Cytoskeleton: Structure & Dynamics Flashcards

Question

Methods for measuring MT polymerisation:

Answer 1

Light Scattering Electron microscopy

Answer 2

MTs can become v long/stiff so give light scattering interference Light scattering (proxy for extent of polymerisation) against time: Have long lag phase of low scattering then rapid extension - quick increase in scattering Then Oscillations at higher steady state (due to GTP hydrolysis)

Answer 3

Use cilium from a Paramecium Remove membrane w detergent giving premade nucleation point for MT growth original Cilium is darker - so can see new Tubulin addition More dimer addition at PLUS end can measure extent of poly- at certain time points BUT have to stop it to measure can only do this once per so need to do many experiments to do diff time points repeat for multiple repeats and organisms so can be issues w continuity between them?

Answer 4

Plot of amount of filament against time: -Lag phase of v low filament for period of time -Then sudden quick growth phase, amount of filament quickly increases here -then the Equilibrium phase - steady state of higher levels of polymer

Answer 5

Need to form a Minimally Stable Nucleus -for actin polymerisation this is 3 subunits (2 in one strand, 1 in the other-beginnings of one strand buttressed by subunit in another) due to the weak bonds along protofilaments needing to be strengthened by lateral bonds and then for more subunits to be added The more subunits attached - the less likely the filament is to disappear and then reach growth phase

Answer 6

AKA Steady state phase where addition rate = rate of depolymerisation Never reaches 100% filamentous there will always be a Critical Concentration of monomers that does not polymerise

Answer 7

-- the conc of monomers that will never polymerise - means can never reach 100% filamentous -- also is the conc beneath which no new polymerisation can occur - as no new nuclei are formed -- also is a measure of affinity for the end of the filament -- greater affinity gets it closer to 100% Filamentous

Answer 8

allows cell to control where actin polymerisation occurs to some degree as it need the nucleus to begin even a small conc of nuclei added obliterates the lag phase

Answer 9

Cc is Kd Kd is given by: Conc of F-actin*Conc of free actin divided by conc of F actin so Kd=conc of free actin = CC at equilibrium addition of subunit to F-actin doesnt change no. of F-actin molecules (just No of subunits in it)

Answer 10

plot two lines Fluorescence intensity against Actin conc one line for low salt, one for high low salt: v small increase in fluorescence with actin conc due to higher proportion of G-actin High-salt: same actin conc gives much higher fluorescence (higher F-actin) These two lines dont cross at 0 but in fact at 0.11 micromolar tells us that Cc/Kd=~0.11 micromolar

Answer 11

is the same at each -use binding proteins-preferentially bind P or B end -so can look at Cc of one end specifically -Plot polymerisation state against Actin conc -lines have diff gradients - so DIFF rates of addition -BUT they cross at same point so have SAME Cc/Kd makes sense as addition at P and B ends both entail making the same No. of lateral and pointed/barbed contacts so subunit affinities are identical so no energy here to make polymerisation go in one direction rather than other at steady state filaments wont grow or shrink subunits come on and off at fixed rates therefore cannot do work here

Answer 12

centre of Actin subunit: Nucleotide (ATP) binding site ATP hydrolysis differences between the ends causes them to be more different

Answer 13

When affinities Differ G-actin: bound ATP is protected by hydrolysis - stays as ATP form F-actin: subunit conformational change when G-actin joins filament, increases hydrolysis rate of the nucleoside triphosphate ATP G-actin predominates ADP form so most actin joining filament is ATP form the longer the subunit stays in the filament the higher the chance of its ATP being hydrolysed Rate of addition higher at Barbed end so hydrolysis catches up to Pointed first this now gives a difference between P and B ends (ATP form vs ADP form)

Answer 14

Repeat the experiment used to find the Cc/Kd of each end (P/B) except with the B in ATP form and P in ADP form the lines cross the [Actin] line at diff points the ADP form has a higher Cc/Kd - hence lower affinity this causes growing at B end and depoly at P end - Allowing treadmilling

