title: Spatiotemporal imaging of small GTPases activity in live cells.
abstract: Ras-like small GTPases function as molecular switches and regulate diverse cellular events. To examine the dynamics of signaling requires spatiotemporal visualization of their activity in the cell. Current small GTPase sensors rely on specific effector domains that are available for only a small number of GTPases and compete for endogenous regulator/effector binding. Here, we describe versatile conformational sensors for GTPase activity (COSGAs) based on the conserved GTPase fold. Conformational changes upon GDP/GTP exchange were directly observed in solution, on beads, and in live cells by Förster resonance energy transfer (FRET). The COSGAs allow for monitoring of Rab1 and K-Ras activity in live cells using fluorescence lifetime imaging microscopy. We found that Rab1 is largely active in the cytoplasm and inactive at the Golgi, suggesting that the Golgi serves as the terminal of the Rab1 functional cycle. K-Ras displays polarized activity at the plasma membrane, with less activity at the edge of the cell and membrane ruffles.
authors.affiliation: Chemical Genomics Centre, Max Planck Society, 44227 Dortmund, Germany.|Max-Planck-Institute of Molecular Physiology, 44227 Dortmund, Germany.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Voss
authors.affiliation: Chemical Genomics Centre, Max Planck Society, 44227 Dortmund, Germany.
authors.forename: Dennis M
authors.initials: DM
authors.lastname: Krüger
authors.affiliation: Faculty of Chemistry and Chemical Biology, TU Dortmund University, 44227 Dortmund, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Koch
authors.affiliation: Chemical Genomics Centre, Max Planck Society, 44227 Dortmund, Germany; yaowen.wu@mpi-dortmund.mpg.de.|Max-Planck-Institute of Molecular Physiology, 44227 Dortmund, Germany.
authors.forename: Yao-Wen
authors.identifier: 0000-0002-2573-8736
authors.initials: YW
authors.lastname: Wu
mesh_terms: D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D004285:Dogs; D005454:Fluorescence Polarization; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D020559:Monomeric GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D015398:Signal Transduction; D020761:rab1 GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020559:Monomeric GTP-Binding Proteins; D020761:rab1 GTP-Binding Proteins; D018631:ras Proteins
keywords: FLIM/FRET; membrane trafficking; protein chemical modification; signal transduction; small GTPases