cell bio exam 3: cytoskeleton

cytoskeleton functions

Cytoskeleton Functions: Strength, Support, Flexibility, Intracellular Transport, Morphology, Motility, Cell Division

Key:  The Cytoskeleton is Dynamic, Not Static!

theme of the cytoskeleton

The Cytoskeleton is Dynamic, Not Static!

• Theme of movement  (transport, protein trafficking, moving within cells/secretion)

• Provides basis for moving within cells and secretion. Provides infrastructure that allows directed and specific movement tof organelles and vesicles along microfilament tracts. Also motor proteins

• Vs need rabs, but the rabs are recruiting the motor proteins

• Makes movement directed 

The 3 Classes of Cytoskeleton Components

microfilaments, microtubules, intermediate filaments

• Have division of labor 

• Each has responsibility. 

• Categorized based on their different size 

• the one time they all work together: mitosis 

intermediate filaments

• Intermediate in size 

• Different organization in cell . organized inside the nucleus around the nuclear membrane 

• Important (type called lamens) during mitosis 

  • Every time cell divides, need to make mitotic spindle. Attach chromosomes in nucleus to microtubules . but the microtubles are not inside the nuc. To make spindle , have to get rid of nuc. Dissolve the nuc . the lamens dissolve the nuc and provide access to microtubules 

• is a gptase 

microfilaments

• Smallest

• Made of a single protein called actin 

  • Actin is very universally conserved aka the same in living beings

  • actin is an atpase

• Are strings, fibers 

• They are distributed throughout the cell , everywhere 

• Cause pm to pinch off and separate in mitosis 

• can come from anywhere (no origin like microtubules)

• highly dynamic, but not as dynamic as microtubules

• polarized

• functions as cellular architecture: comprises microvilli, surrounds the cell for support

• create foundation on PM for cells to attach to . cells need adhesion and need to stick to solid surface

• play role in cell migration

actin

• very universally conserved

• a single unit that makes up microfilament

• an atpase 

• most abundant protein in eurkaryotic cells

• ancient af

• concentration always stays the same, never changes

• tracks for myosins (motor proteins)

• Actin is a globular protein. Very sphericalish 

  • Refer to monomeric form as G actin. G or globulin. Free actin 

  • Polymerizes into microfilaments, called F-actin

  • 4 domains. But not symmetrical. 

  • Is an ATP-binding protein and has a cleft for atp to bind. Can hydrolyze the atp to adp and the adp can exit out of the cleft and a new atp can bind 

  • Cleft allow access for atp and ADP 

  • Also has Mg binding site. Needed to bind atp. Mg will coordinate with P on adp or atp. No Mg on actin = cannot polymerize 

G actin

monomeric form of actin. not polymerized

F actin

polymerized form of actin

Microtubules

• vesicle trafficking

• Very large tubes 

• Hollow 

• made up of protein heterodimer: alpha tubulin and beta tubulin. Just called tubulin, they are never found alone. Is an alpha beta dimer 

  • both are gptase

  • have different funtions. alpha is always GTP bound. beta exchanges and hydroylzes.

  • alpha is the gap for beta

• has polarity —- always polymerizes at beta end

• more dynamic (1000x more) in terms of growing and shrinking than mfs

• have dynamic instability: individual mts can grow and shrink in the same environment , bc of rate of gtp hydrolysis.

• MAIN DIFFERENCE: are gpt driven

• going to be the tracks for vesicles, and does stuff with recruiting motor proteins

• all have the same origin: the centrosome.

  • the centrisome duplicates during mitosis c need two to make spindle. Duplicate when DNA does. so usually one centrosome sometimes two 

  • all MTs have their minus end at the centrisome and the plus end extending towards the PM

• no MTs in the nuc , all out in the cytoplasm

• After GTP hydrolysis, tubulin adopts a curved conformation, weakening lateral interactions between protofilaments and promoting depolymerization.

Cell migration

its giving inch worm: Resculpting mfs to push on pm to make cell move in one direction

will have trailing edge and leading edge of PM

1.  Extracellular signaling molecules like growth factors/inflammation(cytokines) bind to their integral membrane receptors in their vicinity. the receptors are all around the periphery of the cell, but only the ones near the signal are bound to. These are activated

2. receptors transduce that signal to the inside of th cell. 

