Eukaryotic cytoskeleton and mitotic cell division

Question 1

Cytoskeleton overview and the 3 basic polymers that make it up

Answer 1

Cytoskeleton= system of filaments in all euks for shape, locomotion+ trafficking/cell migration. E.g. cancer metastasis, sperm motility, muscle contraction. Important in architecture, mitotic apparatus, contractile ring, organelle partition, establishing polarity, asymmetric position/cell determinants.
Small diffusible subunits mainly held by non-cov interactions. Accessory proteins modulate distribution+ dynamic behaviour, provide interface for signalling. Filaments can span cell.
3 basic polymers
1) Intermediate filaments (Ifs)- restricted distribution, strong, rope like. Role in strength to withstand stretch+ full dev/neurons. Nuclear lamins, vimentin, keratin, neurofilaments. 10nm wide
2) Microfilaments (MFs)- actin monomers. All euks. Rich in cell cortex-> strength. Filaments bundle-> protrusions, like microvilli, stereocilia (inner ear); filopoda+ lamellipoda in cell crawling. Tracks for intracellular transport; contractile structures (stress fibres, myofibrils, actinomyosin ring). Flagellar mvmt. Nuclear actin role-? Tight G-actin helix, 7nm wide.
3) Mictrotubules (MTs)- hollow. Heterodimeric alpha/beta-tubulin subunits. Organelle transport; compartmentalisation. 25nm wide.

Question 2

Intermediate filaments, keratins and Vimentin-like structures

Answer 2

IFs: heterogenous fam. Globular domains @ ends, separated by central rod of extended alpha-helix domain- mediated formation/ stable coiled-coil dimers- arrange antiparallel, staggered-> non-polar tetramers= soluble subunits of IF polymers. Ifs bundle by self-association (neurofilaments) or accessory proteins (filaggrin, plectin)
Keratins= most diverse fam. Tissue-specific forms in epithelia, nails, hair, feathers. May anchor to desmosomes (cell junctions linking epithelial cells). Keratin cables- skin withstand stretch. Epidermolysis bullosa simplex= disease where keratin gene mutations disrupt formation-> skin prone to mechanical injury.
Vimentin-like- vimentin (fibroblasts)+ desmin (smooth muscle)-> cellular meshwork; Neurofilaments: strengthen nerve connections in axons, dendrites; Lamins form nuclear lamina. Mitotic CDK promotes disassembly @prometaphase in open mitosis. Overall provide mechanical strength, critical to axonal org

Question 3

Microfilament overview

Answer 3

Actin in all euk cells, highly conserved. 10% muscle+ 1-5%non-muscle cell protein. Cell motility, polarity, shape. Roles in endocytosis, intracellular traffic, contractility, surface protrusions/adhesions, mitotic spindle org, cytokinesis, division patterning, embryonic dev, infection defence, metastasis, wound healing.
Cycles of polymerisation+ disassembly between globular+ filamentous forms/actin-> cells constantly remodel cytoskeleton, use as force-generating system. Globular (G)-actin crystal structure solved in 90s: 43kDa, 4 subunits has AT/DP nt binding cleft.

Question 4

MF structure, directionality and growth

Answer 4

G-actin-> double-helical filament F-actin- flexible, 7nm diameter. 2 Monomer lobes separated by deep cleft for AT/DP, displays ATPase activity. Filament polar- all monomers added in same orientation, grow to barbed+ end+ disassemble @pointed/arrowhead - end. Polarity/ end shaped revealed by “decoration” exp- decorate w/ myosin heads (proteolytic S1 fragment), elongates 10x faster @ barbed end. Held by non-cov interaction.

Nucleation+ growth: nucleation= initial formation of oligomers- E/ kinetically unfavourable, rate-limiting step of F-actin assembly-> lag phase in polymerisation. Adding stabilised oligos (“seed”) suppresses lag. Filaments elongate-> [free monomer] falls-> critical conc (Cc), when subunits join+ leave @same rate (steady state). Below Cc, depol, no formation. Poly in vitro can be initiated by salts (increasing ionic strength)+ pure G-actin.

Question 5

MF dynamics, high-res cryo-EM and treadmilling

Answer 5

Filament end dynamics: ATP-bound actin monomers T form, preferentially added. Newly poly subunits-> flat conform by ~20o scissor-like rotation/outer domain (faces out in helical filament) wrt inner, enhancing ATP hydrolysis, then slow Pi release-> ADP-actin (D form)- tends to dissociate.
High-res Cryo-EM show differences in actin @ each end. +end retains flat conformation typical of internal subunits, - subunit= twisted, monomer-like conf- hence + favours addition+ vice versa. Rate of T-actin addition > rate of conversion to D form-> + end has T form subunits while – end has D. capping 1 filament end allows observation of just pol/depol-> determine rate. Diff filament dynamics+ ATP cycle-> different Cc values for each end, drives treadmilling. Cc pointed/- >Cc barbed/+ end.
Treadmilling: @ set conc [G-actin] intermediate between Cc/each end, F+G actin @steady state, filament length+ [G-actin] constant: treadmilling= net flow of actin through a filament of constant length. Treadmilling filament has ATP @ + and ADT@-. Dynamic behaviour also depends on existence of multiple polymers conf.s not strictly coupled to bound nt state- structural plasticity impacts dynamics+ recognition by actin-binding proteins involved in actin org+ turnover.