Answer 15

when [Actin] is: Below Cc for pointed end (ADP-actin) Above Cc for barbed end (ATP-actin) cause net addition at B end and net depoly at P end this difference between the ends allows there to be WORK The ENERGY INPUT comes from the ATP hydrolysis - used in the timing mechanism of ADP vs ATP forms likelihood to come off Filament

Answer 16

focus on Same subunits over time subunits coming on more on one side and subunits coming off more from the other looks like filament is "moving forward" relative to these subunits could push against membrane to move it forward

Answer 17

MTs growing and shrinking similar phenomenon to actin stuff this causes the oscillations seen around the MT steady state due to GTP hydrolysis Cc at plus end much lower than at minus end there is a point of [tubulin] between these where the off rate of minus end is matched by in rate of Plus end Ahead of it is region of Growth Region behind it (still between the Ccs) is Region of Filament Instability

Answer 18

there will be overall depolymerisation (catastrophe) this is what causes MT to grow and shrink (dynamic instability)

Answer 19

Tubulin that adds onto filament is GTP form GTP form has v low Cc - so high affinity for ends the longer a subunit is in the MT - Hydrolysis will catch up if it manages to catch up to the Plus end of the MT then affinity of Tubulin addition for that end is reduced this gives CATASTROPHIC DEPOLYMERISATION - rapid shrinkage but while this happens if enough GTP tubulin has managed to bind - it forms a stable GTP cap - rescuing the MT from catastrophe

Answer 20

is a good search mechanism eg could be used by mitotic spindles to feel out and capture chromosomes -capture chromosome = STOP -no chromosome = retreat + try again

Answer 21

When the GTP is hydrolysed causes the individual protofilaments making up the tubule to become curved protofilaments become less stabilised by neighbours so more likely to depoly at this end basically unfurl in catastrophic depoly When GTP cap present there is stronger associations between neighbour protofilaments

Answer 22

-Sequestering proteins -Filament Cleavers -Gel strengtheners

Answer 23

bind monomeric actin prevent it from joining filament used to keep a reserve of G-actin for when it is necessary Profilin: binds polyprolene in PM - brings G-actin to site of action also binds actin at P end preventing addition there funneling it to the more productive end Capz: binds 2 actin monomers at barbed end - prevents addition of subunit to B Caps the B end of older filaments so G-actin is used more productively in newer filaments

Answer 24

cleave filament break down the gel eg Gelsolin can study using viscosity Cofilin preferentially destabilises Pointed end severs them has a greater affinity for ADP subunits so targets older pointed ends -releases ADP actin so it can replenish ATP and join barbed end

Answer 25

Filamin: antiparallel actin binding heads - one end binds one filament and the other end binds the other Alpha-actinin is similar; but has a shorter linker between binding heads so forms actin cables can also study w viscosity ig

Answer 26

microtubule associated proteins have MT binding domains some proteins have larger extensions which emanate from surface and form connectiosn btwn MTs to control spacing Tau protein can fold and form filaments associated w Alzheimer's

Answer 27

Tau often found in neuron axons binds MTs and affects spacing w its spacer transfect Tau expression plasmid into non-neuronal cell causes development of processes that resemble axons

Answer 28

hard to do biochemically as need to fractionate cells - erasing the mechanism Instead use LISTERIA ROCKETING rocket through cells allowing travel between cells w/out going extracellular they subvert actin polymerisation to themselves Fluorescent Phalloidin tag shows this add actin to Listeria (w Rhodamine Phalloidin stain) then add diff fractions of the eukaryotic cell certain fractions allow actin Poly at listeria SO it is binding something in the cell allowing this

Answer 29

complex of 7 proteins (because come out in same fraction) come together form Arp2/3 complex can be recruited toward membrane by activated WASP or WAVE complex

Answer 30

Arp= actin related proteins Arp2 and 3 are more similar to barbed end the other non-Arp proteins in the complex come together to hold them in place so Arp2 and 3 can act as barbed end of stable nucleus and form a template to control where actin nucleation occurs

