A biosensor of S100A4 metastasis factor activation: inhibitor screening and cellular activation dynamics.
S100A4, a member of the S100 family of Ca2+-binding proteins, displays elevated expression in malignant human tumors compared with benign tumors, and increased expression correlates strongly with poor patient survival. S100A4 has a direct role in metastatic progression, likely due to the modulation of actomyosin cytoskeletal dynamics, which results in increased cellular motility. We developed a fluorescent biosensor (Mero-S100A4) that reports on the Ca2+-bound, activated form of S100A4. Direct attachment of a novel solvatochromatic reporter dye to S100A4 results in a sensor that, upon activation, undergoes a 3-fold enhancement in fluorescence, thus providing a sensitive assay for use in vitro and in vivo. In cells, localized activation of S100A4 at the cell periphery is observed during random migration and following stimulation with lysophosphatidic acid, a known activator of cell motility and proliferation. Additionally, a screen against a library of FDA-approved drugs with the biosensor identified an array of phenothiazines as inhibitors of myosin-II associated S100A4 function. These data demonstrate the utility of the new biosensor both for drug discovery and for probing the cellular dynamics controlled by the S100A4 metastasis factor.
D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002118:Calcium / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D002465:Cell Movement / Q000187:drug effects; D002474:Cell-Free System; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004533:Egtazic Acid / Q000737:chemistry; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D005454:Fluorescence Polarization; D006801:Humans; D007460:Iodoacetamide / Q000737:chemistry; D008246:Lysophospholipids / Q000494:pharmacology; D051379:Mice; D008958:Models, Molecular; D015394:Molecular Structure; D024507:Nonmuscle Myosin Type IIA / Q000737:chemistry / Q000378:metabolism; D010640:Phenothiazines / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D011744:Pyrimidinones / Q000737:chemistry; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014268:Trifluoperazine / Q000737:chemistry / Q000494:pharmacology
D008246:Lysophospholipids; D010640:Phenothiazines; D011744:Pyrimidinones; D011994:Recombinant Proteins; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins; C077591:S100A4 protein, human; D014268:Trifluoperazine; D004533:Egtazic Acid; C003954:merocyanine dye; D024507:Nonmuscle Myosin Type IIA; D003545:Cysteine; C032881:lysophosphatidic acid; D002118:Calcium; D007460:Iodoacetamide