Fig. 4: Micron-scale glycocalyx segregation from the outer poles of leading-edge protrusions, blebs and retraction fibers.

a–f Glycocalyx/β1 fluorescence in leading pseudopod (a) and quantification of single (b) and multiple (c, d) pseudopods normalized by average non-contacting membrane fluorescence. Multichannel and single-channel micrographs from 3-slice maximum-intensity projections from Fig. 3a (region 1) showing glycocalyx along each pseudopod (d), vs. β1 enrichment (e) or per glycocalyx intensity category (f). Line in (a), quantification line in (b), with colors in (a) matching shades in (b). Scale bar, 2 µm. Dashed/solid vertical lines, β1 cluster peaks/edges, respectively. Datapoints (c–f): 449 β1 clusters from 22 protrusions, 7 cells. Black lines, linear (d) and logarithmic (e) fit ± 95% CI (ribbon). Calculation (e, f), see Supplementary Fig. 8d. Categorized glycocalyx in 3 content groups based on total cluster number. g–i Glycocalyx/β1 distributions in blebs using 3-slice maximum-intensity projections (g; indicated in Figs. 3a-2, post-rotation), fluorescence intensity in single bleb (h) and multiple blebs (i). Line subsegment colors in (g), shaded areas in (h). Yellow arrowhead, bleb apex. Pseudocolor: Fire-LUT. Scale bar, 2 µm. i Mean glycocalyx intensity normalized to mean collagen-contact-free membrane region; 32 blebs, 12 cells. j Glycocalyx vs. β1 fluorescence in blebs and paired bleb apexes (lines). Datapoints replotted from (i). k–m Glycocalyx/β1 fluorescence micrograph (3-slice maximum-intensity projections) (k; from Figs. 3a–3) and quantification along single (l) and multiple retraction fibers compartments corrected for collagen-contact-free fluorescence (m) and along relative fiber length (n). Line in (k), quantification line matching (l). In (l): Solid/dashed lines, cluster edges/centers, respectively. Datapoints (m): 328 clusters from 13 retraction fibers, 5 cells. Data in (d, n): clusters (dots) on the same protrusion (connected lines distinguished by colors). Line, linear fit ± 95% CI. R² values, adjusted coefficient of determination. P.adj, adjusted p-value (all panels). All panels: Kruskall-Wallis test with Bonferroni correction (ε² = 0.06 (f), indicates moderate effect size; ε² = 0.39 (c), ε² = 0.19 (i) and ε² = 0.39 (m) indicate high effect sizes). β1, β1 integrin. Data present the same 3 independent experiments as Fig. 3. Boxplots: middle-line, median; outlines, 1^st-3^rd quantiles; whiskers, quantiles ±1.5x interquantile range. CI confidence interval. Source data are provided as a Source Data file.