Monitoring the Response of Multiple Signal Network Components to Acute Chemo‐Optogenetic Perturbations in Living Cells

Cells process information via signal networks that typically involve multiple components which are interconnected by feedback loops. The combination of acute optogenetic perturbations and microscopy‐based fluorescent response readouts enables the direct investigation of causal links in such networks. However, due to overlaps in spectra of photosensitive and fluorescent proteins, current approaches that combine these methods are limited. Here, we present an improved chemo‐optogenetic approach that is based on switch‐like perturbations induced by a single, local pulse of UV light. We show that this approach can be combined with parallel monitoring of multiple fluorescent readouts to directly uncover relations between signal network components. We present the application of this technique to directly investigate feedback‐controlled regulation in the cell contraction signal network that includes GEF‐H1, Rho and Myosin, and functional interactions of this network with tumor relevant RhoA G17 mutants.