Figure 8.

SHCA localization to adhesions precedes LPP recruitment. ErbB2-NT/SHCA^low (explant 87) cells expressing SHCA-WT-iRFP and EGFP-LPP were seeded onto fibronectin-coated glass bottom dishes and imaged with TIRF microscopy. A, time-lapse montage of a newly forming adhesion (see Movie S3). White arrowheads indicate the site of adhesion nucleation; green arrowheads indicate the appearance of SHCA; and magenta arrowheads indicate the appearance of LPP. Intensity traces for SHCA and LPP are depicted in the adjacent graph. B, time-lapse montage of a maturing adhesion (see Movie S4). White arrowheads indicate the adhesion of interest. Intensity traces for SHCA and LPP are depicted in the adjacent graph. C, pseudo-colored montage (16-color) of SHCA and LPP in a mature adhesion. The black arrowhead points to a second nucleation point in the SHCA channel. Linescan analysis was performed every 20 s to show the formation of a second SHCA nucleation point at the growing end of the adhesion. Fluorescence intensity of SHCA and LPP are plotted as a function of distance and time. D, time-lapse montage of a disassembling adhesion (see Movie S5). White arrowheads indicate the adhesion of interest; green arrowheads indicate the start of SHCA disassembly; and magenta arrowheads indicate the start of LPP disassembly. Intensity traces for SHCA and LPP are depicted in the adjacent graph. E, time-lapse montage of a short-lived adhesion (see Movie S6). White arrowheads indicate the site of adhesion nucleation, and green arrowheads indicate the appearance of SHCA. Intensity traces for SHCA and LPP are depicted in the adjacent graph. Scale bars, 2 μm.