High-throughput, single-particle tracking reveals nested membrane domains that dictate KRasG12D diffusion and trafficking

Abstract

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 KRasG12D (an active KRas mutant) diffusion and trafficking in U2OS cells. KRasG12D exhibits an immobile state in ~70 nm domains, each embedded in a larger domain (~200 nm) that confers intermediate mobility, while the rest of the membrane supports fast diffusion. Moreover, KRasG12D 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 KRasG12D 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.

Raf promotes dimerization of the Ras G-domain with increased allosteric connections

“…dimer through residue D113 in the loop 4 region could form a kinetic proofreading platform similar to that observed for the LAT/Grb2/SOS complex 28,46-48 . Biocondensates, consisting of concentrated multiprotein assemblies forming distinct fluid structures that separate from surrounding areas, are ubiquitous across signal transduction networks 49 . Furthermore, single particle tracking (SPT) experiments 50,51 , most recently coupled with photoactivated localization microscopy (SPT-PALM) and detailed trajectory analysis 52 , have shown that activated Ras proteins in live cells are found on the membrane in mobile and immobile phases, the latter being consistent with the predicted formation of large macromolecular assemblies that cannot freely diffuse on the membrane. Recent work with the transmembrane receptor LAT and its cytosolic binding partners Grb2 and the Ras guanine nucleotide exchange factor SOS, have shown that these proteins form polymer-like assemblies on supported lipid bilayers 46 , with restricted mobility.…”

“…Other interactions with the membrane from within these condensates, for example involving membrane receptors 53 , SOS [54][55][56] or PLCg 57 , further restrict molecular mobility and may influence Ras. Increasing connectivity with dimerization of the Ras/Raf complex could allosterically prime the Galectin binding site on Raf-RBD, to form a multivalent protein complex on the membrane corresponding to the immobile species observed in the SPT-PALM experiments 52 . This platform of synchronized activated signaling proteins (Fig.…”

RAS Nanoclusters: Dynamic Signaling Platforms Amenable to Therapeutic Intervention

“…The spatial mapping of the state coordinates showed that the slow and intermediate diffusion states cluster within nested nanoscopic domains of 200 nm and 70 nm for the intermediate and slow states, respectively [ 21 ], further confirming that RAS is exploring a hierarchical membrane structure. The formation of these states follows a nonequilibrium steady state where immobile KRAS molecules are endocytosed, and the endomembrane recycling system replenishes the fast-moving KRAS molecules.…”

“…Recently, work using improved imaging techniques and more photostable fluorescent tags indicates that KRAS membrane dynamics can be described by a three-state diffusion model ( Figure 4 A,B) [ 20 , 21 ]. In this model, KRAS4b diffusion in the plasma membrane is described by fast (~1 μm 2 /s), intermediate (~0.2 μm 2 /s) and slow (~0.05 μm 2 /s) diffusion states.…”

Inhibition of Nonfunctional Ras

“…The high-affinity Ras-Raf's RBD interaction, enhanced by CRD membrane attachment (Fang et al, 2020;Li et al, 2018aLi et al, , 2018bOkada et al, 1999;Sarkar and Garcia, 2020;Travers et al, 2018), shifts Raf's equilibrium toward the open state and interaction with Ras (Nussinov et al, 2019a). RBDs' interactions with spatially proximal Ras catalytic domains in Ras dimers or nanoclusters promote Raf's KD dimerization and activation (Holderfield et al, 2014;Lavoie et al, 2013;Lee et al, 2019;Muratcioglu et al, 2015Muratcioglu et al, , 2020. Active Raf phosphorylates MEK1/2 (MAPK kinase 1/2) (De Luca et al, 2012;Young et al, 2013), with signaling propagating down the mitogen-activated protein kinase (MAPK) pathway to activate extracellular signal-regulated kinase (ERK) and transcription factors, such as Elk-1 (Gao et al, 2019;, with the resulting expressed proteins entering the G 1 phase of the cell cycle, thereby linking the cellular environment to cell-cycle progression.…”