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Cytokinesis – the “origin of two-ness”
1. What determines placement of the cleavage furrow
good bad
2. Classical cytokinesis experiments as a paradigm for testing causality of structures
Outline
This i
Cleavage plane always forms halfway between poles, perpendicular to spindle
Oriented spindle axis - oriented cell division
Off-center spindle asymmetric cell division producing different sized daughters
Cleavage plane always correlates with spindle position
This is a CORRELATION
spindle furrow
furrow spindle
spindle X furrow
How do we determine causality?
Mitotic spindle is needed for furrow initiation but not progression
Hiramoto 1956
Metaphase(before furrow starts)
No cleavage
Anaphase(after furrow has started) Cleavage completes
Mitotic spindle is needed for furrow initiation but not progression
Hiramoto 1956
Metaphase(before furrow starts)
No cleavage
Anaphase(after furrow has started) Cleavage completes
Instructive vs. Permissive
Instructive: spindle tells furrow WHERE to form
Permissive:spindle tells furrow WHEN to form(like a checkpoint)
Separating spindle from cortical regions normally destined for furrow formation
Yatsu 1912
Implies spindle sends a signal TO the cortex
Moving the spindle moves the cleavage furrow position
O’Connell and Wang, 2000
Normal cells (NRK)Asymmetric spindle correlates withasymmetric cleavage furrow
Impose new long axis Push spindle and pin at one end
What part of the spindle is instructing the cortex? Maybe it’s the chromosomes!
Zhang & Nicklas 1996
Remove all chromosomes bymicrosurgery
“chromosomes? We don’t need nostinking chromosomes”
What’s left: centrosomes, astral MTs, spindle MTs
What part of the spindle is instructing the cortex? Maybe it’s the centrosomes!
Khodjakov & Rieder
Ablate centrosomeswith “laser”
Centrosomes are also dispensable
Microtubules by themselves are sufficient
Aslop & Zhang 2003
Remove all chromosomes AND centrosomesby microsurgery
What’s left: spindle MTs
Furrow here
Not here
Chromosomes vs. microtubules(maybe MTs are a back-up system only if chromosomes are missing
Zhang 2004
Remove all but onechromatid byMicrosurgery
Cell formsasymmetric spindle MTs dictate furrow placement
EVEN WHENCHROMOSOMES ARE PRESENT
Microtubule Overlap dictates furrow formation – THE Rappaport Experiment 1961
Overlap zone of antiparallel microtubules (midzone) recruits specific proteins
Centralspindlin complex – required to form midzoneMKLP1/Zen4/Pavarotti
+mgcRacGAP/cyk-4
ABI complex – required to recruit centralspindlinAurora B + INCENP + Survivin
rhoGEF/Pebble
Centralspindlin and ABI complexes = “Passenger Proteins”
Midzone components that associate with kinetochores in metaphase
MetaphaseLoad ontokinetochores
AnaphaseDeposit atmidzone
Model:1 chromosome congression locates spindle center
2 passengers jump off and build midzone
3 Midzone induces furrow formation
Predicts that furrow position is dictated by location of kinetochores.
Quiz: Can this be true?
Passenger proteins localize to midzone in the absence of chromosomes
Savoian & Rieder 1999
Electro-fusion
INCENPLocalizesTo all 3 midzones
So why bother putting the passenger proteins on kinetochores?regulation? Sequestration?
Suggests MIDZONE is the key for specifying furrow position.
Midzone necessary to induce furrow Gatti et al.
Pavarotti mutants Asterless mutants
Kinesin needed to build midzone Recruits gamma tubulin to centrosomeNeeded to form astral MT but not midzone
Normal chromosomesNormal astral MTNo midzone
NO FURROW FORMSNormal midzoneNo astral MT
Furrow forms just fine
What’s so special about the midzone? Is it the antiparallel microtubule array?
Canman et al 2003
Treat cells with:Monastrol blocks centrosome separation to make monopolar spindleMad2DC bypass spindle checkpoint
Band of INCENP localization
Furrow here
Conclusion:Antiparallel MTs are NOT needed
So what is?
MTs that determine furrow are unusually stable
Canman et al 2003
Dynamic MT
Stable MTs
How does a microtubule “know” it is aimed at the furrow site?
dynamic
dynamic
stable
stable
stable Model: MT stabilizing factorsProvided by chromosomes
+-
Alternative model:Kinetochore-nucleated MT joint withSpindle MT to form antiparallel bundle
How do the two populations of MT direct furrow placement?
Model 1 – dynamic MT inhibit furrow
But we know astral MTnot necessary
Model 2 - stable (or anti-parallel)MT induce furrow
MT as wires
Antiparallel bundles recruit factors
Aslop et al., 2009 actin recruited to Antiparallel MT bundles during cytokinesis
How does the midzone induce the furrow?
Antiparallel microtubule bundle (midzone)
Centralspindlin complex – required to form midzoneMKLP1/Zen4/Pavarotti
+mgcRacGAP/cyk-4
ABI complex – required to recruit centralspindlinAurora B + INCENP + Survivin
rhoGEF/Pebble
Pebble/RhoGEF
RhoGDP RhoGTP
Diaphanous(a formin) Rho Kinase
Actin polymerization Myosin II activation
Actin in the ring: anchored to cortex by anillin/septin, nucleated by formins and Moved by myosin
What else do you need to make a ring?
Possible answer: NOTHING: J. Alberts, D. Vavylonis
Capture & pull models ring as attractor of a dynamical system
But in many cell types cytokinesis proceeds via an incomplete furrow
e.g. Beroe
We are still lacking a mechanistic model for actin ring assembly
General problem: Determining Causality in Cell Biology
Spatial correlation suggests causal relation but:a. Is it just coincidence?b. Which way does the causality go?
Furrow Spindle
Spindle Furrow
SpindleSomething else
Furrow
Laser Ablation
Main Caveat: Destruction versus dispersal
WWII Ball bearing factory, Stuttgart
Surgical Removal
Main Caveat: What else did you remove?
Mortal Kombat “spine-rip” finishing move
Molecular perturbation(Genetic, RNAi, small molecules….)
Main Caveats:Off-target or Pleiotropic effectsIndirect effects
Drosophila pavarotti mutant
Reposition structure
Main caveat:What else moves with it?
Build synthetic structure
Main Caveat:Is it really the same thing?
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