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In Vivo Single-Molecule Tracking of Voltage-Gated Calcium Channels with Split-Fluorescent Proteins in CRISPR-Engineered C. elegans

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Single Molecule Microscopy in Neurobiology

Part of the book series: Neuromethods ((NM,volume 154))

Abstract

How voltage-gated calcium channels (VGCCs) mediate signal transductions in response to changes in membrane potential is primarily studied in in vitro and ex vivo systems via biochemical and electrophysiological methods. With the emergence of single-molecule (SM) fluorescence microscopy techniques, it is now possible to characterize the molecular organization and the biophysical dynamics of ion channels in cells with precisions on the order of a few nanometers. However, performing such SM measurements within excitable tissues in intact animals is challenging. Here we describe protocols for an in vivo and tissue-specific SM imaging technique called complementation-activated light microscopy (CALM). By combining native expression of CRISPR-engineered split-fluorescent protein (split-FP) fusions and controlled fluorescence activation of split-FPs in vivo, CALM enables researchers to study the dynamics of individual calcium channels with a precision better than 30 nm directly within neuromuscular synapses of adult Caenorhabditis elegans (C. elegans) nematodes or at the sarcolemma of their body-wall muscle cells. With the availability of various split-FP spectral variants and of tissue-specific fluorescent markers, CALM can be extended to multicolor and nanoscale dynamic studies of virtually any membrane proteins and channels expressed at physiological levels in live animals.

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Acknowledgments

We would like to thank Dr. Jean-Louis Bessereau and members from his laboratory for kindly providing the transgenic strains with neuromuscular junction markers, Dr. Hong Zhan for sharing the pHZ043 plasmid and alternative genome editing strategies, and Dr. Thomas Duchaine and Vinay Mayya for many critical advices and reagents for CRISPR-based genome engineering.

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Authors and Affiliations

  1. Department of Biological Sciences, Dana and David Dornsife College of Letters, Arts and Sciences, University of Southern California, Los Angeles, CA, USA

    Yunke Zhao & Fabien Pinaud

  2. Department of Chemistry, Dana and David Dornsife College of Letters, Arts and Sciences, University of Southern California, Los Angeles, CA, USA

    Fabien Pinaud

  3. Department of Physics and Astronomy, Dana and David Dornsife College of Letters, Arts and Sciences, University of Southern California, Los Angeles, CA, USA

    Fabien Pinaud

Authors
  1. Yunke Zhao
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  2. Fabien Pinaud
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Correspondence to Fabien Pinaud .

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Editors and Affiliations

  1. Laboratory of Cellular and Molecular Neurobiology, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan

    Nobuhiko Yamamoto

  2. Laboratory for Cell Polarity Regulation, RIKEN Center for Biosystems Dynamics Research, Suita, Osaka, Japan

    Yasushi Okada

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Zhao, Y., Pinaud, F. (2020). In Vivo Single-Molecule Tracking of Voltage-Gated Calcium Channels with Split-Fluorescent Proteins in CRISPR-Engineered C. elegans. In: Yamamoto, N., Okada, Y. (eds) Single Molecule Microscopy in Neurobiology . Neuromethods, vol 154. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0532-5_2

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  • DOI: https://doi.org/10.1007/978-1-0716-0532-5_2

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