Our goal is to understand the molecular mechanisms controlling assembly and disassembly of actin filaments during cellular locomotion and endocytosis in terms of the rates of specific reactions. We will use a combination of biochemical, genetic and cellular experiments in fission yeast to test how cells maintain a pool of actin subunits, initiate new actin filaments and disassemble aged actin filaments. We will use spectroscopic assays to map the pathway of actin filament branch formation by Arp2/3 complex in the presence and absence of profilin. We will characterize the biochemical properties of a temperature sensitive mutant of fission yeast cofilin for use in experiments with live yeast cells. We will reconstitute a recycling actin motility system from purified proteins and analyze the dynamics of individual filaments by fluorescence microscopy. We will use fluorescence microscopy of fission yeast expressing functional fusion proteins to determine the molecular pathway of actin filament assembly and turnover during endocytosis for comparisons with mathematical models. We will test mechanisms in cells by varying protein concentrations and/or mutagenesis. We will use direct observations of growing actin filaments to determine how elongation is influenced by the length of linker peptides in the FH2 domain, tension on the N-terminus of formin FH1FH2 constructs and the distance between profilin binding sites in the FH1 domain and the FH2 domain. We will use single molecule fluorescence microscopy to study conformational changes in the FH2 domain on the end of actin filaments and to investigate the """"""""rotation paradox"""""""" of FH2 domains on the ends of growing actin filaments. ? ? PUBLIC HEALTH REVELATION: 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. ? ? ?
| Espinoza-Sanchez, Sofia; Metskas, Lauren Ann; Chou, Steven Z et al. (2018) Conformational changes in Arp2/3 complex induced by ATP, WASp-VCA, and actin filaments. Proc Natl Acad Sci U S A 115:E8642-E8651 |
| Arasada, Rajesh; Sayyad, Wasim A; Berro, Julien et al. (2018) High-speed superresolution imaging of the proteins in fission yeast clathrin-mediated endocytic actin patches. Mol Biol Cell 29:295-303 |
| Aydin, Fikret; Courtemanche, Naomi; Pollard, Thomas D et al. (2018) Gating mechanisms during actin filament elongation by formins. Elife 7: |
| Friend, Janice E; Sayyad, Wasim A; Arasada, Rajesh et al. (2018) Fission yeast Myo2: Molecular organization and diffusion in the cytoplasm. Cytoskeleton (Hoboken) 75:164-173 |
| Fujiwara, Ikuko; Zweifel, Mark E; Courtemanche, Naomi et al. (2018) Latrunculin A Accelerates Actin Filament Depolymerization in Addition to Sequestering Actin Monomers. Curr Biol 28:3183-3192.e2 |
| Akamatsu, Matthew; Lin, Yu; Bewersdorf, Joerg et al. (2017) Analysis of interphase node proteins in fission yeast by quantitative and superresolution fluorescence microscopy. Mol Biol Cell 28:3203-3214 |
| Pollard, Thomas D (2017) Nine unanswered questions about cytokinesis. J Cell Biol 216:3007-3016 |
| Pollard, Thomas D (2017) What We Know and Do Not Know About Actin. Handb Exp Pharmacol 235:331-347 |
| Courtemanche, Naomi; Pollard, Thomas D; Chen, Qian (2016) Avoiding artefacts when counting polymerized actin in live cells with LifeAct fused to fluorescent proteins. Nat Cell Biol 18:676-83 |
| Pollard, Thomas D (2016) Actin and Actin-Binding Proteins. Cold Spring Harb Perspect Biol 8: |
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