3. receptors that are activated turn on one or more of three monomeric g proteins that are also along the periphery of the cell 

   1. rho 

   2. rac

   3. cdc42

4. g protein is going to recruit adpator proteins.

5. stress fibers generated bundles of linear mfs near pm that poke it

6. Cells promoting growth/polymerization of linear and branched microfilaments that push the PM in the direction of the signal, cell stretches and elongates . makes stress filaments

   1. linear mfs: (lipoprotein A) = rho. Binds formin protein. Plus end of mf binds to formin, formin binds to rho, rho is attached to pm. So mf pushes pm.

   2. Branched mfs (growth factor) = rab and cdc42. Wave or wasp protein. Recruit arp2 and arp3. Cause linear mfs to branch

7. cell retracts trailing edge (pm) via cofilin chopping microfilaments into tiny pieces . makes cell shrink and catch up 

• stress filaments: Need stretchy cell, long microfilaments, so that it doesn’t tear

• The resculping is rapid and reversible 

the cell appears to be doing nothing

microfilaments and microfilaments are very dynamic and are constantly going between dimerized state and then repolymerizing.

Aka a-b is polymerizing, and actin is polymerizing with itself 

stress fibers

allow cell stretch 

are microfilaments 

• made by rho and formin

polymerization to make MFs

Conversion of G-Actin to F-actin is Polymerization

• does not require ATP hydrolysis

• atp hydrolysis influences polymerization kinetics 

• monomeric form of actin is called G actin 

• G actin polymerizes into F actin, a microfilament 

• actin has Mg binding site. Mg will coordinate with P on adp/atp. no Mg on actin = no polymerization 

microfilament structure

spark notes:

• MFs have polarity: The 2 ends are structurally & functionally distinct

• Myosin "decorates" MFs & "points" towards the (–) end

• ATP cleft "exposed" at (–) end & "hidden" at (+) end

• the plus end is the barbed end

• the minus end is the pointed end

in depth:

• the structure of the mf is two actin chain things that wrap around each other

• then they polymerize, they make a double helix

• has two ends

  • one end is closed: the plus end.

  • one end is open: the left of the last actin is facing out: the minus end

• when actin binds, it always faces the same way 

in vitro experiences on dynamics of polymerization 

Polymerization is dependent on the concentration of actin

1. put actin in tube 

2. over time actin polymerizes into mfs with time lag. 

   1. the lag is from the rate limiting step: nucleation. the first 2-3 g actins coming together

3. growth plateau. steady stage with no net growth or shrinkage . all the actin is polyermized . called treadmilling

if get rid of the rate limiting step:

1. put free actin and some nuclei in tube (already clustered actins)

2. reaches plataeu faster

the kinetics of polyermization is different for each end. Bias built into system. Built into structure of actin. Always favors the plus end

 

Dynamics of actin polymerization & depolymerization in vitro: “Nuclei” limit polymerization

how cell speed up polymerization?

the rate limiting step is nucleation . get rid of this step to increase speed

• cell can make its own nuclei via formins 

• cell can make nuclei via fake actin, called actin relate proteins: arp2 and arp3 

• cell can have cofilin that acts like a pair of scissors and chips mfs into pieces. the fragements function as nuclei 

critical concentration

different for each end of the mf

the concentration of g actin needed to start adding to one end

lower for plus end

If [G-Actin] is < Cc (Critical Concentration) no polymerization occurs 

If [G-Actin] is > Cc net polymerization occurs

At steady state, [G-Actin] = Cc & no net growth occurs, called treadmilling 

Asymmetrical polymerization: what and why

additon to plus end (closed end) of mf is favored. 12x preferences

has lower critical concentration

actin is atp bound when being added. aptase activity of actin is slow. the terminal actin on the plus end is always an atp cap. the terminal actin on on the minus end is adp cap. the intermediate region is adp and p because it hasnt diffused yet

Dynamics of Actin polymerization & depolymerization in vitro: ATP & ADP caps

ATP is hydrolyzed after G-actin polymerizes into F-actin, but Pi does not immediately dissociate

"New" (+) end has ATP cap & low C+c  "Old" (–) end has ADP cap & high C–c

how to cells control actin polymerization?

