We propose to continue the development of quantitative Fluorescent Speckle Microscopy (qFSM). qFSM is a variant of fluorescence microscopy to analyze the dynamics of subunits within macromolecular structures in living cells. In the first round of funding, we developed focus-stabilized TIRF FSM and spinning disk confocal imaging of actin cytoskeleton and focal adhesion dynamics in tissue culture cells, and developed image analysis software that converts the stochastic speckle image signal into high- resolution maps of the assembly, disassembly, and transport of the actin filaments. We exploited the quantitative information delivered by this technology to study the regulation of the actin cytoskeleton in migrating epithelial cells and thus established a new paradigm for how the cell builds specific actin-based structures to drive directed cell migration. Here, we seek to make the next critical steps towards our long term goal of establishing qFSM as a method for the comprehensive analysis of spatiotemporal dynamics and interaction of multiple macromolecular structures in living cells.
Our specific aims are:
Aim 1 : To extend qFSM to the measurement of absolute rates of polymer turnover which will allow us to perform in situ biochemistry of actin dynamics in living cells.
Aim 2 : To extend qFSM to the analysis of microtubule and intermediate filament cytoskeletons.
Aim 3 : To establish correlational qFSM to probe the dynamic interaction between two or more macromolecular structures in living cells. These developments are motivated by the hypothesis-driven cell biological research in our labs on the fundamental mechanisms of cytoskeletal function in cell migration, but our technology is implemented generically with an eye towards its broad use by the biomedical research community. We propose to further develop the technique of quantitative Fluorescent Speckle Microscopy for the analysis of the dynamics and interaction of macromolecular assemblies. Our studies will focus on the spatial and temporal integration of the dynamics of the actin filament, microtubule, and intermediate filament cytoskeleton systems. These interactions are centrally implicated in a wide array of cell functions. Thus, the proposed developments will serve the cell biology community to investigate fundamental aspects of cell physiology and pathological behaviors.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project--Cooperative Agreements (U01)
Project #
5U01GM067230-08
Application #
7921994
Study Section
Microscopic Imaging Study Section (MI)
Program Officer
Deatherage, James F
Project Start
2003-01-01
Project End
2012-08-31
Budget Start
2010-09-01
Budget End
2011-08-31
Support Year
8
Fiscal Year
2010
Total Cost
$377,561
Indirect Cost
Name
Harvard University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Werbin, Jeffrey L; AvendaƱo, Maier S; Becker, Verena et al. (2017) Multiplexed Exchange-PAINT imaging reveals ligand-dependent EGFR and Met interactions in the plasma membrane. Sci Rep 7:12150
Kwon, Mijung; Bagonis, Maria; Danuser, Gaudenz et al. (2015) Direct Microtubule-Binding by Myosin-10 Orients Centrosomes toward Retraction Fibers and Subcortical Actin Clouds. Dev Cell 34:323-37
Mendoza, Michelle C; Vilela, Marco; Juarez, Jesus E et al. (2015) ERK reinforces actin polymerization to power persistent edge protrusion during motility. Sci Signal 8:ra47
Danuser, Gaudenz (2014) Reply to ""Acquisition frame rate affects microtubule plus-end tracking analysis"". Nat Methods 11:220
Smole, Zlatko; Thoma, Claudio R; Applegate, Kathryn T et al. (2014) Tumor suppressor NF2/Merlin is a microtubule stabilizer. Cancer Res 74:353-62
Long, Jennifer B; Bagonis, Maria; Lowery, Laura Anne et al. (2013) Multiparametric analysis of CLASP-interacting protein functions during interphase microtubule dynamics. Mol Cell Biol 33:1528-45
Thievessen, Ingo; Thompson, Peter M; Berlemont, Sylvain et al. (2013) Vinculin-actin interaction couples actin retrograde flow to focal adhesions, but is dispensable for focal adhesion growth. J Cell Biol 202:163-77
Lowery, Laura Anne; Stout, Alina; Faris, Anna E et al. (2013) Growth cone-specific functions of XMAP215 in restricting microtubule dynamics and promoting axonal outgrowth. Neural Dev 8:22
Nishimura, Yukako; Applegate, Kathryn; Davidson, Michael W et al. (2012) Automated screening of microtubule growth dynamics identifies MARK2 as a regulator of leading edge microtubules downstream of Rac1 in migrating cells. PLoS One 7:e41413
Reis, Gerald F; Yang, Ge; Szpankowski, Lukasz et al. (2012) Molecular motor function in axonal transport in vivo probed by genetic and computational analysis in Drosophila. Mol Biol Cell 23:1700-14

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