Figure 3. Observation of RAW264 cells expressing constitutively active mCitrine-Rac1Q61L by fluorescence and scanning electron microscopy.

RAW264 cells were transfected with pmCitrine-Rac1Q61L. After 12 hours, the cells were serum starved for 2 hours and then subjected to live-cell imaging or processed for scanning EM. (a) After confirming the expression of mCitrine-Rac1Q61L by fluorescence microscopy, we added FM4-64 to the extracellular medium and immediately (<1 min) acquired phase-contrast, mCitrine-Rac1Q61L and FM4-64 images. RAW264 cells expressing mCitrine-Rac1Q61L showed remarkable membrane ruffling and phase-bright vacuoles. Most of the phase-bright vacuoles associated with mCitrine-Rac1Q61L (yellow) were labelled with FM4-64 (red), indicating that these structures are unclosed pre-macropinosomes. The bar indicates 10 μm. (b) Scanning EM of RAW264 cells expressing constitutively active Rac1Q61L. We can identify the cell showing numerous ruffles across the whole dorsal surface as a Rac1Q61L expressing cell (arrows), since non-expressing cells lost dorsal ruffles and lamellipodia (open arrow) as observed by fluorescence microscopy (Supplementary Fig. S2). A higher magnification of the ruffling region shows the openings of macropinocytic cups or pockets (arrowheads). The bars indicate 5 μm.