Cytoskeleton and Mitosis Flashcards
Actin filament dynamics
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)
Arp2/3
Activated by WASP
Binds to pre-existing F-actin, resembles F-actin filament end, and nucleates a 70o branched filament
Formin
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
Cofilin
Cofilin –> binds to ADP-F-actin, promotes disassembly of older filaments - by both depolymerisation and severing
Inhibits ADP/ATP exchange on G-actin
Profilin
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
Capping proteins
’-‘end capping proteins stabilise - end
+end capping proteins (CapZ) bind and prevent addition of G-actin
Myosin II
and
Myosin V
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
Signal transduction for chemotaxis
Stimulus binds receptor –> PI3K activates, forms PIP3
PIP3 activates GEF (by binding PH domain) –> Rho-family GTP
Spatial regulation of Rho-GTPase activity
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)
Rac-GTPase
Activates WASP –> Arp2/3
Activates PAK (phosphorylates MHC and MLC kinase, filamins, and LIM kinase which inhibits cofilin)
Antagonises Rho and activates Cdc42
Cdc42-GTPase
WASP –> Arp2/3
Formin
PAK (phosphorylates MHC and MLC kinase, filamins, LIM kinase –> phosphorylates and inhibits cofilin)
Rho-GTP
Formins
ROCK (phosphorylates MLC and MLC phosphatase, LIM kinase which inhibits cofilin)
Taxol
Nocodazole
Taxol binds and inhibits MT depolymerisation - can form monopolar spindle due to inability to separate centrosomes
Nocodazole binds tubulin and prevents MT assembly
Polarity of MTs
B-tubulin exposed at ‘+’ end, and a-tubulin exposed at ‘-‘ end
MT dynamics
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
Dynamic instability
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
MT polymerases and depolymerases
Rescue promoted by XMAP215 (a MT polymerase)
Catastrophe promoted by MCAK, a Kinesin-13
gTuRCs and gTuSCs
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
Centrosome
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
Kinesins
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)
Dyneins
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
Cilia and flagella structure
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
basal bodies
9 triplet MTs arranged in a cartwheel, originating from a mother centriole
In multi-ciliated cells, basal bodies are assembled into deutrerosomes
Spindle preparation in Prophase
Disassemble interphase MTs (increase MT turnover via MCAK activation and inhibition of XMAP215)
Centrosomes separate and recruit more PCM
NEBD
Formation of metaphase spindle
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
Centrosome-independent spindle assembly - evidence and mechanisms
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
Assembly and organisation of the spindle by motors
- organisation of MTs into bipolar spindle
- Focusing of MTs at spindle poles
- 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
MT dynamics in metaphase
Poleward flux - proof by photoactivatable fluorophore in the midzone or by fluorescent speckle microscopy
Anaphase A
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
Anaphase B
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
Centralspindlin regulation
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
Centralspindlin activity
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
