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Mol Biol Cell. 2017 Jul 15;28(15):2135-2145. doi: 10.1091/mbc.E16-09-0665. Epub 2017 May 24.

Spatial analysis of Cdc42 activity reveals a role for plasma membrane-associated Cdc42 in centrosome regulation.

Author information

1
Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA 92697.
2
Department of Biomedical Engineering, University of California, Irvine, Irvine, CA 92697.
3
Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.
4
Centre for Bioactive Discovery in Health and Ageing, School of Science and Technology, University of New England, Armidale, NSW 2351, Australia.
5
Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA 92697 suetterc@uci.edu.

Abstract

The ability of the small GTPase Cdc42 to regulate diverse cellular processes depends on tight spatial control of its activity. Cdc42 function is best understood at the plasma membrane (PM), where it regulates cytoskeletal organization and cell polarization. Active Cdc42 has also been detected at the Golgi, but its role and regulation at this organelle are only partially understood. Here we analyze the spatial distribution of Cdc42 activity by moni-toring the dynamics of the Cdc42 FLARE biosensor using the phasor approach to FLIM-FRET. Phasor analysis revealed that Cdc42 is active at all Golgi cisternae and that this activity is controlled by Tuba and ARHGAP10, two Golgi-associated Cdc42 regulators. To our surprise, FGD1, another Cdc42 GEF at the Golgi, was not required for Cdc42 regulation at the Golgi, although its depletion decreased Cdc42 activity at the PM. Similarly, changes in Golgi morphology did not affect Cdc42 activity at the Golgi but were associated with a substantial reduction in PM-associated Cdc42 activity. Of interest, cells with reduced Cdc42 activity at the PM displayed altered centrosome morphology, suggesting that centrosome regulation may be mediated by active Cdc42 at the PM. Our study describes a novel quantitative approach to determine Cdc42 activity at specific subcellular locations and reveals new regulatory principles and functions of this small GTPase.

PMID:
28539409
PMCID:
PMC5509425
DOI:
10.1091/mbc.E16-09-0665
[Indexed for MEDLINE]
Free PMC Article

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