Mitosis Microtubules and Motors: MT polymer ratchets and motor proteins. F ~ 1-10pN F ~1-50pN Teadmilling/Flux. (Margolis and Wilson, 1978; Mitchison, 1989). Dynamic Instability. (Mitchison and Kirschner, 1984). Microtubule Polymer Dynamics v g, v s, f res, f cat dn/dt = K on [C] - K off 1953: Shinya Inoue uses polarizing light microscopy Dynamic Equilibrium (Inoue, 1967). Poleward Flux (Forer, 1965). Polymerize/push; depolymerize/pull. Steady state fibers polymerize at equator; depolymerize at poles. The Spindle Machine: Drosophila spindle MTs display poleward flux AND dynamic instability. 1.How do microtubules and motors drive mitotic motility. 2.How are smooth, accurate movements produced if the microtubule tracks are constantly growing and shrinking. t 1/2 = 5s (versus Ptk t 1/2 = 400s). E.M. analysis by McIntosh et al, : Sliding Filaments Polarity of MTs in the Spindle Interzone. Force Generation by Spindle Fibers. Nicklas, 1983; 1 nN stall force. Ostegren, 1951; Force balance. 1.Antagonistic poleward forces. 2.Force proportional to distance from pole. 3.Forces balance at metaphase. 4.Poleward forces drive anaphase A Mitotic Force Generators. a. MT polymer Ratchets. b. MT depolymerizing motors. c. MT crosslinking and sliding motors. Mechanism of Anaphase A and B Metaphase Anaphase A Anaphase B Model for Anaphase B 1.Kinesin-5-dependent ipMT sliding pushes poles apart 2.Poleward Flux is an on-off Switch (Brust-Mascher et al, 2004, PNAS,101, 15938; Cheerambathur et al, 2007, JCB, 177, 995) 1. dS/dt = 2 (v sliding - v dep ) 2. dL/dt = 2 (v poly - v sliding ) 3. (dS/dt) / 2 = kNLF m (1 - v sliding / V m ) S(t) Anaphase B Model Equations L 12 f2f2 f2f2 f1f1 v2v2 v3v3 v4v4 v1v1 f3f3 v poly contains v grow, v shrink, f cat, f res (Gul Civelekoglu-Scholey) Model Results Conclude: Anaphase B kinesin-5 motors drive ipMT sliding and flux = on/off switch MT Flux, Sliding and Pacman Metaphase: sliding: KLP61F flux: KLP10A 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 t 1/2 ~ 5 sec Anaphase A: sliding: KLP61F flux: KLP10A pacman: KLP59C Dynein Anaphase B: sliding: KLP61F flux: turned off Mechanism of Anaphase A Metaphase Anaphase A Anaphase B Kinetochore motility - Pacman versus poleward flux. Mechanism of Anaphase A. Coupled kinesin-13 dependent flux-pacman model for anaphase A. (Rogers et al, 2004, Nature; 427, 364.) Cytokinesis in Animal Cells.