Cell Exam 3 (F19)

What happens once you reach the end of a MT?

- kinesin motors on MTs hand off their cargo to myosin motors on MFs - myosin takes the vesicle, grabs on, & brings it to the plasma membrane

structural features of kinesins & dyneins

- kinesin: tail, light chains, stalk w/ linker, 2 heads that bind ATP/ ADP - dynein: dynactin-binding region, intermediate & light chains, linker region, MT-binding domain; shorter & more squat

dynamics of actin polymerization & depolymerization

- "nuclei" limit polymerization: there's a RLS for polymerization of actin- we see this in vitro w/ only purified G-actin - the graph of time vs. mass of filaments shows a lag before any filaments are formed during which a couple of G-actins are crystallizing - if we prime the rxn w/ nuclei it gets rid of this lag & we end up w/ a rectangular hyperbola - once we reach steady state, there's no net growth of MF's b/c once free actin is depleted, its rate of removal= rate of addition

EB1

- (+) end TIP binding protein that binds specifically to the structural end of MTs undergoing catastrophe & creates a high affinity binding site for tubulin --> initiates the rescue process - not a motor protein; hops, doesn't walk from one tubulin to the next, always moving in the (+) direction - hitchhiking proteins that aren't involved in vesicle transport can grab on to EB1 & use it as a free ride; *not all cargo needs to be carried in vesicles* - falls off at the start of another catastrophe event

tubulin

- globular protein made of alpha & beta heterodimers; you'll NEVER find alpha and beta tubulin separate in a cell (highest affinity protein-protein interaction in all cells) - different isoforms of alpha & beta confer different properties & stability depending on the cell type - most highly PTM protein in cells - GTP-binding proteins: alpha ALWAYS has nonexchangeable GTP bound (tucked away at its interface w/ beta); the GTP in beta is exchangeable and acts as the site of GTP hydrolysis in growing MT's

microtubules (MTs)

- hollow rod composed of tubulin proteins that makes up part of the cytoskeleton in all eukaryotic cells -larger & more rigid than MF's - found in cilia and flagella - support/ highway system that vesicles are trafficked on; directional motors are used to move things - important in separating chromosomes in mitosis

actin treadmilling

- if 0.12 micromolar<[G-actin]<0.6 micromolar, there's no net growth, only treadmilling - only the (+) end grows & the (-) end shrinks at an equivalent rate

kinesin-1 (conventional) vs. kinesin-5 (bipolar)

- in general, most transport cargoes (vesicles, organelles) on stationary MTs, but in the mitotic spindle, some can transport another MT (sliding requires bipolar kinesins: the feet on either side walk in opposite directions on two different MT's --> the cell divides as the poles are pushed further apart)

HW 10/14 #1 Which of the following statements correctly describes the role of ATP hydrolysis in MF dynamics? A. ATP hydrolysis is not required for the conversion of F-Actin to G-Actin, but influences the polymerization kinetics of converting F-Actin to G-Actin. B. ATP hydrolysis is not required for the conversion of G-Actin to F-Actin, but influences the polymerization kinetics of converting G-Actin to F-Actin. C. ATP hydrolysis does not influence MF dynamics D. ATP hydrolysis is required for the conversion of F-Actin to G-Actin E. ATP hydrolysis is required for the conversion of G-Actin to F-Actin

B.

WASp

- stands for Wiskott-Aldrich syndrome, a rare X-linked disorder characterized by immune dysregulation, recurrent infections, & small platelets - like Rho, WASp has a RBD, but Cdc42 binds to it, the WASp structure unfolds, exposing a nucleation site from which the MF can grow - as the protein elongated, the Arp 2/3 complex is attached at any point, acting as a nuclei that begins growth of a new MF off of the original MF, at an angle - WAVE, which binds to Rac is a member of the WASp family

How does a cell "transduce" the binding of an extracellular ligand to elicit both short-term and long-term intracellular physiological responses?

- short-term: immediate effects due to altering activities of previous proteins, no new proteins are made; REVERSIBLE; ex. changes in glucose metabolism by GPCR signaling, changes in phosphorylation, inc. in intracellular Ca2+ - long-term: effects due to altering gene expression & protein synthesis; ex. changes in cellular proliferation by RTK signaling (re-entry into cell cycle)

wound closure experimentation

- take confluent cells (normal cells that can stop growth when triggered by contact inhibition), scratch the surface, over time the cells will migrate back tgt b/c they release growth factors that help them close the wound - dominant negative G-protein mutants can't exchange GDP for GTP, thus preventing cell-migration: Rac & Cdc42 have ~ the same dec. in % wound closure, but if you only knock out one or the other they are somewhat redundant vs. dom. neg. Rho is reduced even further below these two

GPCR morphology

- the human genome encodes ~800 GPCR's: all of them have 7 membrane-spanning alpha-helices - at least 100-200 of these are orphan receptors that we don't know their effect - two "business ends": E1, E2, & E3 are the extracellular ligand-binding portion; C3 & C4 are in the cytosol , they interact w/ the G-protein (remember that the G protein & GPCR aren't the same thing, they are just coupled) - *we can make chimeric receptors by fusing the ligand-binding module from 1 receptor (ex. BAR) w/ the activation module from a different receptor (ex. AchR)*

the role of G-proteins in dynamically altering MF organization & cellular architecture during cell migration

1. ligand binds a GPRC that acts as a GEF for a monomeric G protein (Rho, Rac, or Cdc42) 2. the G-protein acts as an intermediary btwn the plasma membrane & MF: the activated GTP bound G-protein targets effector proteins that have the net effect of polymerizing MFs 3. MF pol. only occurs at the portion of the plasma membrane that's exposed to the signal; this results in growth at the "leading edge" of the cell while the other edge, the "trailing edge" depolymerizes & retracts its MFs so that the cell doesn't rip apart

What are two problems that arise with GPCR signaling that the cell has evolved mechanisms to solve?

