Our long range goal is to understand the molecular mechanisms by which actin and its associated proteins participate in cellular movements and the structure of cytoplasm. We will use Acanthamoeba as our model system. To clarify the mechanism of actin polymerization, we will characterize nucleotide hydrolysis during polymerization, reconcile the mechanism of elongation that we deduced from measurements in the electron microscope with observations on bulk samples, analyze the mechanism of nucleation by direct methods, characterize the dynamics of actin filaments at seady state and study the mechanisms of action of cytochalasin and phalloidin. To understand the functions of the actin monomer binding protein called profilin, we will determine the 3-D structure of Acanthamoeba profilinI, profilin-II and ventebrate profilin at atomic resolution by X-ray crystallography, identify amino acid contacts between profilin and actin by chemical crosslinking and protein sequencing so that we can establish how the proteins bind together, characterize the dynamics of the binding of profilin to actin monomers and filaments and search for factors that might regulated this interaction, localize the 2 profilin isoforms in Acanthamoeba and characterize a library of monoclonal antibodies to profilins with the goal of identifying inhibitors of actinprofilin interaction for use in other experiments. Actophorin is an actin monomer binding and filament severing protein. We will determine the primary structure of actophotin from a cloned cDNA, determine the 3-D structure of actophorin at atomic resolution, identify amino acid contacts between actophorin and actin by chemical crosslinking and protein sequencing so that we can show how the proteins bind together, characterize the interaction of actophorin with actin monomers and filaments as a model for the mechanisms of other severning proteins, search for factors that might regulate the interaction of actophorin and actin and localize actophorin in Acanthanoeba. To characterize the actin filament crosslinking protein called alphaactinin, we will study the mechanical properties of mixtures of alphaaclinin and actin filaments as a function of filament length and concentation, analyze the dynamics of the interaction of alphaactinin with actin filaments by fluroescence recovery after photobleaching, stopped flow kinetics, equilibrium binding and electron microscopy, search for factors regulating the interaction of alphaactinin with actin filaments and document the intracellular dynamics of alphaactinin by microinjection of labeled protein and photobleaching recovery.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Cellular Biology and Physiology Subcommittee 1 (CBY)
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Johns Hopkins University
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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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