Structure based design of a Ca2+-sensitive RhoA protein that controls cell morphology

The Rho proteins are important regulators of cell morphology, and the prototypical protein RhoA is known to regulate contraction, blebbing and bleb retraction. We have identified and experimentally confirmed that RhoA has a binding site for calmodulin, a ubiquitous transducer of the Ca2+ second messenger. Using structural modeling, a fusion protein was designed wherein RhoA activity was controlled by Ca2+ via calmodulin. Living cells transfected with this synthetic protein underwent Ca2+ sensitive and calmodulin-dependent bleb retraction within minutes. Further, the modularity of Ca2+ signaling was exploited to induce bleb retraction in response to blue light (using channelrhodopsin-2) or exogenous chemicals (with acetylcholine receptor), showing input signal versatility. The widespread use of Ca2+ signaling in nature suggests that fully exploring its signaling potential may allow powerful applications to other synthetic biological systems.