Presentation on theme: "Cytokinesis – the “origin of two-ness” 1. What determines placement of the cleavage furrow goodbad 2. Classical cytokinesis experiments as a paradigm for."— Presentation transcript:
Cytokinesis – the “origin of two-ness” 1. What determines placement of the cleavage furrow goodbad 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
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 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
Furrow here Not here Chromosomes vs. microtubules (maybe MTs are a back-up system only if chromosomes are missing Zhang 2004 Remove all but one chromatid by Microsurgery Cell forms asymmetric spindle MTs dictate furrow placement EVEN WHEN CHROMOSOMES ARE PRESENT
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 Metaphase Load onto kinetochores Anaphase Deposit at midzone Model: 1 chromosome congression locates spindle center 2 passengers jump off and build midzone 3Midzone 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 INCENP Localizes To 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 furrowGatti et al. Pavarotti mutantsAsterless mutants Kinesin needed to build midzoneRecruits 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 Mad2 C 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 stable Model: MT stabilizing factors Provided by chromosomes + - 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 But we know astral MT not 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 midzone MKLP1/Zen4/Pavarotti + mgcRacGAP/cyk-4 ABI complex – required to recruit centralspindlin Aurora 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 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
Molecular perturbation (Genetic, RNAi, small molecules….) Main Caveats: Off-target or Pleiotropic effects Indirect effects Drosophila pavarotti mutant
Reposition structure Main caveat: What else moves with it?
Build synthetic structure Main Caveat: Is it really the same thing?