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

摘要: The GTPases RhoA and Rac1 are key regulators of cell spreading, adhesion, migration, they exert distinct effects on the actin cytoskeleton. While classically stimulates stress fiber assembly contraction, promotes branched polymerization membrane protrusion. These competing influences reinforced by antagonistic crosstalk between Rac1, which has complicated efforts to identify specific mechanisms each GTPase regulates behavior. We therefore wondered whether intrinsically coupled or can be manipulated independently. To address this question, we placed constitutively active (CA) under a doxycycline-inducible promoter CA an orthogonal cumate-inducible promoter, stably introduced both constructs into glioblastoma cells. found that doxycycline addition increased activity without altering similarly cumate RhoA. Furthermore, co-expression mutants enabled high activation simultaneously. When cells were cultured collagen hydrogels, prevented spreading motility, whereas stimulated migration dynamic protrusions. Interestingly, induced third phenotype, in migrated at intermediate speeds similar control but also aggregated large, contractile clusters. In addition, demonstrate reversible switching phenotypes. Overall, approach represents unique way access different combinations levels single may serve as valuable tool for multiplexed dissection mechanobiological signals.