High-throughput single-particle tracking reveals nested membrane domains that dictate KRas G12D diffusion and trafficking.

Membrane nanodomains have been implicated in Ras signaling, but what these domains are and how they interact with Ras remain obscure. Here, using single particle tracking with photoactivated localization microscopy (spt-PALM) and detailed trajectory analysis, we show that distinct membrane domains dictate KRas(G12D) (an active KRas mutant) diffusion and trafficking in U2OS cells. KRas(G12D) exhibits an immobile state in similar to 70 nm domains, each embedded in a larger domain (similar to 200 nm) that confers intermediate mobility, while the rest of the membrane supports fast diffusion. Moreover, KRas(G12D) is continuously removed from the membrane via the immobile state and replenished to the fast state, reminiscent of Ras internalization and recycling. Importantly, both the diffusion and trafficking properties of KRas(G12D) remain invariant over a broad range of protein expression levels. Our results reveal how membrane organization dictates membrane diffusion and trafficking of Ras and offer new insight into the spatial regulation of Ras signaling.