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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

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


Cytokinesis the origin of two ness

Cleavage plane always forms halfway between poles, perpendicular to spindle

This i


Cytokinesis the origin of two ness

Oriented spindle axis - oriented cell division


Cytokinesis the origin of two ness

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


Cytokinesis the origin of two ness

Cleavage plane always correlates with spindle position

This is a CORRELATION

spindle furrow

furrowspindle

spindle  X  furrow

How do we determine causality?


Cytokinesis the origin of two ness

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


Cytokinesis the origin of two ness

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


Cytokinesis the origin of two ness

Separating spindle from cortical regions normally destined for furrow formation

Yatsu 1912

Implies spindle sends a signal TO the cortex


Cytokinesis the origin of two ness

Moving the spindle moves the cleavage furrow position

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


Cytokinesis the origin of two ness

What part of the spindle is instructing the cortex?

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


Cytokinesis the origin of two ness

What part of the spindle is instructing the cortex?

Maybe it’s the centrosomes!

Khodjakov & Rieder

Ablate centrosomes

with “laser”

Centrosomes are also dispensable


Cytokinesis the origin of two ness

Microtubules by themselves are sufficient

Aslop & Zhang 2003

Remove all chromosomes

AND centrosomes

by microsurgery

What’s left: spindle MTs


Cytokinesis the origin of two ness

Chromosomes vs. microtubules

(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


Cytokinesis the origin of two ness

Microtubule Overlap dictates furrow formation – THE Rappaport Experiment 1961


Cytokinesis the origin of two ness

Overlap zone of antiparallel 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


Cytokinesis the origin of two ness

Centralspindlin 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


Cytokinesis the origin of two ness

Passenger proteins localize to midzone 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.


Cytokinesis the origin of two ness

Midzone 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


Cytokinesis the origin of two ness

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 spindle

Mad2DC  bypass spindle checkpoint

Band of INCENP localization

Conclusion:

AntiparallelMTs are NOT needed

So what is?

Furrow here


Cytokinesis the origin of two ness

MTs that determine furrow are unusually stable

Canman et al 2003

Stable MTs

Dynamic MT


Cytokinesis the origin of two ness

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


Cytokinesis the origin of two ness

How do the two populations of MT direct furrow placement?

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


Cytokinesis the origin of two ness

How does the 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


Cytokinesis the origin of two ness

Myosin II activation

Actin polymerization

Diaphanous

(a formin)

Rho Kinase

RhoGTP

RhoGDP

Pebble/RhoGEF


Cytokinesis the origin of two ness

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


Cytokinesis the origin of two ness

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


Cytokinesis the origin of two ness

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

Spindle

Something else

Furrow


Cytokinesis the origin of two ness

Laser Ablation

Main Caveat: Destruction versus dispersal

WWII Ball bearing factory, Stuttgart


Cytokinesis the origin of two ness

Surgical Removal

Main Caveat:

What else did you remove?

Mortal Kombat “spine-rip” finishing move


Cytokinesis the origin of two ness

Drosophila pavarotti mutant

Molecular perturbation

(Genetic, RNAi, small molecules….)

Main Caveats:

Off-target or Pleiotropic effects

Indirect effects


Cytokinesis the origin of two ness

Reposition structure

Main caveat:

What else moves with it?


Cytokinesis the origin of two ness

Build synthetic structure

Main Caveat:

Is it really the same thing?


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