Abstract
Rab GTPases (>60 members in human) function as master regulators of intracellular membrane trafficking. To fulfill their functions, Rab proteins need to localize on specific membranes in cells. It remains elusive how the distinct spatial distribution of Rab GTPases in the cell is regulated. To make a global assessment on the subcellular localization of Rab1, we determined kinetic parameters of the spatial cycling of Rab1 in live cells using photoactivatable fluorescent proteins and live cell imaging. We found that the switching between GTP- and GDP-binding states, which is governed by guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), GDP dissociation inhibitor (GDI) and GDI displacement factor (GDF), is a major determinant for Rab1’s ability to effectively cycle between cellular compartments and eventually for its subcellular distribution. Herein, we describe the method for monitoring Rab1 dynamics in live cells. This approach can be used to study spatial cycling of other Rab GTPases.
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Acknowledgments
We thank Sven Müller for technical support with Microscopy . This work was supported by the Deutsche Forschungsgemeinschaft, DFG (Grants SPP 1623 and SFB 642), the European Research Council (ERC, ChemBioAP), Vetenskapsrådet (Nr. 2018-04585), the Knut and Alice Wallenberg Foundation and Goran Gustafsson Foundation for Research in Natural Sciences and Medicine (to Y.W.W.).
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Li, F., Wu, YW. (2021). Imaging of Spatial Cycling of Rab GTPase in the Cell. In: Li, G., Segev, N. (eds) Rab GTPases. Methods in Molecular Biology, vol 2293. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1346-7_8
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DOI: https://doi.org/10.1007/978-1-0716-1346-7_8
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