Polymerization/depolymerization motors

Transcription

1 Polymerization/depolymerization motors

2 Movement formation Kuo Lab, J.H.U a0_S3.mov

3 Beads movement From Welch Lab. RickA-coated beads in Xenopus egg extract that was supplemented with rhodaminelabelled actin and visualized by fluorescence microscopy

4 Movement of rickettsia Picture From Welch Lab

5 Actin polymerization generates protrusive force Miyata et al. 1999: giant liposomes containing monomeric actin (100 or 200 microm) and introduced KCl into individual liposomes by an electroporation

6 G-actin structure

7 G-actin structure

8 F-actin

9 Nucleation of F-actin

10 Nucleation of F-actin Nucleation by Arp2/3 complex Nucleation by Formin

11 Steady status

12 Treadmilling

13 How movement is generated? Cc = Koff/Kon

14 Force generation

15 Force generation

16 Force generation Load affects kon or koff or both, it will most likely increase Cc: Cc(loaded) = Cc(unloaded) exp (df/kτ) d is the length of the subunit; F the force; k the Boltzmann's constant; Τ the absolute temperature Fmax = (kt/d) ln ( kon [C]/ koff ) For actin at 50 µm one microfilament can generate a Fmax = 9 pn (equivalent to several myosins) ATP is not required for force generation, mechanical force is derived from the chemical potential of protein polymerization.

17 Classic Brownian ratchet Single polymer Peskin, Odell & Oster 1993, Biophys J 65: Rigid actin polymer 2. Gap generation (at least 2.7nm) between polymer tip and the cell surface by Brownian motion 3. Intercalation of monomer lls.swf

18 Classic Brownian ratchet According the brownian motion, the magnitude of the "wiggles" are inversely proportional to the size of the bacterium and to the viscosity of its environment. Diffusion is time-dependent, the longer you wait, the larger the magnitude of Brownian motions and intercalation eventually occurs. The high speed of Listeria motility ( nm/s) implies that bacteria diffuse very readily. Rapid diffusion means that its Brownian motions are sufficiently large at the right time scales so that the rates of actin monomer intercalation can explain Listeria's high speed.

19 The fact First, fluctuations of bacteria are much smaller than the intercalation size of G-actin (20 X less). They must be binding their F-actin tails. Kuo and McGrath 2000, Nature 407:1026-9

20 Elastic Brownian Ratchet Single polymer Mogilner & Oster 1996, Biophys J 71: If filament tips flex sufficiently far from the bacterial surface, actin monomers can intercalate. Also the longer filament applies increased pressure to the bacterial surface. Increased pressure will eventually cause bacteria to move. In bead experiments, symmetry breaking can be explained by stochastic theory with this EBR model. Van Oudenaarde and Theriot,

21 EBR and Tethered filaments Meshwork

22 Model fits in the artificial bead movement 1. Density of coating and percent of extract don t affect velocity. 2. Smaller beads move slower 3. Tiny beads don t move 4. Force-Velocity dependence on the tail density

23 VASP effect

24 VASP effect

25 VASP effect

26 Shape of moving lipid vesicles

27 Challenge Listeria have episodes of motility with pauses spaced at about 5.4 nm, the bacteria probably step along growing actin filaments. Kuo and McGrath 2000, Nature 407:1026-9

28 Listeria move and pause Kuo and McGrath, 2000

29 1. Sambeth, R., Baumgaertner, A. (1999). Rectification of random motion by asymmetric polymerization. Physica A 271(1-2): van Oudenaarden, A., Theriot, J. (1999). Cooperative symmetrybreaking by actin polymerization in a model for cell motility. Nature Cell Biol. 1( Giardini, P. A., Fletcher, D. A., Theriot, J. A. (2003). Compression forces generated by actin comet tails on lipid vesicles. PNAS 100(11): Mogilner, A., Oster, G. (2003). Force generation by actin polymerization ii: The elastic ratchet and tethered filaments. Biophys. J. 84(3): Daniels, D., Turner, M. (2004). The force generated by biological membranes on a polymer rod and its response: Statics and dynamics. J. Chem. Phys. 121(15): Plastino, J., Olivier, S., Sykes, C. (2004). Actin filaments align into hollow comets for rapid vasp-mediated propulsion. Current Biology 14 (19): Burroughs, N. J., Marenduzzo, D. (2005). Three-dimensional dynamic monte carlo simulations of elastic actin-like ratchets. The Journal of Chemical Physics 123(17): Kuo, S.C., and McGrath, J. L. (2000) Steps and fluctuations of Listeria monocytogenes during actin-based motility. Nature, 407: