title: A new genetically encoded single-chain biosensor for Cdc42 based on FRET, useful for live-cell imaging.
abstract: Cdc42 is critical in a myriad of cellular morphogenic processes, requiring precisely regulated activation dynamics to affect specific cellular events. To facilitate direct observations of Cdc42 activation in live cells, we developed and validated a new biosensor of Cdc42 activation. The biosensor is genetically encoded, of single-chain design and capable of correctly localizing to membrane compartments as well as interacting with its upstream regulators including the guanine nucleotide dissociation inhibitor. We characterized this new biosensor in motile mouse embryonic fibroblasts and observed robust activation dynamics at leading edge protrusions, similar to those previously observed for endogenous Cdc42 using the organic dye-based biosensor system. We then extended our validations and observations of Cdc42 activity to macrophages, and show that this new biosensor is able to detect differential activation patterns during phagocytosis and cytokine stimulation. Furthermore, we observe for the first time, a highly transient and localized activation of Cdc42 during podosome formation in macrophages, which was previously hypothesized but never directly visualized.
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York, United States of America.
authors.forename: Samer
authors.initials: S
authors.lastname: Hanna
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York, United States of America.
authors.forename: Veronika
authors.initials: V
authors.lastname: Miskolci
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York, United States of America; Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York, United States of America.
authors.forename: Dianne
authors.initials: D
authors.lastname: Cox
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York, United States of America; Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York, United States of America.
authors.forename: Louis
authors.initials: L
authors.lastname: Hodgson
mesh_terms: D000818:Animals; D015374:Biosensing Techniques; D002478:Cells, Cultured; D031541:Fluorescence Resonance Energy Transfer; D005470:Fluorometry; D051379:Mice; D041681:NIH 3T3 Cells; D020764:cdc42 GTP-Binding Protein / Q000032:analysis*
chemical_list: D020764:cdc42 GTP-Binding Protein