Answer 31

occurs by side binding of the complex to an existing filament at 70degree angle causes formation of reticulated network/gel -maximally non-commital about direction in which cell will go -net movement is the angle which bisects the processive 70 degree angles is signal came that stimulated movement to the left then would just need to favour the filaments going toward the left makes for better poised machinery that can move should a signal its following change direction

Answer 32

fluorescent label actin photobleach spot on it looks like the mark is moving backwards as new subunits are added at barbed / removed at pointed relative to this point it the cell is moving forwards getting productive movement requires the actin filaments to stay stationary relative to substrate done using FOCAL CONTACTS since the filament is stationary relative to substrate new polymerisation at the membrane pushes the membrane FORWARDS

Answer 33

using interference microscopy look from beneath at an attached cell thorugh interference phenomena places where membrane is close to slide show as black destructive interface then look at same cell through fluorescence of actin see actin cables all end at places where membrane is close to substratum at FOCAL CONTACTS actin attaches non covalently via receptors

Answer 34

actin filaments held together by alpha-actinin Vinculin - an adapter between Talin + the inside of the integrins AND the actin filament then Talin Then integrins which connect to Fibronectin in the basement membrane means that actin is connected to the outside of the cell any polymerisation at the ends of the filaments that are cross-linked and connected to these stress fibres causes the membrane to be pushed forwards -because filament is connected to larger network attached to substrate allows actin polymeriasation work to push the membrane

Answer 35

produce contractile forces contract to cause hydrostatic pressure within cell and push cell contents forward Have ATPase domain attached to : -Tail which can bind cell membranse (Myosin I) -or to longer alpha helical coiled coil tails the coiled coil allows formation of Bipolar Filaments

Answer 36

occur in folded soluble complexes if the light chains are phosphorylated: there is a folding back of the coiled coil connecting to the light chains making the folded diffusable molecule

Answer 37

myosin light chain kinase -activated by Rho G-protein phosphorylates the light chains interrupting the interaction unfolds the molecule into bipolar filaments 1/2 of molecules face one way, other 1/2 the other way -attached at each end to different actin filaments in a gel -by each head walking over the filaments it can squeeze the gel -causing it to contract - creating hydrostatic pressure on the more fluid cytoplasm in the centre of the cell -pushing cell contents forwards as the cell geos forwards

Answer 38

mostly in back of cell contraction hydrostatic pressure in back pushes against the lamellipodium pushed cell forward

Answer 39

rich in the leading edge of cell connected to membrane by its C-terminus walks the membrane towards the barbed end of the filaments i.e. walking the filament forward

Answer 40

Dictystelium KO of Myosins (not yeast as they chitinous cell wall and not normal eukaryotic cell motility) though in Dictystelium mutations are not as stable KO myosins - cells just move uncontrollabnle in all directions at once w no direction/polarity of movement suggests WT myosin is necessary for proper cell motility

Answer 41

speckles of Rhodamine Phalloidin fluorescence in Fish keratinocyte watch movement w respect to cell appears as if the cells stay in one place while membrane is pushed forward but if look relative to cell boundary: -there is a backward FLUX of actin -due to new poly ahead of that pushing the membrane -the actin speckles are attached to actin, indirectly attached to substratum -speckles move back and then disappear in the regions where there is myosin II at the back (some evidence that tension on actin filaments by myosin II facilitates Actin interaction w other proteins that cause depoly) backward flux is FASTER at front of lamellipodium slows down as go back Blebbistatin inhibits Mysoin II adding it stops cell movement AND the backward actin flux presumably due to the actin not being disassembled at back of cell to provide new monomers for the backward flux and push membrane forwards

Answer 42

-Gel like actin cortex just beneath PM is most developed in Lamellipodium -less actin near the more fluid centre -connections connect actin cytoskeleton to underlying substratum through focal contacts -Lamellipoium moves forward through treadmilling and preferential nucleation of actin at leading edge of cell -at same time Myosin II contracts actin cortex at back placing hydrostatic pressure on more fluid central region of cytoplasm pushing it forward -and MIGHT also be involved in removing Focal adhesions at the back of the cell allowing new ones to form at the front of cell (specific proteases involved) by the two mechanisms of: CORTICAL TENSION created by myosin and NEW POLYMERISATION at lamellipodium the cell moved forward

