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We study the molecular basis of cellular motility and cytokinesis, 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 use fission yeast for much of this work because they use the same molecular mechanisms at humans, but are much more amenable to experimentation using molecular genetics. We find it advantageous to use a combination of biochemical, biophysical, cellular and genetic experiments to test hypotheses about molecular mechanisms and biological functions. 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 and vesicles formed by endocytosis. 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.
Cell surface receptors activate WASp/Scar proteins, which stimulate Arp2/3 complex to nucleate new actin filaments as branches on the sides of pre-existing filaments. 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. We use biophysical methods including x-ray crystallography in parallel with real time observations of proteins in live cells to study the pathway.
The mechanism of actin filament disassembly is less well understood. Our evidence suggests that ADF/cofilin proteins control the tempo of disassembly. We study how ADF/cofilins promote dissociation of phosphate after ATP hydrolysis by polymerized actin, dissociate Arp2/3 complex from the sides of filaments and sever ADP-actin filaments, providing more ends for subunit dissociation.
2. Cytokinesis: Cytokinesis remains one of the most mysterious of vital biological processes. Fission yeast is the ideal organism to learn how cells control the assembly of an equatorial contractile ring composed of actin filaments and myosin-II and trigger its constriction once chromosomes have separated. We study the key cytokinesis proteins. The formin protein Cdc12p nucleates actin filaments for the contractile ring and remains attached to their fast growing ends, so it can also anchor the filaments to the membrane. Myosin-II is the motor protein that applies tension to the actin filaments. Our time-lapse movies of live cells expressing fluorescent fusion proteins revealed the temporal and spatial pathway for contractile ring assembly. Numerical simulations show that a simple mechanism involving random searches by growing actin filaments, capture of these filaments and pulling by myosin-II and frequent release of these attachments accounts for contractile ring assembly. Mathematical modeling has also directed our attention to many unanswered questions to be explored at the cellular and molecular levels.
Selected Publications
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.
Wu JQ, Pollard TD (2005). Counting cytokinesis proteins globally and locally in fission yeast. Science 310:310-314.
Kovar DR, Harris ES, Mahaffy R, Higgs HN, Pollard TD (2006) Control of the assembly of ATP- and ADP-actin by formins and profilin. Cell 124:423-435.
Nolen B and Pollard TD (2007) 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.
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