title: Predictive Spatiotemporal Manipulation of Signaling Perturbations Using Optogenetics.
abstract: Recently developed optogenetic methods promise to revolutionize cell biology by allowing signaling perturbations to be controlled in space and time with light. However, a quantitative analysis of the relationship between a custom-defined illumination pattern and the resulting signaling perturbation is lacking. Here, we characterize the biophysical processes governing the localized recruitment of the Cryptochrome CRY2 to its membrane-anchored CIBN partner. We develop a quantitative framework and present simple procedures that enable predictive manipulation of protein distributions on the plasma membrane with a spatial resolution of 5 μm. We show that protein gradients of desired levels can be established in a few tens of seconds and then steadily maintained. These protein gradients can be entirely relocalized in a few minutes. We apply our approach to the control of the Cdc42 Rho GTPase activity. By inducing strong localized signaling perturbation, we are able to monitor the initiation of cell polarity and migration with a remarkable reproducibility despite cell-to-cell variability.
authors.affiliation: Laboratoire Physico-Chimie, Institut Curie, Centre National de la Recherche Scientifique UMR168, Paris-Science Lettres, Université Pierre et Marie Curie-Paris 6, Paris, France.
authors.forename: Leo
authors.initials: L
authors.lastname: Valon
authors.affiliation: Center for Studies in Physics and Biology, The Rockefeller University, New York, New York.
authors.forename: Fred
authors.initials: F
authors.lastname: Etoc
authors.affiliation: Laboratoire Physico-Chimie, Institut Curie, Centre National de la Recherche Scientifique UMR168, Paris-Science Lettres, Université Pierre et Marie Curie-Paris 6, Paris, France.
authors.forename: Amanda
authors.initials: A
authors.lastname: Remorino
authors.affiliation: Institut de Biologie de l'École Normale Supérieure, Ecole Normale Supérieure, Paris, France.
authors.forename: Florencia
authors.initials: F
authors.lastname: di Pietro
authors.affiliation: Institut de Biologie de l'École Normale Supérieure, Ecole Normale Supérieure, Paris, France.
authors.forename: Xavier
authors.initials: X
authors.lastname: Morin
authors.affiliation: Laboratoire Physico-Chimie, Institut Curie, Centre National de la Recherche Scientifique UMR168, Paris-Science Lettres, Université Pierre et Marie Curie-Paris 6, Paris, France.
authors.forename: Maxime
authors.initials: M
authors.lastname: Dahan
authors.affiliation: Laboratoire Physico-Chimie, Institut Curie, Centre National de la Recherche Scientifique UMR168, Paris-Science Lettres, Université Pierre et Marie Curie-Paris 6, Paris, France. Electronic address: mathieu.coppey@curie.fr.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Coppey
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D056931:Cryptochromes / Q000378:metabolism*; D004058:Diffusion; D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008027:Light; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D041681:NIH 3T3 Cells; D062308:Optogenetics / Q000379:methods*; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*; D000097573:Red Fluorescent Protein
chemical_list: D056931:Cryptochromes; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D020764:cdc42 GTP-Binding Protein; D000097573:Red Fluorescent Protein; C538966:CRY2 protein, human