. This process is known as phosphorylation, where the substrate gains a phosphate group and the high-energy ATP MOLECULE DONATES A PHOSPHATE GROUP.

What evidence suggests a common ancestry for all organisms on earth?

...The fact that the fundamentals of life processes (DNA, RNA, protein as well as certain metabolic pathways) are similar in all cells argues that there was a "common ancestor cell." If there were other cells, their progeny did not survive to present.

What is RNA interference (RNAi)?

...a mechanism eukaryotic cells have evolved to: (1) protect against viral RNA (2) regulate the stability of the cell's own mRNAs via microRNAs (miRNAs). • We can exploit these mechanisms to knock down specific gene function by engineering small interfering RNAs (siRNAs), that when transfected into cells, target the inhibition or destruction of the mRNA.

Cellular Compartment

...all of the closed parts within the cytosol of a eukaryotic cell, usually surrounded by a single or double lipid layer membrane. These compartments are often, but not always, defined as membrane enclosed regions. - The formation of cellular compartments is called compartmentalization.

Electron Microscopy (what are the two types?)

...another means of getting around the resolution limits of light. Electron beams have a shorter wavelength and thus a higher resolution (Wavelength is 0.004 nm--as opposed to 400-500nm for light) • Transmission electron microscope (TEM)- images electrons that pass THROUGH a thin specimen - Actual resolution = 0.1-2 nm (100-1000x better than light microscope) - If lenses were as good as optical ones, resolution would be 0.002 nm (100,000x better than light) but NA of magnetic lenses is much worse • Scanning Electron microscope (SEM)- images electrons scattered by an intact object. Depth of focus gives images a three-dimensional quality. - Resolution of SEM is about 5 nm

Proteases

...cleave or digest accessible protein regions and can be used to deduce the topology of a protein in the membrane. • Some, like trypsin, are digestive; they break the protein into many small, often non-functional, peptide fragments. • Others are highly specific and only cleave protein substrates at a certain specific sequence, thus generating larger, intact, potentially functional fragments.

Phospholipid Bilayer

...consists of two layers of phospholipids, with a hydrophobic, or water-hating (nonpolar), interior and a hydrophilic, or water-loving (polar), exterior.

Action Potentials (Definition. What do they do? What initiates them? What does this initiation do? What prevents APs from firing?)

...electrical signals; rapid changes in Vm (membrane potential) • Involves coordinated gating of voltage-gated Na+ channels and voltage-gated K+ channels • They are all-or-none • They propagate actively down nerve axons • Action potentials can travel down axons at speeds of up to 200 miles per hour • Neurons encode information by the frequency of action potentials • The action potential does not propagate backwards because the Na+ channels have not yet recovered from inactivation An action potential is initiated by a variety of stimuli... - another nerve (synapses) - a mechanical stimulus (hearing, touch) - a sensory stimulus (smell, taste) • There are also inhibitory signals that are feeding into neurons, hyperpolarizing (making super negative) Vm, working to prevent action potentials from firing. • Whether a neuron fires an action potential or not depends on whether the net sum of the stimulatory and inhibitory current inputs causes Vm to reach threshold. • ***After many thousands of rapid action potentials, enough Na+ has entered that it must be pumped out by increased activity of the Na/K ATPase.

Transport Proteins

...enzymes that catalyze movement of specific substances across the membrane

Epigenetic Phenomena

...heritable (or propagated), alternative states of gene expression, molecular function, or organization specified by the same genetic instructions (DNA sequence).

Chaperones and Chaperonins (What's the difference between the two. Name a famous example of Chaperones.)

...proteins that bind to proteins during OR after synthesis and help them fold properly. Both use ATP. • There are many possible ways to fold a protein, but only one is "right" for proper function. • *Some proteins can fold properly on their own, others do not. • Protein folding can occur co-translationally. Sometimes, an N-terminal hydrophobic domain should pair with one at the C-terminus. • Because protein synthesis occurs over time, the C-terminus may not even exist for a while, creating a situation where the N-terminal pairs incorrectly. Chaperonins = protein complexes that are called in when even more help is needed. Chaperones: Hsp70 are a family of conserved ubiquitously expressed heat shock proteins. Proteins with similar structure exist in virtually all living organisms. The Hsp70s are an important part of the cell's machinery for protein folding, and help to protect cells from (heat and oxidative) stress.

Contrast (what are two limiting factors? How can we counteract them?)

...the difference in intensity between an object and its background. • Cells are mostly transparent. They neither reflect nor absorb much light (unless colored, e.g. chloroplasts, or stained) so contrast is poor and little detail can be made out. • To bring out detail (enhance contrast), we need to exploit changes in the phase of light (i.e., modulate phase of light using optical tools) or chemically stain the object to make it darker.

Transfection (Definition. Constitutively active? Dominant negative?)