Answer 43

even though Cc is 0.11 micromolar certain ABP (profilin, Thymosin B) sequester it keep it in reserve

Answer 44

profilin sequesters G-actin for when needed binds the barbed end of the monomer so it directs addition at barbed end of filament Nucleation of new filaments at 70degrees from last filament by Arp2/3 complex localises nucleation toward receptor sites following external stimulus also makes sure polymerisation is localised near the PM Filament barbed end binding proteins (CapZ eg) prevent additions of Profilin:Actin to older filaments so instead more likely go new filament cross linkers (Filamin eg) connect actin filaments to each other so that they are all at indirectly associated w focal contacts then as filaments become older ATP hydrolysed to ADP Cofilin/gelsolin (prefer bind ADP actin) sti,ulate depoly actin released from filament as ADP-G-actin promotes exchange for ATP ATP has larger affinity and greater ON rate for Barbed ends

Answer 45

integrating chemokine gradient signals affecting the polarity and growth of the CS through Small GTP binding proteins eg RHO normally binds myosin light chain kinase Lysophosphatidic acid (LPA) binds the TM receptor, indirectly leads to Rho activation leading to Myosin II activation Rac G-protein affects lamellipodium formation activates Arp2/3 and cofilin Cdc42 g-protein causes filipodia formation, Formin localisation

Answer 46

are prenylated at C-terminal cysteine (for Cdc42 its a Geranylgeranyl modification) is a lipophilic molecule Interacts with membrane so they only diffuse in plane of membrane In GDP form when receptor inactive (default) receptor activation: leads to GTP exchange factor localisation close to site of activation at the membrane leads to exchange of G protein of GDP for GTP GTP form G protein has diff conformation allowing it to bind an effector leads to effector response near the membrane (due to membrane localisation of G-protein) This allows local effects on cytoskeleton in response to chemokines/other signals

Answer 47

N-WASP Wave complex

Answer 48

is an intermediary necessary for listeria movement? not present in platelets is autoinhibited in inactive form has an acidic and basic region that bind to each other causing protein to fold back on itself CRIB domain is adjacent to Basic domain is the binding site for activated G-protein Acidic domain binds/activates Arp2/3 complex but is cryptic in inactive form active G-protein at membrane near active receptor CRIB domains binds breaks interaction btwn acidic/basic regions maked acidic Arp2/3 binding domain accessible Arp2/3 binding to A region changes its conformation causing the two actin like proteins to be in Filament conformation available for G-actin addition to form filaments

Answer 49

WASP family Verprolin-homologous protein complex of many proteins has acidic binding region sequestered in similar way to N-WASP Binding to Rac1 g-protein displaces the: -V-helix -and C-helix so that they are available in cytoplasm (Acidic region follows C-helix) where they can bind and activate Arp2/3 WAVE complex is the most important on found from sequence homology to a more minor actor N-WASP

Answer 50

intermediaries: N-WASP WAVE complex are activated by g-protein then activate Arp2/3 that then nucleates Formins are directly activated by G-protein and also direclt nucleate Actin

Answer 51

dont have an adapter protein are directly activated by G-protein in Budding yeast (S. cerevisiae) budding is indicative of Haploid form have A and Alpha mating types also diploid form after A/Alpha cell fusion fusion occurs after repolarisation of actin cytoskeleton causing cells to grow towards each other after which nuclei fuse controlled by a/alpha mating factor secretion cause opposite type cell to repolarise in that direction forming a Shmoo -patches of actin form at shmoo tip -pointed ends towards nucleus -MTs also orient w minus end towards nucleus, provide track for them to move towards each other -this allows trafficking of vesicles of membrane components for building the extending shmoo

Answer 52

look for cells that grow as haploid but cannot form shmoo (or for temperature sensitive mutant that cannot do either at restrictive temp) identified all the Smoo formation actors this way named the mutants Sterile mutants

