title: Performing Chromophore-Assisted Laser Inactivation in Drosophila Embryos Using GFP.
abstract: Chromophore-assisted laser inactivation (CALI) is an optogenetic technique in which light-induced release of reactive oxygen species triggers acute inactivation of a protein of interest, with high spatial and temporal resolution. At its simplest, selective protein inactivation can be achieved via the genetic fusion of the protein to a photosensitizer such as EGFP, and using standard optical setups such as laser scanning confocal microscopes. Although use of CALI in Drosophila is relatively recent, this technique can be a powerful complement to developmental genetics, especially in vivo as it allows visualization of the immediate consequences of local protein inactivation when coupled to time-lapse microscopy analysis. In addition to providing examples of protocols, this chapter is intended as a conceptual framework to support the rational design of CALI experiments.
authors.affiliation: Université Toulouse III-Paul Sabatier, 118 route de Narbonne, 31062, Toulouse, France.|CNRS, UMR5547, Centre de Biologie du Développement, 31062, Toulouse, France.
authors.forename: Anne
authors.initials: A
authors.lastname: Pélissier-Monier
authors.affiliation: Department of Physiology, Development and Neuroscience, University of Cambridge, Anatomy Building, Downing Street, Cambridge, CB2 3DY, UK. bs251@cam.ac.uk.
authors.forename: Bénédicte
authors.initials: B
authors.lastname: Sanson
authors.affiliation: Université Toulouse III-Paul Sabatier, 118 route de Narbonne, 31062, Toulouse, France.|Laboratoire de Biologie Cellulaire et Moléculaire du Contrôle de la Prolifération, CNRS, UMR5088, 31062, Toulouse, France.
authors.forename: Bruno
authors.initials: B
authors.lastname: Monier
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D061885:Chromophore-Assisted Light Inactivation / Q000379:methods*; D029721:Drosophila Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics* / Q000378:metabolism; D004625:Embryo, Nonmammalian; D036681:Fluorescence Recovery After Photobleaching; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism*; D018613:Microscopy, Confocal; D024462:Myosin Type II / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D017319:Photosensitizing Agents / Q000737:chemistry / Q000378:metabolism*; D059008:Time-Lapse Imaging
chemical_list: D029721:Drosophila Proteins; D017319:Photosensitizing Agents; C485184:enhanced green fluorescent protein; C508771:killer red protein, Anthomedusae; D049452:Green Fluorescent Proteins; D024462:Myosin Type II
keywords: Chromophore-assisted laser inactivation (CALI); Conditional protein inactivation; Drosophila; Genetic photosensitizer; Green fluorescent protein (GFP); Live imaging; Optogenetics