title: Ca2+-mediated synthetic biosystems offer protein design versatility, signal specificity, and pathway rewiring.
abstract: Synthetic biosystems have been engineered that enable control of metazoan cell morphology, migration, and death. These systems possess signal specificity, but lack flexibility of input signal. To exploit the potential of Ca(2+) signaling, we designed RhoA chimeras for reversible, Ca(2+)-dependent control over RhoA morphology and migration. First, we inserted a calmodulin-binding peptide into a RhoA loop that activates or deactivates RhoA in response to Ca(2+) signals depending on the chosen peptide. Second, we localized the Ca(2+)-activated RhoA chimera to the plasma membrane, where it responded specifically to local Ca(2+) signals. Third, input control of RhoA morphology was rewired by coexpressing the Ca(2+)-activated RhoA chimera with Ca(2+)-transport proteins using acetylcholine, store-operated Ca(2+) entry, and blue light. Engineering synthetic biological systems with input versatility and tunable spatiotemporal responses motivates further application of Ca(2+) signaling in this field.
authors.affiliation: Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College Street, Toronto, ON M5S 3G9, Canada.
authors.forename: Evan
authors.initials: E
authors.lastname: Mills
authors.forename: Kevin
authors.initials: K
authors.lastname: Truong
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D002147:Calmodulin / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement; D002522:Chlorocebus aethiops; D006801:Humans; D008027:Light; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D002147:Calmodulin; D010455:Peptides; D011993:Recombinant Fusion Proteins; D019284:Thapsigargin; D020742:rhoA GTP-Binding Protein; D000109:Acetylcholine; D002118:Calcium