Fig. 3: Focal adhesion proteins and cytoskeleton (actin and microtubules) are enriched at β1-integrin clusters on SLBs. | Nature Communications

Fig. 3: Focal adhesion proteins and cytoskeleton (actin and microtubules) are enriched at β1-integrin clusters on SLBs.

From: Cell adhesion and spreading on fluid membranes through microtubules-dependent mechanotransduction

Fig. 3

All cells are Mn2+-treated and imaged 45 min – 1 h after seeding in the chamber. A Main panels: fluorescence multi-channel images showing β1-integrin (cyan) and paxillin (magenta) cluster organization in cells adhering on SLBs coated with RGD (left) and Invasin (right). Images are taken as max intensity z projections (in Fiji) of the volume with 1 µm height (z coordinate) centered at the focal plane of the SLB. Lower  left corners: corresponding brightfield images. Medium panels: zooms corresponding to the white squares on the main panels, showing regions with β1-integrin and paxillin clusters. Bottom: β1-integrin and paxillin intensity profiles along the lines (white rectangles in the zoomed panels). Scale bars: 5 µm (main panels); 1 µm (zoomed panels). B Relative enrichment of focal adhesion (FA) proteins in β1-integrin clusters in cells on RGD- (blue) and Invasin-SLBs (red). It is calculated as an increase in fluorescence of a FA protein in β1-integrin clusters compared to the mean cell fluorescence normalized by the mean cell fluorescence. These measurements were conducted on fixed cells to examine FA proteins, with β1-integrin clusters identified using a threshold of 100 arbitrary units (‘arb. units’) (Methods). Violin plots with the median represented by a central line and the interquartile range (25th-75th percentiles) indicated by the upper and lower lines. Data from: Talin: Ncells = 87, Nexp = 4 (RGD-SLBs); Ncells = 65, Nexp = 4 (Invasin-SLBs). Kindlin-2: Ncells = 42, Nexp = 3 (RGD-SLBs); Ncells = 87, Nexp = 4 (Invasin-SLBs). Paxillin: Ncells = 53, Nexp = 3 (RGD-SLBs); Ncells = 128, Nexp = 3 (Invasin-SLBs). Vinculin: Ncells = 59, Nexp = 4 (RGD-SLBs); Ncells = 56, Nexp = 3 (Invasin-SLBs). VASP: Ncells = 42, Nexp = 4 (RGD-SLBs); Ncells = 66, Nexp = 4 (Invasin-SLBs). Zyxin: Ncells = 61, Nexp = 4 (RGD-SLBs); Ncells = 76, Nexp = 4 (Invasin-SLBs). Statistical significance for cells on RGD- vs. Invasin-SLBs (two-tailed Mann-Whitney tests): Talin: p-value = 0.1030. Kindlin-2: p-value < 0.0001. Paxillin: p-value = 0.0002. Vinculin: p-value = 0.0131. VASP: p-value = 0.0147. Zyxin: p-value < 0.0001. Source data are provided as a Source Data file. C, D Experiments focused on F-actin and microtubule recruitment to β1-integrin clusters performed on live cells in which the clusters were quantitatively defined using a threshold of 100 integrins/μm2 (Methods). D Relative enrichment of the actin in β1-integrin clusters in cells on RGD- (blue) and Invasin-SLBs (red). Violin plots with the median represented by a central line and the interquartile range (25th-75th percentiles) indicated by the upper and lower lines. Data from Ncells = 58, Nexp = 3 for RGD and Ncells = 75, Nexp = 3 for Invasin. Statistical significance (two-tailed Mann-Whitney tests): p-value < 0.0001. Source data are provided as a Source Data file. E Relative enrichment of the microtubules in β1-integrin clusters in cells on RGD- (blue) and Invasin-SLBs (red). Violin plots with the median represented by a central line and the interquartile range (25th-75th percentiles) indicated by the upper and lower lines. Data from Ncells = 53, Nexp = 3 for RGD and Ncells = 54, Nexp = 3 for Invasin. Statistical significance (two-tailed Mann-Whitney tests): p-value < 0.0001. Source data are provided as a Source Data file. E Fluorescence multi-channel images showing β1-integrin (yellow), actin (magenta) and microtubules (cyan) in cells adhering on SLBs coated with RGD (blue frame, top) and Invasin (red frame, bottom). Images are taken in the focal plane of the SLB. β1-integrin density, actin and microtubules intensity profiles along the lines (black rectangles in the zoomed panels) are plotted for both cells. Scale bars: 5 µm, 1 µm.

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