Abstract
In this chapter, we report a protocol to perform correlative light electron microscopy (CLEM) on adult Caenorhabditis elegans. We use a specific fixation protocol, which preserves both the GFP fluorescence and the structural integrity of the samples. Thin sections are first analyzed by light microscopy to detect GFP-tagged proteins and, subsequently, with transmission electron microscopy (TEM) to characterize the ultrastructural anatomy of cells. The superimposition of light and electron images allows determining the subcellular localization of the fluorescent protein.
We used CLEM to characterize the subcellular localization of the C. elegans ESCRT-II component VPS-36. VPS-36 protein localization in C. elegans muscle cell is strongly correlated with the sarcoplasmic reticulum network. Together with genetic evidences, the CLEM data support a role for ESCRT-II proteins in sarcoplasmic reticulum membrane shaping.
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23 January 2020
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Acknowledgments
The authors would like to thank the C. elegans community and the Caenorhabditis Genetic Center which is funded by the NIH National Center for Research Resources (NCRR) for sharing C. elegans strains. We are grateful to Christophe Lefebvre for Fig. 2 and Claire Boulogne for critically reading the manuscript. The present work has benefited from the expertise of Béatrice Satiat-Jeunemaitre, Cynthia Gillet, Jessica Marion, and Claire Boulogne and the core facilities of Imagerie-Gif (http://www.i2bc.paris-saclay.fr) member of IBiSA (http://www.ibisa.net), supported by “France-BioImaging” (ANR-10-INBS-04-01) and the Labex “Saclay Plant Science” (ANR-11-IDEX-0003-02). This work was supported by the Agence Nationale de la Recherche (project EAT, ANR-12-BSV2-018) and the Association pour la Recherche contre le Cancer (SFI20111203826).
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Largeau, C., Culetto, E., Legouis, R. (2019). Subcellular Localization of ESCRT-II in the Nematode C. elegans by Correlative Light Electron Microscopy. In: Culetto, E., Legouis, R. (eds) The ESCRT Complexes. Methods in Molecular Biology, vol 1998. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9492-2_4
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DOI: https://doi.org/10.1007/978-1-4939-9492-2_4
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