BIOL 3000 Exam 3 Big Questions

What are the major features of the 3 cytoskeletal systems and what cellular processes do they mediate? (Note that mitosis involves all 3!)

Microfilaments: movement of cells, made up of actin, 3 regulatory proteins: profilin, cofilin, and thymosin-B4

Microtubules: movement of cargo and vesicles, structure of cell, and necessary for mitosis, especially anaphase

Intermediate filaments:

What are the primary structural and biochemical properties of G-Actin and F-Actin? Why do the (+) and (-) ends have different critical concentrations (Cc)? How do these differences govern microfilament polymerization and depolymerization?

G-actin is the monomeric version of actin, and there is a significantly high concentration of it in the cell. F-actin is filamentous actin; It is actin that is polymerized in the form of a microfilament. Critical concentration is the minimum concentration of G-actin required for a microfilament to begin polymerization. Nuclei are required for this process to start. The initial forming of F-actin from G-actin does not require ATP hydrolysis, but as polymerization occurs, ATP is hydrolyzed to ADP (middle region of MF has actin-ADP +P). The minus end has the actin-ADP with an exposed ATP cleft, while the plus end has actin-ATP. G-actin (free and bound to ATP) has a higher affinity to bind to actin-ATP, so the Cc for the (+) end is much lower than the Cc for the (-) end (high affinity) This also means that the rate of polymerization on the (+) is faster than the assembly rate on the (-) end, given that the disassembly rate on both ends is essentially equivalent.

How do cells regulate preferential assembly or disassembly at either the (+) or (-) ends of microfilaments independently of [G-Actin]?

Cap proteins

- CapZ: binds to actin-ATP on (+) end when [G-actin] > Cc+. this prevents assembly at this end.

- Tropomodulin: binds to actin-ADP on (-) end when [G-actin is < Cc-]. This prevents disassembly at this end.

How can toxins that target the polymerization cycle of actin and are therefore toxic to cells, be used to study actin dynamics?

Toxins can target polymerization by either inhibiting it or preventing depolymerization at both ends. The phalloidin toxin targets F-actin and prevents Rac, Cdc, and Rho from hydrolyzing GTP, which prevents depolymerization on both ends and thus continuous growth of actin. This over-stretches the cell and kills it.

How do extracellular signals activate small, monomeric G proteins to mediate and coordinate microfilament dynamics at the leading edge of a migrating cell?

these signals bind to receptors on the PM that activate monomeric G proteins that are lipid-anchored in the PM adjacent to the receptors. Binding domains on the G proteins interact with adaptor proteins in the cytosol, stimulating a pattern of growth that polymerizes at the (+) end of actin filament that pushes against the G-protiens that push against the PM (leading edge). The minus end is attached to

Monomeric G-proteins:

1. Rho: responsible for linear microfilament movement

- adaptor protein called formin links Rho with MF and pushes against PM

- formin is like a collar that surrounds the (+) end of existing MF (currently not growing)

- formin has high affinity binding site for profilin actin

-interacts with Rho through a RBD (Rho will be bound to GTP in active state at this point)

- GDP was exchanged for GTP on Rho when the signal bound to PM receptors

- as actin is added, formin slides up to the next one, this is what pushes against the PM (more Rho = increased level of pushing)

2. Rac & Cdc 42: works the same for our purposes, responsible fore branched microfilament movement.

- like formin, Wasp binds to its G-protein (Cdc42) with use of a RBD

- Arp2/3 attach to current MF and act as new nuclei; new MF shoots off at an angle

How do dominant negative and dominant active alleles of Rac, Cdc42 & Rho affect cell migration and morphology?

Dominant negative Rho: largest affect on cell migration; nearly no wound closure. Linear movement is the most essential for all movement, branched movement must start with linear

Dominant negative Cdc42 & Rac: some wound closure, but branching still affected.

Dominant active Rho: With non-hydrolyzable GTP, continuous growth occurs and stretches cell PM until bursts.

Dominant active Cdc42 & Rac: continuous growth occurs on the entire circumference of cell PM and breaks PM.

What are the primary structural and biochemical properties of Tubulin and MTs?

MTs are composed of an inseparable alpha and beta subunit. Alpha subunits are unable to hydrolyze ATP because they are permanently bound to B subunits, which block the binding domain for GTP. Can't exchange either. A subunits will protrude at the (-) end. B subunits have a free binding domain for GTP, where it can exchange GDP for GTP and hydrolyze GTP. Free tubulin has GDP and exchanges it for GTP, so it can bind to the previous tubulin on the growing MT.

What governs microtubule polymerization and depolymerization?