Answer 53

is a G-protein coupled receptor a TM spanning receptor when inactive binds to heterotrimeric G protein complex of Alpha, Beta, and Gamma Beta and gamma are lipidated so stay by membrane activation causes Alpha subunit to sever its connection to beta and gamma Beta and gamma can only diffuse by the membrane and only close to where they are activated cdc42-GDP also prenylated- stays membrane localised Released beta/gamma subunits localise the GEF cdc24 to cdc42 converting it to its GTP form Active cdc42-GTP binds Formins - which nucleate actin

Answer 54

Crib domain - G-protein binding domain Acidic domain next to it Basic domain at other end looping back to bind Acidic domain Between A and B domains: Formin homology regions 1 and 2 (FH1, FH2) when acidic and basic domains bind each other FH1/2 are sterically inhibited cdc42-GTP (or other active G-protein) binds the Crib domain (only happens close to membrane, close to active receptor eg Ste2) -Acidic and basic domains' interaction interrupted - FH1/2 exposed FH1 is proline rich - which profilin binds to - this localises Profilin/G-actin complexes to the active receptor to create a pool for nucleation FH2 domain forms a dimer which is site of nucleation

Answer 55

strange way to do it as when subunits are added - need to then follow the new end which has just been created by subunit addition (is not an issue for Arp2/3 as nucleates from other end?) How does FH2 begin nucleation at Barbed end and continue it even though filament barbed end is growing away

Answer 56

due to the helical twist between subunits they are at 167degrees (13 short of 180 deg two fold axis between them) FH2 dimer forms ring is a symmetrical dimer with 180 degrees between them if subunit no. 1 is the furthest to barbed end then Subunit 1, 2, and 3 are forced by the dimer into 180deg conformation (strained from normal 167) No. 3 then goes into 167deg conformation to better bind into filament so no longer interacts well w the formin so one of the FH2 domains in the dimer ring so no longer binds the actin This frees up space for new subunit to bind to the freed up formin domain previouslt bound to 2/3 Subunit 0 can bind filaments formed like this are STRAIGHT not BRANCHED

Answer 57

axons melanocytes Golgi ER

Answer 58

done over cytoskeleton as regular diffusion takes too long - need motor proteins Transcription/translation in cell body Inject large conc radiolabelled/hot amino acids then after inject large conc non-labelled/cold to dilute out signal leaves pulse of radiolabel (pulse chase) cut up squid axon after certain time points can see how far diff proteins moved diff cargos that go diff speeds slow and fast done by same motors, just more or less continuously

Answer 59

Colour change in african Chiclid males fight start black change to white when submit melanocytes contain melanin vesicles dispersed vesicles = Black aggregated to cell interior = white Kinesins used to move them out Dyenins create white colour by collecting the vesicles in by the MT organisation centre manipulated via cAMP levels decrease - outward movement increase - inward movement

Answer 60

in cell about to divide: two foci around the two MT organisation centres and spindle poles as that have duplicated before prophase prophase: Golgi evenly distributed between the two poles interphase: after division golgi is only on one side close to MT organising centre suggests dyenin moving them to minus end of MTs ensures even segregation of golgi into daughters depolymerise MTs - golgi end up all over cell active process with ATP hydrolysis to keep them there

Answer 61

stain for both ER tracks along the shape of the MTs - is coincident with them consisten w kinesin motors moving the tubular reticular ER network over MTs

Answer 62

Kinesin - from minus to plus end Dyenin - from plus to minus

Answer 63

Alpha helical coiled coil globular domain at N terminus: -Has ATPase and MT binding -is the motor domain Adapter proteins at C terminus that bind vesicles other sorts of kinesins with slightly diff organisations motor at C terminus/ middle too allows diff sorts of cargo binding and diff movement

Answer 64

work in hand over hand direction move over protofilaments in the tubule is important to have 13 protofilaments in the tubule ring so that they are parallel to MT axis means kinesin doesnt have to go around helical path

Answer 65

from giant squid axon big sample of axoplasm axon is process, extended out of cell so is already somewhat purified in contents fractionate squid axoplasm and add to purified MTs see which direction they go in