Question 6

Actin poisons

Answer 6

Actin poisons: help uncover cytoskeletal contribution to cell function
1) Phalloidin: binds, stabilises filament. Block depolymerisation (Rhodamine-conjugated phalloidin= popular reagent to stain F-actin by fluorescence microscopy)
2) Cytochalasin: caps + end, prevent elongation leading to depoly
3) Latrunculin: sequester monomers (binds nt-binding cleft), stop poly. LatA promotes subunit dissoc. From ends+ severing (create fragments w/ no growth ends). Reversible.
Actin dynamics key to cell function. E.g., latrunculin stops keratocyte migration, showing actin depol importance for generating protrusions @ leading edge+ associated mvmt.

Question 7

Actin binding proteins and the cell cortex

Answer 7

Actin-binding proteins (ABPs) modulate actin filament organisation+ dynamics in vivo: spontaneous nucleation strongly inhibited by G-actin sequestering proteins (e.g. thymosin beta4)-> >40% actin monomeric in non-muscle (well over Cc). Signalling-> pool released rapidly. Gen/new filaments by deploying nucleators- profilin promoted AD/TP exchange, delivers ATP-actin. Elongation limited by proteins capping + end/ cause fragmentation/severing/ favour depol (-) ends (cofilin)/ cap (-) end for stability. Myosins move along filaments. Cell cortex rich in actin- ABPs link F-actin in loose network (crosslinking proteins)/tight bundle (bundling proteins)/anchor to membranes (ERM)- combinations-> higher order structure-> shape+ f(x). leading edge protrusions (lamellipodia, filopodia)-> crawling. Tight bundles support persistent structures (microvilli, stereocilia).

Question 8

Actin nucleation: spire proteins and ARP2/3

Answer 8

Requires facilitating proteins, suppressing lag. Localisation, activation, mechanism of nucleators dictates spatial distribution+ nature of structures made. 3 nucleator classes:
Spire proteins promote linear assoc/4 actins- act as scaffold for poly/unbranched filament. May also cap - end
ARP2/3 mimics actin di/trimer, initiate new filament branching off existing one-> Y-branched networks. (-) end of new filament/branch capped by Arp2/3 complex. Can’t form filament on its own. Activated in vivo by assoc w/ WASP family proteins (nucleation promoting factors) released from autoinhibitory conf (response to signalling) to open conf (permits binding+ activation/ Arp)- 1 end binds Arp, other brings actin (increase effective conc). WASP= Wiskott-Aldrich Syndrome protein- syndrome=sex-linked disorder w/ wrong platelet f(x)-> immunity, cell motility, increased risk of cancer.

Question 9

Actin nucleation: formins and the rocket launcher model

Answer 9

Formins promote unbranched filament nucleation. Formin dimer stabilises actin di/trimer, remain associated w/ growing barbed ends by sequential binding/release. Best-studied members= Dia-related formins. Open conf upon GTP-bound-Rho-like GTPases (bind domain adjacent to Diaphanous-inhibitory domain (DID), disrupting DID-DAD (autoreg-domain) autoinhibitory interaction) recruitment. Dimeric formin-homology 2 (FH2) domain processively tracks + end, protecting from capping proteins. Adjacent FH1s recruit profilin-actin complexes, can increase rate of elongation. Single-molecule formin assoc to barbed end, followed by reconstitution experiments by TIRFM+ high-res cryo-EM of actin bound to formins.
Rocket launcher mech/model: Formins activated in presence of nucleation promoting factor APC (tumour suppressor)+ actin, acts as ‘leaky cap’ to promote elongation.

Question 10

Actin nucleation: cofilin and profilin

Answer 10

Cofilin/ADF bind actin (G/F), induce depol from end+ severing (can occur @ interface between cofilin-decorated and non-decorated regions)-> increase conc/free ends, accel turnover. Higher affinity to D form, stimulated depol/older filaments. Inhibits spontaneous AD/TP exchange on G-actin. Cofilin Pi releases D subunit- can then undergo profilin-stimulated AD/TP exchange.
Profilin binds monomers opposite ATP cleft, block – end assoc, but can add to free + end-> flat conf, releases profilin. Binding PIP2 or FH1 locally modulates profilin delivery of actin to poly sites near mem

Question 11

MF motor proteins and myosin

Answer 11

Motor proteins/ mechanochemical enzymes (myosin superfam) move unidirectionally along cytoskeleton, couple ATP use w/ conf changes. Track polarity+ motor properties dictate direction. Motor domain/head region hydrolyses ATP, cycles between binding/releasing filament- structure dictates choice of filament+ direction/ mvmt. Tail region interacts cargo, determining specific f(x)/protein.
Myosins: move along F-actin. Superfam/18 members, including 1-headed (e.g., myosin I) and 2-headed ones. Carry organelles/ cause adjacent filaments to slide. All except VI move toward + end. 1st myosin ID’d= myosin II in skeletal muscle (assoc w/ contraction- elongated w/ 2 heavy, 2 essential+ 2 light chains. Each heavy chain has globular head domain @ N-term w/ force-generating machinery, then seq forming extended coiled-coil (mediated heavy chain dimerization). Light chains bind close to N-term head, coiled-coil bundles w/ tails of other myosin molecules. Bundling-> bipolar “thick filaments” w/ 100s/ heads oriented in opposite directions @ ends (away from middle). Myosin II req for cytokinesis (actomyosin ring). Spends major fraction of cycle detached. Myosin V (vesicular transport)- hand-over-hand mvmt- processive motor travels long distance w/out detaching. Myosin VIIa in stereocilia, mutations-> deafness.