...to "infect" a cell with foreign DNA/RNA; to cause a foreign protein to be expressed in a cell. • In addition to GFP-tagged versions of molecules present in a cell, you can express (either tagged or not): - Molecules that aren't normally present in a cell - Mutant molecules that are constitutively active- i.e. active all the time - Mutant molecules that are dominant negative- i.e. don't function right and block the function of the cell's own version of the molecule. (*So many mutant molecules, overwhelm wild type --> cell follows mutant molecules although wild type is STILL BEING EXPRESSED, just in small amounts). • Transfection can be transient (expression from plasmid) or stable (DNA integrates into genome; heritable). • Transgenic lines of animals can be generated by stable transfection of germ cells. Protein expression can be general, in all tissues, or under the control of a tissue-specific promoter (brain, heart, etc.). Proteins can also be "knocked out". - "Knocked out" = a genetic technique in which one of an organism's genes is made inoperative ("knocked out" of the organism). Used in learning about a gene that has been sequenced, but which has an unknown or incompletely known function. Researchers draw inferences from the difference between the knockout organism and normal individuals.

Functional Domain (aka Protein Domain)

..A combination of helices and sheets with turns and connecting regions can fold into a functional domain that acts as a unit (i.e., can evolve/be folded, function, and exist independently) but is still only part of a protein. - Tertiary structure - Many proteins consist of several structural domains. One domain may appear in a variety of different proteins. Functions include: - ATP binding sites (myosin motor) - Ca++ binding sites - Enzyme activity of a particular sort - Regulation via interactions with another protein It is increasingly possible to analyze primary sequences and deduce the function of a protein by analyzing its domain structure.

Sympoter Example (Why does it work the way it does?)

2Na+/Glucose 1 Transporter • Concentrates glucose from intestine into epithelium cells • Works against the glucose gradient using Na+ gradient • Can work against a 30,000 fold gradient- i.e. accumulate glucose 30K-fold (because charge is more important than chemical concentration) **Note that for uncharged solutes like glucose, transport is only subject to the chemical concentration gradient. So UNLIKE IONS, uncharged molecules CANNOT move PASSIVELY AGAINST their chemical gradient.

Differential Centrifugation vs. Gradient Centrifugation

A great deal of what we know about cells comes from biochemical experiments conducted with organelles isolated and purified by gradient centrifugation. Differential Centrifugation: Lipids and other molecules with lighter densities may never filter out as they are too light, they will float at the top (they're bouyant). Gradient Centrifugation: going down tube, there's increasing density of sucrose. Molecules will filter out with sucrose match. Fine-tune centrifugation.

What is a key difference between prokaryotic and eukaryotic cells? How does multi-cellularity differ between the two?

A key difference between prokaryotic and eukaryotic cells is in the elaboration of internal membranes. Multi-cellularity doesn't differ between the two. They can both be multicellular.

Ligand

A molecule which produces a signal by binding to a site on a target protein. - The binding typically results in a change of conformation of the target protein. - Include substrates, inhibitors, activators, and **neurotransmitters

Membrane Domain

A particular region of the membrane. • Some membrane proteins are restricted in their location to this and cannot move freely within the membrane In a single cell, some proteins may be evenly distributed while others are restricted. • The same protein may be restricted in one type of membrane and not in another • Cells employ a variety of mechanisms to non-randomly distribute proteins: - Link them to other membrane proteins - Link them to outside molecules - Link them to inside molecules - Prevent their diffusion to parts of the membrane

Myxobacteria

A prokaryote that can form a multicellular organism • Atypically large genome for prokaryote • Can aggregate to form swarms and fruiting bodies via cell-cell signals

Promoter (What is the down side of using a viral one? How is this combated by other promoters?)

A region of DNA that initiates transcription of a particular gene. Viral = "promiscuous" -- wherever it finds that sequence in organism it will promote • CMV promoter (a strong, ubiquitously expressed, promoter) driving expression of a GFP-Tubulin fusion protein in all cells. - As a result, GFP is expressed everywhere there's Tubulin (entire organism will glow) Tissue-specific/muscle-specific/etc. = targeted • Ex: Only tails of tadpole

Dictyostelium

A single-cell amoeboid protist that can transform into a multicellular organism by active intercellular signaling. Forms migrating "slugs" and fruiting bodies.

Passive Diffusion (what molecules diffuse easily vs not as easily? What influences the ease with which they diffuse? What protein is used here? How does it function?)

AKA "facilitated diffusion ...when molecules diffuse directly through the lipid bilayer- no protein involvement • Some molecules diffuse through easily - Small, uncharged, non-polar - Gasses (O2, CO2) cross easily • Small, uncharged polar such as H2O and Urea cross (but slowly) • The rate is much slower as molecules get larger or more polar or charged. - Sugars, Amino Acids very slow - Ca2+, K+, Na+, Cl-; effectively impermeable - Proteins- not at all • Uniporter - Multi-pass transmembrane proteins that act more like an enzyme than a pore: - Substrate binding induces reversible conformational changes to bring about transport from one side of the membrane to the other; no energy other than random thermal fluctuation of carrier is necessary to drive the change - A passive carrier protein can work in either direction (functionally bidirectional), but the net direction of transport is down an electrochemical gradient. • 102-104 molecules/sec; slower than channels due to substrate-carrier interactions and significant conformational changes during translocation.

