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

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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?