40% of total actin is unpolymerized G-Actin instead of it all being polymerized

• concentration of actin is always way above CC of both plus end and minus end

• the concentration of actin never changes

changing polymerization mechanism

• capping proteins. makes plus or minus end disapear. protein called cap z (plus end capping protein) hides the plus end. the minus end could still grow. alternatively, stop the minus end from growing via tropomodulin (minus end capping protein)

• changing pool of available actin via binding to actin

  • increase pool: cofilin = molecular scissors. chops existing mf into tiny pieces from minus end. the pieces depolymerize into free actin (adp actin). since it is adp actin, it loses specificity (not higher affinity for plus end). in order for the actin to be recycled, need to exchange the adp for atp. can exchange on its own but very picky. speed up this exchange with profilin. this actin will now ONLY add to the plus end. now have a new pool of actin.

  • decrease pool: take actin out of process needed to make mf. thymosin beta 4 binds to atp actin and takes it out of the pool

making a linear mf

• rho = lipid anchored G protein, on pm . 

• Rho is bound to gtp. Gdp form does not do anything . 

• forms linear mfs , together with formin

• Formin . inactive form = paperclip configuration. Rho has region called the rbd. Rho binding domain. Can only interact with rho when rho has gtp bound do it. Activates the two other domains fh1 and fh2. The binding site for profilin. Brings profiling actin up from the cytsol. Loads up fh1 domain where it is handed off to fh2 where the mf is bound. 

• Formin is like a collar. Fits around plus end of mf. Profilin actin comes in and binds with very high affinity. Every time an actin is bound, the collar is pushed up another notch. 

• Rho activity is contingent of recepetor being active 

• When rho hydroylizses gtp to gdp 

  • Formin falls off 

  • No mf growth 

  • Cell signal activates the receptor ( and rho?)

Linear mfs (lipoprotein A) = rho. Binds formin protein. Causes linear mfs to polymerize. Generates stress fibers, bundles of linear mfs near pm that poke it. Plus end of mf binds to formin, formin binds to rho, rho is attached to pm. So mf pushes pm. Branched mfs (growth factor) = rab and cdc42. Wave or wasp protein. Recruit arp2 and arp3. Cause linear mfs to branc

making a branched mf 

• need some linear ones 

  • Starting with a linear mf. Want to push on pm at an angle 

  • Rac and cdc42 recruit a different adaptor. Called wasp or wave. 

  • Lacking one of these = cannot localize . immunocompromised. Cannot migrate. Inflammation continues 

  • Wasp and wave have variant rbd . a rho like binding domain but dont bind rho. They bind cdc42 or rac. Have a long hinge region. The activated g protein recruits rbd and hinge region interacts with mf at an angle. A 70 degree angle. G protein binds a new nuceleus. Recruit arp2 and arp3 to nuclear formation of banr d new mf. Nucelus is attached to existing mf. Will eventually hit the pm. Push in two directions at once. 

Linear mfs (lipoprotein A) = rho. Binds formin protein. Causes linear mfs to polymerize. Generates stress fibers, bundles of linear mfs near pm that poke it. Plus end of mf binds to formin, formin binds to rho, rho is attached to pm. So mf pushes pm. Branched mfs (growth factor) = rab and cdc42. Wave or wasp protein. Recruit arp2 and arp3. Cause linear mfs to branc

leading edge

• part of pm where extracellular signal is activating the receptors. Pushed by mfs. 

sequence to make cell move

• receptor activated by signal (only the ones near signal)

• those receptors activate g proteins, also on pm, through physical connection.

• the g proteins exchange gdp for gtp and are lipid anchored to the pm (rho, rac, cdc42)

• activation of g protein promotes binding of adpator protein (ex formin, wave, wasp ).

• they make the mfs (linear through rho and branched through rac and dcd42) and stress fibers (rho) and the mfs push on the pm

• need to detach trailing edge: rho also activates myosin. makes the adjacent mfs shrink and contract, and they are then fragmented by cofilin. rho phosphorylated integrins that causes them to release from fibronectin

spark notes:

• mfs are built at leading edge and chopped up at trailing edge

what if rac, rho, and cdc42 cannot exchange gdp for gtp?

called ocminant negative

aka what if they dont have enough g protein function to have the normal phenotype

in a normal dish: 

• Normal cells grow until form confluent monolayer.