Secondary Active Transport

AKA co-transport (aka completed by symporter) Use energy of an electrochemical gradient generated by primary active transporters to couple movement of one molecule down its gradient to movement of another molecule up its gradient. (NOT USING ATP DIRECTLY) - Symporters (Cotransporters) move two molecules in same direction. - Antiporters (Exchangers) move two molecules in opposite directions https://biology.stackexchange.com/questions/31462/difference-between-facilitated-diffusion-and-secondary-active-transport-in-cells

What determines the way proteins bind to other proteins or DNA?

Ability to form bonds - Charges (-/+, +/- = ionic bond) - OH, NH (hydrogen bond) - CH3, CH3 (hydrophobic and van der Waals interactions)

How do we show equilibrium in a Kd equation? What does Kd stand for? What are its units?

Achieving Equilibrium • Put A and B together and let them come to equilibrium; on rate starts high and decreases as [A] and [B] fall, off rate starts low and increases as [AB] rises. • Equilibrium is reached when the concentrations of A, B and AB are not changing; A and B are associating to AB as fast as AB is dissociating to A and B. • Higher concentration = higher frequency of interactions = higher off rate • Given the same starting concentrations, complexes will stay bound longer, and thus there will be more of them at equilibrium, for a higher affinity interaction (smaller koff and off rate) vs. a lower affinity interaction. [A] x [B] x kon= [AB] x kof Kd = koff/kon = [A]x[B]/[AB] This ratio of the equilibrium concentrations of reactants to reaction products, which is equal to the ratio of the koff and kon rate constants, is the equilibrium dissociation constant, Kd. (M = units) **NOTE: • Higher affinity interaction = smaller koff = smaller Kd. - Higher concentration of AB complexes (products) at equilibrium = smaller Kd • Lower affinity interaction = higher koff = higher Kd. - Lower concentration of AB complexes (products) / Higher concentration of separate [A] [B] at equilibrium = higher Kd At fixed [B], If [A] is well below Kd, there will be relatively little AB. If [A] is well above Kd, there will be a lot of AB.

Selectivity Filter (what is it? What does it do?)

Acts as temporary hydration shell in vestibule. It makes ion willing to give up its shell. **Diagram: http://slideplayer.com/slide/8994554/27/images/61/The+Structure+of+bacterial+K++channel.jpg

Ramifications

An object 200 nm or smaller will appear to be 200 nm regardless of its actual size. You can still see the object, but it will appear to be 200 nm. If you see a 200 nm spot in a microscope, it could be: • 1, 200 nm or smaller object • Multiple smaller objects close together

Differential Interference Contrast (DIC)

An optical microscopy technique used to enhance the contrast in unstained, transparent samples. • Used to image, living or stained specimens, which contain little or no optical contrast when viewed using brightfield illumination. http://www.microscopemaster.com/differential-interference-contrast.html

What effect does an increase or decrease in extracellular K+ concentration have on resting membrane potential?

Because the concentration gradient of potassium ions (K+) across the plasma membrane has a major influence on the resting membrane potential of electrically excitable cells, K+ concentrations are tightly regulated. An increase in extracellular K+ concentration leads to depolarization, and a decrease in extracellular K+ concentration leads to hyperpolarization of the resting membrane potential.

Beta sheets and membranes?

Beta sheets can also interact with membranes • The R groups from the sheet are organized so that the non-polar are on one side and polar on the other • The sheet is rolled into a tube (Beta-barrel) • The hydrophobic are out toward the bilayer and the polar are inside • Forms a pore through the membrane that is a hydrophilic environment

Method #1 for measuring membrane mobility

Cell Fusion • Label proteins of one cell with red dye • Label those of a second cell with green dye • Fuse the membrane of the two cells to form a heterokaryon - **NOTE: cell membranes normally do not fuse. You have to force it to happen • Watch what happens to the two dyes • Result- over time they become mixed

Methylation? Acetylation?

DNA and its associated histone proteins can become chemically modified (i.e. by methylation or acetylation respectively), causing the underlying genes to be held in a transcriptionally active or inactive, semi-permanent state. - Methylation = gene switched "off" - Acetylation = gene switched "on" (Sometimes these modifications can be stably inherited from one cell to another).

Biological Membrane Composition (and what does this composition affect?)

Different cellular membranes are composed of different amounts of lipids and cholesterol, and have very different protein composition • The differences in composition relate to differences in function • Each biological membrane has different properties based upon the different molecules used to make it • Lipid composition affects bilayer thickness and membrane curvature. - Cholesterol increases length/thickness of bilayer when with unsaturated fatty acid chains

Epifluorescence Microscopy

Dramatically improves contrast and allows specific cellular structures to be labeled. • Fluorophores (fluors)--fluorescent chemical compounds that can re-emit light upon light excitation--can be linked to various chemicals or biological molecules so as to label specific structures. A variety of fluorophores allows multi-color labeling of multiple cellular structures. • Because specific molecules glow brightly against a dark background, the contrast of epifluorescence microscopes is excellent. • **Fluorescent proteins are genetically encoded fluorescence markers that can be fused to proteins of interest at the DNA sequence level. **Allows for LIVE imaging. **NOTE: Using tissue-specific promoters can drive tissue-specific expression

Domain Shuffling

Evolutionarily, new proteins can be formed by putting together new combinations of domains.

