Our laboratory investigates the molecular basis of cellular motility and cytokinesis. Actin-based cellular movements are essential for 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. Cytokinesis is essential for the replication of all cells and is still one of the least understood aspects of cell division. Half of the lab studies how assembly of actin filaments pushes forward the leading edge of motile cells and moves coated vesicles into cells. We discovered that Arp2/3 complex is both the central integrator of inputs from signaling pathways and initiator of actin filament assembly. Half of the lab uses fission yeast to study cytokinesis, still one of the most mysterious of cellular processes. 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 and are investigating how a formin protein mediates actin assembly and how cells trigger constriction of the ring at the end of mitosis.
Extensive Research Description
We use biochemical, biophysical, cellular, and genetic experiments to study the molecular mechanisms of actin-based cellular movements.
Actin-based movements: We study how cells control the assembly and disassembly of actin filaments during cellular movements. We have projects on the structure and function of actin, Arp2/3 complex, activators of Arp2/3 complex (such as the Wiskott-Aldrich syndrome protein, WASp), profilin, ADF/cofilin and capping protein. We use fluorescence microscopy of proteins tagged with fluorescent fusion proteins to follow the time course of the interactions of these proteins during endocytosis in fission yeast.
Cytokinesis: We study the mechanism of cytokinesis using the fission yeast S. pombe as a favorable model organism to learn how cells pinch themselves in two when they divide. We investigate the early steps in the assembly of the cytokinetic contractile ring, so we study the structures and functions of the proteins that organize the precursors of the contractile ring, including anillin, myosin-II, a formin, an IQGAP and an F-BAR protein. Anillin and IQGAPs appear to be adapter proteins that form a scaffold for the other proteins. Formins grow actin filaments while remaining attached to the end of the elongating polymer. Myosin-II pulls together the precurors of the contractile ring and later constricts the contractile ring to pinch the daughter cells in two.
We use computer simulations of mathematical models to compare our ideas regarding mechanisms with observations in live cells.
- Effect of profilin on actin filament assembly
- Actin filament severing by cofilin
- Mechanism of actin filament nucleation by Arp2/3 complex
- Structural studies of nucleation promoting factors binding Arp2/3 complex
- Assembly and disassembly of actin patches at sites of endocytosis
- Role of F-BAR proteins in clathrin-mediated endocytosis
- Pathway of assembly of the contractile ring in fission yeast
- Role of anillin in organizing the contractile ring
- Role of IQGAP in organizing the contractile ring
- Role of F-BAR proteins in organizing the contractile ring
- Mechanism of actin filament nucleation and elongation by formins
- Mechanism of constriction and disassembly of the contractile ring
- Vavylonis, D., Wu, J.-Q., Hao, S., O'Shaughnessy, B. and Pollard, T.D. (2008) Assembly mechanism of the contractile ring for cytokinesis by fission yeast. Science 319:97-100.
- 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.
- Wu, J-Q., Kuhn, J. R., Kovar, D. R. and Pollard, T. D. Spatial and temporal pathway for assembly and constriction of the contractile ring in fission yeast cytokinesis. Devel. Cell 5, 723-734 (2003)
- Paul, A. and Pollard, T. D. The role of the FH1 domain and profilin in formin-mediated actin filament elongation and nucleation. Curr. Biol. 18, 9-19 (2008)
- Nolen, B. and Pollard, T. D. Insights into the influence of nucleotides on the actin family of proteins from seven crystal structures of bovine Arp2/3 complex. Mol. Cell 26, 449-457 (2007)