Polymerization/depolymerizationmotors

Movement formation

Kuo Lab,J.H.U.

http://www.nature.com/nature/journal/v407/n6807/extref/4071026a0_S3.mov

http://www.bme.jhu.edu/~skuo/movies/MacrophChase.mov

http://www.bme.jhu.edu/~skuo/movies/GC_filo.mov

Beads movement

From Welch Lab. RickA-coated beads in Xenopusegg extract that was supplemented with rhodamine-labelled actin and visualized by fluorescencemicroscopyhttp://mcb.berkeley.edu/labs/welch/jenggoley2004_1.mov

Movement of rickettsia

Picture From Welch Lab

Actin polymerization generatesprotrusive force

Miyata et al. 1999: giant liposomes containingmonomeric actin (100 or 200 microM) and introducedKCl into individual liposomes by an electroporation

G-actin structure

G-actin structure

F-actin

Nucleation of F-actin

Nucleation of F-actinNucleation by Arp2/3complex Nucleation by Formin

Steady status

Treadmilling

How movement is generated?

Cc = Koff/Kon

Force generation

Force generation

Force generation• Load affects kon or koff  or both , it will most likelyincrease Cc:Cc(loaded) = Cc(unloaded) exp (dF/kΤ)

d is the length of the subunit; F the force; k the Boltzmann'sconstant; Τ the absolute temperature

• Fmax = (kT/d) ln ( kon [C]/ koff )• For actin at  50 µM one microfilament can generate aFmax  = 9 pN (equivalent to several myosins)• ATP is not required for force generation, mechanical forceis derived from the chemical potential of proteinpolymerization.

Classic Brownian ratchetSingle polymer

Peskin, Odell & Oster 1993, Biophys J 65:316-324

1. Rigid actin polymer

2. Gap generation (at least 2.7nm) betweenpolymer tip and the cell surface by Brownianmotion

3. Intercalation of monomer

http://www.bme.jhu.edu/~skuo/anim/cBRatchet_balls.swf

Classic Brownian ratchetAccording the brownian motion, the magnitude of the"wiggles" are inversely proportional to the size of thebacterium and to the viscosity of its environment.Diffusion is time-dependent, the longer you wait, the largerthe magnitude of Brownian motions and intercalationeventually occurs. The high speed of Listeria motility (10-100 nm/s) implies thatbacteria diffuse very readily.  Rapid diffusion means that itsBrownian motions are sufficiently large at the right timescales so that the rates of actin monomer intercalation canexplain Listeria's high speed.

The fact First, fluctuations of bacteria are much smallerthan 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

Elastic Brownian RatchetSingle polymer

Mogilner & Oster 1996, Biophys J 71: 3030-3045If filament tips flex sufficiently far from the bacterialsurface, actin monomers can intercalate.  Also thelonger filament applies increased pressure to thebacterial surface.  Increased pressure will eventuallycause bacteria to move.In bead experiments, symmetry breaking can beexplained by stochastic theory with this EBR model.Van Oudenaarde and Theriot, 1999http://www.bme.jhu.edu/~skuo/anim/eBRatchet2.swf

EBR and Tethered filamentsMeshwork

Model fits in the artificial beadmovement

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

VASP effect

VASP effect

VASP effect

Shape of moving lipid vesicles

ChallengeListeria have episodes of motility with pausesspaced at about 5.4 nm, the bacteria probablystep along growing actin filaments.

Kuo and McGrath 2000, Nature 407:1026-9

Listeria move and pause

Kuo and McGrath, 2000

1. Sambeth, R., Baumgaertner, A. (1999). Rectification of random motion by asymmetric polymerization. Physica A 271(1-2):48-62.

2. van Oudenaarden, A., Theriot, J. (1999). Cooperative symmetry-breaking by actin polymerization in a model for cell motility. Nature Cell Biol. 1(493-499.

3. Giardini, P. A., Fletcher, D. A., Theriot, J. A. (2003). Compression forces generated by actin comet tails on lipid vesicles. PNAS 100(11):6493-6498.

4. Mogilner, A., Oster, G. (2003). Force generation by actin polymerization ii: The elastic ratchet and tethered filaments. Biophys. J. 84(3):1591–1605. 5. 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):7401–7407.

6. Plastino, J., Olivier, S., Sykes, C. (2004). Actin filaments align into hollow comets for rapid vasp-mediated propulsion. Current Biology 14(19):1766-1771.

7. Burroughs, N. J., Marenduzzo, D. (2005). Three-dimensional dynamic monte carlo simulations of elastic actin-like ratchets. The Journal of Chemical Physics 123(17):174908-11.

8. Kuo, S.C., and McGrath, J. L. (2000) Steps and fluctuations of Listeria monocytogenes during actin-based motility. Nature, 407: 1026-9.