Precision Optogenetic Tool for Selective Single- and Multiple-Cell Ablation in a Live Animal Model System

Makhijani, Kalpana; To, Tsz-Leung; Ruiz‐González, Rubén; Lafaye, Céline; Royant, Antoine; Shu, Xiaokun

doi:10.1016/j.chembiol.2016.12.010

Cited by 61 publications

(63 citation statements)

References 42 publications

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“…The transgenic tools like Gal4 drivers, fluorescently tagged proteins and other markers, which were originally developed and established for studies in embryos can also be used for larvae. In addition, larvae are resilient to heat-shock or UV irradiation, the methods used to induce mosaic clones and to control temperature sensitive protein function and optogenetic tools 34,[36][37][38] .…”

Section: Advantages Of Drosophila Larvae As a Model Systemmentioning

confidence: 99%

Long-termin vivoimaging ofDrosophilalarvae

Kakanj

Eming

Partridge

et al. 2019

Preprint

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The Drosophila larva has been used to investigate many processes in cell biology, including morphogenesis, physiology, responses to drugs and new therapeutic compounds. Despite its enormous potential as a model system, it has technical limitations in cases where longer-term live imaging is necessary, because of the lack of efficient methods for immobilising larvae for extended periods. We describe here a simple procedure for anaesthetisation and long-term in vivo imaging of the epidermis and other larval organs including gut, imaginal discs, neurons, fat body, tracheae and haemocytes, and show a procedure for probing cell properties by laser ablation. We include a survey of different anaesthetics, showing that short exposure to diethyl ether is the most effective for long-term immobilisation of larvae. This method does not require specific expertise beyond basic Drosophila genetics and husbandry, and confocal microscopy. It enables high-resolution studies of many systemic and subcellular processes in larvae.

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Section: Advantages Of Drosophila Larvae As a Model Systemmentioning

confidence: 99%

Long-termin vivoimaging ofDrosophilalarvae

Kakanj

Eming

Partridge

et al. 2019

Preprint

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“…miniSOG (mini Singlet Oxygen Generator), an engineered LOV domain from Arabidopsis phototropin 2 (PHOT2), and subsequent variants such as miniSOG2 have similarly been used to photoablate cells in Caenorhabditis elegans and Drosophila (Fig. 3d–e) 30-32 .…”

Section: Photoactivatable Probes Of Cellular Functionmentioning

confidence: 99%

“…For example, directed evolution has been used to optimize LOV2 domain-caged peptide ligands and to obtain miniSOG mutants with greater in vivo efficacy 32,73 . Large libraries of photoreceptor-effector chimeras with differing structures and connectivities could be similarly screened for light-dependent activities.…”

Section: Lighting the Way Forward With Chemistrymentioning

confidence: 99%

Illuminating developmental biology through photochemistry

Kowalik

Chen

2017

Nat Chem Biol

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Developmental biology has been continually shaped by technological advances, evolving from a descriptive science into one immersed in molecular and cellular mechanism. Most recently, genome sequencing and “omics” profiling have provided developmental biologists with a wealth of genetic and biochemical information; however, fully translating this knowledge into functional understanding will require new experimental capabilities. Photoactivatable probes have emerged as particularly valuable tools for investigating developmental mechanisms, as they can enable rapid, specific manipulations of DNA, RNA, proteins, and cells with spatiotemporal precision. In this perspective, we describe optochemical and optogenetic systems that have been applied in multicellular organisms, insights gained through these probes, and their current limitations. We also suggest how chemical biologists can expand the reach of photoactivatable technologies and bring new depth to our understanding of organismal development.

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“…A new version of miniSOG, miniSOG2, involves seven point mutations: G22S, G40P, Q44R, R57H, L84F, H85R, M89I. Mutations R57H, Q44R, G40P, L84F directly interacting with the chromophore FMN are likely responsible for the red-shifted excitation and emission spectra, together with enhanced singlet oxygen generation, but the quantum yield remains to be measured (Makhijani et al, 2017). …”

Section: Protein Photosensitizer For Nanoscopically-confined Photodynmentioning

confidence: 99%

Photodynamic Physiology—Photonanomanipulations in Cellular Physiology with Protein Photosensitizers

Jiang

Cui

2017

Front. Physiol.

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Singlet oxygen generated in a type II photodynamic action, due to its limited lifetime (1 μs) and reactive distance (<10 nm), could regulate live cell function nanoscopically. The genetically-encoded protein photosensitizers (engineered fluorescent proteins such as KillerRed, TagRFP, and flavin-binding proteins such as miniSOG, Pp2FbFPL30M) could be expressed in a cell type- and/or subcellular organelle-specific manner for targeted protein photo-oxidative activation/desensitization. The newly emerged active illumination technique provides an additional level of specificity. Typical examples of photodynamic activation include permanent activation of G protein-coupled receptor CCK1 and photodynamic activation of ionic channel TRPA1. Protein photosensitizers have been used to photodynamically modulate major cellular functions (such as neurotransmitter release and gene transcription) and animal behavior. Protein photosensitizers are increasingly used in photon-driven nanomanipulation in cell physiology research.

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Precision Optogenetic Tool for Selective Single- and Multiple-Cell Ablation in a Live Animal Model System

Cited by 61 publications

References 42 publications

Long-termin vivoimaging ofDrosophilalarvae

Long-termin vivoimaging ofDrosophilalarvae

Illuminating developmental biology through photochemistry

Photodynamic Physiology—Photonanomanipulations in Cellular Physiology with Protein Photosensitizers

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