Abstract
Microtubules (MTs) form dynamic cytoskeletal scaffolds essential for intracellular transport, organelle positioning, and spatial organization of signaling. Their architecture and function are continuously remodeled through the concerted actions of microtubule-associated proteins (MAPs), post-translational modifications (PTMs), and molecular motors. To precisely interrogate these processes in living systems, we developed a genetically encoded optogenetic toolkit for spatiotemporal control of MT organization and dynamics. By replacing native multimerization motifs with a blue light-responsive oligoermization domain, we have engineered single-component probes, OptoMT and OptoTIP, that reversibly label MT polymers or track plus-ends with tunable kinetics from seconds to minutes. When coupled to enzymatic effectors, these modules enable localized tubulin acetylation or detyrosination, directly linking PTMs to MT stability. We further engineered OptoMotor, a light-activatable kinesin platform that reconstitutes tail-dependent cargo transport along MTs, and OptoSAW, a light-triggered severing actuator for controlled MT disassembly. Using these tools, we reveal how local MT integrity governs lysosomal trafficking and ER-associated signaling dynamics. Collectively, this versatile single-component toolkit bridges molecular design with cytoskeletal function, offering new avenues to illuminate how dynamic cytoskeletal architectures coordinate intracellular organization, transport, and signaling.