Fig. 7: ROCS microscopy of LecA dynamics at the outer cell cortex.

a TIR-ROCS image (10 ms integration time) of a H1299 lung carcinoma cell in gray colors overlaid with TIR- image (50 ms integration time) of LecA clusters (Cy5) in orange colors. b Difference images averaged over 10 s show dynamics of LecA clusters (TIR-F) and (TIR-ROCS). The difference histogram is asymmetric, negative biased for TIR-F indicating enhanced endocytosis. Negative (positive) differences in black (white) correspond to endocytosis (exocytosis). c TIR-ROCS activity map over 10 s and full 100 Hz bandwidth show cell cortex dynamics relative to edges of LecA TIR-F images. d Regions of interest ROI 3, ROI 2 (see Supplementary Movies) and ROI 1 from figure above show cell cortex details (ROCS), the occurrences of Cy5-Lec A clusters (edge enhanced) and the LecA dynamics by TIR-F difference averaged over 10 s. e Sketch of membrane invagination and transport of LecA along actin fibers through myosins. Outside the membrane LecA binds to Gb3 and CD59, which loosely connects through the membrane to long saturated fatty acid chains of PIP3 and myosin. f Imaging of LecA with 2 Hz TIR-F after 5 min and 11 min of LecA addition. TIR-F differences averaged over 10 s reveal several uptake events (black patches and corresponding white edges, right-shifted). g TIR-ROCS imaging of H1299 cell after 5/11 min of LecA addition with white edges in ROI 1 from LecA uptake areas in f. ROI 2 shows one (foc)ussed particle (yellow arrow) at membrane and two (def)ocussed particles inside the cell, which cause activity as well (dashed circles in activity ROI below). The kymographs along t1 and t2 depict stop-and-go diffusion with maximal velocities v₁ and v₂ (white arrows). Activity plots at 5/11 min show high particle dynamics at the membrane and in the cortex. Source data are provided as a Source Data file.