title: Chromophore-assisted laser inactivation--towards a spatiotemporal-functional analysis of proteins, and the ablation of chromatin, organelle and cell function.
abstract: Chromophore-assisted laser or light inactivation (CALI) has been employed as a promising technique to achieve spatiotemporal knockdown or loss-of-function of target molecules in situ. CALI is performed using photosensitizers as generators of reactive oxygen species (ROS). There are two CALI approaches that use either transgenic tags with chemical photosensitizers, or genetically encoded fluorescent protein fusions. Using spatially restricted microscopy illumination, CALI can address questions regarding, for example, protein isoforms, subcellular localization or phase-specific analyses of multifunctional proteins that other knockdown approaches, such as RNA interference or treatment with chemicals, cannot. Furthermore, rescue experiments can clarify the phenotypic capabilities of CALI after the depletion of endogenous targets. CALI can also provide information about individual events that are involved in the function of a target protein and highlight them in multifactorial events. Beyond functional analysis of proteins, CALI of nuclear proteins can be performed to induce cell cycle arrest, chromatin- or locus-specific DNA damage. Even at organelle level - such as in mitochondria, the plasma membrane or lysosomes - CALI can trigger cell death. Moreover, CALI has emerged as an optogenetic tool to switch off signaling pathways, including the optical depletion of individual neurons. In this Commentary, we review recent applications of CALI and discuss the utility and effective use of CALI to address open questions in cell biology.
authors.affiliation: Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
authors.forename: Yukimi
authors.initials: Y
authors.lastname: Sano
authors.forename: Wataru
authors.initials: W
authors.lastname: Watanabe
authors.forename: Sachihiro
authors.initials: S
authors.lastname: Matsunaga
mesh_terms: D000818:Animals; D002468:Cell Physiological Phenomena; D002843:Chromatin / Q000502:physiology*; D061885:Chromophore-Assisted Light Inactivation; D003598:Cytoskeletal Proteins / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D006801:Humans; D024101:Mitochondrial Proteins / Q000502:physiology; D015388:Organelles / Q000502:physiology*; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D002843:Chromatin; D003598:Cytoskeletal Proteins; D024101:Mitochondrial Proteins; D017382:Reactive Oxygen Species; C485184:enhanced green fluorescent protein; C508771:killer red protein, Anthomedusae; D049452:Green Fluorescent Proteins
keywords: Chromophore; Imaging; Photosensitizer; Reactive oxygen species; Spatiotemporal inactivation