Fig. 3: Submicron glycocalyx segregation in cell-matrix adhesions.
a Single- and multi-channel micrographs (maximum intensity projections) of migrating cells. ROIs: 1) leading edge protrusion, 2) membrane bleb, 3) retraction fiber and 4) collagen contact-free membrane. White arrow, migration direction. Scale bar, 5 µm. b Zoom of leading pseudopod from (a). White arrowheads and insets (A, B), β1 clusters outward-segregated from glycocalyx. Scale bar, 2 µm. c Size of 499 β1 clusters from 22 leading edge protrusions (7 cells, 3 independent experiments). d Representative micrographs (from inset A, panel b) of β1-glycocalyx segregation. White arrowhead and line denote β1 cluster and ROI used for outer cluster analysis. Blue line/arrowhead, lateral ROI/boundaries for β1 cluster-adjacent inner zone. Yellow arrowheads, β1 cluster-associated collagen fibers. Collagen channel, Fire pseudocolor. Asterisk, intersection point of both line ROIs. Scale bar, 1 µm. o, outer cluster; i, inner cluster. e Quantification of β1-glycocalyx distance segregation in individual contact to collagen fibril. Magenta/yellow dashed lines, cluster /glycocalyx enrichment middle, determined by maximum β1/glycocalyx levels for outer clusters and corresponding peak in the lateral ROI (inner zone). Blue box, β1 cluster edges, based on the peak-adjacent lateral minima. f, g paired β1 (f) and glycocalyx (g) enrichment in outer β1 cluster and corresponding lateral membrane zone, normalized to matched membrane region lacking β1 clustering (“nonfocal”). 25 (cell body) and 38 (inner-outer matched) line ROIs from 9 cells of 3 independent experiments. Wilcoxon Rank-Sum test with Bonferroni correction (ε2 = 0.25 (f) and ε2 = 0.54 (g), large effect size). h Segregation distance of β1 and glycocalyx in outer β1 clusters. Data show 25 individual perpendicular membrane regions and 38 focal outward clusters from 11 cells of 3 independent experiments. Wilcoxon Rank-Sum test (ε2 = 0.56, large effect size). i Correlation of local glycocalyx density and β1 enrichment in outward β1 clusters (R-squared = −0.02, adjusted p-value = 1). Data replotted from (h). Line, logarithmic fitting curve with 95% confidence interval (ribbon). All data derive from the same 3 independent experiments. Cells (all panels): MV3. Boxplots: middle-line, median; outlines, 1st-3rd quantiles; whiskers, quantiles ±1.5x interquantile range. ROI region of interest. β1, β1 integrin. Source data are provided as a Source Data file.
