title: Optical Tools To Study the Isoform-Specific Roles of Small GTPases in Immune Cells.
abstract: Despite the 92% homology of the hematopoietic cell-specific Rac2 to the canonical isoform Rac1, these isoforms have been shown to play nonredundant roles in immune cells. To study isoform-specific dynamics of Rac in live cells, we developed a genetically encoded, single-chain FRET-based biosensor for Rac2. We also made significant improvements to our existing single-chain Rac1 biosensor. We optimized the biosensor constructs for facile expression in hematopoietic cells and performed functional validations in murine macrophage sublines of RAW264.7 cells. Rac2, Rac1, and Cdc42 have been implicated in the formation of actin-rich protrusions by macrophages, but their individual activation dynamics have not been previously characterized. We found that both Rac1 and Rac2 had similar activation kinetics, yet they had distinct spatial distributions in response to the exogenous stimulus, fMLF. Active Rac1 was mainly localized to the cell periphery, whereas active Rac2 was distributed throughout the cell, with an apparent higher concentration in the perinuclear region. We also performed an extensive morphodynamic analysis of Rac1, Rac2, and Cdc42 activities during the extension of random protrusions. We found that Rac2 appears to play a leading role in the generation of random protrusions, as we observed an initial strong activation of Rac2 in regions distal from the leading edge, followed by the activation of Rac1, a second burst of Rac2 and then Cdc42 immediately behind the leading edge. Overall, isoform-specific biosensors that have been optimized for expression should be valuable for interrogating the coordination of Rho family GTPase activities in living cells.
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461;
authors.forename: Veronika
authors.initials: V
authors.lastname: Miskolci
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461; Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY 10461.
authors.forename: Bin
authors.initials: B
authors.lastname: Wu
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461;
authors.forename: Yasmin
authors.initials: Y
authors.lastname: Moshfegh
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461; Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY 10461 Louis.hodgson@einstein.yu.edu Dianne.cox@einstein.yu.edu.
authors.forename: Dianne
authors.initials: D
authors.lastname: Cox
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461; Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY 10461 Louis.hodgson@einstein.yu.edu Dianne.cox@einstein.yu.edu.
authors.forename: Louis
authors.identifier: http://orcid.org/0000-0001-9188-7580
authors.initials: L
authors.lastname: Hodgson
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D002460:Cell Line; D022081:Cell Surface Extensions / Q000502:physiology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D008264:Macrophages / Q000276:immunology; D051379:Mice; D009479:Neuropeptides / Q000235:genetics*; D020033:Protein Isoforms / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D020764:cdc42 GTP-Binding Protein / Q000235:genetics*; D020744:rac GTP-Binding Proteins / Q000235:genetics*; D020830:rac1 GTP-Binding Protein / Q000235:genetics*; D000097766:RAC2 GTP-Binding Protein
chemical_list: D009479:Neuropeptides; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D000097766:RAC2 GTP-Binding Protein; C581598:Cdc42 protein, mouse; C484602:Rac1 protein, mouse