A Modular Optogenetic Split-Small GTPase Platform Reveals Long-Range RhoA Signaling

Small GTPases are critical regulators of cellular processes such as cell migration. However, no generalizable method exists for directly controlling their activity with subcellular precision, limiting the ability to dissect long-range signaling effects. To address this, we pair fragmented small GTPases with improved light-inducible dimerization (iLID) domains, optimizing for low dark-state background and efficient light-induced reassembly, affording spLIT-small GTPases. Demonstrating modularity, we control filopodia formation (spLIT-Cdc42), cell movement (spLIT-Rac1), and membrane retraction (spLIT-RhoA) with subcellular precision. Applying this platform to long-range signaling, we provide the first demonstration that localized RhoA activation is sufficient to drive membrane retraction at the opposite cell pole, with a directional dependence set by actin cytoskeleton polarization, a result inaccessible to global perturbations used previously. These results suggest a mechanism for coordinated establishment of cell polarity and highlight spLIT-small GTPases as a versatile platform for the direct, spatial control of small GTPase signaling in living cells.