Cytoskeleton and Mitosis

Question 1

Actin filament dynamics

Answer 1

Polarity of filaments - myosin S1 heads decoration experiment

Nucleation is rate-limiting (addition of oligomers will get rid of RLS)

Treadmilling occurs at Cc (Loss of ADP-actin at ‘-‘ end = gain of ATP-actin subunits at ‘+’ end)

Question 2

Arp2/3

Answer 2

Activated by WASP

Binds to pre-existing F-actin, resembles F-actin filament end, and nucleates a 70o branched filament

Question 3

Formin

Answer 3

Dimers with modular GBD, DID-FH1-FH2-DAD domains

Nucleates unbranched actin filament assembly, leaky cap binds processively to ‘+’-end

DID-DAD interaction is autoinhibitory
Activated by Rho-family GTPases

FH1 recruit and delivers G-actin:ATP bound to profilin

FH2 remains processively attached to F-actin

Question 4

Cofilin

Answer 4

Cofilin –> binds to ADP-F-actin, promotes disassembly of older filaments - by both depolymerisation and severing

Inhibits ADP/ATP exchange on G-actin

Question 5

Profilin

Answer 5

Binds to G-actin-ADP; promotes ADP-ATP exchange

Recruits it to growing F-actin, eg. via formins)

Blocks G-actin association with ‘-‘ end, so its added to + end

Dissociates after addition of G-actin to F-actin filament

Question 6

Capping proteins

Answer 6

’-‘end capping proteins stabilise - end

+end capping proteins (CapZ) bind and prevent addition of G-actin

Question 7

Myosin II
and
Myosin V

Answer 7

Bundling of myosin II tails forms bipolar thick filaments of myosin within actomyosin filaments. Cross-bridge cycling drives non-processive sliding, causing contraction

Transports cargo via hand-over-hand movement, remains processively attached

Question 8

Signal transduction for chemotaxis

Answer 8

Stimulus binds receptor –> PI3K activates, forms PIP3

PIP3 activates GEF (by binding PH domain) –> Rho-family GTP

Question 9

Spatial regulation of Rho-GTPase activity

Answer 9

Rho-GTP active in trailing edge

Rac and Cdc42 active in leading edge.

Rac and Rho behave antagonistically, while Rac and Cdc42 behave synergistically (activate their GEFs and inhibit their GAPs)

Question 10

Rac-GTPase

Answer 10

Activates WASP –> Arp2/3

Activates PAK (phosphorylates MHC and MLC kinase, filamins, and LIM kinase which inhibits cofilin)

Antagonises Rho and activates Cdc42

Question 11

Cdc42-GTPase

Answer 11

WASP –> Arp2/3
Formin
PAK (phosphorylates MHC and MLC kinase, filamins, LIM kinase –> phosphorylates and inhibits cofilin)

Question 12

Rho-GTP

Answer 12

Formins

ROCK (phosphorylates MLC and MLC phosphatase, LIM kinase which inhibits cofilin)

Question 13

Taxol

Nocodazole

Answer 13

Taxol binds and inhibits MT depolymerisation - can form monopolar spindle due to inability to separate centrosomes

Nocodazole binds tubulin and prevents MT assembly

Question 14

Polarity of MTs

Answer 14

B-tubulin exposed at ‘+’ end, and a-tubulin exposed at ‘-‘ end

Question 15

MT dynamics

Answer 15

Nucleation is rate-limiting

Shown by in vitro polymerisation using flagellum fragment as a seed for MT extension
GTP-a/B tubulin dimers added to existing protofilaments, and enhances GTP hydrolysis of the preceding a/B tubulin dimer

Hence mostly GDP-tubulin down its length with a cap of GTP-tubulin

Dynamic instability

Question 16

Dynamic instability

Answer 16

Abrupt switch between polymerisation and disassembly (catastrophe and rescue).

Due to GTP hydrolysis by B-tubulin subunits, which promotes a curved conformation of protofilaments

Catastrophe: when lattice stress is too great, ‘+’end loses its GTP-tubulin cap and opens into a fountain-like spread of protofilament

Rescue: Binding of GTP-tubulin to the ‘+’ end forms a cap of GTP-tubulin at the ‘+’ end that stabilises the lattice strain

Question 17

MT polymerases and depolymerases

Answer 17

Rescue promoted by XMAP215 (a MT polymerase)

