Actin filaments are essential structural elements in cells and actin filament assembly produces forces for some types of cellular movements such as locomotion and endocytosis. Cellular movements are essential for cell division, embryonic development and defense against microorganisms. Movement of cells out of primary tumors is the chief cause of mortality in cancer. This grant supports basic research on the structures and functions of actin-binding proteins and their roles in live cells. The long-term goal is to understand molecular mechanisms that control the dynamics of the actin cytoskeleton. The work focuses on formin proteins and proteins that coordinate actin filament turnover during endocytosis and cellular motility: Arp2/3 complex, WASp/Scar proteins (activators of Arp2/3 complex), capping protein (terminator of actin filament elongation), ADF/cofilin (recycles actin subunits) and profilin (nucleotide exchange factor for actin and cofactor for formins). Simulations of mathematical models based on quantitative biochemical parameters are compared with quantitative measurements in live cells, not only to impose rigorous thinking but also to identify features that are most likely to distinguish competing hypotheses. The fission yeast, S. pombe, is our experimental organism. Motility of its cortical actin patches depends on the same proteins used by motile amoebas and animal cells to advance their leading edges. The fission yeast offers outstanding genetics, molecular genetics and spectacular cytology, all of which are required to reach the goals of the project. Proteins of interest are manipulated through site directed mutagenesis and studied in live cells after tagging with a fluorescent fusion protein (and verifying their functionality with genetic tests). Methods are available to isolate sufficient quantities of most proteins of interest for structural and biophysical studies. The project has three wide goals for the next five years. The first is to understand how formins change their shape during each cycle that adds a subunit to the end of an actin filament and how force influences the process. The second is to understand how Arp2/3 complex changes its shape from the inactive form free in solution to the active form anchoring an actin filament branch to the side of another actin filament. The third is to use clathrin-mediated endocytosis as the model system to test hypotheses about how actin filaments are assembled and disassembled in live cells. The experiments depend on quantitative fluorescence microscopy to measure dynamics, super-resolution fluorescence microscopy to determine the positions of molecules over time and mathematical modeling to test the ability of biochemical hypotheses to account for events in live cells.

Public Health Relevance

Actin forms filaments to support the structure of the cell and their assembly produces forces for cellular movements that are essential for cell division, embryonic development and defense against microorganisms. Movement of cells out of primary tumors is the chief cause of mortality in cancer. This grant supports basic research on the structure of actin-binding proteins, the dynamics of their interactions and their functions in live cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM026338-36
Application #
8538983
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Gindhart, Joseph G
Project Start
1978-09-01
Project End
2016-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
36
Fiscal Year
2013
Total Cost
$490,510
Indirect Cost
$195,909
Name
Yale University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Baker, Joseph L; Courtemanche, Naomi; Parton, Daniel L et al. (2015) Electrostatic interactions between the Bni1p Formin FH2 domain and actin influence actin filament nucleation. Structure 23:68-79
Berro, Julien; Pollard, Thomas D (2014) Local and global analysis of endocytic patch dynamics in fission yeast using a new "temporal superresolution" realignment method. Mol Biol Cell 25:3501-14
Akamatsu, Matthew; Berro, Julien; Pu, Kai-Ming et al. (2014) Cytokinetic nodes in fission yeast arise from two distinct types of nodes that merge during interphase. J Cell Biol 204:977-88
Arasada, Rajesh; Pollard, Thomas D (2014) Contractile ring stability in S. pombe depends on F-BAR protein Cdc15p and Bgs1p transport from the Golgi complex. Cell Rep 8:1533-44
Courtemanche, Naomi; Pollard, Thomas D (2013) Interaction of profilin with the barbed end of actin filaments. Biochemistry 52:6456-66
Courtemanche, Naomi; Lee, Ja Yil; Pollard, Thomas D et al. (2013) Tension modulates actin filament polymerization mediated by formin and profilin. Proc Natl Acad Sci U S A 110:9752-7
Pollard, Thomas D; De La Cruz, Enrique M (2013) Take advantage of time in your experiments: a guide to simple, informative kinetics assays. Mol Biol Cell 24:1103-10
Courtemanche, Naomi; Pollard, Thomas D (2012) Determinants of Formin Homology 1 (FH1) domain function in actin filament elongation by formins. J Biol Chem 287:7812-20
Chen, Qian; Nag, Shalini; Pollard, Thomas D (2012) Formins filter modified actin subunits during processive elongation. J Struct Biol 177:32-9
Ti, Shih-Chieh; Jurgenson, Christopher T; Nolen, Bradley J et al. (2011) Structural and biochemical characterization of two binding sites for nucleation-promoting factor WASp-VCA on Arp2/3 complex. Proc Natl Acad Sci U S A 108:E463-71

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