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Mitosis

Mitosis. Microtubules and Motors: MT polymer ratchets and motor proteins. F ~1-50pN. F ~ 1-10pN. Microtubule Polymer Dynamics. Dynamic Instability. (Mitchison and Kirschner, 1984). Teadmilling/Flux. (Margolis and Wilson, 1978; Mitchison, 1989). v g , v s , f res , f cat.

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Mitosis

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  1. Mitosis

  2. Microtubules and Motors: MT polymer ratchets and motor proteins. F ~1-50pN F ~ 1-10pN

  3. Microtubule Polymer Dynamics Dynamic Instability. (Mitchison and Kirschner, 1984). Teadmilling/Flux. (Margolis and Wilson, 1978; Mitchison, 1989). vg , vs, fres, fcat dn/dt = Kon[C] - Koff

  4. 1953: Shinya Inoue uses polarizing light microscopy

  5. Dynamic Equilibrium (Inoue, 1967).Poleward Flux (Forer, 1965). Steady state fibers polymerize at equator; depolymerize at poles. Polymerize/push; depolymerize/pull.

  6. The Spindle Machine: Drosophila spindle MTs display poleward flux AND dynamic instability. t1/2 = 5s (versus Ptk t1/2 = 400s). • How do microtubules and motors drive mitotic motility. • How are smooth, accurate movements produced if the microtubule tracks are constantly growing and shrinking.

  7. E.M. analysis by McIntosh et al, 1969-80: Sliding Filaments

  8. Polarity of MTs in the Spindle Interzone.

  9. Force Generation by Spindle Fibers. Ostegren, 1951; Force balance. Nicklas, 1983; 1 nN stall force. • Antagonistic poleward forces. • Force proportional to distance • from pole. • Forces balance at metaphase. • Poleward forces drive anaphase A

  10. Mitotic Force Generators. b. MT depolymerizing motors. a. MT polymer Ratchets. c. MT crosslinking and sliding motors.

  11. Metaphase Anaphase A Anaphase B Mechanism of Anaphase A and B

  12. Model for Anaphase B • Kinesin-5-dependent ipMT slidingpushes poles apart • Poleward Flux is an “on-off” Switch (Brust-Mascher et al, 2004, PNAS,101, 15938; Cheerambathur et al, 2007, JCB, 177, 995)

  13. f3 S(t) f2 f2 v1 v2 v3 f1 v4 L12 vpoly contains vgrow ,vshrink , fcat , fres Anaphase B Model Equations 1. dS/dt = 2 (vsliding- vdep) 2. dL/dt = 2 (vpoly - vsliding) 3. μ·(dS/dt) / 2 = k·N·L·Fm(1 - vsliding / Vm) (Gul Civelekoglu-Scholey)

  14. Model Results Conclude: Anaphase B – kinesin-5 motors drive ipMT sliding and flux = on/off switch

  15. Sharp et al. Nature Cell Biol, 2000 • Brust-Mascher & Scholey. MBC, 2002 • Brust-Mascher et al. PNAS, 2004 • Rogers et al. Nature, 2004 Rapid MT turnover: FRAP t1/2 ~ 5 sec MT Flux, Sliding and Pacman sliding: KLP61F flux: KLP10A Metaphase: sliding: KLP61F flux: KLP10A pacman: KLP59C Dynein Anaphase A: sliding: KLP61F flux: turned off Anaphase B:

  16. Metaphase Anaphase A Anaphase B Mechanism of Anaphase A

  17. Mechanism of Anaphase A. Kinetochore motility - Pacman versus poleward flux.

  18. Coupled kinesin-13 dependent flux-pacman model for anaphase A. (Rogers et al, 2004, Nature; 427, 364.)

  19. Cytokinesis in Animal Cells.

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