Membrane Potential (Vm)

Example situation - As K+ ions flow out, down their electrochemical gradient, Cl- ions that helped maintain charge balance are left behind. - These Cl- ions distribute themselves beneath the membrane, attracted to cations outside. ***The separation of charges exerts a force across the membrane. That force is an electrical potential, the membrane potential (Vm). - A very small number of ions need to move to create the membrane potential. Concentration = essentially unchanged.

What happens when the combined electrochemical gradient is at 0?

Example situation - As K+ ions flow out, down their electrochemical gradient, Cl- ions that helped maintain charge balance are left behind. - These Cl- ions distribute themselves beneath the membrane, attracted to cations outside. When the density of negative charges increases enough, it exerts a negative inside electrical force that is sufficient to counter the concentration gradient, so the K+ ions stop flowing out. K+ is now at equilibrium, as its combined electrochemical gradient is now 0.

Method #2 for measuring membrane mobility

FRAP of fluorescently labeled protein SEE "How can microscopy be used to determine the lateral mobility of lipids in the plane of the membrane?" FOR MORE INFO

How can microscopy be used to determine the lateral mobility of lipids in the plane of the membrane?

Fluorescence recovery after photobleaching (FRAP) • Label phospholipids with a fluorescent probe • Shine a bright laser on a small spot of membrane to bleach & destroy the fluorescence on those lipids • Measure how long it takes for other fluorescent lipids to diffuse into the bleached region until it is as bright as the rest of the membrane **NOTE: Bleaching PERMANENTLY breaks covalent bonds of individual phospholipids. If fluorescence is STILL THERE, other phospholipids must've diffused into that area.

Transgenic Organism

Fusion protein will be in ALL of organism's cells. • Expression depends on what "on gene" is targeted

Nernst Equation (What is it? What is it used for? What are its components and what do they mean?)

Gives the membrane potential that is sufficient to counter a given concentration gradient for an ion. - How we determine what is needed to reach equilibrium potential. Eion = (60/z) * log ([ion]out/[ion]in) Eion = ion equilibrium z = charge of the ion, 60 = combination of a bunch of constants

Resolution (and what does it depend on?)

How far apart two objects have to be to be seen as two separate objects. (AKA How much detail you can make out.) • Dependent upon the properties of light and how it interacts with the specimen. |> The wavelength of light (**shorter wavelength=better resolution -- V of ROYGBIV) - **Resolution of conventional light microscopes (assuming a perfect lens) is ~½ the wavelength of light being used |> Properties of the lenses used (specifically the numerical aperture, (N.A.), the width of the light-cone the objective gathers). • It is not directly related to magnification, although magnification has to be high enough to appreciate resolution.

Conductance

How much of a given substance flows throught a channel. / How many ions enter the cell in a period of time.

Antibodies (how do you make them? What are the two types of antibodies)

Immune proteins that bind to specific proteins. To make an antibody, you obtain pure protein of interest and inject it into an animal. The animal recognizes it as foreign and mounts an immune response. Individual B-cells will each produce a unique antibody that recognizes a specific ~ 8-12 amino acid sequence (the epitope). (1) Monoclonal antibodies are made by isolating and cloning a single antibody-producing cell, and thus recognize a single epitope. All the antibody molecules produced are identical. (2) Polyclonal antibodies are a mixture of different antibodies produced by the host animal's B-cells against various epitopes of the target protein, and isolated from blood serum.

How can you label structures in EM?

Immuno-electron microscopy • You can't see antibodies in the EM, but you can attach dense particles to antibodies to make them visible in the EM (gold beads). • Allows you to visualize the localization of specific proteins in the EM. • Very difficult technique! NOTE: Upside down gold toothbrush image

Name three methods that use antibodies. Describe. Which one uses fixatives. Why?

Important methods in cell biology that use antibodies include: |> Immunocytochemistry/Immunofluorescence • To immunolabel intracellular structures, you must fix and permeabilize the cell. - Fix: add chemicals (fixatives) that cross-link everything in the cell to nearby molecules --> Done to lock cell in place - Permeabilize: add a detergent to perforate the membrane so antibodies can enter --> The cell membrane won't let just anything through. Because antibody proteins are very large, the only way to get them through the membrane is to KILL the cell. (**As such, this technique is NOT for LIVE cells). • Add the antibody to the fixed, permeabilized cell and allow it to bind its target. • Use a fluorescent "secondary antibody" (an antibody against antibodies) to bind to the primary antibody if the primary is not directly labeled ("direct" vs. "indirect" Immunofluorescence). |> Immunoblotting (Western blotting) • Lysis via detergents to obtain protein sample • Add sample to polyacrylamide gel (separates based on size) • Transfer to polymer sheet • Lab-manufactured monoclonal primary antibody added. Antibody only binds to protein of interest. • Fluorescently marked lab-manufactured monoclonal secondary antibody added. Antibody only binds to matching primary antibody. |> Immunoprecipitation/Immunoisolation • Using antibodies to "pull down" proteins, complexes or organelles from cell extracts

Transmitted Light Microscopy

In a transmitted light microscope, white light passes through the specimen before being collected.