Answer 66

Spin down MTs - brings down Motor proteins and also MAPs w no motor activity can Stall Motor proteins if add AMP-PNO non hydrolysable can restart if add ATP - lets them be removed so: -spin down MTs - remove axoplasm contaminants -take pellet - resuspend in ATP presence -then spin again to separate Kinesins and non-motor MAPs that didnt separate from ATP addition -preferentially purifies kinesins -Then take Pure MTs + AMP-PNO and add to this supernatant -only kinesins will bind in AMP-PNO presence -throw away supernatant - further purify -then add ATP to release Kinesins repeat cycle to further purify

Answer 67

many heavy and light chains 2 motor heads - AAA+ repeat protein hexamer ATPase: -hydrolyses in cyclic fashion (domain looks like donut) -in doing so affects an insert into one of the AAA+ domain proteins -connection to MT is localised to small domain at the top - antiparallel alpha helical coiled coil which undergoes conformational changes in the ATPse cycle and circles around the Hexameric AAA+ domain -end of the AH coiled coil contacts the MT -Cargo attached at other end

Answer 68

Can use taxol to stabilise it (expensive) OR use high GTP in buffer (ends of MTs now capped so no catastrophe) complex doesnt come off in pure MT in presence of GTP - remains bound so use impure MT because in impure purification can get enzymatic activity that takes gamma phosphate of GTP and transfers it to ADP -> ATP converts GTP to ATP which REMOVES dyenin GTP alone will not (but it will remove kinesin) so have MT w kinesin and Dyenin bound add GTP -removes kinesin and leaves dyenin spin down to leave kinesin in supernatant then resuspend pellet add ATP to release dyenin into the supernatant when spun

Answer 69

discovered from mouse breeding mutation in myosin V affecting pigment distribution giving dilute coat moves along actin network transports melanin vesicle into hair shaft gene found because breeders keeping good records

Answer 70

Kinectin - vesicles to kinesin Dynactin - connects dyenin to vesicles Rab27a and melanophilin connect mysoin V to melanin vesicles

Answer 71

binds kinesins also evidence that may bind dyenin too could be that vesicles have more than one motor protein and when there is movement to plus end dyenins inactivate and then same vesicles can be moved back by dyenin by reciprocal dyenin activation/kinesin inactivation discovered by: detergent treat membrane - makes microsomes purify then column with Ab against kinesin pulled down with 160kd protein kinectin take kinectin Ab another microsomal fraction gradient Ab staining for western blot for kinesin and kinectin show up in same fraction suggesting presence in same vesicle/association

Answer 72

Dyenin adaptor has 7 actin related proteins forming a mini helix - Arp1 purifying dyenin as seen before its moving activity is lost so sucrose gradient fractionate cell contents see which fractions restore dyenin movement - going longer and longer before falling off -identified dynactin has: -short Arp filament capped at both end by proteins -p150 protein associated too

Answer 73

dilute mouse is deficient in Exon F of this which locates the adapter for melanin vesicles -melanophilin complex between melanophilin and Rab27a (small G-protein) melanophilin mutant Myosin V diffused throughout cell Myosin and melanophilin no longer coincident order: F-actin Myosin Va bound to it by motor domain Myosin Va exon F bound to melanophilin melanophilin binds Rab27a-GTP which binds vesicle

Answer 74

move down axon via kinesin on MTs carry myosin along too use myosin to span actin cytoskeleton at end to dock w synapse

Answer 75

- centrosomes have pair of centrioles arranged perpendicular - in prophase centriole pairs separate to opposite poles - in prometaphase spindles from them attach to proteinaceous plaques on centromeres on chromatids - in metaphase once all aligned, sister chromatids disconnected from each other in anaphase sisters move apart Purse string action of actin and myosin at midpoint draws in creating two pouches which separate into daughters in telophase

Answer 76

these cell cycle events in mitosis are deiven by cyclin/Cdks Cyclin E important in early phases Cyclin B important in MITOSIS

Answer 77

Centrosomes go on to become Foci for spindles the spindle poles duplicate to form these Cyclin E important

