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 Ca(2+) second messenger. Using structural modeling, a fusion protein was designed wherein RhoA activity was controlled by Ca(2+) via calmodulin. Living cells transfected with this synthetic protein underwent Ca(2+) sensitive and calmodulin-dependent bleb retraction within minutes. Further, the modularity of Ca(2+) 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 Ca(2+) signaling in nature suggests that fully exploring its signaling potential may allow powerful applications to other synthetic biological systems.
Mills
Evan
E
Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario M5S 3G9, Canada. e.mills@utoronto.ca
Pham
Elizabeth
E
Truong
Kevin
K
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D048430:Cell Shape; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D017434:Protein Structure, Tertiary; D020742:rhoA GTP-Binding Protein / Q000737:chemistry* / Q000378:metabolism
D002147:Calmodulin; D020742:rhoA GTP-Binding Protein; D002118:Calcium