Question 12

Video imaging of myosin 5

Answer 12

Video imaging myosin V by atomic force microscopy- single molecule fluor, microscopy using TIRFM (observe individual protein-fluor attachments (spots))- but not high res. EM/Xray static. AFM-> direct observation of both structure+ dynamics: mica surface coated w/ biotin-containing lipid bilayers; streptavidin deposited on substrate; biotinylated F-actin immobilised through streptavidin. M5-HMM (tail truncated myo V) deposited- directly visualise walking in successive images to corroborate hand over hand model.

Question 13

Actin polarisation+cell motility: crawling mechanism and polarisation

Answer 13

Crawling to: est nerve connections w/ neuron bodies stationary; white blood cells; fibroblasts to wound; osteoclasts-> bone remodelling; metastasis. All animal cells except sperm locomote by crawling.
Crawling mech: cell protrudes a front, attach substrate, retreat rear. Cell must be polarised. + ends face membrane- branched actin-> lamellipoda, bundled-> filopoda; stress fibres= actin+ myosin contractile bundles ending in focal contacts. Myosin II drives rear retraction. Reorg./actin coupled to substratum adhesion by linkage of cytoskeleton to transmem receptors for extracel. matrix (integrins) @specialised focal contacts.
Polarisation: mvmt direction signalled by chem gradient (chemotaxis), e.g. for neutrophils (-> bacteria), D discoideum (-> cAMP-> aggregation+ dev/fruiting bodies) or Non-diffusible cues from ECM (nerve projections). Long distance gradients too shallow- cells may break down chemoattractant (cell surface enzymes, receptor-ligand endocytosis)-> steeper self-gen gradient. Models ring attractant depletion+diffusion predict self-gen gradients allow navigation of complex paths- tested w/ microfluidic mazes in vivo using D discoideum (self-gen cAMP gradient)/ cancer (LPA). Cells sensed upcoming junctions, chose live channels, ID’d optimum paths.

Question 14

Signal transduction directing cell movement (MFs and crawling)

Answer 14

Signal transduction directs cell mvmt: Rho/Rac/Cdc42 of Ras superfam/GTPases= ~21kDa proteins, weak intrinsic GTPase activity, act as switches in signal transduction cycling between GTP+ GDP-bound. Vital to transduce ext/internal signals to effector ABPs, imparting directionality (principles also apply to cell polarity in embryo dev/patterning, underlie biased spindle orientation+ cell division patterning, asymmetric partitioning/ fate determinants/organelles).

Question 15

Cell crawling (MFs) overall model

Answer 15

Crawling: Asymmetrical activation/ small GTPases @ mem according to external gradient: GTP-bound form-> mem protrusions- Rac+Cdc42 activate Arp2/3-> lamellipodia +/formins-> filopodia. Rho activates actin org by formins/myosin II (Pi of regulatory light chains on myosin II, mostly via Rho kinase (ROCK)-> slide)-> contraction. ROCK Pi’s MLC (reg light chain)+ MLC phosphatase (deactivating to stop de-Pi). Focal adhesion dev+ maintenance links myosin II contractility+ traction @ focal contacts to transport cell body. Increase in anchorage-> retard migration (hence directional mvmt coupled to polarised adhesion-site turnover)
Extracellular signals that locally activate Rho/Rac/Cdc42 trigger v. polarised F-actin nucleation-> leading edge. F-actin + ends toward leading edge+ cofilin depol.s actin further back in cell-> treadmilling forward coupled to leading edge progression. Overall model of polarised actin network @ leading edge: GTPases activate WASP (free from autoinhibition)-> activate Arp2/3. Capping proteins bind growing ends, terminate elongation. Formins-> filopodia, cofilin binds ADP-actin, profilin-> AD/TP exchange. Rho GTPases activate p21-activated protein kinase (PAK)-> LIM kinase Pi’s cofilin (inhibitory).

Question 16

Evidence for cell crawling mechanism

Answer 16

Evidence: biochem+ EM studies. In vitro assays demo ability of Arp2/3 to form branch points, correlated w/ EM images showing richly branched networks underlying protruding mem (“dendritic network model”). Some aspects of model (existence, density+ stability of branch points) challenged by e- tomography+ alternative fixation protocols- later studies detected fewer branches, showed long, linear fibres-> relative importance of Arp2/3 (vs formins, e.g.) questioned. Consensus that F-actin grows @ lamellipodium tip, forming treadmilling network. Contrasting views on mechanistic impact of cofilin severing- one suggests indirect contribution to treadmilling by increase/ monomer pool, other suggests cofilin-> network formation by severing-> short filaments providing new sites of Arp2/3 initiation. Molecular mech still under investigation.

Question 17

MT overview

Answer 17

Strong polymers of tubulin. Alpha/beta tubulin heterodimers arr head-to-tail protofilaments in parallel->hollow tube of ~13 protofilaments, polar (beta tubulin exposed @+ end), dynamic structure w/12nm helical pitch+ tubulin 8nm repeats along protofilament-> lattice seam where alpha+beta subunits make lateral contacts, interrupting helix. Adjacent filaments staggered ~1nm in helical tubulin array Subunits held by non-cov interactions. Alpha tubulin bound irreversibly to GTP. Beta (E site) GTPase (in polymer)+ GD/TP exchange as free monomer.

