Single molecule imaging reveals actin-dependent diffusive states of IQGAP1 molecules in Jurkat T cells

Cytoskeletal elements of T cells such as actin and microtubules are known to regulate T cell function. The Ras GTPase-activating-like protein, IQGAP1 has been known to directly bind F-actin and serves as a scaffold for multiple microtubule and actin regulators. In T cells, IQGAP1 is recruited to the immune synapse where it serves as a negative regulator of signaling. A key step in understanding how IQGAP1 functions is to precisely define its mobility and the kinetics of its interactions with actin filaments and microtubules. We used single molecule imaging of IQGAP1 to track its accumulation and dynamics during the early stages of T cell activation. Using perturbation expectation-maximization (pEM), a machine learning based method, we characterized IQGAP1 trajectories into distinct diffusive states. Using pharmacological inhibitions and genetic perturbations, we determine the links between actin polymerization, microtubule dynamics and IQGAP1 mobility. Our results indicate that actin polymerization regulates single molecule dynamics of IQGAP1 and provide information about the timescale of actin-IQGAP1 interactions. Overall, our work sheds light on the mechanisms underlying IQGAP1 accumulation at the immune synapse and its coordination of signaling and cytoskeletal dynamics.