• Called contact inhibition: will continue profilerating until each cell is touching a neighbor on each cell. Then stop. No longer proliferating 

• Mimic wound healing: scratch away a row of cells on dish. Cells near the scratch are no longer touching 

• Cell perceives the gap and causes cells to start proliferating. Takes 24 hours before actually have doubled in number . slow process. 

• BEFORE HEALING: Cells will migrate towards each other to fill in the gap until they can generate new cells 

• Cell migration is a short term response to proproliferative signals 

in cancer cells: 

• Cancer cells will proliferate without extracellular growth signal. Do not have contact inhibition. Would pile up on top of each other. Would have foci (?) cell clumps 

in no functional g protein cells: 

• No functional g proteins = less percent wound closure (aka less migration).

• comparing Rho, rac, and cdc42. Most compromised cell mirgration = rho.

  • Need the linear ones to make the branched mfs. No rho = no other ones either 

• No cdc42 vs no rac . rac has a bit higher effect but not really. Cell that express both cdc42 and rac have a but of redundancy. they can somewhat rescue each other if one is missing 

• dominant negative form of rho, rac, cdc42

microtubules vs microfilaments

microfilaments:

• bigger

• more dynamic (1000x more) in terms of growing and shrinking

• individuals mts can grow and shrink in the same environment , bc of rate of gtp hydrolysis

• MAIN DIFFERENCE: are gpt driven

• made of heterodimers, alpha and beta subunits. have different function

• has polarity —- always adds to beta

• going to be the tracks for vesicles, and does stuff with recruiting motor proteins

• all have the same origin: the centrisome.

  • the centrisome duplicates during mitosis c need two to make spindle. Duplicate when dna does and replicate with same mechanism . so usually one centrisoms sometimes two 

• no MTs in the nuc , all out in the cytoplasm

• hydroylzes gtp way faster than actin hydrolyizes atp

microtubules:

• atp driven

• Actin hydrolyzes gtp very slowly, so a couple at th eplus end stil, have atp.

• has polarity — prefer to add plus end

microtubule structure (more in depth)

• made of an alpha and beta subunit

• alpha - beta dimer = Tubulin

• Subunits are never separated. Have extremely high affinity to each other 

• subunits Are both gpt binding proteins. Alpha unit binds one and keeps it forever bc it is trapped on the inside of the heterodimer. Never hydroylzed 

• The action is always in the beta subunit. Has a cleft where the gtp binds. Tubulin is gtpase and can hydrolyze it. Basically a g protein. It is also exchanges, does not need a gef though.

polyermization

• Plus end has exposed beta tubulun

• Gtp immediately hydrolyzed when added 

• Have gtp cap, but the minute another added, will be hydrolyzed to gdp. Makes them way more dynamic 

  • so there is only a single gtp bound beta tubilin, and it is the one at the very plus end

  • if there is a lag in this and there is gdp but and no gtp cap, will cause catastrophe 

• Are built by making linear polymers called protofilaments. 13 of these = 1 mt. that is a singlet. Most prevalent form of mt 

• All mts have minus ends tucked away at end point, the centisome. the plus ends extend out towards PM

• Each minus point has two centrioles. They are always perpendicular to each other, and have mother daughter relationship 

  • The very base of the centrioles there is a gamma tubulin. the minus cap. Gamma turc ring complex. Makes the minus end invisible. 

taxol

•  inhibits gtp hydrolysis on beta tubulin. Binds irreversibly to beta tubulin.

• Cancer inhibition drug.

• makes that tubulin unable to polymerize. Stops cell from dividing, anti mitotic. Kills the cells. But no specificity. Horrific side effects 

• causes catastrophe/depolymerization event

what if rac, rho, and cdc42 cannot hydrolyze gtp to gdp?

change the morphology

are always on

they go crazy and the microfilaments fills up the entire cell

express a dominant active form of rho, rac, cdc42

what form are MTs in the cytoplasm

singlets

made of 13 protofilaments

this is the majority of MTs

what form are MTs in the cilia/flagella

doublets

13 protoilmanets, 10 protofilaments

what form are MTs in hte basal bodies/centrioles

triplets

13 protofilmanets, 10 protofilmanets, 10 protofilaments

How does tubulin concentration control the dynamic instability of MTs?