- the ligand binds extracellularly, but activity needs to occur in the cytosol --> 2nd messengers produced by effector enzymes transmit the signal - the initial hormone is only present in low [ ] --> output is amplified at certain steps of the pathway, resulting in a response that's orders of magnitude larger than the initial signal

HW 10/14 #2 What distinguishes the plus (+) and minus (-) ends of a MF? A. The (+) and (-) ends of a MF are indistinguishable from each other. B. The (+) end has exposed ATP cleft and myosin decorates with a "barbed" end. C. The (-) end has exposed ATP cleft and myosin decorates with a "barbed" end. D. The (-) end has exposed ATP cleft and myosin decorates "pointing" towards it. E. The (+) end has exposed ATP cleft and myosin decorates "pointing" towards it.

D.

HW 10/18 #1 Which of the following directly causes the formation of branched MFs at the leading edge of a cell exposed to an extracellular growth factor or LPA? A. Rac-GTP•WAVE•Arp2/3 B. Rho-GTP•Formin C. Cdc42-GTP•WASP•Arp2/3 D. (a) and (c) E. (a) (b) & (c)

D.

HW 10/14 #5 What does profilin do? A. It converts ADP-F-Actin to ATP-F-Actin that can only add to the (-) end of a MF. B. It converts ADP-F-Actin to ATP-F-Actin that can only add to the (+) end of a MF. C. It converts ADP-G-Actin to ATP-G-Actin that can only add to the (-) end of a MF. D. It binds ATP-G-Actin so it cannot be added to either the (-) or (+) end of a MF. E. It converts ADP-G-Actin to ATP-G-Actin & remains bound so that this complex can only add to the (+) end of a MF.

E.

HW 10/21 #4 EB1 is a + end binding protein associated only with growing MTs. How can EB1 potentially stabilize MTs when it rescues them? A. EB1 increases the affinity of tubulin to bind to the (+) of a MT that had undergone catastrophe. B. EB1 increases the interactions between curved protofilaments. C. EB1 decreases the interactions between curved protofilaments. D. EB1 accelerates the rate of GTP hydrolysis on β-tubulin. E. (a) & (b) F. (c) & (d)

E.

HW 10/25 #1 What do Phalloidin and Taxol have in common despite their interaction with different cytoskeletal proteins? A. Both prevent the movement of motor proteins. B. Both stimulate rates of nucleotide hydrolysis C. Both increase rates of polymerization at the (-) ends. D. Both increase rates of polymerization at the (+) ends. E. Both inhibit depolymerization at both the (+) and (-) ends.

E. Phalloidin binds to F-actin and prevents MF depolymerization. vs. Taxol binds β-tubulin and prevents MT depolymerization.

MFs vs/ MT's

SIMILARITIES - both have directional polarity: the (+) end has an ATP cap in MFs vs. GTP cap in MTs - same rectangular hyperbola on a graph of mass vs. [dimers & polymers] graph (no growth until you reach Cc) - b/c of Cc, there should be no free actin or tubulin in cells - majority of the pol. action occurs at the (+) end DIFFERENCES - GTP hydrolysis occurs more quickly in MTs than ATP hydrolysis in MFs - in MTs, depol. occurs at (+) end during catastrophe - there's no spontaneous nucleation required for the formation of MT's b/c the gamma-TuRC acts as the nucleation site

[T/F]: alpha-tubulin acts as a GAP for beta-tubulin

TRUE! - as soon as the newest dimer adds to the plus end, alpha tubulin stimulates GTP hydrolysis on the beta that was previously the end cap - only an actively elongating MT has "new" GTP*Tubulin at its (+) end; if growth stops, slow GTP hydrolysis eventually converts the (+) end to a GDP cap & catastrophe results - a growing MT tends to keep growing, vs. a static MT tends to shrink

[T/F]: Cell signaling is a complex, pathway driven process. Over the years, research in this area has led to the greatest number of Nobel prizes of any topic in science.

TRUE! - discoveries: hormone signaling, peptide hormones, growth factors, protein phosphorylation, G-proteins, NO, & GPRC's - cell signaling evolved as MCO's arose b/c there was a need for communication btwn cells that are no longer in contact

HW 10/21 #5 In addition to rescuing a MT that had undergone a catastrophe, what other function does EB1 have? A. EB1 binds certain proteins and transports them towards the plasma membrane through its binding to the elongating (+) end of a MT. B. EB1 is a neuronal specific protein that facilitates anterograde transport. C. EB1 is also a bidirectional motor protein that transports secretory vesicles towards the plasma membrane and transports late endosomes towards the lysosome. D. EB1 is also a kinesin that transports secretory vesicles towards the plasma membrane. E. None of the above. EB1 has no function other than rescuing a MT that had undergone catastrophe.

A.

HW10/28 #1 Which of the following statements correctly describes the function of the Gα subunit of Gs, Go, and Gi heterotrimeric G protein complexes? A. Gαs increases cAMP levels, Gαo increases IP3 and DAG levels, Gαi reduces cAMP levels. B. Gαs and Gαi both increase cAMP levels, Gαo increases IP3 and DAG levels. C. Gαi increases cAMP levels, Gαo increases IP3 and DAG levels, Gαs reduces cAMP levels D. Gαsi, Gαs & Gαs all reduce cAMP levels E. Gαsi, Gαo & Gαs all increase cAMP levels

A.