Since the (-) end is typically anchored into the MTOC or centriole, polymerization and depolymerization primarily applies to the (+) end. The plus end will have a B subunit bound to GTP (GTP cap), which binds the next aB subunit to the MT at the a position. GTP hydrolysis at the plus end is what governs poly and depoly.

How does GTP hydrolysis control the dynamic instability of MTs?

Question: in normal MT, how does the cell time it so that the hydrolysis of GTP at the plus end is quickly replaced by a new aB dimer of tubulin so that the protofilaments don't disassemble? GTP hydrolysis determines how stable or unstable the (+) end is: the faster the rate of GTP hydrolysis, the more unstable MT becomes and cause it to undergo catastrophe. Slower rates contribute to increased growth because of prolonged GTP caps, that maintain the more straight structure of tubulin. When tubulin is bound to ADP, the structure adopts a much more curved formation, and these begin to peel away from the linear structure of the MT, causing rapid depolymerization.

What are the functions of the Microtubule Associated Proteins (MAPs) EB1, MAP2 and Tau?

MAP2: spacer protein that increases distance between microtubules in a given cell, found the the cell body portion of neuronal cells and entirely in non-neuronal cells. Spacing is necessary for bidirectional transport to fit

Tau: spacer protein specific to neuronal cells, predominantly within the axon due to a lack of space, Tau has a shorter extending arm than MAP2, thus spaces out the MTs less

EB1: protein that binds just below the B-tubulin GTP region on MT (binds to B-tubulin-GDP) and assists in depolymerization during catastrophe.g

How does Tau contribute to the progression of neurodegenerative diseases such as Alzheimer's?

When Tau aggregates or misfolds, they accumulate to excessive levels and become toxic. Cell accumulates "tangles" of Tau that can't be removed and interfere with cell function in neurons. Higher age leads to an increased level of ApoE4 which enhances effect of Tau.

What is the link between Tau and cholesterol uptake in neurons by ApoE4?

ApoE4 is a neuronal-specific form of LDL particle that binds to neuronal specific receptors. Since neurons can't synthesize their own cholesterol, they have a high demand (formation of myelin sheaths). When there are increased levels of ApoE4, it accelerates the formation of aggregates of Tau mutants (P301S). Intake of ApoE4 accelerates accumulation of Tau.

How does MT polarity necessitate the use of directional motor proteins?

By using the (+) and (-) ends of a MT, there is an established directionality of motor proteins to go to one end or the other. Kinesins are + end motors and dyneins are - end motors (they move in opposite directions). Vesicles will know to bind to specific motor proteins to take them towards the + end or - end depending on where their Rab-Gap tells them they need to go. In neuronal cells, only the cell body has MTs that originate in centrosome, but in the axon the MTs are fragmented (+ end towards end of axon). Kinesins and Dyneins still have the same polarity here.

What are the primary structural and biochemical features of kinesins and dyneins?

Kinesins: tall/stalky, ATP dependent, has a tail that binds to vesicle/cargo (space it out from MT with their length), 2 heads

Dyneins: short, ATP dependent, made up of multiple other proteins

How do kinesins walk?

Kinesins can only bind to B-tubulin on microfilaments. They walk along the same protofilament all the way down the MT. Each head by default bound to ADP (won't move). Once one head exchanges for ATP, it can bind to MT. This becomes the leading head, the trailing head is bound to ADP and loosely associated with the MT. When binding to ATP, the leading head changes conformation and swivels the kinesin stalk so that the trailing head becomes the leading head. The new trailing head hydrolyzes ATP and detaches from the MT. Now the same thing can happen again with the new leading head to swivel/walk.

Why does vesicle transport to the plasma membrane require both MTs and MFs?

MTs do not reach the PM sufficiently for the Rab on the vesicle/cargo to bind to its GAP on the PM. The MFs are close enough/articulate with the PM, so the MTs will hand off their cargo to the MFs to take it to the PM. Kinesin on MT hands of cargo to myosin on the MF. Myosin is the motor that allows the cargo to travel up MF to PM to engage with its GAP.

What is the fundamental problem of cell signaling?

How can a cell take and transduce an extracellular signal and elicit an intracellular response to have a physiological effect for the cell?

What are the main components involved in GPCR signaling?

GPCR is comprised of 7 membrane spanning alpha-helices with cytosolic face and extracellular face

- cytosolic face couples the receptor to the G-protein

- EC face has the binding site for the stimuli

Stimuli: a hormone will be released from an organ to elicit a cellular response/change

Receptor: a PM receptor binds to hormone such as epinephrine, which activates the receptor. It can now bind to resting G-protein complex and act as a GEF.

