Thomas D. Pollard, M.D.

Sterling Professor of Molecular, Cellular & Developmental Biology, of Cell Biology and of Molecular Biophysics and Biochemistry Chair, Molecular, Cellular & Developmental Biology Molecular mechanisms of cellular motility and cytokinesis.
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

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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. We use fission yeast for genetic tests of physiological function as well as biochemistry and microscopy. 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 and moves coated vesicles into cells. We discovered 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-existing filaments. Signals, which include Rho-family GTPases, SH3 domain proteins and polyphosphoinositides, 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 initiate 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. ADF/cofilin also dissociates branches and 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 equatorial ring composed of actin filaments and myosin-II and trigger its contraction once chromosomes have separated. We mapped the spatial and temporal pathway for the assembly of more than a dozen proteins in the equatorial contractile ring of actin filaments and myosin-II.  We also developed methods to count protein molecules at any place in live cells. We are now investigating the biochemical and cellular reactions along this pathway including the spatial signals that determine the position of the contractile ring, assembly of “nodes” (the precursors to the contractile ring), the mechanism of actin nucleation and elongation by formins, condensation of nodes into the ring, the regulation of myosin-II and the mechanisms that trigger the constriction and disassembly of the ring at the end of mitosis.

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

 

Selected Publications

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.

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.

Kovar, D.R. and Pollard, T.D. (2004) Insertional assembly of actin in association with formins produces piconewton forces. Proc. Nat. Acad. Sci. USA 101:14725-14730.

Lord, M. and Pollard, T.D. (2004) UCS protein Rng3 activates actin filament gliding by fission yeast myosin-II. J. Cell Biol. 167:315-325.

Nolen, B., Littlefield, R.S. and Pollard, T.D. (2004) Crystal structures of bovine Arp2/3 complex with bound ATP or ADP. Proc. Nat. Acad. Sci. USA 101:15627-15632.

Sirotkin, V., Beltzner, C.C., Marchand, J.B. and Pollard, T.D. (2005) Interactions of WASp, myosin-I, and verprolin with Arp2/3 complex during actin patch assembly in fission yeast. J. Cell Biol. 170:637-648.

Wu, J.-Q. and Pollard, T.D. (2005) Counting cytokinesis proteins globally and locally in fission yeast. Science 310:310-314.

Kovar, D.R., Harris, E.S., Mahaffy, R.E. Higgs, H.N. and Pollard, T.D. (2006) Control of the assembly of ATP- and ADP-actin by formins and profilin. Cell 724:423-435.

Vavylonis, D., Kovar, D.R., O'Shaughnessy, B. and Pollard, T.D. (2006) Mathematical model of the assembly of ATP- and ADP-actin by formins and profilin. Molec. Cell 21:455-466.

Wu, J.-Q., Sirotkin, V., Kovar, D., Lord, M., Beltzner, C., Kuhn, J.R. and Pollard, T.D. (2006) Assembly of the cytokinetic contractile ring from a broad band of nodes in fission yeast. J. Cell Biol. 174:391-402.

Andrianantoandro, E. and Pollard, T.D. (2006) Mechanism of actin filament turnover by severing and nucleation at different concentrations of ADF/cofilins. Molec. Cell 24:13-23.