HW 10/14 #4 How does Thymosin β-4 affect MF dynamics and/or structures? A. Thymosin β-4 depolymerizes MFs into ADP-G-Actin. B. Thymosin β-4 binds ATP-actin & inhibits its incorporation at either the (-) or (+) end of a MF. C. Thymosin β-4 exchanges ADP for ATP & recruits ATP-Actin to the "barbed end" of a MF. D. Thymosin β-4 exchanges ADP for ATP & recruits ATP-Actin to the "pointed end" of a MF. E. Thymosin β-4 cleaves MFs into short fragments of ADP-F-Actin.

B.

HW 10/18 #1 What is the primary function of cofilin during directed cell migration? A. Cofilin binds to Rho•GTP to promote directed (+) end MF polymerization at the leading edge of a migrating cell. B. Cofilin cleaves linear MFs in the trailing edge of a migrating cell resulting in their rapid fragmentation enabling the trailing edge to "catch up" with the leading edge. C. Cofilin binds to G-Actin•ATP and prevents its incorporation into elongating MFs at the trailing edge of a migrating cell. D. Cofilin accelerates ATP hydrolysis in F-Actin causing its depolymerization at the trailing edge of a migrating cell. E. Cofilin accelerates the exchange of ADP for ATP on G-Actin and directs its specific addition to the (+) of a MF bound to either Formin, WASP or WAVE at the leading edge of a migrating cell.

B.

HW 10/21 #3 How does the addition of tubulin at the (+) end of an elongating MT paradoxically cause it to be more susceptible to undergo catastrophe? A. Newly added β-tubulin activates GTP hydrolysis on the α-tubulin at the (+) end of the pre-existing MT. B. Newly added α-tubulin activates GTP hydrolysis on the β-tubulin at the (+) end of the pre-existing MT. C. Newly added β-tubulin promotes exchange of GDP for GTP on the α-tubulin at the (+) end of the pre-existing MT. D. Newly added α-tubulin promotes exchange of GDP for GTP on the β-tubulin at the (+) end of the pre-existing MT.

B.

What happened to D/ E residues over time in enzymes?

- *at one time, enzymes may have been constitutively active, but phosphorylation allowed for regulation of activity*: over time, S, T, or Y were put in to restore functionality of an E/D in an enzyme's AS; i.e. activity isn't stuck on or off, sites of reversible phosphorylation can result in the enzyme either being off or on - glycogen synthase (GS) has a Ser residue that gets phosphorylated: when non-phosphorylated it's on, but when phosphorylated, it shuts off= negative regulation by phosphorylation vs. - glycogen phosphorylase (GP) is normally off, it has an Arg & a Ser residue that can't interact until Ser is phosphorylated, then the two come into contact= positive regulation by phosphorylation

4 general schemes of intercellular signaling

- *endocrine*: site of synthesis & release is distinct from the target site of action; most common signaling in MCO's; ex. hormones secreted by their glands into the bs travel a long distance to their target organs - *paracrine*: signaling & responding cells in close proximity; applicable to growth factors, nerve impulse transmission, & embryogenesis - *autocrine*: positive feedback growth responses for individual cells; most applicable to cell proliferation & tumorigenesis - *membrane-attached proteins*: signaling & responding cells in direct physical contact; applicable to embryogenesis & tissue formation

microfilaments (MFs)

- *simplest structure of the cytoskeleton*: small rodlike structures, about 4-7 nm in diameter, present in the cytoplasm of many eukaryotic cells - made of actin - give shape to microvilli, provide contractile function in muscle, motility in macrophages, contractile ring in cytokinesis

cytoskeleton functions

- 3-D filamentous protein network - strength/ support/ flexibility - mvmt of cellular structures & materials - morphology - motility - cell division -*the cytoskeleton is dynamic, not static* - made up of MFs, MTs, & intermediate filaments - IFs are the only thing w/ no directional polarity & thus no known associated motor protein

connection btwn LDL RME & ALZ

- APOE4 is the strongest genetic risk factor for late-onset Alz --> it binds to Tau, promoting neuroinflammation, & tau-mediated neurodegeneration independently of amyloid-beta-pathology - *ApoE4 exerts a toxic gain-of-function* mutation whereas a knockdown form is protective - neurons are dependent on the production & secretion of LDL from astrocytes; ApoE 2, 3, & 4 are carriers that transport LDL from astrocytes to neurons - individuals w/ a higher expression of ApoE4 have an extreme predisposition for Alz - in mice that express the gain-of-function mutation, you end up w/ Tau aggregation that was accelerated by ApoE4 - *these findings illustrate a connection btwn 2 seemingly unrelated processes that we learned about*

How are the second messengers degraded or inactivated?

- Ca2+ goes back into the ER/ SR or gets exported via Ca2+ ATPase - cyclic nucleotides are inactivated by cyclic phosphodiesterase: AMP or GMP - gases diffuse out

critical concentration (Cc)

- MF polymerization is [G-actin]-dependent": if [G-actin] < Cc, no polymerization occurs vs. if [G-actin] > Cc, net polymerization occurs; at steady state, [G-actin]=Cc, no net polymerization occurs - C+c= 0.12 micromolar - C-c= 0.60 micromolar - *the (+) end grows ~12x faster than the (-) end when [G-actin]>0.6 micromolar*; the forward rate constant is 12x faster than the reverse rate constant --> HUGE BIAS to add to (+) end - the off rate (1/s) is relatively the same at both ends - cells can regulate preferential assembly/ disassembly at (+) or (-) ends independently of [G-actin] b/c in normal cells the physiological [G-actin] is much greater than 0.12 micromolar

tau aggregation & Alzheimer's

- P301-->S mutation causes tau to aggregate - when you look at immunostained cerebral cortex sections of patients w/ Alzheimer's, you see brown clumps- these are the tau aggregates

The 1st World Cell Race (2012)

- a bunch of scientists got tgt, they set up rules & a racecourse to compete different mobile cells - the race track was made of fibronectin lines to which cells could adhere - cancer cells were highly migratory, but embryonic cells actually won the race - we learned that not all cells have the same capability/ rate of migration

microtubule-associated proteins (MAPs)

