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Cytokinesis – the “origin of two- ness ”. Outline. 1. What determines placement of the cleavage furrow. good. bad. 2. Classical cytokinesis experiments as a paradigm for testing causality of structures. Cleavage plane always forms halfway between poles, perpendicular to spindle. This i.

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Cytokinesis – the “origin of two-ness”

Outline

1. What determines placement of the cleavage furrow

good

bad

2. Classical cytokinesis experiments as a paradigm for testing causality of structures



Oriented spindle axis - perpendicular to spindle oriented cell division


Off-center spindle perpendicular to spindle asymmetric cell division producing different sized daughters


Cleavage plane always correlates with spindle position perpendicular to spindle

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

Instructive vs. Permissive

Instructive:

spindle tells furrow WHERE to form

Metaphase

(before furrow starts)

No cleavage

Permissive:

spindle tells furrow WHEN to form

(like a checkpoint)

Anaphase

(after furrow has started)

Cleavage completes


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 for furrow formation

O’Connell and Wang, 2000

Normal cells (NRK)

Asymmetric spindle

correlates with

asymmetric cleavage furrow

Impose new long axis

Push spindle and pin at one end


What part of the spindle is instructing the cortex? for furrow formation

Maybe it’s the chromosomes!

Zhang & Nicklas 1996

Remove all chromosomes by

microsurgery

“chromosomes? We don’t need no

stinking chromosomes”

What’s left: centrosomes, astral MTs, spindle MTs


What part of the spindle is instructing the cortex? for furrow formation

Maybe it’s the centrosomes!

Khodjakov & Rieder

Ablate centrosomes

with “laser”

Centrosomes are also dispensable


Microtubules by themselves are sufficient for furrow formation

Aslop & Zhang 2003

Remove all chromosomes

AND centrosomes

by microsurgery

What’s left: spindle MTs


Chromosomes vs. microtubules for furrow formation

(maybe MTs are a back-up system only if chromosomes are missing

Zhang 2004

Furrow here

Not here

Remove all but one

chromatid by

Microsurgery

Cell forms

asymmetric spindle

MTs dictate furrow placement

EVEN WHEN

CHROMOSOMES ARE PRESENT


Microtubule Overlap dictates furrow formation – for furrow formationTHE Rappaport Experiment 1961


Overlap zone of for furrow formationantiparallel microtubules (midzone) recruits specific proteins

Centralspindlin complex – required to form midzone

MKLP1/Zen4/Pavarotti

+

mgcRacGAP/cyk-4

ABI complex – required to recruit centralspindlin

Aurora B + INCENP + Survivin

rhoGEF/Pebble


Centralspindlin for furrow formation and ABI complexes = “Passenger Proteins”

Midzone components that associate with kinetochores in metaphase

Model:

1 chromosome congression locates spindle center

2 passengers jump off and build midzone

Midzone induces furrow formation

Predicts that furrow position is dictated by

location of kinetochores.

Quiz: Can this be true?

Metaphase

Load onto

kinetochores

Anaphase

Deposit at

midzone


Passenger proteins localize to for furrow formationmidzone in the absence of chromosomes

Savoian & Rieder 1999

INCENP

Localizes

To all 3 midzones

Electro-

fusion

So why bother putting the passenger proteins on kinetochores?

regulation? Sequestration?

Suggests MIDZONE is the key for specifying furrow position.


Midzone for furrow formation necessary to induce furrow

Gatti et al.

Pavarotti mutants

Asterless mutants

Kinesin needed to build midzone

Recruits gamma tubulin to centrosome

Needed to form astral MT but not midzone

Normal chromosomes

Normal astral MT

No midzone

NO FURROW FORMS

Normal midzone

No astral MT

Furrow forms just fine


What’s so special about the for furrow formationmidzone?

Is it the antiparallel microtubule array?

Canman et al 2003

Treat cells with:

Monastrol blocks centrosome separation to make monopolar spindle

Mad2DC  bypass spindle checkpoint

Band of INCENP localization

Conclusion:

AntiparallelMTs are NOT needed

So what is?

Furrow here


MTs for furrow formation that determine furrow are unusually stable

Canman et al 2003

Stable MTs

Dynamic MT


How does a microtubule “know” it is aimed at the furrow site?

dynamic

stable

stable

stable

Model: MT stabilizing factors

Provided by chromosomes

dynamic

+

-

Alternative model:

Kinetochore-nucleated MT joint with

Spindle MT to form antiparallel bundle


How do the two populations of MT direct furrow placement? site?

Model 1 – dynamic MT inhibit furrow

Model 2 - stable (or anti-parallel)

MT induce furrow

MT as wires

Antiparallel bundles recruit factors

But we know

astral MT

not necessary

Aslop et al., 2009  actin recruited to

Antiparallel MT bundles during cytokinesis


How does the site?midzone induce the furrow?

Antiparallel microtubule bundle (midzone)

Centralspindlin complex – required to form midzone

MKLP1/Zen4/Pavarotti

+

mgcRacGAP/cyk-4

ABI complex – required to recruit centralspindlin

Aurora B + INCENP + Survivin

rhoGEF/Pebble


Myosin II activation site?

Actin polymerization

Diaphanous

(a formin)

Rho Kinase

RhoGTP

RhoGDP

Pebble/RhoGEF


Actin in the ring: anchored to cortex by site?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 site? 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 site?

Spatial correlation suggests causal relation but:

a. Is it just coincidence?

b. Which way does the causality go?

Furrow Spindle

Spindle Furrow

Spindle

Something else

Furrow


Laser Ablation site?

Main Caveat: Destruction versus dispersal

WWII Ball bearing factory, Stuttgart


Surgical Removal site?

Main Caveat:

What else did you remove?

Mortal Kombat “spine-rip” finishing move


Drosophila site?pavarotti mutant

Molecular perturbation

(Genetic, RNAi, small molecules….)

Main Caveats:

Off-target or Pleiotropic effects

Indirect effects


Reposition structure site?

Main caveat:

What else moves with it?


Build synthetic structure site?

Main Caveat:

Is it really the same thing?


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