Catastrophe promoted by MCAK, a Kinesin-13

Question 18

gTuRCs and gTuSCs

Answer 18

In yeast: gTuSCs formed of GCP2/3 and 2 g-tubulins
Oligomerisation of 6 and a half gTuSCs, matching protofilament number.
Docking of gTuSCs to MTOCs and oligomerisation is facilitated by Spc110

gTuRCs: GCP2-6 subunits and g-tubulin subunits arrange into a ring structure that resembles the gTuSC oligomer

Promote polymerisation by associating with and stabilising the ‘-‘ end of MTs

Question 19

Centrosome

Answer 19

PCM contains g-TuRCs, and centrosomes recruit more PCM as the cell enters mitosis

Some cells exit cell cycle and mother centriole forms a basal body that gives rise to flagella or cilia

Question 20

Kinesins

Answer 20

14 families, +-end tracking except kinesin-14

Kinesin-1 is a processive hand-over-hand motor

Kinesin 5 can self-associate forming a bipolar motor

Kinesin 13 can function as MT depolymerases (Eg. MCAK)

Question 21

Dyneins

Answer 21

Cytoplasmic:
Associates with dynamitin, Arp1 (actin-like filament), p150Glued to form DYNACTIN

Used for vesicle trafficking, localisation of Golgi via SPECTRIN ARRAYS on Golgi that bind to Arp1 filaments

Question 22

Cilia and flagella structure

Answer 22

Axoneme - 9+2 array
9 doublets and 2 central singlets

Axonemal dynein forms bridges between neighbouring doublets, and attempts to slide. Adjacent doublets are joined by nexin, which resists sliding and translates this sliding into bending of the axoneme

Question 23

basal bodies

Answer 23

9 triplet MTs arranged in a cartwheel, originating from a mother centriole

In multi-ciliated cells, basal bodies are assembled into deutrerosomes

Question 24

Spindle preparation in Prophase

Answer 24

Disassemble interphase MTs (increase MT turnover via MCAK activation and inhibition of XMAP215)

Centrosomes separate and recruit more PCM

NEBD

Question 25

Formation of metaphase spindle

Answer 25

Search-and-capture: MTs nucleated by centrosomes, via dynamic instability search for kinetochores- stabilised by binding kinetochore

Self-organisation: MTs nucleated near chromatin, incorporated into spindle by motors

Question 26

Centrosome-independent spindle assembly - evidence and mechanisms

Answer 26

Xenopus egg extracts can form bipolar spindles around DNA-coated beads - without kinetochores or centrosomes

Animal cells with centrosomes removed by laser = can still form bipolar spindle

Chromatin-mediated nucleation (Ran-GTP is chromatin-localised, releasing TPX2 from importins, which bind gTURCs)

MT-mediated MT nucleation via Augmin - recruits gTuRCs

Dynein focuses ‘-‘ ends into forming spindle

Question 27

Assembly and organisation of the spindle by motors

Answer 27

1. organisation of MTs into bipolar spindle
2. Focusing of MTs at spindle poles
3. Positioning of chromosomes at midzonoe

Overlapping interpolar MTs - outward spread by kinesin-5, inward overlap by kinesin 14

’-‘ ends focused into spindle poles by NMA recrutied by dynein/dynactin complexes

Chromosomes move towards midzone by chromokinesins 4/10

Astral MTs anchored by cortical dynein/dynactin

Question 28

MT dynamics in metaphase

Answer 28

Poleward flux - proof by photoactivatable fluorophore in the midzone or by fluorescent speckle microscopy

Question 29

Anaphase A

Answer 29

Poleward flux, but net depolymerisation due to MCAK activity

Kinetochores remain tethered by NDC80, CENP-E (kinesin-7) and dynein

Interpolar distance remains constant
Chromosomes delivered to poles by dynein along shortened MTs,
Shortened MTs carrying chromosomes can be delivered as cargo by dynein

Question 30

Anaphase B

Answer 30

Poles move further apart :
Kinesin-5 causes outward sliding of antiparallel MTs
Dynein/dynactin at the cortex bind to astral MTs
Chromokinesins pushes poles apart

Question 31

Centralspindlin regulation

Answer 31

Kinesin-6 dimer and a Rac-GAP dimer

Temporal regulation:
Phosphorylated and inactivated by CyclinB/CDK1
Phosphorylated and activated by AuroraB and PLK-1

Spatial regulation:
‘+’-end movement to the midzone by Kinesin6, anchored at cell membrane

Question 32

Centralspindlin activity

Answer 32

Rac-GAP activates Rho-GEF, which activates RhoGTP:
Promotes formation of contractile actomyosin ring that promotes furrow ingression

Contractile ring compresses central spindle to form the midbody within the intercellular ridge

ESCRT-III mediates abscission on either side, forming midbody remnant