- a class of proteins that participate in the regulation of microtubule assembly & function - in nerves, the MAP2 & Tau bind to the surface of MTs & modify the "caliber" of the axon that contains a fixed # of MTs; stabilize MTs by spacing them out - MAP2 has long spacer arms- found in the cell body - Tau has shorter "spacer" arms- found in the narrower axon where MTs are more densely packed

formins & actin-related proteins (ARPs)

- act as nuclei to promote actin polymerization - prevents a lag in polymerization - cofilin chops up existing MF's to recycle the nuclei to the (+) end

beta-adrenergic receptor (BAR)

- activated by EP (fight or flight response) or glucagon in order to inc. levels of blood glucose via 2 pathways: inhibit glycogen synthesis & converts glycogen to glucose - muscle & liver are the 2 primary target tissues in which the 2 simultaneous metabolic responses are elicited - glucose is the energy source for muscle contraction - in the liver, you get the largest response b/c PLC is involved (generates DAG & IP3) as well as cAMP, this makes sense b/c the largest stores of glycogen for the body are here - in the muscle cells, neural stimulation generates calcium vs. hormonal stimulation generates cAMP; the muscle tissue stores glycogen for its own use - cAMP inc. PKA activity --> this inhibits GS, but activates GPK which then activates GP (kinase cascade)

the kinesin strut

- each step covers 16nm; it. binds only to beta-tubulin - each kinesin walks along the same protofilament on the same MT 1. leading head binds ATP 2. binding of ATP induces a conformational change causing the neck linker to swing forward & dock on the next open beta-tubulin (the former trailing head is now the leading head) 3. new leading head binds, releasing ADP; the trailing head coordinately hydrolyzes ATP to ADP + Pi; Pi is released & the linker becomes undocked - the head domain binds the MT weakly in ATP & ADP + Pi states, but not nearly as well in ADP state (trailing head always has ADP attached)

How do vesicles move around the cell?

- all thanks to MT polarity - single MTOC in interphase has all the MT's radiating out of it w/ the (+) ends reaching towards the plasma membrane - you can have motors carrying things in opposite directions on the same MT - in normal cells, MTs aren't fragmented, but in nerve cells, they are: in the dendrites, MTs face in different directions vs. in the axon all the (+) ends of the fragments point towards the synapse; vesicles can jump from one fragment to the next (the same is true in all cell types if the MT undergoes catastrophe) - motor proteins will often carry the mitochondria w/ them b/c the energy demand for vesicle trafficking is so high - MTs don't go all the way to the edge of the plasma membrane, so we have to hand off the vesicle to a myosin motor on an MF once it reaches the end of the MT

concentration dependance of tubulin polymerization

- all the action happens at the (+) end: C+c= 0.03 micromolar, but like for actin, polymerization is independent of this concentration b/c [tubulin]cell= 10-20 micromolar - no #'s for the (-) end b/c it's anchored in MTOC so there's virtually no growth or shrinkage here

capping proteins

- allow the cells to create an environment where only a specific end can grow - make growth independent of Cc so that the [Actin] doesn't oscillate; technically, since [Actin] is so high (50-200 micromolar), the cells shouldn't have any free G-actin, it should all be polymerized; in reality, 40% of total actin is unpolymerized G-actin

recognition sites of kinases

- any given residue's consensus sequence has a lot of redundancy w/ the phosphorylation sites for other enzymes (ex. BARK & PKA response in desensitization) - given this degeneracy, it's common for a protein to be regulated by multiple kinases

examples of GPCR's

- beta-adrenergic receptor (BAR): ligand= epinephrine (EP) & glucagon; effect= inc. blood glucose for locomotive activity vs. inc. blood glucose when blood sugar is low (same effect but for different reasons) - Ach receptor: ligand= Ach & adenosine; effect= relaxes the body; opposite response of BAR

examples of second messengers

- cAMP (pictured): activates PKA -cGMP: activates PKG & opens cation channels in rod cells - DAG: activates PKC - IP3: also required to ~indirectly~ activate PKC; opens Ca2+ channels in the ER - DAG & IP3 are both derived by cleavage of plasma membrane phosphatidyl inositol, but DAG remains in the membrane (b/c it's the FA tails) & inositol diffuses away (b/c it's the polar head) - nitric oxide (NO): a "biological neutrino"; it's a gas, not a hormone (there's no receptor); only signal that can diffused from the cell it was produced in to other cells where it has an effect - Ca2+: primary biological function is to mediate intracellular signaling

Cap Z

- caps + end of actin, preventing assembly when [G-actin] >/= C+c - depol. can still occur at the (-) end

What enables motile cells to change shape?

- cells can respond to extracellular chemotaxins (signals that direct mvmt) by changing the polymerization/ depolymerization of MFs - small G proteins play a role in directed assembly of MFs

melanocytes

- cells that produce the pigment melanin - they exhibit an interesting ex. of bidirectional transport that allows the organism to camouflage - when placed against a light background, the pigment disperses out so that the cell appears lighter vs. when placed against a dark background, the pigment clusters in the center so that the cell appears darker - in secretory trafficking, GFP-tagged proteins show bidirectional transport

tauopathies

- class of neurodegenerative diseases involving aggregation of abnormal Tau protein --> collapse of the cytoskeleton --> halts neurosecretory trafficking

cofilin cycle

- cofilin fragments ADP-F-actin --> inc. # of (-) ends & enhances depolymerization - the ADP G-actin that is now free in the cytosol quickly picks up ATP, since this is more abundant - "cofilin cuts" & creates new nuclei

thymosin-beta-4 cycle

- coordinated actin regulation cycle where thymosin-beta-4 binds to ATP actin & makes it invisible --> dampens addition to both ends