• More tubulin = nothing happens until hit critical concentration 

• Then convert tubulin dimers into mts 

• The CC at the plus end is really low . concentration of tubulin is not as abundant as actin, but usually 10-20 micromoles. Theoretically, no free tubulin in cells

• Mts are designed to be intrinsically unstable. Grow MT and make longer, at the same time also make it more and more unstable, so easier to get rid of it when the cell has to 

• Lag: nuclear event. Longer than in mfs. Bc mfs only need 3-4 actins put together. Slower here bc have to start growing individual protofilaments first, grow until 5-6 filaments, and then wrap around and generate a mt. Protofilaments are linear. Ones mt made, only then can they elongate them 

• There will be nuclei in cells for mts. The mts are much longer. Will never depolymerize into tiny pieces
  

dynamic instability of MTs

• How can have one growing, one not doing anything, one shrinking inthe same conditions? Called dynamic instability. 

• Any given mt is alternating between polymerization and depolymerization. Over minutes. 

• Transition between growing and shirnking/poly and depoly is called catastrophe . 

• Rescue = shrinking to growing. Deploy to poly . remnants used as nucleus. 

• Mts have no control over how long it will be before it hits catastrophe, or how much it is going to shorten before rescue 

• Shrinking is faster than growing. 

• All of this is determined by gtp or gdp . mt growing normally requires there to be the gtp cap on the plus end. if hydroylze it before another GTP tubulin is added, will have catastrophe. 

what causes MT catastrophe and why

• Taxol prevents beta tubulin from hydroylzing gtp. Makes stable mts. Keeps all of the protofilaments in gtp GTP-bound state, and will all be linear. Cannot depolymerize. 

• If for some reason pause adding gtp to plus end or existing mt, the whole thing will fall apart, catastrophe .

• Tubulin is  gap for the tubulin that is already there

• Every time add, replacing gtp with gdp at the plus end and then adding gtp 

• Growing mt = less and less capable of holding itself together. If gtp not at plus end = catastrophe. Protfilanets seperate and break off into pieces that dissociate back into tubulin dimers. Has gdp bound to it. Gdp dissociates and binds gtp

• Rate of gtp hydrolysis on temrina beta tubulin = important thing 

-tubulin on the “newest” dimer is a GAP that stimulates GTP hydrolysis on the “older” -tubulin (+) end

Therefore, only an actively elongating MT has “new” GTP•Tubulin at its (+) end

If growth stops, then slow GTP hydrolysis ultimately converts (+) end to GDP cap & catastrophe results

In summary: A growing MT tends to keep growing while a static MT will tend to shrink

so 

• if mt hydroylizes its gtp cap before another is added behind it = catastrophe 

  • hydroylzing too fast

• if mt does not add next gtp cap in time = catastrophe

  • not adding next one fast enough

microtubule associated protein (list)

EB1/3, MAP2 and Tau

EB1 and 3

• only bind to plus end. EB1 and EB3 can also move along length of mts but are not motor proteins. EBs can move over to new tubilin added. Moves to plus end . give ability to carry cargo outside of vesicle. Takes cargo with it towards the plus end, which is at the PM. 

• Altnerative way to deliver cargo to PM other than secretary pathway 

• Also promote rescue and catastrophe. 

• EB1: destabilizes bc every time boudn to plus end, pulls the protofilament away from the others. Eventually fall off when it shrinks and binds to another if delivering cargo to pm . 

• EB3: on plus end . stops depolymerization. Stabilizes the protofilament. Holding the ring of protofilaments togethe rlong enough fo new tubulin to add (rescue)

• Hops from one end to the next, no atp reqreuiment 

• Both can take cargo 

MAP2 and Tau

microtubule associated protein

play role in neurons 

MAP2 & Tau bind to the surface of MTs & stabilize them

MAP2 has a long "spacer" arm & increases spacing between MTs near cell body

Tau has a shorter "spacer" so MTs are more closely packed closer to axon terminus