Question 18

MT polymerisation dynamics

Answer 18

Poly dynamics: nucleation rate limiting. + end grows faster, w/lower Cc. poly/deplo cycle driven by beta tubulin E site GTPase. GTP-alpha/beta tubulin (T form) added to MT, GTPase activated by next dimer addition. Glu-254 in alpha of incoming subunit completes E-site catalytic pocket of prior beta. kinetic race of dimer addition vs GTP hydrolysis (possible up to penultimate + end dimer) determines modest GTP cap @+ end. Following GTP->GDP, Pi release slowly-> D form. Treadmilling possible due to GTP cycle+ structural end differences. Protofilaments tend to curve-> lattice sheet under stress-> dynamic instability. Individual + ends stochastically switch between pol/depol, protofilaments peel outward, releasing stress on GTP cap (traps polymer in straight conf despite preference for curve loss. In vivo, - ends capped by gamma-tubulin ring complex gamma-TuRC.

Question 19

Allosteric model of MT polymerisation and dynamic instability

Answer 19

Allosteric model-changes in curvature governed by GD/TP bound. + end is open sheet, but MT stores E of GTP hydrolysis in lattice (mechanical stress), staging dynamic instability. E release-> switch to + end depoly as curved protofilaments burst open outward (not simple reversal of poly). Closed tube may exist transiently when MTs “pause”. Switch from growth to depoly= catastrophe (opposite=rescue)
Dynamic instability described by growth/shrinkage rates+ freq of catastrophe+ rescue. Treadmilling+ dynamic instability regulated by MT-assoc proteins. Cell division depends on dynamic instability, blocked by drugs like colchicine/colcemid/nocodazole/benomyl/vinblastine/vincristine (bind subunits, prevent incorporation, enhance depoly- reversible)/taxol (binds+ stabilises MTs, preventing depoly)- MT poisons used to probe f(x), also used in some cancer drugs to target proliferating cells.

Question 20

MT tip structure and speckle microscopy

Answer 20

Tip structure: consensus on depoly MTs exists but not poly. Cryo-EMs show multiple structures of growing tips-> alternative models.
1) Allosteric model: Straight protofilaments. GTP-tubulin dimers straight, curve upon GTP->GDP
2) Sheet-like structures roll up+close into tube
3) Curved protofilaments @tip grow by addition/curved T-tubulin dimers which also have curved conf inspired by lattice model (subunits straighten upon incorporation into lattice). Curved protofilaments straighten by thermal fluctuations to form lateral bonds. GTP->GDP has no effect on curvature, but weakens lateral bonds +/ strengthen tendency to curve
Fluor speckle microscopy (FSM)- limiting amount/fluor labelled tubulin (~1.25%) microinjected. Incorp w/ endogenous tubulin to MTs generates speckles forming “barcode” stochastically- each unique, can be followed. Show retention of initial MT by rescue (rather than full breakdown).MT - ends typically anchored in vivo, + ends explore by instability. Grow faster+ have more catastrophe+ rescue in cells than MTs from pure tubulin due to nucleating, (de)stabilising factors acting in spatially+ temporally specific ways-> diff MT assemblies along cell cycle

Question 21

Nucleation of MTs

Answer 21

Nucleation @ MT-org centres (MTOCs) containing gamma tubulin in small complex w/ GCP-2+3 (gamma-TuSC). Gamma-TuSC= yeast nucleating complex. Other species- gamma-TuRCs large ring complex w/GCP-2,3+ GCPs 4,5,6. TuR/SCs= (part of) MTOCs. In vivo nucleation only@ MTOCs (centrosome in animals=main MTOC). Diff MTOCs depending on cell type. Gamma-tubulin-> complexes of diff sizes- only large rings=effective MTOCs. Ran-GTP-> MT assembly near mitotic chromosomes, Augmin complex-> MT-based nucleation- both req gamma-TuRCs. Golgi also MTOC. Template model- y TuRC ring accommodates - end/13 protofilaments.

Question 22

Nucleation of MTs: TuSCS and TuRCs

Answer 22

Yeast y-TuSC: cryo-EM structure/ purified yeast gamma-TuSC bound to attachment factor Spc110 is solved, goves clues for y-TuRC assembly mech in other species. Spc110 promotes oligomerisation-> ring-like structures. “closed state” found @ MT - end (cryo-e- tomography of capped MT in situ)- req for efficient nucleation. Y-TuSC+Spc110=MT template. ½ yTuSC overlap between 1st+ 7th-> Y-TuSC oligomer structure has 6 ½ y-TuSCs/ helical turn= 13 y-tubulins/turn, matching in vivo MT protofilament # w/ similar helical pitch, supporting ‘template model’- y-tubulin symmetry not perfect match w/MTs- docking @ MTOC via attachment factors/further regulatory event may adjust ring to closed state to fit 13 protofilaments perfectly, -> nucleation.
y-TuRC: Spindle-enriched fraction-> analyse native y-TuRC capping MT – end by cryo-e- tomography- structure matches MR. Series of long coiled-coils “staple” 1st row a/b-tubulin, y-tubulin+ Spc98- protein not ID’d but Stu2 (in TOG fam/MT polymerases, localises to yeast spindle pole) implicated.

