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Reconstitution of Organelle Transport Along Microtubules In Vitro

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Dynein

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2623))

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Abstract

In this chapter, we describe methods for reconstituting and analyzing the transport of isolated endogenous cargoes in vitro. Intracellular cargoes are transported along microtubules by teams of kinesin and dynein motors and their cargo-specific adaptor proteins. Observations from living cells show that organelles and vesicular cargoes exhibit diverse motility characteristics. Yet, our knowledge of the molecular mechanisms by which intracellular transport is regulated is not well understood. Here, we describe step-by-step protocols for the extraction of phagosomes from cells at different stages of maturation, and reconstitution of their motility along microtubules in vitro. Quantitative immunofluorescence and photobleaching techniques are also described to measure the number of motors and adaptor proteins on these isolated cargoes. In addition, we describe techniques for tracking the motility of isolated cargoes along microtubules using TIRF microscopy and quantitative force measurements using an optical trap. These methods enable us to study how the sets of motors and adaptors that drive the transport of endogenous cargoes regulate their trafficking in cells.

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Authors and Affiliations

  1. Department of Bioengineering, McGill University, Montreal, QC, Canada

    Daniel Beaudet & Adam G. Hendricks

Authors
  1. Daniel Beaudet
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  2. Adam G. Hendricks
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Correspondence to Adam G. Hendricks .

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Editors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA

    Steven M. Markus

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Beaudet, D., Hendricks, A.G. (2023). Reconstitution of Organelle Transport Along Microtubules In Vitro. In: Markus, S.M. (eds) Dynein. Methods in Molecular Biology, vol 2623. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2958-1_8

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  • DOI: https://doi.org/10.1007/978-1-0716-2958-1_8

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2957-4

  • Online ISBN: 978-1-0716-2958-1

  • eBook Packages: Springer Protocols

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