MAP2 & Tau alter the spacing between MTs along the length of an axon

neurons also move cells long their axon to the terminal branches btw 

• Near cell body , the circumference of neuron is much bigger and thens shrinks toward terminus. All the Vs are being transported by motor proteins on mts. Need to space them to keep them far enough apart so that they dont aggregate and run into each other . if this happens = AD. perturb vescile transport to axon terminus 

• MTs are nice and evenly space because of MAP2. keeps them far enough apart . binds to seam betwee npart of protofilaments. Tau also binds there, but it smaller so can bind further down there the axon gets skinnier 

• Map 2 is very large, tau is very short 

• Map2 can work i nnon neuronal cells and larger part of neuron. . until reaches transition zone. Where the axon is narrowing and map2 is too fat. Replace with tau 

• tau Has a shorter arm. MTs are still separated but are packed more tightly. 

• Motor proteins carrying the Vs are using so much atp, that the  neurons have to bring a mechanism to make atp with them. Needs to bring mitochondria with it up and down the mts. So they mTS need enough space to move things up and down 

• This dumbass bug im gonna crash out  

what contributes to the progression of Alzheimer’s Disease (AD)?

• Two proteins that are implicated in early and late onset: both needed to stop aggregating 

• 1: Tau group. Mutation in tau (non-conservative AA substitution) creates site that can be phosphorylated that isn’t usually there. Makes tau aggregate and condense, MTs near axon terminus are no longer spaced. Tau creates tangles. Causesthe cytoskeleton to collapse . cell starts filling up with Tau and Vs that cant be delivered. Proteosome cannot keep up , lysosome cannot keep but. Cell dies. Toxic form of tau 

• 2: beta ammuloid. Secreted protein that can enter cells and also can lead to aggregation. 

faster gtp activity in MT

more unstable

more likely to have catastrophe/depolymerize

correctly describe the role of ATP hydrolysis in MF dynamics

-not required

-but influences the polymerization kinetics of converting G-Actin to F-Actin.

What distinguishes the plus (+) and minus (-) ends of a MF?

The (-) end has exposed ATP cleft and myosin decorates “pointing” towards it.

what accounts for the initial lag in the conversion of G-Actin to F-Actin?

A small # of individual G-Actin proteins (~3) must first polymerize to form nuclei which can then be rapidly elongated to longer MFs.

graphing definition of C_c

G-Actin is converted to F-Actin once its concentration exceeds the C_c

Cytochalasin D binds specifically to the (+) end of F-Actin and prevents further polymerization at this end. The C_c of  ATP-Actin for the pointed end of F-actin is 0.6 µM and the C_c of G Actin for the barbed end of F-actin is 0.12 µM. At what concentration of ATP-Actin would only the pointed end of a MF elongate if incubated in the presence of excess cytochalasin D?

Anything over .6 

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

How does Thymosin β-4 affect MF dynamics and/or structures?

Thymosin β-4 binds ATP-actin & inhibits its incorporation at either the (–) or (+) end of a MF.

How does Profilin affect MF dynamics and/or structures?

It rapidly converts ADP-G-Actin to ATP-G-Actin & remains bound so that this complex can only add to the (+) end of a MF.

What is the primary function of cofilin during directed cell migration?

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

profilin doesnt work. what happens?

ALS cells have reduced ratio of F-Actin:G-Actin.

aka more g actin idk why he makes everything as convoluted as possible

when cofilin chops off, they are gdp, and they will not be converted and will not be added back. therefore, there is less F actin and more G actin

Toxin covalently modifies Rho. MF cytoskeleton collapses and no cell migration in response to extracellular growth factors .skin abrasions in Pa infected individuals heal very slowly. how does PaTox affect Rho function?

PaTox prevents Rho from exchanging GDP for GTP

Cells expressing a dominant active form of Rho. what cause? 

Phalloidin inhibits the conversion of F-actin to G-actin.

What is never found “free” in a cell?

the two tubilin dimers

Why is ApoE4 a risk determinant for AD & what is the link between TauP301S & ApoE4?

• Apolipo protein E = carrier , neuronal specific carrier for HDL> neurons need C but cant make their own. Predominantly hdl: astrocytes package the HDL and secrete it so the neurons can uptake it 

• there is ApoE2, 3, and 4

  • 3 is the prevalent form and contributes average risk to alzehimers. Arg158 and cys112 

  • 2 is much less common. Cys158,cys112. Protects against alzheimers. Increases risk of hypercholesterolemia bc the variant does not bind to receptor as well. Lower affinity of binding hdl to hdlr. Higher C in blood

  • 4 is 20% of pop. risk raising. Arg158 and arg112. 