Question 23

MTs: Human TuRC

Answer 23

Human y-TuRC: cryo-EM-> ~2.2MDa vertebrate y-TuRC, 14 spokes from 7 subcomplexes: 4x GCP2-3, GCP4-5, GCP4-6, GCP2-3 (half-overlaps 1st subcomplex). Has ‘luminal bridge’ containing actin molecule. Mozart/Mzt1 (mitotic-spindle organising protein assoc. w/y-tub ring) conserved in y-TuRC, absent in yeast. Identical TuSC subcomplexes in ‘closed side’- almost matched MT symmetry. Diverse subcomplexes interface has open conf, incompatible w/ MT arr. Activation/transition to all-closed conf w/1/14 overlap (match MT) may req other factors interacting y-TuRC/ MT, may involve forcing closed conf, post-translational mods, adding incoming tubulin (lattice-driven). More recent high-res cryo-EM/ human y-TuRCs capping MTs nucleated in vitro show TuRCs can assemble but remain inactive unless bound to extra factors e.g. CDK5RAP2- activator @ centrosome; binding may force partial closure, initiate nucleation. MT nascent lattice may cooperate for full closure. Bridge structure in free y-TuRCs no longer fits in lumen of closed ring, therefore proposed to be obstacle for spontaneous closure+ nucleation. (In yeast, nucleating complex assembles solely @’yeast centrosome’ (SPB) in assoc. w/ Spc110)

Question 24

MT nucleation: Centrosome and its structure

Answer 24

Centrosome: (-) ends buried inside, + ends reach cell cortex. Provide spatial coordinates to cellular structures, ctrl assembly+ position/mitotic spindle. Proposed as actin-organising centre too, + thus a spatial integrator/cytoskeletal networks (still badly understood). Main MTOC/fungi+ diatoms= spindle pole body (SPB). Centrosome structure=mature (mother)+ immature (daughter) centrioles @ right angles, surrounded by centrosome matrix/pericentriolar material (PCM) rich in y-tubulin (components structurally org, as shown by super-res microscopy). Mother has distal (anchor @ plasma mem, acts as basal body)+ subdistal (docking sites for cytoplasmic MTs) appendages. Doublet MT has typical 13+ 10 extra protofilaments forming 2nd (B) tubule fused to singlet (A) MT wall. +10 more to B-> C tubule, triplet structure (as at proximal wall/ centriole). Triplet stability in mammals depends on delta-/epsilon-tubulins- f(x)/structural detail to be decided.
Centriole initially built around 9-fold symmetry scaffold (cartwheel). Only daughters maintain this @ proximal ½ in human cells. Procentriole= early stage/duplication)
Sequestering proteins (e.g. stathmin) limit available tubulin dimers. Severing factor katanin fragments MTs. MAPs (e.g. MAP2 in axon) promote overall stability, binding MT

Question 25

MT motor proteins: kinesins

Answer 25

MT motor protein superfams incl kinesin-like proteins (KLPs)+ dyneins- cellular transport, org/MT-based machines for cell locomotion (cilia, flagella), chromosomal segregation. Kinesin superfam has (=/>)14 fams, grouped according to N-term, C-term or internal position/motor domain. Standard nomenclature= numbered, specific names depend on history/ studies in model system. Most have tail binding site for a mem-enclosed organelle/ MT. General KLP properties: most have N-term motor domains, move to + end. Kinesin-1 ID’d 1st- found in squid giant axon, carries organelles neuronal cell body-> + ends @ axon terminals; 2 globular head motor domains, elongated coiled-coil for dimerization, ending in pair of small globular tail domains; highly processive: mechanochemical cycles/ motor heads coordinated, hand-over-hand motion preventing cargo diffusing away from MT, spends majority of ATPase cycle bound to MT. Kinesin-14 fam have C-term, are – end directed. Kinesin -13s have internal motor domain, f(x) as MT depolymerases (bind+ induce protofilament curling). Kinesin-5 can self-assoc via tail (tail-tail assoc)-> bipolar motor, slides antiparallel MTs past one another. Many have roles in mitosis.

Question 26

MT motor proteins: dyneins and tripartate complex formation

Answer 26

Dyneins: - end directed. 2/3 heavy chains, some intermediate+ light chains (6 polypeptides, each in 2 copies- ~1.4MDa total). 2 major fam branches: Axonemal dyneins- in cilia+ flagella (Incl heterodi/trimers w/ 2/3 motor domain heads) and Cytoplasmic dynein- largest known molecular motor, among fastest. typically heavy-chain homodimers, 2 large motor domain heads. In all euk cells. Vesicle traffic, localisation/Golgi near cell centre. Req assoc w/dynactin (23-subunit complex of 11 diff polypep, ~1.1MDa total), incl p50 dynamitin, p150Glued(contains ATP-insensitive MT-binding domain)+ a short, capped actin-like filament made of Arp1. Spectrin (actin-binding protein) arrays assoc. w/Golgi mem may bind Arp1 filament (link golgi to transport by dynein). Forming Tripartate complex (ultraprocessive) of dynein, dynactin, specific protein adapter for cargo-> complex stability, motor activity, high processivity (i.e. only active once cargo+ adaptor acquired). Some adaptors recruit 2nd dynein to further increase processivity. Pure dynein doesn’t move processively. Several adaptors characterised, but whether this mech applies to all cargoes unclear.

Question 27

MT end-binding proteins

Answer 27

MT end-binding proteins recognise conformational features of end, affect parameters of dynamic instability. Depolymerizing kinesins (Kin-13 fam, e.g. MCAK) force protofilament peeling-> catastrophe. + end-tracking proteins (+TIPs) assoc + end, incl conserved XMAP215/TOG, CLIP-170, EB1 fams,-> Growth, rescue (+ reduce catastrophe), attachments for growing/shortening MTs to kinetochores, organelles, cell cortex; mediate interactions w/ actin cytoskeleton, cooperate w/ motors (CLIP170 promotes recruitment of dynein-dynactin @ + ends); some +TIPs transported to +end in assoc. w/ KLPs.