Intracellular

Inside the cell.

What blurs microscope images? How is this generally combatted? Name two methods that use anti-blur techniques.

Light above and below the focal plane you are interested in blurs images. Methods to deblur/get better, crisper images remove out of focus light. **NOTE: Resolution DOESN'T change, just what light is filtered out. (1) (Laser-scanning) Confocal microscopy uses pinholes to deblur, generating an "optical section" (2) Digital deconvolution uses computational methods to deblur

Membranes & Their Important Cellular Functions

Membranes are fluid lipid bilayers studded with proteins and often contain areas of differing composition called rafts that float around like icebergs on the ocean Functions • Separate one compartment from another -membranes are selectively permeable barriers across which solutes are transported • Provide a scaffold for biochemical activities -one key example is energy transduction in mitochondria and chloroplasts. • Mediate some kinds of cell-cell interactions. • Key element of many signal transduction pathways

Lipid Rafts

Microdomains (areas)with locally high concentrations of certain lipids and proteins that float around like icebergs on the ocean. • Form because lipids are not distributed randomly in the plane of the membrane. Some lipids, especially sphingolipids, like to cluster relative to other membrane lipids • Raft components include cholesterol, sphingolipids, and proteins. Rafts tend to accumulate different proteins than the non-raft areas (for ex., GPI-linked proteins).

Most transmembrane regions: hydrophobic or hydrophilic?

Most transmembrane regions are hydrophobic alpha helices. • You can predict that a protein has a domain that associates with membranes by looking at the amino acid sequence • A stretch of hydrophobic amino acids means the protein is likely to associate with membranes Dark green positive peaks = hydrophobic portions (counted as "passes") Light green positive peaks/negative lulls = hydrophillic portions

New "Superresolution" Fluorescence Techniques

New "superresolution" fluorescence techniques are breaking the resolution limits of light microscopy. (1) PALM (PhotoActivated Localization Microscopy) (2) STORM (Stochastic Optical Reconstruction Microscopy) • Use lasers to switch on a sparse subset of very bright, photoactivatable fluorphores that then switch back off. • Repeating this allows the center of the 200nm light spot of the individual fluorescent molecules to be calculated and "mapped" onto a digital image. *Think dotted line map image, X marks the spot

Would a cation continue infinitely across the cell's membrane as it attempts to balance an overall negative charge on the other side?

No. On the side from which these cations leave, the anion concentration would increase causing an overall negative charge. This would cause SOME K+ (now on the other side) to be attracted back to the side they left.

Light Microscopy (what is it limited in?)

One of the core tools of cell biology. Light microscopes magnify a specimen using one or more lenses. Light microscopes are limited in contrast, magnification and resolving power, limiting what can be detected with them.

Leak Channels (Definition. Conductance? What channel has the higher conductance? Why? Equation? What determines Vm? What if there are only conductances for only one ion? Steady state vs. equilibrium? What is run down? Why does it happen? How is this "run down" combated?)

Open channels that resting cells have that are always open. (Often find some K+, Na+, and Cl- channels). - Typically, resting cells have about 10 times the open K+ channels than Na+ and Cl-, and thus the K+ conductance (GK) is 10 times higher than the Na+ and Cl- conductances. The open K+ channels make the cell 10 times more permeable to K+ than to Na+ or Cl-. The current equation can be used to estimate Vm under these conditions: Vm = [(GK x EK) + (GCl x ECl) + (GNa x ENa)]/(GK+GCl+GNa) Plugging in the values for relative conductance (G) and the Nernst potentials for the various ions gives a value for Vm of ~-75 mV - Vm will tend to be close to the Eion for the ion that has the most conductances open in the membrane. **NOTE: If conductances for only one ion are present, Vm is equal to the equilibrium potential for that ion. - Vm reflects a steady state, not an equilibrium. There is ion flow (Na+ is flowing in and K+ is flowing out) but net current is zero (Na+ current and K+ current are equal, but opposite). The strong driving force and low conductance of Na+ is balanced by the weaker driving force but high conductance of K+. Over time, the Na+ and K+ gradients will run down (aka run out). This is because there's only a limited amount of Na+ and K+ on each side and, because neither ever return to their "homeland" (don't replenish that side), there's no more left to leave. - To prevent this from happening, cells use energy from ATP hydrolysis to pump Na+ out and pump K+ in. This allows the steady state membrane potential to persist. **NOTE: Not all Na+ is "leaking in" via channels to contribute to Vm. Some Na+ is used by secondary active transporters to other work. (aka not all Na+ enters the cell). (The resting Na+ conductance in part represents the activity of thousands of these transporters).

Name 3 other ATP-dependent pumps. State their functions.