• apoe3 worsens affect of tau mutation

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

MT polarity necessitates the use of directional motor proteins

(–) end motors (e.g., dyneins) carry cargoes towards (–) end (retrograde transport)

(+) end motors (e.g., kinesins) carry cargoes towards (+) end (anterograde transport)

• MTS radiate out towards PM with plus end

• while they are moving, the MTS may be lengthening and shortening.

• motor proteins go along the MTs. kinesins cant make it all the way to the PM in antergrade transport bc there is a gap. go along mfs instead via myosin motor protein

  • conversaely, in retrograde transport, the vesicles go along mfs first, and then get hitched by motor proteins (dyenin) on mts towards the centrisome

• Motor proteins walk along one protofilament, so they can have 13 on one mt. they dont collide. They can go in opposite directions 

troubles:

• Mts are growing and shrinking

  • v has to skip between them while they are moving,

• MTS dont go all the way to the PM (there is a gap). Cannot be carried by MT motor all the way to the PM. but there are mfs and myosin motors instead

  • V has to be handled off from kinin to myosin motor.

name of directional motor protein towards plus end of mt

Kinesin

• Motor proteins move very quickly 

• Consume a lot of ATP

• Tall and skinny and have heads that interact with mts. other portion binds to the Vesicle. 

name of directional motor protein towards minus end of mt

dyenin

• Motor proteins move very quickly 

• Consume a lot of ATP

• fatter than kinesins 

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

• The dendrite has a bunch of mts that are free and not connected to centrosome. Are much more dynamic bc have free minus end. They are randomly arranged. 

• Axonal transport requires generation and production of neurosecretory vesicles. anterograde transport.

  • Made in ER/golgi and then transported by kinesins down the length of MTs.

  • all the MTS still have plus end towards PM but only some of them are attached to the centrisome / are in fragments.

• V are transported via motor proteins (kinesins) that hop on mts until they make it to the pm. 

troubles:

• NEURON SPECIFIC: have to hop along fragments bc mts would never be so long as to span the entire axon

• NEURON SPECIFIC: Have a bunch of possible presynatpic sites (where

• NEURON SPECIFIC: Has a bunchof cargo

• Mts are growing and shrinking

  • v has to skip between them while they are moving,

• MTS dont go all the way to the PM (there is a gap). Cannot be carried by MT motor all the way to the PM. but there are mfs and myosin motors instead

  • V has to be handled off from kinin to myosin motor.

squid giant axon research 

found a motor protein (kinesins)

• 1) reconstitute anterograde vesicle trafficking . add taxol, add MT, add Vs . nothing happens. V dont even bind to mts. 

• 2) take MTS, Vs, axoplasm (axon goo). Nothing happens. 

• 3) need ATP. take MT. axoplams, ATP, Vs. V will bind to MT and move in the same direction (turns out to be a kinesi) 

• Non hydrolyzable atp = v can bind to motor protein/mt but cannot move. 

• 4) isolate axoplasm and purify

How does v know that it needs to bind kinesin or dynein?

• Because rab. Allows binding of kinesin

What distinguishes GTP binding and/or hydrolysis by α and β tubulin?

Both α- and β-tubulin bind GTP, but only β-tubulin hydrolyzes GTP and exchanges GDP for GTP.

What is the function of

-tubulin?

To terminate the (-) ends of MTs within the centrosome.

How does the addition of tubulin at the (+) end of a growing MT paradoxically cause it to be more susceptible to undergoing catastrophe?

Newly added a-tubulin activates GTP hydrolysis on the b-tubulin at the (+) end

EB3 is a + end MT binding protein. How can EB3 potentially stabilize MTs that underwent catastrophe when it rescues them?

 

EB3 increases the interactions between curved protofilaments.

EB3 increases the affinity of a MT that had undergone catastrophe for tubulin to bind to the (+) .

Tubulin isoforms or post-translational modifications that would destabilize MTs could do so by:

Increasing α  tubulin GAP activity and/or increasing β  tubulin GTPase activity

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.