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A Chemically Inducible Organelle Rerouting Assay to Probe Primary Cilium Assembly, Maintenance, and Disassembly in Cultured Cells

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Cilia

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2725))

Abstract

The primary cilium is a conserved, microtubule-based organelle that protrudes from the surface of most vertebrate cells as well as sensory cells of many organisms. It transduces extracellular chemical and mechanical cues to regulate diverse cellular processes during development and physiology. Loss-of-function studies via RNA interference and CRISPR/Cas9-mediated gene knockouts have been the main tool for elucidating the functions of proteins, protein complexes, and organelles implicated in cilium biology. However, these methods are limited in studying acute spatiotemporal functions of proteins as well as the connection between their cellular positioning and functions. A powerful approach based on inducible recruitment of plus or minus end-directed molecular motors to the protein of interest enables fast and precise control of protein activity in time and in space. In this chapter, we present a chemically inducible heterodimerization method for functional perturbation of centriolar satellites, an emerging membrane-less organelle involved in cilium biogenesis and function. The method we present is based on rerouting of centriolar satellites to the cell center or the periphery in mammalian epithelial cells. We also describe how this method can be applied to study the temporal functions of centriolar satellites during primary cilium assembly, maintenance, and disassembly.

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Acknowledgments

This work was supported by the EMBO Installation Grant 3622 and Young Investigator Award to ENF and TUBITAK BIDEB 120C148 grant to ENF.

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Author notes

  1. F. Basak Turan and M. Erdem Ercan contributed equally with all other contributors.

Authors and Affiliations

  1. Department of Molecular Biology and Genetics, Koç University, Istanbul, Turkey

    F. Basak Turan, M. Erdem Ercan & Elif Nur Firat-Karalar

  2. Koc University School of Medicine, Istanbul, Turkey

    Elif Nur Firat-Karalar

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  1. F. Basak Turan
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  2. M. Erdem Ercan
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  3. Elif Nur Firat-Karalar
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Correspondence to Elif Nur Firat-Karalar .

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Editors and Affiliations

  1. Medical Research Council Toxicology Unit, School of Biological Sciences, University of Cambridge, Cambridge, UK

    Vito Mennella

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Turan, F.B., Ercan, M.E., Firat-Karalar, E.N. (2024). A Chemically Inducible Organelle Rerouting Assay to Probe Primary Cilium Assembly, Maintenance, and Disassembly in Cultured Cells. In: Mennella, V. (eds) Cilia. Methods in Molecular Biology, vol 2725. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3507-0_3

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  • DOI: https://doi.org/10.1007/978-1-0716-3507-0_3

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3506-3

  • Online ISBN: 978-1-0716-3507-0

  • eBook Packages: Springer Protocols

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