Other ATP-dependent pumps and their functions • K+/H+ ATPases - Stomach acidification - P-type • Ca++ ATPases - Pump Ca++ out of the cell (PMCA ATPase) or into the ER (SERCA ATPase). - P-type

Passive vs. Active Transport (name types)

Passive - Simple diffusion - Channel-mediated - Carrier mediated (uniporter) Active - Carrier mediated (sympoter and antiporters) |> Relatively large conformational changes, unlike channels which undergo minor shape changes.

TRANSPORT OVERVIEW

Passive Transport - (Simple) Diffusion - Facilitated Diffusion |> Uniporter • Channels --> Ions/Voltage --> Ligand • Carrier proteins Active Transport - Primary active |> ATP-dependent • P-type --> Na+/K+ ATPase • V-type (Vesicular H+ ATPases) • ABC Transporters |> Light-dependent - Secondary active |> Symporter • Na+2/Glucose 1 Transporter |> Antiporter

What are the three classes of membrane lipids?

Phospholipids, Glycolipids and Sterols • These are all amphipathic molecules. Hydrophilic end and hydrophobic end. (1) Phospholipids • All have a phosphate linkage to a "head" group, and 2 fatty acid chain "tails". • Phosphoglycerides - Major component of most membranes - Consist of two fatty acids linked to glycerol, with differing chemical groups added to glycerol phosphate in the head group. - One fatty acid chain is saturated, one unsaturated* - Many different types with different structures • Sphingomyelin - Sphingosine amino group, instead of glycerol, links to phosphate in head - Two saturated fatty acid chains* (2) Glycolipids • Sphingosine amino group links directly to a sugar head group (no phosphate) • Have two saturated fatty acid chains* (3) Sterols are amphipathic, four-ring hydrocarbons. Cholesterol can increase or decrease membrane fluidity depending on conditions. *Saturated (linear structure, aka single bonds = more atoms) fatty acid chains give rise to thicker and less fluid bilayers.

What can cause conformational changes that affect interactions with other proteins?

Protein-Protein, Protein-Ligand, Protein-DNA interactions, chemical modifications, or mutations to the primary AA sequence can cause conformational changes that affect interactions with other proteins.

Proteins (and their 3 basic roles)

Proteins are the basic machinery of cells. They serve three basic roles: • Enzymatic • Structural • Regulatory

Stochastically

Randomally determined

Phosphate

Removes a phosphate group from the phosphorylated amino acid residue of its substrate protein.

Method #3 for measuring membrane mobility

Single particle tracking • Particle maps drawn "without taking pen off paper"

Name a famous P-type pump. What does it do? Why is it considered a P-type? (hint: What drives this mechanism?) Describes what happens (in order). What is something that can block this pump? What happens when you block this pump? Why?

The Na+/K+ ATPase In resting cell, K+ moves outward while Na+ moves in. The Na+/K+ ATPase moves K+ INWARD and Na+ OUTWARD using energy from ATP - Both are moving AGAINST their electrochemical gradients (b/c more K+ inside than out and more Na+ outside than in) • Key role in maintaining the distribution of these ions in cells • Mechanism involves a conformational change in shape of protein driven by ATP and binding of ions - ATP cleavage is used to actually phosphorylate the transporter - P-type • Exchanges 3 Na+ for each 2 K + ORDER: Na+ enters transporter (from inside), ATP hydrolyzed = ADP and P attaches to transporter, Na+ is released onto other side, K+ enters transporter (from outside), P disappears, more K+ is added, K+ is released onto other side. OUABAIN = a plant compound that blocks activity of Na+/K+ ATPase. Blocking Na+/K+ ATPase will slowly lead to a depolarization (when membrane potential becomes less negative) and swelling of the cell, as gradients run down (aka run out) and Na+ accumulates in the cell (because the 2Na+/1 Glucose symporter is still at work so Na+ is still being pumped into the cell). This increase in Na+ in the cell causes water from outside to rush in, in an attempt to balance the osmotic concentration (osmolarity).

Magnification (and what does it depend on?)

The amount you blow up the initial image. It's dependent on the lenses you use.

Electrochemical Gradient

The combination of the chemical concentration gradient and the electrical gradient determines the rate and direction of transport of a charged molecule. - Provides the "driving force" for ions to move. This electrochemical potential (units are millivolts mV) can be calculated: Vm-Eion Vim = membrane potential Eion = equilibrium potential

Differential Gene Expression

The concept that all cells in the body have the same genome, but each cell type expresses different parts of it ("differential expression").

Patch Pipette

The electrical isolation of a small patch of membrane from the rest of the cell. To achieve this isolation, the patch pipette is placed against the cell membrane, and a slight suction (1) Charge? Voltage? --> Open channel (or not)? (2) Single channel? - Based on unit of voltage being picked up on

Proteome

The entire complement of proteins that is or can be expressed by a cell, tissue, or organism.

Transcriptome

The genes that are being transcribed.

