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. 2011 May 23;108(24):E201–E210. doi: 10.1073/pnas.1101929108

Fig. 4.

Fig. 4.

SM imaging and tracking of intracellular cav1-GFP 1–10(h) proteins by CALM. (A) A U2OS cell coexpressing cav1-GFP 1–10(h) and the nucleus-localized CFP-LacI-NLS coexpression marker (+) are microinjected with biotin-M3 peptides (∼5 μM final intracellular concentration) together with a biotin-Alexa 647 injection marker (star). A region of interest (white square) is then imaged for GFP fluorescence by TIRF microscopy. (Scale bar: 10 μm.) (B) Pixel-based maximum intensity projection (ΣImax) TIRF images of all complemented cav1-GFP(h) detected at the ventral intracellular plasma membrane for the region of interest in A 3, 5, and 10 min after injection of M3 peptides (Movie S8). When overlaid with the wide-field fluorescence image, the cumulative 3- to 10-min maximum intensity projection image shows the high specificity of complementation. (Scale bar: 5 μm.) (C) 3D rendering of raw diffraction-limited spots corresponding to membrane-associated single cav1-GFP(h) proteins (white square in B). (D) Representative trajectories from single cav1-GFP(h) diffusing in the cytoplasmic side of the plasma membrane of U2OS cells during CALM imaging.