ARHGAP18-ezrin functions as an autoregulatory module for RhoA in the assembly of distinct actin-based structures

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Abstract

The location of different actin-based structures is largely regulated by Rho GTPases through specific effectors. We use the apical aspect of epithelial cells as a model system to investigate how RhoA is locally regulated to contribute to two distinct adjacent actin-based structures. Assembly of the non-muscle myosin-2 filaments in the terminal web is dependent on RhoA activity, and assembly of the microvilli also requires active RhoA for phosphorylation and activation of ezrin. We show the RhoGAP, ARHGAP18, is localized by binding active microvillar ezrin, and this interaction enhances ARHGAP18's RhoGAP activity. We present a model where ezrin-ARHGAP18 acts as a negative autoregulatory module to locally reduce RhoA activity in microvilli. Consistent with this model, loss of ARHGAP18 results in disruption of the distinction between microvilli and the terminal web including aberrant assembly of myosin-2 filaments forming inside microvilli. Thus, ARHGAP18, through its recruitment and activation by ezrin, fine-tunes the local level of RhoA to allow for the appropriate distribution of actin-based structures between the microvilli and terminal web. As RhoGAPs vastly out-number Rho GTPases, this may represent a general mechanism whereby individual Rho effectors drive specific actin-based structures.

Data availability

Supplemental document 1a provides all DNA plasmid information, 1b all DNA Oligos used and their sequence, and 1c all data used in statistical analysis and quantification. Full images of gels or blots shown are provided as supplements to the associated figure. All figures were assembled in Adobe Illustrator 2023 (Adobe, Mountain View, CA).

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Andrew T Lombardo

Department of Molecular Biology and Genetics, Cornell University, Ithaca, United States

For correspondence
ATL73@cornell.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-1814-5748
Cameron AR Mitchell

Department of Molecular Biology and Genetics, Cornell University, Ithaca, United States

Competing interests
The authors declare that no competing interests exist.
Riasat Zaman

Department of Molecular Biology and Genetics, Cornell University, Ithaca, United States

Competing interests
The authors declare that no competing interests exist.
David J McDermitt

Department of Molecular Biology and Genetics, Cornell University, Ithaca, United States

Competing interests
The authors declare that no competing interests exist.
Anthony Bretscher

Department of Molecular Biology and Genetics, Cornell University, Ithaca, United States

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-1122-8970

Funding

National Institute of General Medical Sciences (R35GM131751)

Andrew T Lombardo
Cameron AR Mitchell
Riasat Zaman
David J McDermitt
Anthony Bretscher

The Sam and Nancy Fleming Research Fellowship

Andrew T Lombardo

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: A custom ARHGAP18 antibody was produced in Rabbit by Pocono Rabbit Farm & Laboratory, Inc (Canadensis, PA) using the animal use protocol approved by Cornell University IACUC number 2014-0109 to A. Bretscher.

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.