Answer 78

9-3 config of MTs but these in the centriole are NOT CONTINUOUS with the MTs dont form direct template 9 fold symmetry due to protein SAS-6 forms homdimers that form the 9 fold symmetric complex star -around this form template for cartwheels of triplet MTs that then form the centriole end up with 2 perpendicular centrioles amorphous material around minus ends in the material plus ends emanate out

Answer 79

MTs emanating out of pericentriolar material within this is complexes made partly from ring isoform of tubulin: Gamma Tubulin binds the Beta tubulin (odd as minus end ends with Alpha T) Gamma-tubulin complex forms MT nucleation template preferentially nucleates MTs at centrosome 3D starburst of MTs radiating out

Answer 80

CDK2-Cyclin E complex initiates duplication

Answer 81

nuclear envelope breakdown btwn Pro/Metaphase (lamin phosphorylation-> depoly) Centrosomes then start to form half spindles from each pole which interdigitate some capture sister chromatids (catastrophe search mechanism) need large reorganisation of cells MT network so Microtubule stability Decreases before M-phase

Answer 82

introduce fluorescent tubulin photobleach MT w laser see recovery time half lives: -interphase: ~5mins -metaphase: ~15secs -so much more dynamic in meta than inter a MAP that stabilises MTs: unphosphorylated XMAP 125 CDK2-CyclinB phosphorylates it so no longer binds MTs so they become more dynamic as are stabilised less in tubule conformation by XMAP 125

Answer 83

more dynamic MT cytoskeleton in metaphase than inter MINUS directed kinesins (weird) go toward MT organising centre These kinesins are connected in a homotetramer, but are bound to diff MTs so as approach organising centre cause MTs to splay out affect whole network Kinesin C (aka BimC)

Answer 84

Plus end multimeric motors (kinesins) bind to opposite oriented MTs from opposing half spindles walk to plus end on both pushes them out ALSO Membrane bound dyenins walk Astral MTs toward minus end pulling the centrosomes toward opposite sides of cell toward membrane multimeric-kinesins and membrane bound dyenins working together to setup chromosome separation axis

Answer 85

Dynamic instability search mechanism If MT captured by proteinaceous plaque - gets stabilised preventing catastrophe not captured: catastrophe, then rescue, can grow out and search again MTs that capture kinetochore on sister chromatid become are kinetochore MTs some never find one

Answer 86

MTs emanating from centosomes that are not connected to kinetochores not spindles

Answer 87

prometaphase -> metaphase chromosmes are randomly attached by MTs due to the higher dynamicity of MTs in metaphase (XMAP 125 phosphorylation eg) spindles are dynamic the MTs shift POLEWARDS to centrosomes (stain/bleach parts - can see them move back) -poleward force occurring through MT attached to chromosome

Answer 88

parts of chromosome without kinetochore eg the arms are pushed away from the poles ASTRAL EJECTION FORCE from plus end directed motor proteins ound to these parts of chromosome + the MT so there is a pull from kinetochore MTs to the poles and a push away from the astral MTs these 2 forces align the chromosomes along the metaphase plate

Answer 89

-factors in cell need to be imported into nucleus before pole is set up -are transported through Nuclear pore complex by Importin -released by small g-protein action -compete for binding w importin in active GTP form -no release of cargo outside nucleus as no GEFs in cytoplasm, most Ran is GDP form -GEFs in nucleus so cargo offload in there

Answer 90

-in mitosis: Cyclin B/CDK2 phosphorylates near kinetochore -leads to localisation of RCC1 (a GEF) next to kinetochore -catalyses exchange of GDP to GTP -> Ran-GTP in nucleus localised by the kinetichore -so SAF is released from importin close to needed site -Free un-occluded SAF leads to setup of half spindle in this region near chromosomes

Answer 91

FRET reporter constructs w Ran binding domain fused to FRET pair: -CFP- as long as no Ran bound - its emission wavelength excites YFP next to it Ran-GTP binds interferes w association of the FPs in the FRET pair no longer any yellow fluorescence (too far apart for FRET to occur) lowest Yellow intensity nearest where the chromosomes are where Ran-GTP is highest also other way round similar reporter but w an importin binding domain no yellow where no Ran-GTP higher Ran-GTP =more reporter outcompeted for importin binding =more yellow