Question 28

Motile cilia and flagella, basal bodies

Answer 28

Motile cilia/flagella: cilia/flagella both= core bundle/stable MTs for E-driven bending. Mvmt by bending MT core (axoneme), arr. In pattern w/9 MT doublets around central pair/MT singlets. ‘9+2’ array characteristic for all motile cilia+ euk flagella. Accessory proteins cross-link MTs @ regular positions. Ciliary (/axonemal) dynein bridges neighbouring doublets around axoneme circumference; upon activation, dynein on 1 doublet attempt to walk along adjacent doublet- other links prevent sliding, so force-> bending. Basal bodies root euk cilia/flagella @ cell surface, originate @ centrioles, typical arr 9 fused MT triplets. #/centrioles per cell-># cilia/flagella. Dividing cell centriole formation coordinated w/ cell cycle- 1 centriole forms next to each of 2 parental ones. Cells that exited the cell cycle- mother centriole docks below plasma me, triggers cilium/flagellum formation. In Multiciliogenesis, centriole biogenesis linked to deuterosomes (electron-opaque globular entities, platforms for de novo assembly) Primary cilium- 1 on apical surface of most human cells. Lack central pair/MTs (9+0 config)+ dynein-> non-motile. Sensory organelles- e.g. in kidney sense urine flow-> modulate duct morphogenesis; Olfactory receptors on cilia, rods+ cones=highly modified cilia

Question 29

Flagella+ cilia- physiology and disease

Answer 29

Physiology+ disease: cilium/flagellar-driven motility-> airways, oviducts, cerebrospinal fluid (brain ventricles), sperm. Cilium-driven flow in node/dev embryo-> determine left-right asymmetry/viscera. Non-motile cilia-> sensory roles, transduce Hedgehog signalling in dev. Motility defects-> bronchiectasis, chronic sinusitis. Node defects-> sinus inversus (viscera asymmetry inversion). Motion defects in brain-> hydroencephalus. Primary cilia defects in kidney-> polycystic kidney disease; other tissues-> anosmia, retinal degeneration, obesity. Cilia in Hedgehog signalling defects-> polydactyly.

Question 30

Mitosis and cell division overview

Answer 30

M-phase entry-> cytoskeletal remodelling. Regulated vesiculation+ fusion ctrls segregation/ endomembraneous structures. Cytoskeleton may help partition mt. Prophase- as MT network disassembles, centrosomes begin nucleating v dynamic MTs, nuclear env breakdown-> MT capture @kinetochores begins. Metaphase- bi-orientation (chromosomes under tension @ metaphase plate). Anaphase A- each sister chromatid becomes chromosome, kinetochore MT shortening poleward. Anaphase B- interpolar MTs elongate, separating sprindle poles. Telophase- chrs reach poles, nuc envs reform, chrs decondense. Cytokinesis- contriction/actinomyosin ring in animals, constriction of ring+ sptum deposition in yeast, deposition of specialise vesicle-based structure @ cell plate/site of division in plants. In fungi, spindle formed in intact nucleus (closed mitosis). Female meiosis of many animals- bipolar spindles form in centrosome absence. 1st mitotic divisions of mouse embryo w/out centrosomes.

Question 31

The spindle

Answer 31

Spindle: self-org bipolar array/ MTs of 3 classes. – ends @spindle poles set up by centrosomes. Kinetochore MT + ends interact kinetochores. Interpolar MT +ends interact @ overlaps w/ non-kinetochore MTs from opposite pole (become crosslinked, generating spindle midzone). Astral MT +ends extend to cell cortex. Budding yeast- single kinetochore MT attach each sister kinetochore. Animal- 1 kinetochore: bundle/~20kMTs (kinetochore/k-fibre). 3D microscopy analysis, incl 3D reconstruction/entire spindles challenges 3 MT view, suggests: more complex MT-based modules; MTs branch off from other MTs, not all (-) ends buried near centrosome; MTs form antiparallel bridges between k-fibres (contribute to dynamics); Human k-fibres contain MTs from centrosome+ MTs that don’t reach spindle pole- may be crosslinked/interact MT-assoc components. MT sets arise from parallel pathways for nucleation in M-phase (centrosome-dependent+ independent pathways)- dynamic instability+ MT-interacting proteins-> generation/bipolar structure+ ‘search+ capture’/chrs. Once all chrs bi-oriented, they’re aligned @ spindle equator

Question 32

Centrosome dependent pathway

Answer 32

Centrosome-dependent pathway: centrosome duplication in interphase. G1- q centrosome w/ mother+ daughter centrioles. S- duplication begins- semiconservative (new centrosome in M carries 1 of 2 original centrioles from G1). Reorg./MT toward spindle assembly initiated by rapid increase in MT turnover (early prophase). Centrosomes separate (driven by MT motors), complete ‘maturation’. Progressively recruit more PCM, increasing nucleation capacity-> asters form, astral MTs interact cell cortex-> aster separation (driven by dynein/dynactin anchored @cortex). Dis1 fam/MAPs (XMAP215,HsTOG) promote MT stability+ growth, counteracted by kinesin-13. Balance shifts-> favour depoly by -ve reg/MAP by CDK-mediated Pi as enter M- shown in vitro by exposing MTs to mitotic vs interphase extracts+ measure ensuing MT dynamics. Taxol stabilises MT, prevent bipolarity, centrosomes fail to separate-> assemble ‘monopolar’ spindle. ‘search+ capture’-> dynamic MT nucleated @centrosome+ contacting kinetochore captured, dynamics suppressed- capture typically begins by lateral kinetochore-MT attachments converted to end-on ones or reorient kinetochore to favour end-on attachment. Mathematical modelling shows unbiased ‘search+ capture’ exceed duration observed- extra mechs present, esp density of MT ends generated by centrosomes= critical determinant for efficient search, decreases w/ increasing distance from poles…

