Delineation of signaling microdomains from live-cell movies

Many molecular signals are known to be organized in subcellular regions termed microdomains. One example entails Rho GTPases, which control a range of cell behaviors through their functional patterning in spatiotemporal units. While fluorescent biosensors have allowed precise measurement of Rho GTPase activity in living cells, computational tools to delineate and track signaling microdomains in time-lapse image sequences of such biosensors do not exist. Here, we introduce a method that centers on the notion of activation time series coordination to identify signaling microdomains in space and time. After validating the algorithm with simulated microdomains, we show that our method identifies domains that support current hypotheses on the signaling architecture governing Rho GTPase organization and also respond to perturbation with optogenetics. A record of this paper's transparent peer review process is included in the supplemental information.