Answer 92

MTs depolymerise at anaphase no poleward flux now - can be seen with photobleaching strips instead the region between the bleached stripe and kinetochore reduces in size -suggests depolymerisation from plus end (the one at the kinetochore)

Answer 93

kinetochore has CENP-E a plus end directed kinesin forms fibrous corona around the kinetochore MT allows kinetichore to keep binding to the dynamic end of the depoly plus end MCAK protein on the kinetochore displays MT depolymerisation activity

Answer 94

may not be general to other organisms -forms ring complex binding around + end of MT -GTP hydrolysis reaches plus end -Now GDP tubulin at end -causes end to depoly and SPLAY OUT -creates BIAS where ring cannot diffuse off backwards toward the splaying -BUT under thermal motion can only move other way to the poles -biases kinetochore movement to poles and allows tracking

Answer 95

when the chormosomes are tracking MTs to the spindle poles spindle poles not moving w respect to each other at this stage

Answer 96

MT based Gap between spindle poles increases so moving chormosomes pulled even further apart in overlap zone between ASTRAL MTs (not connected to chormosomes): -proteins like BimC act as homotetramers to work on opposite orientated MTs from opposing spindle poles -Tubulin added to the ends of these to increase astral MT overlap extension even more - push even further apart membrane bound dyenins also pull poles apart like before - indirectly pulling chromos apart The overlap then decreases over Anaphase B

Answer 97

sets up midpoint - perpendicular bisector formation of Anulus around this bisection pinch to form two pockets forms daughters

Answer 98

Astral stimulation Polar Relaxation Central Spindle

Answer 99

only at midpoint do you get overlapping Astral MTs somehow this delivers some factor toward the cell actin cortex causing the setting up of the Anulus ring of straight F-actin and bipolar myosin

Answer 100

opposite of Astral Stim around cell actin is under tension tension only released at poles something moves out along astral MTs to inhibit this tension leading to the drawing in of the actin in the middle

Answer 101

signalling from central spindle signals to mid zone to set up actinomyosin anulus structure central spindle starts as astral MTs then becomes separate array of opposite oriented MTs foming the central spindle some proteins left behind from chromatids after separation: -CHROMOSOMAL PASSENGER PROTEINS -eg kinase Aurora B could set up central tension

Answer 102

overlap of astral MTs in midpoint sets up the cleavage furrow Take urchin oocytes glass needle - push through centre of cell exclude cytoplasm into anulus shape -got cleabage furrow forming, because of needle could not go through entire cell - reaches one side of midpoint - but blocked by the "donut hole" end up w one cell, 2 nuclei (no full division, just the cleavage on bottom half) -these nuclei then duplicate -2 pairs on each side -got cleavage furrows between them (expected) -BUT also got a third one where the astral MTs from 1/2 of each pair overlapped at top of anulus shaped cell -EVIDENCE for astral MT overlap causing cytokinetic cleavage furrows

Answer 103

signal from central spindle leads to cleavage furrow forming Plus end directed kinesin ZEN-4 can form complex w CYK-4 (a row gap? idk what that means) form a tetramer Cyk-4 organises kinesin into a tetrametic kinesin complex bind oppositely oriented MTs and so locates itself at midpoint of central spindle mechanism for cell being able to tell where this is

Answer 104

Aurora B kinase - chromosomal passenger left behind on central spindle at mid point where sisters separate -phosphorylates protein bound to the CYK-4:Zen4 and releases them into cytosol at the central spindle (ONLY here) -then later in cell cycle Polo kinase activates Cyk4 to bind and activate ECT2 (a Rho GEF) -causes Rho activation to GTP form -Rho activates Formins -Activated formins located at midpoint (as everyting began here) -Forms the Anulus of Straight actin filaments at the mid point -Rho G-protein also activates myosin Light chain kinases - causes unfolding of Myosin II - bipolar myosin II filaments - causes contraction of actin ring