Question 33

Centrosome independent pathways and their molecular basis

Answer 33

Centrosome-independent pathways rely on alternative pathways for nucleation, req y-TURCs, promote MT end density near kinetochores, form sufficient MTs to permit assembly/functional bipolar spindles in absence/centrosomes in vitro+vivo. Demonstrated in cell-free systems. Bipolar spindle can assemble w/out centrosomes, triggered in cell-free system by DNA-coated beads (convert to chromatin when added to Xenopus egg extract in centrosome absence)- works even w/ bacterial DNA- kinetochore not req. seen in cells too. Motor protein function probed in cell-free systems using specific inhibitors/antibody-based depletion. Monastrol-> kinesin-5 inhibited, monopolar spindle. P50 dynamitin of dynein-dynactin inhibited-> bipolar but poles unfocused. Xkid (a chromokinesin/KLP) depletion-> bipolar but congression-defective. MT self-organisation driven by motors. Nucleate near chromatin, aligned by + end directed chromokinesins (chromatin cargo- Kinesins 4+10), pushing parallel MT (-) ends away of each side of chromatin mass-> 2 half-spindles. Bipolar, tetrameric + directed Kinesin 5 crosslinks+ slides antiparallel MTs, org halves of spindle+ supporting overlap @midzone. Multimeric (-) directed dynein moves along parallel MTs, focuses poled by bringing (-) ends together. Molecular basis for centrosome-independent pathways: 1) Chromatin-dependent nucleation: RanGEF RCC1 binds chromatin, RanGAP cytoplasmic-> gradient/active RanGTP near chromatin-> TPX2 release-> promotes nucleation w/y-TuRCs 2) MT-dependent nucleation: octameric Augmin complex recruits y-TuRCs to pre-existing MT, creating new MT (MT branching)-> MT amplification. New MTs travel along existing MTs to join spindle 3) MT nucleation near kinetochores- more recently proposed, possible mech for initiating kinetochore capture through non-centrosomal MTs that join spindle via dynein-mediated transport/interactions w/ other crosslinkers.

Question 34

bipolarity at metaphase, kinesin and dyneins

Answer 34

Bipolarity @ metaphase: balance of pulling+ pushing (overlapping interpolar MTs crosslinked by + directed kinesin-5, -end directed Kinesin-14. Dynein/dynactin+ NuMA (nuclear mitotic apparatus- an adaptor. May also be present @ cortex to promote dynein recruitment) crosslink MTs, focus poles by -end directed mvmt. Dynein/dynactin -end directed generates force from cortex to pull poles apart via astral MTs. Chromosome assoc, +directed kinesin 10 mediate interactions between chromosome arms+ spindle, chr drawn away from spindle poled- this force disappears in anaphase A by proteolysis/relocation/ chromokinesin)-> chr alignment under tension @ ‘metaphase plate’, sets length of spindle (pole-pole distance). Mt attachments to sister kinetochores elicit tension, stabilised. No tension-> correction mechanism until chromatid pair bi-oriented.

Question 35

Poleward flux in mitosis and experimental evidence for it

Answer 35

Poleward flux= continuous treadmilling of metaphase spindle- tubulin addition @+ ends balances loss @-. Demonstrated by allowing MTs to add tubulin cov coupled to photoactivatable ‘caged’ fluorescin. Shine UV from laser to mark midzone-> marks move toward spindle poles, dim as tubulin incorporated @ +, lost @-. Evidence from FSM: imaging of low density rhodamine-labelled tubulin (speckles followed as fiduciary marks) in Xenopus extract spindle- strips from images taken @ 10s intervals displayed side by side-> kymograph- show diagonal streaks (individual speckles moving to pole by flux)- slope reflects flux speed.

Question 36

SAC, Anaphase A, CENP-E and MCAK

Answer 36

SAC: anaphase onset subject to checkpoint, can’t proceed unless spindle assembled correctly, chrs bioriented+ under tension (sister kinetochores attached to MTs from opposite poles)-> spindle assembly checkpoint (SAC) satisfied. Checkpoint inhibits APC/C. Activation of E3 APC/C targets anaphase inhibitor securing for UQ-dependent proteolysis-> release of active separase (cleaves cohesins). Anaphase A: Ndc80 complex (kinetochore subcomplex), Dam1 (fungi)+Ska1 (vertebrates, plants, nematodes) complexes key to kinetochore-MT interactions. kMTs shorten w/kinetochore acting like ‘pacman’ (+directed CENP-E (a kinesin-7) tethers kinetochore to MT; -directed dynein opposes this, leading kinetochore to pole; MCAK (kinesin-13) drives depoly) @+end while MT poleward flux continues-> chr mvmt to poles while pole-pole distant stays constant. Kinetochore can be labelled in FSM (diff label to MTs)- as kMTs shorten, kinetochore mark accelerates, overtakes MT speckles- shows +end pacman-style depoly/kMTs overlapping poleward flux. Proposed that chrs attached to short kMTs may also move to poles by dynein-driven transport w/ short kMTs ac cargo- Laser ablation to cut spindle MTs-> severed MTs linked to chrs delivered to pole by dynein walking along intact MT. Questions remain (how does MT depoly @kinetochore/+end drive chr mvmt, how kinetochores retained despite MT shinkage).