G-protein heterotrimer complex: Has GDP bound at rest (alpha component has GDP bound), but when binding to active receptor, is pried open to release GDP and bind a higher affinity, higher conc. of GTP. GTP causes a conformational change in the a subunit that causes it to dissociate from B and y. A subunit will diffuse laterally in PM (lipid anchored) and turns on IMP enzyme.

target enzyme: will be activated once bound by alpha G-protein subunit and produces 2nd messengers into the cytosol to activate protein kinases --> intracellular response.

How did phosphorylation evolve as a mechanism to regulate enzymatic activities?

More primitive, simple organisms had enzymes that required electrostatic interaction, so they had an

E or D on their phosphorylation site. They would be constitutivley active in their non-phosphorylated, basal form. Phosphorylation of that site would deactivate the protein and glucose would cease being converted to glycogen. Evolution and complication replaced E with S/T and the enzyme lost its ES interaction and the basal state became inactive. Phosphorylation restores this ES interaction by replacing a conserved charged AA with a phosphoric acid (works sufficiently). Essentially phosphorylation reverts the enzyme back to its primitive/ancestral form.

What provides the specificity for S/T kinases vs Y kinases?

Binding sites/clefts. For Y kinases, there is a deep cleft to properly fit the Y AA in order to phosphorylate (Y is a larger amino acid). S/T would not reach deep enough in the cleft of the Y kinase to be phosphorylated. S/T kinases have a shallow cleft to fit S/T, and Y is too large to fit into this cleft for S/T kinases.

How does b-adrenergic receptor signaling alter glucose metabolism and how do various steps in this pathway "amplify" the initial signal?

Binding of signal epinephrine at the BAR ends up causing a 10^4 amplification of signal in the cell. BAR activates the G-proteins to activate target enzyme adenylyl cyclase. Adneylyl cyclase is responsible for producing cAMP from ATP. 1 epinephrine molecule elicits a response for the G-protein to activate 100 AC on the PM. That's the first amplification of signal. These 100 AC produce 100 cAMP each, further amplifying the original signal.

How can FRET be used to experimentally assay real-time changes in protein-protein interactions?

By pairing fluorescent proteins, one can follow protein-protein interactions in real time and see when they associate or disassociate (We use CFP as donor and YFP as acceptor for our purposes). When proteins are not interacting and the cell is irradiated, CFP is seen but YFP is not. When proteins are interacting, the emission wavelength of the donor overlaps with the excitation of the acceptor and we see YFP. Interaction is called FRETON. Using CFP and YFP one can see how quickly proteins come together or dissociate.

How can phosphorylation sites be altered to either prevent phosphorylation (S/T to A; Y to F) or mimic it (S/T/Y TO E)?

By changing an S/T site to an A, the absence of a hydroxyl group prevents any interaction with the S/T protein kinase and thus the enzyme cannot be phosphorylated. A is a similar size to S/T, so the protein function is not disturbed. F works for Y sites because it is also a large, aromatic molecule, but functions like A in this case by lacking a hydroxyl group.

How do second messengers function both independently and synergistically to drive different physiological outputs in the b-adrenergic receptor pathway?

Independently, 2nd messengers can elicit certain physiological responses at 50% activity, assuming they are the only ones in the process. When synergistically combined with other second messengers or kinases that phosphorylate the same enzyme, they can elicit a 100% physiological response. In the BAR pathway, GPK can be 50% activated by both PKA or calcium-calmodulin, and activation by either will induce the same response from GPK. When both in the mix, GPK can be at 100% activity and thus increased phosphorylation and activation of GP to break down glycogen.

Why is receptor desensitization an important aspect of cell signaling pathways?

When a receptor is bound to a hormone for an extended period of time, PKA is also hyper-activated. The cell builds in this mechanism for PKA to phosphorylate the receptor to recruit B-arrestin and its associated proteins to prevent engagement of the receptor with Gas subunit of GCPR. This, in turn, shuts off the production of cAMP which will reverse the whole process and PKA will become inactive with lowering levels of cAMP. This can be rapidly reversed. In extreme cases though, B-arrestin will recruit AP2 and Clathrin to be endocytosed (along with the receptor) and degraded. This process shuts off cAMP production, but takes a much longer time for the cell to recover when in need of increased glucose production since new receptors need to be synthesized.

How does Acetylcholine receptor signaling in vascular endothelial cells promote vascular smooth muscle relaxation (aka vasodilation)?

Ach binding to Ach receptors triggers a Gq to activate PC, PC produces DAG and IP3 (DAG not relevant in vascular endothelial cells). IP3 activates Ca2+ and Ca2+ binds to calmodulin to activate it. Calcium-calmodulin activates NOS to generate new 2nd messenger (Nitric Oxide) as well as citruline. Then

Why do NO and Viagra make a bad combination?