dynamic instability

- cycles of shrinkage & growth in microtubules; in normal cells, we see both occurring at the same time - stages: assembly/ growth, catastrophe, disassembly/ shrinkage, rescue - the slope for disassembly is steeper than for assembly, depol. occurs rapidly once it starts - we can't predict when catastrophe will occur or how far it will fall, but we know that it will recover

major classes of cell-surface receptors

- cytosolic kinases: *GPCR*= most abundant; activated when its ligand binds; the receptor itself is a GEF to turn on a G-protein, which activates effector enzymes, produces small diffusible molecules (2nd messengers)/ activates protein kinases, then these phosphorylate other proteins, either activating or repressing them - receptor-associated kinases: *RTKs*= RTK phosphorylates itself , activates PKs, kinase cascade (no 2nd messengers) Ultimately activates genes in the nucleus that cause the cell to start dividing again

How do PK's distinguish btwn S/T & Y?

- depends on cleft depth - a shallow cleft better accommodates Ser/ Thr vs. a deep cleft accommodates Tyr - if something too small enters the cleft, it can't get close enough to ATP to transfer the phosphate vs. large things get pushed back out

3 classes of protein kinases

- depends on the amino acids that are phosphorylated in the target protein 1. Serine/ Threonine Kinases can phosphorylate S/T but they prefer S 2. "dual specificity" kinases can phosphorylate S/T or Y 3. tyrosine kinases can only phosphorylate Y; we'll come back to these when we learn about the cell cycle

taxol

- drug that inhibits GTP hydrolysis on beta---tubulin - cytotoxic: the binding site is right next to the normal GTP binding site - *anti-mitotic*- prevents MT depolymerization in anaphase --> cells can't resume normal interphase or divide so they undergo apoptosis; makes it a good cancer drug, BUT it's not cell specific, which is why it could also lead to hair loss, anemia, & gut pain (therapeutic target= develop an antibody that will deliver taxol just to tumor cells) - b/c it stabilizes MT's, it can be used in vitro to study vesicle trafficking

microtubules organizing center (MTOC)

- in non-neuronal cells, all MT's have 1 origin that acts as their organizing center - a.k.a. "centrosome"= pair of centrioles (mother & daughter undergo semi-conservative replication in S phase); comes from the sperm. - the (-) ends of all MTs terminate within the centrosome w/ a ring of gamma-tubulin associated w/ other proteins= gamma-TuRC (gamma-tubulin ring complex) --> this is the capping protein that makes the (-) ends invisible - in nerve cells. there are multiple origins for MTs b/c they are fragmented along their length, but they all tend to terminate in the dendrites or axon terminals

wound healing

- injury causes platelets to release growth factor, triggering re-entry into the cell cycle --> cell division to replace damaged cells, but this is a slow process - while this longer process occurs, the cells surrounding the cut are stimulated to migrate towards the site of injury - fibroblast cells are responsible for the formation of fibers and aid in the production of collagen and elastin; we can hook them up to chimeric actin GFP to see their growth

expansion/ contraction cycle of elongating cells

- mechanism of mvmt for macrophages - stress fibers= linear MF's that elongate as the cell migrates, allowing it to stretch; at the same time, we still need branched MF's; some of these have capping proteins attached to keep them stable for as long as the growth factor is present - at the leading edge: Cdc42 & Rac activation vs. Rho is activated at the trailing edge - stress fibers at the trailing edge anchor themselves to integrins in the plasma membrane to remain attached to the fibronectin ECM (external integral domain binds to ECM, internal domain links them to MFs) - when the cell contracts, integrins are phosphorylated, causing the contractile fibers to be releasing --> we see a snapping/ inching forward where the back end of the cell catches up to the front end

heterotrimeric G proteins

- membrane associated proteins w/ 3 subunits: alpha, beta, and gamma - alpha is the important subunit b/c it binds GDP (inactive) or GTP (active); when active, it dissociates away from the other two units - there are different families, all depending on the alpha subunit: Gs (activates adenylyl cyclase, inc. cAMP), Go/Gq (activates phospholipase C, inc. IP3 & DAG), & Gi (inhibits AC, dec. cAMP) - alpha and gamma are both lipid-anchored in the plasma membrane - cells may be exposed to multiple stimuli that elicit opposing effects & they must balance these out to maintain function

How does actin polarity contribute to MF structure?

- monomeric G-actin assembles into chains of F-actin, 2 of which twist helically about each other forming MF's --> "right-handed double-helix" - the difference in structure influences binding affinity of one subunit to another - the two ends of MFs are structurally & functionally distinct: the (+)/ barbed end vs. (-)/ pointed end --> we can biochemically & experimentally distinguish them - intrinsic ATPase activity: polymerization of G to F-actin doesn't require ATP hydrolysis, but ATP hydrolysis influences polymerization kinetics

actin

- most abundant protein in eukaryotic cells (10% by weight of muscle cells; 1-5% in non-muscle cells= 5x10^8 molecules/ cell) - *ancient, highly conserved protein* that arose from the bacterial ancestral protein MreB; 80% of the AA's are IDENTICAL btwn amoebas & humans - a *globular, asymmetric protein that has intrinsic polarity, caused by the ATP-binding cleft* - also binds Mg2+ (ATP salt) under physiological conditions

anterograde transport

- movement down the axon away from soma (in nerves) or mvmt from the ER --> Golgi --> plasma membrane (in non-nerve cells) - direction: towards the (+) end - motor: kinesins

retrograde transport

- movement up the axon toward the soma (in nerves), endocytosis, or mvmt from Golgi --> ER (in non-nerve cells) - direction: towards the (-) end - motor: dyneins

