Probing the spatiotemporal dynamics of Ras-associated membrane nanodomains with high-throughput single particle tracking via photoactivated localization microscopy (spt-PALM)

Membrane nanodomains have commonly been implicated in biological processes. However, what these nanodomains are and how they participate in the processes of interest are still unclear, primarily due to challenges in probing these nanoscopic and dynamic structures in cells. Using high-throughput single particle tracking via spt-PALM and detailed trajectory analysis, here we demonstrate that membrane nanodomains associated with the small GTPase KRas could be detected and analyzed in live cells. By stochastically activating and tracking single PAmCherry1-KRas molecules on the membrane, spt-PALM yields 5,000 to 100,000 single-molecule diffusion trajectories of KRas. Analysis of these trajectories with variational Bayes SPT (vbSPT) revealed that KRas exhibits an immobile state in domains ∼70 nm in size, each embedded in a larger domain (∼200 nm) that confers intermediate mobility, while the rest of the membrane supports fast diffusion. By analyzing the transition kinetics among the three states, we found that KRas is continuously removed from the membrane via the immobile state and replenished to the fast state, likely coupled to internalization and recycling. Our results demonstrate the utility of high-throughput SPT in uncovering the impact of nanoscopic landscape of the membrane on the spatiotemporal dynamics and potentially multimer formation and signaling of membrane-bound biomolecules.