A biosensor of S100A4 metastasis factor activation: Inhibitor screening and cellular activation dynamics
dc.contributor.author | Garrett, Sarah C. | |
dc.date.accessioned | 2018-07-12T17:34:51Z | |
dc.date.available | 2018-07-12T17:34:51Z | |
dc.date.issued | 2008 | |
dc.description.abstract | S100A4, a member of the S100 family of proteins, binds nonmuscle myosin-IIA in a calcium-dependent manner and regulates the monomer-polymer equilibrium of myosin-IIA filaments. S100A4 displays elevated expression in malignant human tumors compared with benign tumors, and increased expression correlates strongly with poor patient survival. S100A4 plays a direct role in metastatic progression, due to the modulation of actomyosin cytoskeletal dynamics, which results in increased cellular motility. To examine S100A4 activity in living cells, we developed a fluorescent biosensor that reports on the Ca 2+-bound, activated form of S100A4. Direct attachment of a novel solvatochromatic reporter dye to S100A4 results in a sensor (Mero-S100A4) that, upon activation, undergoes a 3-fold enhancement in fluorescence, thus providing a sensitive assay for in vitro and in vivo use. Biochemical analysis demonstrates that the calcium and myosin binding properties of Mero-S100A4 are similar to wild-type S100A4 and validate its use for a wide range of studies. This biosensor shows that in fibroblasts, localized activation of S100A4 at the cell periphery is observed during stimulation with lysophosphatidic acid, a known activator of cell motility and proliferation. Live cell imaging also revealed S100A4 activation in cellular regions undergoing retraction. In MDA-MB-231 human breast cancer cells crawling into a wound, S100A4 activation was detected in regions protruding toward the wound with the highest activation localized to dorsal ruffles. Additionally, a screen against a library of FDA approved drugs with the Mero-S100A4 identified an array of phenothiazines as inhibitors of myosin-IIA associated S100A4 function. These drugs are the first reported inhibitors of S100A4 and provide the foundation for future drug development. Together these data demonstrate the utility of the Mero-S100A4 biosensor for both drug discovery and for probing the cellular dynamics controlled by the S100A4 metastasis factor. | |
dc.identifier.citation | Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 9950.;Advisors: Anne R. Bresnick. | |
dc.identifier.uri | https://ezproxy.yu.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqm&rft_dat=xri:pqdiss:3303430 | |
dc.identifier.uri | https://hdl.handle.net/20.500.12202/958 | |
dc.publisher | ProQuest Dissertations & Theses | |
dc.subject | Biochemistry. | |
dc.subject | Cellular biology. | |
dc.title | A biosensor of S100A4 metastasis factor activation: Inhibitor screening and cellular activation dynamics | |
dc.type | Dissertation |