Journal: Nature Communications

Article Title: Glycocalyx micro- and nanodomains in cell-cell and cell-matrix interactions revealed by enhanced click chemistry

doi: 10.1038/s41467-026-69242-1

Figure Lengend Snippet: 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 (ε 2 = 0.25 ( f ) and ε 2 = 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 (ε 2 = 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, 1 st -3 rd quantiles; whiskers, quantiles ±1.5x interquantile range. ROI region of interest. β1, β1 integrin. Source data are provided as a Source Data file.

Article Snippet: For β1 integrin staining, collagen-embedded cells were incubated in blocking buffer (1 % bovine serum albumin, Sigma-Aldrich, Cat# A9647; 10 % normal goat serum, Thermo Fisher Scientific, Cat# 10000 C; PBS, 1 h, 20 °C), incubated with a mixture of two mouse anti-human β1 integrin antibodies (clone K20, Novus Biochemicals, NBP2-52708; clone 4B4LDC9LDH8, Beckman Coulter, 6603113; both 10 ug/mL in 50 μl, blocking buffer, 24 h, 4 °C, mild agitation), washed 3 times (blocking buffer, 15 min, 4 °C) and incubated with secondary antibody mouse IgG (H + L) highly cross-adsorbed AlexaFluor647 (2 μg/ml in 50 μl, Thermo Fisher Scientific, Cat# A21236, 24 h, 4 °C), 1 μg/mL DAPI (Merck, Cat# D9542), and when non-fluorescent collagen was used, with 2U/ml Phalloidin-Alexa Fluor 568 (Thermo Fisher Scientific, Cat# A12380) (washed again 3x, PBS, 15 min, 4 °C).

Techniques: Membrane, Migration

Journal: Nature Communications

Article Title: Glycocalyx micro- and nanodomains in cell-cell and cell-matrix interactions revealed by enhanced click chemistry

doi: 10.1038/s41467-026-69242-1

Figure Lengend Snippet: 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. (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. . 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. a- , 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. a– ) 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. R values, adjusted coefficient of determination. P.adj, adjusted p-value (all panels). All panels: Kruskall-Wallis test with Bonferroni correction (ε = 0.06 ( f ), indicates moderate effect size; ε = 0.39 ( c ), ε = 0.19 ( i ) and ε = 0.39 ( m ) indicate high effect sizes). β1, β1 integrin. Data present the same 3 independent experiments as Fig. . 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.

Article Snippet: For β1 integrin staining, collagen-embedded cells were incubated in blocking buffer (1 % bovine serum albumin, Sigma-Aldrich, Cat# A9647; 10 % normal goat serum, Thermo Fisher Scientific, Cat# 10000 C; PBS, 1 h, 20 °C), incubated with a mixture of two mouse anti-human β1 integrin antibodies (clone K20, Novus Biochemicals, NBP2-52708; clone 4B4LDC9LDH8, Beckman Coulter, 6603113; both 10 ug/mL in 50 μl, blocking buffer, 24 h, 4 °C, mild agitation), washed 3 times (blocking buffer, 15 min, 4 °C) and incubated with secondary antibody mouse IgG (H + L) highly cross-adsorbed AlexaFluor647 (2 μg/ml in 50 μl, Thermo Fisher Scientific, Cat# A21236, 24 h, 4 °C), 1 μg/mL DAPI (Merck, Cat# D9542), and when non-fluorescent collagen was used, with 2U/ml Phalloidin-Alexa Fluor 568 (Thermo Fisher Scientific, Cat# A12380) (washed again 3x, PBS, 15 min, 4 °C).

Techniques: Fluorescence, Membrane

Journal: Nature Communications

Article Title: Glycocalyx micro- and nanodomains in cell-cell and cell-matrix interactions revealed by enhanced click chemistry

doi: 10.1038/s41467-026-69242-1

Figure Lengend Snippet: a Nanoscale segregation of glycocalyx from β1 integrin cluster in a perpendicular direction. The two-compartment zone consists of an outer β1 integrin cluster with low glycocalyx content, segregating perpendicularly from a glycocalyx-rich region at the cell body with variable β1 integrin enrichment, yet a lack of glycocalyx segregation at the base of this interaction. The outer β1 integrin outer cluster interacts with fibrillar collagen and is connected to the actin cytoskeleton, consistent with a glycocalyx-deficient nanoprotrusion. b I) Micron-scale glycocalyx depletion across a leading edge protrusion from the base towards the apical direction. Dashed rectangle, inset II), which illustrates that glycocalyx-depleted zones of the tip of the leading edge protrusion form a zone of high-integrin clustering sensitivity. c Micron-scale glycocalyx depletion in blebs towards the bleb apex. d Micron-scale glycocalyx depletion towards the tip of retraction fibers. e Micron-scale glycocalyx underrepresentation in cell-cell contacts and gradient-like redistribution out of cell-cell contact along single-cell membrane segments and interconnecting transition zone. In all panels, solid arrows indicate migration direction, and dashed arrows indicate glycocalyx depletion direction.

Article Snippet: For β1 integrin staining, collagen-embedded cells were incubated in blocking buffer (1 % bovine serum albumin, Sigma-Aldrich, Cat# A9647; 10 % normal goat serum, Thermo Fisher Scientific, Cat# 10000 C; PBS, 1 h, 20 °C), incubated with a mixture of two mouse anti-human β1 integrin antibodies (clone K20, Novus Biochemicals, NBP2-52708; clone 4B4LDC9LDH8, Beckman Coulter, 6603113; both 10 ug/mL in 50 μl, blocking buffer, 24 h, 4 °C, mild agitation), washed 3 times (blocking buffer, 15 min, 4 °C) and incubated with secondary antibody mouse IgG (H + L) highly cross-adsorbed AlexaFluor647 (2 μg/ml in 50 μl, Thermo Fisher Scientific, Cat# A21236, 24 h, 4 °C), 1 μg/mL DAPI (Merck, Cat# D9542), and when non-fluorescent collagen was used, with 2U/ml Phalloidin-Alexa Fluor 568 (Thermo Fisher Scientific, Cat# A12380) (washed again 3x, PBS, 15 min, 4 °C).

Techniques: Single Cell, Membrane, Migration