Real-Time Visualization and Quantification of Native Ras Activation in Single Living Cells

Members of the Ras family of small guanosine triphosphate phosphohydrolases are GDP/GTP-binding proteins that function as pivotal molecular switches in multiple cell biological processes. The prototypical Ras family members K-Ras, N-Ras, and H-Ras, in particular, have been the focus of intense research for the last 30 years owing to their critical function as signalling nodes in the control of cell growth and proliferation and as drivers of oncogenic transformation. One aspect that has attracted much attention in recent times is the spatial control of Ras activity, which is dictated largely by a series of posttranslational modifications that do effectively govern the subcellular distribution and trafficking of Ras. Accordingly, strong emphasis has been placed on developing methodological microscopy-based approaches for the visualization of active Ras-GTP complexes at subcellular resolution. Here we describe the use of a collection of fluorescent affinity probes for the real-time visualization of Ras-GTP in live cells. These probes are multivalent and thus feature high avidity/affinity to Ras-GTP, which obviates the over-expression of Ras and enables one to image endogenous Ras-GTP formation. In addition, this chapter details the use of automated segmentation strategies for the unbiased quantification of probe-derived fluorescence at individual subcellular sites like the plasma membrane and endomembranes.