Thomas D. Pollard, M.D.

Eugene Higgins Professor of Molecular, Cellular & Developmental Biology, and Cell Biology Chair, Molecular, Cellular & Developmental Biology
Pollard lab website Phone: (203) 432-3565 Lab: (203) 432-3194 Fax: (203) 432-6161 e-mail: thomas.pollard@yale.edu		Department of Molecular Cellular and Developmental Biology 219 Prospect Street PO Box 208103 New Haven, CT 06520-8103 <Courier Address> 219 Prospect Street, KBT 548 (Lab: KBT 538) New Haven, CT 06511-2106

The molecular basis of cellular motility and cytokinesis

We study the molecular basis of cellular motility, particularly the roles of actin filaments and myosin motors. Actin-based movements are essential for cell division, shaping organs during embryonic development, defense against microorganisms and wiring the nervous system. Movement of cells out of primary tumors is the chief cause of mortality in cancer. We find it advantageous to use combination of biochemical, biophysical, cellular and genetic experiments to test hypotheses about molecular mechanisms and biological functions. Fission yeast are useful for genetics tests of physiological function as well as biochemistry, human cells allow us to investigate the mechanisms of disease-causing mutations and Acanthamoeba provide abundant material for biochemical and cellular studies. We divide our effort between two main projects:

1. Actin filament dynamics: We aim to understand how assembly of actin filaments pushes the leading edge of motile cells forward. We discoverd that Arp2/3 complex is both the initiator of actin filament assembly and the central integrator of inputs from signaling pathways. Arp2/3 complex is a stable, ubiquitous assembly of two actin-related proteins and five novel subunits.

In animal cells signals from cell surface receptors activate WASp/Scar proteins, which stimulate Arp2/3 complex to nucleate new actin filaments as branches on the side of pre-exisiting filaments. Signals, which include Rho-family GTPases, SH3 domain proteins and polyphospho-inositides, overcome autoinhibition of WASp. In fungi myosin-I cooperates with WASp to regulate Arp2/3 complex. WASp brings together an actin monomer and Arp2/3 complex on the side of a filament to intiate a branch. Arp2/3 complex is incorporated into the network and new filaments are capped rapidly, so activated Arp2/3 complex must be supplied continuously to keep the network growing. Immediate goals are to determine the atomic structures of the key proteins and to trace the activation pathway and branching mechanism by kinetic and genetic dissection.

The mechanism of actin filament disassembly is less well understood. Our hypothesis is that hydrolysis of ATP bound to polymerized actin, followed by phosphate dissociation marks older filaments for depolymerization. Our evidence suggests that ADF/cofilin proteins control the tempo of disassembly by promoting dissociation of phosphate from polymerized actin after ATP hydrolysis. Phosphate dissociation weakens the attachment of daughter filaments to Arp2/3 complex and promotes debranching. ADF/cofilin also severs ADP-actin filaments, providing more ends for subunit dissociation. Profilin catalyzes exchange of ADP for ATP on the dissociated actin subunits, recycling ATP-actin back to a pool of unpolymerized monomers bound to profilin that is poised for rapid elongation of new ends. This whole process of assembly and disassembly runs automatically once triggered by a signal to WASP or other activators of Arp2/3 complex.

2. Cytokinesis: Cytokinesis remains one of the most mysterious of major biological processes. Fission yeast is the ideal organism to learn how cells control the assembly of an equitorial ring composed of actin filaments and myosin-II and trigger its contraction once chromosomes have separated. We characterized the two myosin-II proteins of fission yeast and have used them to screen for genes required for cytokinesis. Others have found a number of other genes required for cytokinesis. The field is growing rapidly and we expect major progress to be made in the near future as the inventory of essential genes is identified and the mechanisms of the proteins are determined by genetics and biochemistry.

Ribbon diagraom of the crystal structure of bovine Arp2/3 complex.

Selected Publications

Lee, W.-L., Bezanilla, M., and Pollard, T.D. (2000). Fission yeast myosin-I, Myo1p, stimulates actin assembly by Arp2/3 complex and shares functions with WASp. J. Cell Biol. 151:789-800.

Marchand, J.-B., Kaiser, D.A., Pollard, T.D., and Higgs, H.N. (2001). Interaction of WASp/Scar proteins with actin and vertebrate Arp2/3 complex. Nature Cell Biol. 3:76-82.

Robinson, R.C., Turbedsky, K., Kaiser, D.A., Higgs, H.N., Marchand, J.-B., Choe, S. and Pollard, T.D. (2001) Crystal structure of Arp2/3 complex. Science 294:1679-1684.

Amann, K.J. and Pollard, T.D. (2001) Direct real-time observation of actin filament branching mediated by Arp2/3 complex using total internal reflection microscopy. Proc. Nat. Acad. Sci. (U.S.A.) 98:15009-15013.

Kovar, D.R., Kuhn, J.R., Tichy, A. and Pollard, T.D. (2003) The fission yeast cytokinesis formin Cdc12p is a barbed end actin filament capping protein gated by profilin. J. Cell Biol. 161(5): **.

Pollard, T.D. and Borisy, G.G. (2003) Cellular motility driven by assembly and disassembly of actin filaments. Cell 112:453-65.

Panchal, S.C., Kaiser, D.A., Torres, E., Pollard, T.D. and Rosen, M.K. (2003) A conserved amphipathic helix in WASP/Scar proteins is essential for activation of Arp2/3 complex. Nature Struct. Biol. 10:591-598.

Wu, J.-Q., Kuhn, J.R., Kovar, D.R. and Pollard, T.D. (2003) Spatial and temporal pathway for assembly and constriction of the contractile ring in fission yeast cytokinesis. Devel. Cell 5:723-734.

Beltzner, C.C. and Pollard, T.D. (2004) Identification of functionally important residues of Arp2/3 complex by analysis of homology models from diverse species. J. Molec. Biol. 336:551-565.

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