The Fluid Mosaic Model of Membranes

The lipid bilayer is a flexible 2-dimensional fluid sheet. • Membrane proteins "float" in this sheet • Proteins can move laterally in the plane of the membrane, but... - A protein cannot easily leave the membrane once inserted. --> Too much energy is required to tear the hydrophobic region out of the hydrophobic bilayer. --> The topology of a protein cannot easily change once inserted in the membrane. |> If it is made with 7 transmembrane regions, it will stay that way. --> The conformation can change |> Shape changes allow proteins to pass signals from outside to inside

Membrane Depolarization

The loss of the difference in charge between the inside and outside of the cell membrane to due to a change in permeability and migration of Na+ (sodium ions) into the cell. AKA when the membrane potential becomes less negative (more positive). - Opens voltage-gated K+ channels.

Electrochemical Potential

The mechanical work done in bringing 1 mole of an ion from a standard state to a specified concentration and electrical potential. - Can be calculated: Vm-Eion. (units are millivolts mV) EXAMPLE: In our initial conditions before opening the K+ channel the membrane potential was 0mV. 0 - (-88)= +88mV So initially there was a large driving force for K+ to leave the cell. As K+ leaves, the membrane potential changes, so the driving force changes. When the membrane potential reaches -88mV then: -88 - (-88) = 0mV Under standard ionic conditions, and a normal resting membrane potential of -75 mV, the driving forces (-75 mV - Eion ) for various ions are: Ion Driving force Direction of flow K+: +13 mV out Na+: -135 mV in Cl- : 0 mV none Ca2+: -195 mV in (if Ca2+ channels open) In real cells then, Vm reflects a steady state, not an equilibrium. There is ion flow (Na+ is flowing in and K+ is flowing out) but net current is zero (Na+ current and K+ current are equal, but opposite). The strong driving force and low conductance of Na+ is balanced by the weaker driving force but high conductance of K+. Importantly, over time the Na+ and K+ gradients will run down (aka run out). To prevent this from happening, cells use energy from ATP hydrolysis to pump Na+ out and pump K+ in. This allows the steady state membrane potential to persist.

Equilibrium Potential (Definition. Describe what occurs. What is the combined electrochemical gradient?)

The membrane potential at which an ion is at equilibrium. - Represents a true equilibrium - no energy is required to maintain the status quo. There is no net ion flow (although equal amounts of the ion can stochastically pass in and out) and no currents are flowing. - Even though there is still a concentration gradient, and still an electrical gradient, the combined electrochemical gradient is 0. - The Nernst equation **NOTE: The membrane is much more permeable to K+ than to Na+, so the resting potential is close to the equilibrium potential of K+ (the potential that would be generated by K+ if it were the only ion in the system). -- SEE "Leak Channels" INDEX CARD

Epitope

The specific target that an individual antibody binds to.

Epigenetic

The study of heritable changes in gene expression (active versus inactive genes) that does not involve changes to the underlying DNA sequence — a change in phenotype WITHOUT a change in genotype — which in turn affects how cells read the genes.

Leaflets

Two layers--an outer leaflet and an inner leaflet--make up the lipid bilayer. • The outer leaflet and inner leaflet of the membrane are asymmetrical in their composition. - The lipids are synthesized in the ER and inserted into one or the other faces of the bilayer - Membrane proteins (flipases) flip-flop the lipids back to their normal sides to maintain the asymmetry! • Certain proteins and lipids rest only on one surface of the membrane and not the other. - Formed by biological phospholipids

What are the reasonable values for the Nernst equilibrium potentials for K, Cl (Na, Ca)?

Under standard ICF/ECF conditions, reasonable values for the Nernst equilibrium potentials for various ions are: EK = -88 mV ECl = -75 mV ENa= +60 mV ECa= +129 mV

What are the three stages in which voltage-activated (Na+) channels can exist?

Voltage-gated Na+ channels can exist in a (1) closed, (2) open or (3) inactivated state

Fluorescence

When a molecule absorbs light of one wavelength and then re-emits it at a longer wavelength (longer wavelength = R of ROYGBIV).

Is it possible for molecules to cross down the concentration gradient if there is some negative charge on the other side? Why/why not?

Yes, it is. This would occur if the concentration gradient was strong enough (i.e. there as such a large difference in concentration between the two sides) that partially overcame the influence of the charge on transport.

Why are there two types of structures lipids in water can form? What are they?

You need to get the hydrophobic tails of phospholipids away from water. There are two common ways of accomplishing this... - Micelles • Small sphere with tails pointed in - Bilayers • Two layers of lipids with tails pointed toward each other • Which structure is formed is dependent on the type (chemistry- charge on head, tail length, shape, number) and concentration of the lipid

Unit prefixes

giga G 109 1000000000 mega M 106 1000000 kilo k 103 1000 hecto h 102 100 deca da 101 10 ------ deci d 10−1 0.1 centi c 10−2 0.01 milli m 10−3 0.001 micro µ 10−6 0.000001 nano n 10−9 0.000000001 pico p 10−12 0.000000000001

Properties of Lipid Bilayers (what's energetically unfavorable?)

