Ca2+-Mediated Synthetic Biosystems Offer Protein Design Versatility, Signal Specificity, and Pathway Rewiring

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 Ca2+ signaling, we designed RhoA chimeras for reversible, Ca2+-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 Ca2+ signals depending on the chosen peptide. Second, we localized the Ca2+-activated RhoA chimera to the plasma membrane, where it responded specifically to local Ca2+ signals. Third, input control of RhoA morphology was rewired by coexpressing the Ca2+-activated RhoA chimera with Ca2+-transport proteins using acetylcholine, store-operated Ca2+ entry, and blue light. Engineering synthetic biological systems with input versatility and tunable spatiotemporal responses motivates further application of Ca2+ signaling in this field.