Question 37

Anaphase B

Answer 37

Anaphase B: poles move aparts pushed by bipolar +directed kinesin-5-> sliding antiparallel MTs @overlap. (-) directed dynein helps pull poles apart. Integrity of spindle midzone depends of additional MAPs. After anaphase B, spindle gives way to postmitotic bridge which connects separating daughter cells- remnants form midbody (v dense) during cytokinesis- role in abscission.

Question 38

Cytokinesis

Answer 38

Cytokinesis: coordination of mitotic apparatus+ division plane prevents uneven chr distribution/damage- division plane orthogonal w.r.t spindle axis, terminal stages/ cytokinesis don’t occur before chr sep complete. Cleavage furrow forms, encircling cell; deformation/ plasma mem, insertion/ new mem components. Force for furrow ingression from contractile ring 9arrays of F actin w/ myosin II filaments)- cytokinesis fails after injection of anti-myosin antibodies. Cytokinesis suppressed in early embryonic divisions/ Drosophila-> syncytium; in drosophila oogenic cyst, cytokinesis incomplete, cells connected by cytoplasmic bridges. Yeast (budding+ fission) cytokinesis by actomyosin contractile ring AND septum deposition.

Question 39

Asymmetric divisions

Answer 39

Asymmetric divisions key in dev- in animals, cleavage plane specified by mitotic apparatus. Produce genetically identical cells of diff size+/ cytoplasmic content. Polarised org/cytoskeleton couples distribution/ cell fate determinants w/ spindle alignment (1st embryonic division C elegans). Cell polarity by same principles as w/ directional cell motility (above), imparted by ext/internal cues. Budding yeast use internal cues to determine new budding site relative to preceding division site. Baker’s yeast asymmetric division/ budding relies on spindle orienting+ elongating across bud neck- polarised cytoskeletal org also supports asymmetric segregation of other cellular factors between mother+ bud. Factors assoc asymmetrically w/ domains of cortex attract aMTs, dictate position/ spindle poles. Many factors linking polarised actin org w/ biased orientation/ spindle ID’d by genetic analyses/yeast, C elegans, Drosophila, found conserved in humans.

Question 40

aMT and central spindle positioning at the central plane

Answer 40

aMT/central spindle and position of division plane: aMTs possibly sufficient to localise division plane/large embryonic cells (Rappaport 1961). Other exp- glass bead pushed into dividing egg cell, displacing spindle; furrow forms only on one side/cell-> binucleate egg (donut-shaped); both nuclei enter mitosis; cleavage both between centrosomes linked by mitotic spindles and between centrosomes adjacent- 4 daughters form (cytokinesis still occurs, showing plane of cleavage determined by aMTs rather than anything pole to pole)). By contrast, inactivation/ MAPs contributing to org/ midzone+ central sprindle disrupts cytokinesis-> importance in establishing division plane. Unifying mech centred on conserved motor complex centralspindlin accounts for role/ MTs from both location to specify animal cell div plane.

Question 41

Centralspindilin

Answer 41

Centralspindlin= key regulator/cytokinesis in animal cells- tetrameric complex made of 2kinesin-6+ 2RacGAP. Recruits+ reg proteins promoting cleavage furrow ingression+ mem abscission. Inactivation affects central spindle org, disrupts cytokinesis. Assembly+ MT binding+ RhoGEF interaction inhibited until anaphase. Then enriched @ overlapping MT +ends. Model for action: centralspindlin-> midzone along MTs, bind+ activate RhoGEF (RacGAP also downreg Rac, stops activation/ Arp2/3)-> cortical ring of RhoGTP (promoted formation/contractile ring). Overlapping aMTs/opposite poles also contribute to conc/centralspindlin @ equator, explaining “Rappaport furrow”

Question 42

Rho-GTP effectors and furrow ingression

Answer 42

RhoGTP effectors+ furrow ingression: RhoGEF recruitment-> active Rho-GTP accumulates @ equator cortex+ activates its effectors formin (-> actin poly)+ ROCK (Rho kinase, Pi’s myosin light chain+ myosin phosphatase (inactivated), activating myosin)-> furrow ingression. Centralspindlin mem-binding domain keeps it tethered during ingression. RhoGEF+ centralspindlin stay assoc. w/ central spindle until late-stage cytokinesis. Contraction ingresses furrow-> diameter <2um. Nascent daughter cells remain connected by intercellular bridge (persists until abscission machinery splits mem)

Question 43

Bridge maturation and abscission

Answer 43

Bridge maturation+ abscission: cortical F-actin disassembles, bridge processively narrows. Vesicle fusion on both sides of midbody. Bridge has bundles/antiparallel MTs from central spindle, overlapping @midbody (e- dense structure, assembly platform for abscission machinery). Midbody tethered to mem, stabilising furrow after ring disassembly. Cortex adjacent to midbody constricts, appears rippled. E- tomography show constriction zones contain mem-assoc filaments~17nm diameter encircling bridge adjacent to midbody.

Question 44

Abscission by sequential constriction

Answer 44

Abscission by sequential constriction on each side of midbody- mem splits-> 2 daughters+ midbody remnant. 17nm abscission filaments may have endosomal sorting complex req for transport III (ESCRT-III)- mediates mem constriction+ fission from cytosolic face, incl in budding/endosomal vesicles+ viruses; essential for abscission, accumulates in late-stage bridges @constriction zones. ESCRT-III component depletion supresses 17nm filament formation+ constriction, suggests it may either directly polymerise them or reg their assembly. Disassembly of MTs lateral to midbody also during abscission.