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

摘要

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