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Simultaneous and independent tuning of RhoA and Rac1 activity with orthogonally inducible promoters

Content Provider	Royal Society of Chemistry (RSC)
Author	Kumar, Sanjay MacKay, Joanna L.
Copyright Year	2014
Abstract	The GTPases RhoA and Rac1 are key regulators of cell spreading, adhesion, and migration, and they exert distinct effects on the actin cytoskeleton. While RhoA classically stimulates stress fiber assembly and contraction, Rac1 promotes branched actin polymerization and membrane protrusion. These competing influences are reinforced by antagonistic crosstalk between RhoA and Rac1, which has complicated efforts to identify the specific mechanisms by which each GTPase regulates cell behavior. We therefore wondered whether RhoA and Rac1 are intrinsically coupled or whether they can be manipulated independently. To address this question, we placed constitutively active (CA) RhoA under a doxycycline-inducible promoter and CA Rac1 under an orthogonal cumate-inducible promoter, and we stably introduced both constructs into glioblastoma cells. We found that doxycycline addition increased RhoA activity without altering Rac1, and similarly cumate addition increased Rac1 activity without altering RhoA. Furthermore, co-expression of both mutants enabled high activation of RhoA and Rac1 simultaneously. When cells were cultured on collagen hydrogels, RhoA activation prevented cell spreading and motility, whereas Rac1 activation stimulated migration and dynamic cell protrusions. Interestingly, high activation of both GTPases induced a third phenotype, in which cells migrated at intermediate speeds similar to control cells but also aggregated into large, contractile clusters. In addition, we demonstrate dynamic and reversible switching between high RhoA and high Rac1 phenotypes. Overall, this approach represents a unique way to access different combinations of RhoA and Rac1 activity levels in a single cell and may serve as a valuable tool for multiplexed dissection and control of mechanobiological signals.
Starting Page	885
Ending Page	894
Page Count	10
File Format	HTM / HTML PDF
ISSN	17579694
Volume Number	6
Issue Number	9
Journal	Integrative Biology
DOI	10.1039/c4ib00099d
Language	English
Publisher	Royal Society of Chemistry
Access Restriction	Open
Subject Keyword	RHOA Actin Cytoskeleton Stress fiber Polymerization Crosstalk GTPase Orthogonality Glioblastoma multiforme Doxycycline Collagen Phenotype Multiplexing
Content Type	Text
Resource Type	Article
Subject	Medicine Biochemistry Biophysics

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