link btwn profilin & ALS

- mutations in the profilin 1 (PFN1) gene cause familial ALS, a late-onset neurodegenerative disorder, resulting from motor neuron death --> eventually results in paralysis - ~10% of cases are familial (FALS), w/ a dominant inheritance mode - PFN1 is critical for the conversion of G-actin into F-actin, esp. in the growth of axons in nerve cells - cells expressing this mutation contain ubiquinated insoluble aggregates that contain the ALS-associated protein TDP-43 --> aggregates cause problems w/ proteostasis - profilin can no longer bind actin --> axons withdraw & degenerate

formin

- nucleates linear MFs in response to extracellular signals that activate Rho - formin looks like a paperclip, it has 3 domains, 1 of which is a rho binding domain --> active Rho*GTP binds to RBD, exposing both of the forming homology domains - FH1 has a high affinity for profilin-ATP-actin: it acts as a loading site and then hands off the profilin-ATP-actin to the elongating MF on the FH2 domain (still only adds to (+) end)

challenges that motor proteins have to overcome

- only MTs at the initial segment are continuous w/ the centrosome - the total axon length is 10mm-100m - there is a staggering volume of trafficking in any single neuron per day, so axonal transport must be extremely efficient (a single neuron in our brains require 4.95x10^14 ATP per day -> each kinesin walks 2.6 km/day & must hydrolyze 1 ATP per 16 nm step= 0.4-0.7 trillion ATP per day per kinesin) - kinesin & dynein are both attached to the vesicle, myosin can also try and latch on to pull the vesicle off course; if you have kinesin vs. dynein, kinesin always wins b/c it has more brute force

tropomodulin

- prevents assembly & disassembly at (-) end when [G-actin] < C-c

profilin cycle

- profilin acts as an exchange factor by binding ADP*actin & helping it exchange GDP for GTP - it remains bound, converting it to an all-or-nothing bias for the plus end b/c profilin-ATP-actin can only bind to (+) end - profilin prevents (-) end growth >C-c - can bind other proteins that are rich in Pro

redundancy vs. additivity

- redundancy: activation by one mechanism/ pathway or another generates a fully 100% active protein; ex. in liver cells, PKC & PKA both inhibit GS - additivity: activation by one mechanism/ pathway or another generates only 50% activity, activation from both generates 100% activity; ex. GPK in liver & muscle cells is activated 50% by Ca2+*Calmodulin & 50% by PKA

signal transduction

- the linkage of a mechanical, chemical, or electromagnetic stimulus to elicit a specific cellular response - steroids, retinoids, & thyroxine are sufficiently hydrophobic that they can diffuse across the plasma membrane --> their receptor is in the cytosol & takes them to the nucleus where they act as a TF - hydrophilic signals, small molecules, peptides, & proteins are involved in *cell-surface mediated signaling* --> the signal binds to the extracellular domain on an integral membrane protein, activates the receptor (may undergo a conformational change), this activates cytosolic signaling molecules (may involve G-proteins & phosphorylation cascades)

What determines the dynamic instability of MTs?

- the presence of GTP or GDP caps - newly added beta-tubulin on a growing MT has GTP [tubulin] > Cc & slow GTP hydrolysis, favors continued growth & (+) end has GTP "cap" - GTP cap maintains linear protofilaments - GTP is hydrolyzed to GDP after polymerization; "internal" beta-tubulin has GDP - GDP protofilaments curve outward when the GTP cap is lost --> looks more & more like a frayed string until it falls apart - the longer an MT grows, the more unstable it becomes - the GTP cap breaking off is the initiating event for catastrophe

What experimental system was used to analyze axonal transport & identify motor proteins?

- the squid giant axon - experiment: dissect out the axon, roll out the axoplasm (cytoplasm of the axon) & use this jelly to reconstruct vesicle trafficking in a cell free system - purify MT's & add taxol to stabilize them, now add vesicles (nothing happens), add in axoplasm --> vesicles bind MTs & transport occurs - discovery: *binding & mvmt of vesicles requires ATP-hydrolysis* --> the search for these ATP-dependent proteins began - kinesin & dynein were 1st discovered in axons, but were found eventually in all cells

3 G-protein pathways involved in cell migration

- these are the intermediaries btwn extracellular signals & MFs; only active when GTP bound - the signals are growth factors (wound healing) or proteins associated w/ immune response - Cdc42: triggered by fibroblast growth factor (FGF) or platelet-derived growth factor (PDGF): WASP & Arp 2/3 --> filopodia formation (branched MF pol.) - Rac: triggered by FGF or PDGF: WAVE & Arp 2/3 --> lamelliopodia formation (branched MF pol.) - Rho: triggered by lipoprotein A/ pro-inflammatory cytokine --> stress fiber formation & contraction (linear MF pol.) - *growth at any edge depends on which receptors are exposed to growth factor & how many there are in that section of the plasma membrane* - coincidence detection: 2-signal input (binding of growth hormone to GPRC & binding of G-protein to RBD or NPF) ensures that the protein is activated at the right place & time

similarities in the mechanism & generic structure of PK's

- they all have a 3 shape: cleft that binds the substrate protein & ATP --> transfers gamma-phosphate onto S/T or Y - mechanism: PK binds ATP, then substrate, transfers gamma phosphate to the substrate, then the phosphorylated protein & ADP dissociate - kinase-substrate complexes are relatively long-lived; this means they can be biochemically isolated by co-immunoprecipitation involving an antibody that is specific to that kinase; allows you to determine what substrate they bind - substrate specificity is largely due to differences in charge & hydrophobicity of the surface residues within the ATP binding cleft - no single naming convention: may be based on the proteins they phosphorylate, what activates them, the location in which they were discovered, or the ligand that binds to them

