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

Cleavage plane always forms halfway between poles, perpendicular to spindle This i

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 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 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? 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? Maybe it’s the centrosomes! Khodjakov & Rieder Ablate centrosomes with “laser” Centrosomes are also dispensable

Microtubules by themselves are sufficient Aslop & Zhang 2003 Remove all chromosomes AND centrosomes by microsurgery What’s left: spindle MTs

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

Microtubule Overlap dictates furrow formation – THE Rappaport Experiment 1961

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

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

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.

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

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

MTs 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? 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 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 Actin polymerization Diaphanous (a formin) Rho Kinase RhoGTP RhoGDP Pebble/RhoGEF

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 Spindle Something 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

Drosophila pavarotti mutant Molecular perturbation (Genetic, RNAi, small molecules….) Main Caveats: Off-target or Pleiotropic effects Indirect effects

Reposition structure Main caveat: What else moves with it?

Build synthetic structure Main Caveat: Is it really the same thing?

Cytokinesis – the “origin of two- ness ”