H-Ras nanocluster stability regulates the magnitude of MAPK signal output.

H-Ras is a binary switch that is activated by multiple co-factors and triggers several key cellular pathways one of which is MAPK. The specificity and magnitude of downstream activation is achieved by the spatio-temporal organization of the active H-Ras in the plasma membrane. Upon activation, the GTP bound H-Ras binds to Galectin-1 (Gal-1) and becomes transiently immobilized in short-lived nanoclusters on the plasma membrane from which the signal is propagated to Raf. In the current study we show that stabilizing the H-Ras-Gal-1 interaction, using bimolecular fluorescence complementation (BiFC), leads to prolonged immobilization of H-Ras.GTP in the plasma membrane which was measured by fluorescence recovery after photobleaching (FRAP), and increased signal out-put to the MAPK module. EM measurements of Raf recruitment to the H-Ras.GTP nanoclusters demonstrated that the enhanced signaling observed in the BiFC stabilized H-Ras.GTP nanocluster was attributed to increased H-Ras immobilization rather than to an increase in Raf recruitment. Taken together these data demonstrate that the magnitude of the signal output from a GTP-bound H-Ras nanocluster is proportional to its stability.

Rasosomes spread Ras signals from plasma membrane 'hotspots'.

Ras proteins regulate cell growth, differentiation, and apoptosis from various cellular platforms. We have recently identified a novel potential signaling platform, the rasosome, which moves rapidly near the plasma membrane (PM) and in the cytosol, carrying multiple copies of palmitoylated Ras proteins. In the present study we demonstrate that rasosomes are unique entities distinct from PM nanoclusters or from endocytotic compartments. In addition, we examine whether rasosomes can act as regulated Ras signaling platforms. We show that a single rasosome simultaneously carries different types of Ras molecules in their active and inactive state, suggesting that rasosomes can upload and download Ras signals. Total internal reflection fluorescence (TIRF) microscopy combined with fast time-lapse and a new spatial analysis algorithm demonstrate that rasosome movement near the PM is restricted to distinctive areas, rasosomal 'hotspots', localized between actin filament cages. In addition, Ras-binding domain of Raf-1 (RBD) is recruited to Ras in rasosomal hotspots as revealed by bimolecular fluorescence complementation experiments. Interestingly, epidermal growth factor stimulates H/NRas activation on rasosomes and the subsequent recruitment of RBD to rasosomes. Moreover, we show that rasosomes are loaded with Ras downstream effectors and modulators. These findings establish that physiological stimulation originating from PM hotspots is transduced to rasosomes, which appear to serve as robust Ras signaling platforms that spread signals across the cell.