(+) end of MFs

- this is where all the action of polymerization occurs - newer, has ATP end cap b/c it hasn't had time to hydrolyze the ATP yet - explains the higher affinity of G-actin*ATP for the (+) end

(-) end of MFs

- this is where depolymerization occurs - the ATP-binding cleft is exposed - older, has ADP cap

evidence of phosphosite evolution

- topoisomerase II (DNA unwinding): in bacteria, Lys--Glu electrostatic bridge is seen when looking at AA sequence- this is constitutively active vs. in eukaryotes, the same sequence has a non-conservative substitution of Glu--> Ser/ Thr - enolase (glycolysis) - Raf (involved in cell proliferation): in simple eukaryotes: Glu/ Asp -- Arg vs. in humans there are 3 iso forms of Raf w/ Tyr--Arg --> this allows for the enzyme to only be active in certain conditions, preventing continuous cell division (thinking back to the beginning of the year, this could be one of the ways in which cancer retro-evolves back to a more prokaryotic genome)

phalloidin

- toxic mushroom that binds & stabilizes MFs, preventing their depolymerization --> paralyzes the cell b/c it prevents MF turnover --> cell death - lowers Cc - dominant active Rac, Cdc42, & Rho cannot hydrolyze GTP --> perturbs MF morphology - images of these mutant cells show MF's stretching out in all directions (characteristic of linear vs. branching)

progression of MT assembly

- tubulin dimers are polymerized into protofilaments that begin to associate into ring structures - GTP on beta-tubulin is hydrolyzed to GDP immediately after polymerization of the new subunit - singlet= 1 ring, most common type, found in cytoplasm - doublet= 2 fused rings, found in cilia & flagella - triplet= 3 fused rings, found in basal bodies & centrioles

Why did evolution pick protein phosphorylation as the major regulatory function?

- ~20-30% (1/3) of proteins present in a cell are regulated by altering their phosphorylation states; the majority are activated when phosphorylated and inactivated when dephosphorylated; main mechanism of PTM - the evolution of phosphorylation sites from Glu & Asp provides a rationale for why phosphorylation sometimes activates proteins - *a phosphate group* provides a large (-) charge that can create steric hindrance, but it can also *mimick D or E in the enzyme AS*

How many protein kinases & protein phosphatases are encoded by the genome?

- ~400 S/T PK's, ~40 dual specificity PKs, ~90 Y PKs vs. - ~40 S/T PP's, ~50 dual specificity PKs, ~100 Y PKs - there are fewer PP's to reverse the effects of phosphorylation than there are PKs --> goal= shift equilibrium to more phosphorylated protein (typically means more activity), ex. in order to do this, when cAMP activates PKA it also phosphorylates PP, deactivating it - extremely rare to find Y phosphorylated b/c it has profound effects on cell proliferation

HW 10/23 #5 If ATP was replaced with AMPPNP, what would you observe in an in vitro anterograde vesicle transport system derived from axoplasm? A. In the presence of AMPPNP, vesicles would bind to MTs and would exhibit anterograde movement. ATP hydrolysis is not required for dynein-dependent transport. B. In the presence of AMPPNP, vesicles would bind to MTs, but would not exhibit anterograde movement. ATP hydrolysis is required for kinesin-dependent transport. C. In the presence of AMPPNP, vesicles would bind to MTs and would exhibit anterograde movement. ATP hydrolysis is not required for kinesin-dependent transport.

B.

HW 10/23 #1 Microtubule (MT) dynamics in a given cell type can be influenced by the expression of different isoforms of α and β tubulin and/or by various post-translational modifications. Tubulin isoforms or post-translational modifications that would destabilize MTs could do so by: A. Increasing α tubulin GTPase activity and/or increasing β tubulin GAP activity B. Increasing α tubulin GAP activity and/or increasing β tubulin GTPase activity C. Reducing α tubulin GAP and/or reducing β tubulin GTPase activity D. Reducing α tubulin GTPase activity and/or increasing β tubulin GAP activity E. Reducing β tubulin GTPase activity and/or reducing β tubulin GAP activity

B. Any post-translational modification or expression of an α or β tubulin isoform that would increase β tubulin GTPase activity and/or increase α tubulin GTPase activity would promote MT destabilization.

HW 10/18 #4 Photorhabdus asymbiotica is a naturally bioluminescent bacterium that normally infects soil nematodes. However, Pa is an emerging human pathogen that causes skin and soft tissue lesions in unprotected farm workers exposed to nematode-infested soil. The responsible toxin (PaTox) covalently modifies different G proteins. in this case, PaTox glycosylates a tyrosine on Rho. This causes the MF cytoskeleton to collapse and prevents cell migration in response to extracellular growth factors. Thus, skin abrasions in Pa infected individuals heal very slowly. Based on this information, how does PaTox affect Rho function? A. PaTox causes Rho to constitutively bind WAVE or WASP. B. PaTox causes Rho to constitutively bind formin. C. PaTox prevents Rho from exchanging GDP for GTP D. PaTox prevents Rho from hydrolyzing GTP.

C.

HW 10/23 #2 Which two processes are integrated in late-onset Alzheimer's disease? A. Phalloidin-dependent MF stabilization and Tau aggregation. B. Profilin aggregation and impaired leading edge migration during axon outgrowth. C. Receptor-mediated endocytosis of ApoE4 and Tau aggregation. D. Tau aggregation and impaired leading edge migration during axon outgrowth. E. Taxol-dependent inhibition of MT depolymerization and Tau aggregation.

C.

HW 10/23 #4 Axoplasm derived from the squid giant axon was used as the biological system to first discover: A. Tubulin B. Taxol C. MT motor proteins D. EB1 E. MAP2 and Tau

C.

HW 10/25 #4 Which of the following intercellular signaling schemes would be most applicable to the situation described in question #3? A. Autocrine B. Endocrine C. Paracrine D. Plasma membrane attached protein

C.

