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.