• Bilayers close upon themselves to make a continuous surface interacting with water. • No "edges" are left exposed; water interacting with hydrophobic tails = unfavorable. • Membranes try to reseal if broken or punctured - A cell will die if the seal does not reform fast enough

microRNA (miRNA)

• Doesn't code for proteins, regulates stability of mRNA. • Found in plants, animals and some viruses, that functions in RNA silencing and post-transcriptional regulation of gene expression

How does signal get to rest of the body?

• Electrical signal is transmitted down an axon and now must jump to the next neuron • When it reaches the nerve terminal it causes the opening of voltage-gated Ca++ channels. Ca++ enters the cell. |> Ca++ acts as a chemical messenger that causes fusion of synaptic vesicles with the presynaptic membrane. • A chemical neurotransmitter is thus released into the space between the neurons. • This neurotransmitter diffuses to the next neuron and binds to and opens a ligand-gated Na+ channel. • This begins the action potential in the next neuron which then propagates the length of the neuron in one direction. • In the body, the signal is transmitted in one direction from one end of the cell to the other.

How is the action potential generated? What is the typical membrane potential at threshold? What happens to membrane potential as the action potential is generation? Why?

• Initially, voltage gated Na channels are closed, with open inactivation gates • (Electrical) stimulation starts to depolarize the membrane • The depolarization opens voltage-gated Na+ channels, causing further depolarization as Na+ ions flow in • At threshold (typically ~-50 mV), opening of voltage-gated Na+ channels triggers a runaway positive feedback cycle, triggering further depolarization and channel opening. **NOTE: Membrane potential rockets up towards ENa (the Nernst equilibrium potential for Na), because GNa (the conductance for Na) now dominates the current equation: Vm = ((GK x EK) + (GCl x ECl) + (GNa x ENa)) / (GK+GCl+GNa)

Movements of Phospholipids in Membrane

• Lateral shift/diffusion = uses least amount of energy (more thermodynamically favorable) • Flexion (bending) • Rotation • Transverse diffusion ("flip-flop") = RARELY occurs, uses the MOST amount of energy (least thermodynamically favorable)

What are the three primary components of biological membranes?

• Lipids - These form the membrane bilayer itself • Cholesterol (a special type of lipid) - Affects bilayer properties - Found only in eukaryotes • Proteins - These are associated with the bilayer or inserted into it to add function

How is the action potential terminated? **What would happen if there were no voltage gated K+ channels? Why?

• Na+ channel inactivation gates after a time delay following depolarization. In this state, no Na+ current flows even if the activation gates stay open. (This is because Na+ channel inactivation gates close slowly, halting Na+ entry for a short time period, while K+ channels get ready to open. This keeps the potential, ENa, from rising any further.) • With Na+ channels closed, Vm would tend to return to rest. However, voltage gated K+ channels open, speeding the repolarization and causing a slight undershoot of Vm. • Repolarization causes Na+ channel inactivation gate to reopen, and causes the voltage gated K+ channels to close. • The system is now reset, and able to fire another action potential. **If there were no voltage gated K+ channels, the cell would be un-able to repolarize (become more negative). -- https://answers.yahoo.com/question/index?qid=20080708020342AAHFvVl

Passive Diffusion: What is the rate of movement related to? What is the solution to this movement limitation?

• Rate of movement is directly related to solubility of molecule in lipid bilayer (partition coefficient) - The more soluble a molecule is in the lipid bilayer, the faster it will diffuse across • The solution to the limited permeability of the membrane for some substances is for cells is to have transport proteins in their membranes.

What is the structure of the transmembrane region of proteins?

• Typically hydrophobic or amphipathic alpha helix, or amphipathic beta sheet • If alpha helix-based, can span one or more times - Single Pass Transmembrane: crosses the bilayer once; 1 helix. - Multi-pass Transmembrane: crosses 2 or more times; 2+ helices. (Example of a multi-pass transmembrane protein: bacteriorhodopsin) • The length of an alpha helix needed to cross the membrane is about 20-30 amino acids (~4 nm). Remember though that different lipid composition gives rise to different bilayer thickness, so the length of a membrane protein's hydrophobic alpha helix will influence what kind of lipid composition that protein "wants" to be in. • Most amino acids in single pass alpha helical domain of a transmembrane protein are non-polar (hydrophobic) to associate with the inner hydrophobic region of the membrane.

How do you show a protein is associated with the membrane?

• Use immunofluorescence or immuno-EM to immunolocalize the protein to the membrane • Purify the membrane and determine which proteins are present - You can break cells by homogenization - The membranes have a different density than other molecules allowing them to be separated via sucrose gradient centrifugation - Different membranes of the cell have different compositions and so can be separated from each other - Special detergents can be used to dissolve the membrane but keep the membrane protein active for immunoblotting or biochemical assay • Purify the protein and show that association with membrane lipids is required for its function

Scanning Electron Microscope (SEM) (How is it different from TEM?)

• Used to look at surfaces of structures • Samples are fixed, passed through series of alcohols, and dried. Surface of sample is coated with a layer of metal (platinum). • The electron beam is scanned across the surface and the reflection of electrons at each point measured. It is different from TEM because SEM's images are 3D and you only visualize the outside.