HW 10/28 #2 What is a "dual specificity" kinase? A. A kinase that phosphorylates either tyrosines or serines. B. A kinase that phosphorylates either threonines or tyrosines. C. A kinase that phosphorylates serines or threonines or tyrosines. D. A kinase that phosphorylates either serines or threonines.

C.

HW 10/18 #3 Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease also known as Lou Gehrig's disease. A report in Nature by Wu et al showed that missense mutations in the profilin (PFN1) gene cause familial ALS by causing PFN1 protein to aggregate & lose its normal function. Which of the following would you predict to observe in the motor neurons of an ALS individual expressing one of these mutant alleles of PFN1? A. ALS cells have reduced ratio of Arp2:Arp3 B. ALS cells have reduced ratio of WASP:WAVE. C. ALS cells have reduced ratio of Rho:Rac. D. ALS cells have reduced ratio of F-Actin:G-Actin. E. ALS cells have reduced ratio of G-Actin:F-actin.

D.

HW 10/18 #5 Normal cells treated with phalloidin, a toxin derived from the angel death mushroom, Amanita phalloides, resemble cells expressing a dominant active form of Rho. Based on this observation, which of the following explains how phalloidin alters MFs? Fig.pdf 713 KB A. Phalloidin inhibits the conversion of Rho•GDP to Rho•GTP B. Phalloidin inhibits the conversion of Rac•GDP to Rac•GTP C. Phalloidin inhibits formin. D. Phalloidin inhibits the conversion of F-actin to G-actin. E. Phalloidin inhibits profilin.

D.

HW 10/21 #1 Which of the following is never found "free" in a cell? A. G-actin•ATP B. α-Tubulin•GTP C. β-Tubulin•GTP D. Neither (b) nor (c) are found free in a cell E. None of the above are found free in a cell

D.

HW 10/21 #2 Tumor cells often become resistant to taxol by expressing a unique isoform of β-tubulin (β3). How does taxol-resistant β3 tubulin most likely differ from "normal" taxol-sensitive β-tubulin? A. β3 tubulin cannot bind to α-tubulin when it is bound to taxol B. β3 tubulin cannot exchange GDP for GTP when it is bound to taxol C. Tubulin containing β3 tubulin cannot form MTs when bound to taxol. D. β3 tubulin can still hydrolyze GTP when it is bound to taxol E. β3 tubulin cannot hydrolyze GTP when it is bound to taxol

D. Taxol inhibits GTP hydrolysis by β-tubulin which prevents MT depolymerization during mitosis. β3 tubulin can still hydrolyze GTP in the presence of taxol thereby countering its cytoxicity.

HW 10/14 #3 Cytochalasin D is a fungal alkaloid that binds specifically to the (+) end of F-Actin and prevents further polymerization at this end. The Cc of ATP-Actin for the pointed end of F-actin is 0.6 µM and the Cc of ATP-Actin for the barbed end of F-actin is 0.12 µM. At what concentration of G-Actin would only the pointed end of a MF elongate if incubated in the presence of excess cytochalasin D? A. The pointed end would not elongate at any of these concentrations. B. 0.15 µM ATP-Actin C. 0.4 µM ATP-Actin D. 0.7 µM ATP-Actin

D. The pointed end of a myosin decorated MF is the (-) end. Since cytochalasin D blocks the (+) end, no polymerization can occur there. However, [G-Actin] must be above the (-) end Cc which is 0.6 µM in order for polymerization to occur there so the correct answer is 0.7 µM.

HW 10/25 #3 Which of the following is/are required for the transport of a neurosecretory vesicle from the cell body to its fusion with the plasma membrane at the axon terminus? A. Dynein B. Kinesin C. Myosin D. (a) & (b) E. (a) & (c) F. (b) & (c)

F.

HW 10/25 #5 What combination of plasma-membrane associated proteins do each of the α, β, and γ subunits of heterotrimeric G protein complexes represent? A. The α, β, and γ subunits are all peripheral proteins B. The α, β, and γ subunits are all lipid-anchored proteins C. The α, β, and γ subunits are all integral membrane proteins. D. The α and γ subunits are lipid-anchored proteins, β is an integral membrane protein. E. The α and γ subunits are integral membrane proteins, β is a peripheral protein. F. The α and γ subunits are lipid-anchored proteins, β is a peripheral protein.

F.

HW 10/25 #2 As depicted in the Hoogenraad video "A Day in the Life of a Motor Protein", which of the following can potentially impede the directional transport of a neurosecretory vesicle from the cell body to the axon terminus? A. A "tug of war" between kinesin and dynein motors bound to the same vesicle B. Prematurely diverting the vesicle from a MT to a MF by a myosin motor bound to the vesicle C. MT catastrophe D. (a) & (b) E. (a) & (c) F. (b) & (c) G. (a), (b) & (c)

G.

HW 10/23 #3 Which of the following would be affected by rates of GTP hydrolysis in vivo under the indicated conditions? A. Interactions between adjacent protofilaments in non-neuronal cells B. Conversion of G-Actin to F-Actin at the leading edge of a migrating cell C. The movement of a kinesin motor on an individual microtubule towards the plasma membrane in a non-neuronal cell D. (a) and (b) would both be affected E. (a) and (c) would both be affected F. (b) and (c) would both be affected G. (a) (b) and (c) would all be affected

G. A is correct because GTP hydrolysis on β-tubulin reduces interactions between adjacent protofilaments and results in MT depolymerization. This would also result in the dissociation of a kinesin motor transporting a vesicle towards the plasma membrane and require it to bind to another MT that is elongating making C also correct. Finally, GTP hydrolysis by Rho, Rac and Cdc42 influence directed MF polymerization on the leading edge of a migrating cell also making B correct.