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meos2 paxillin construct  (Addgene inc)
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Addgene inc meos2 paxillin construct
Left: Super‐resolution PALM intensity images of <t>β1-WT-mEos2</t> ( a ), β1-Y795A-mEos2 ( b ), β3-WT-mEos2 ( c ), β3-Y759A-mEos2 ( d ) in MEFs obtained from sptPALM sequences (50 Hz, >80 s). Inset: low resolution image of GFP-paxillin, which was co-expressed for FAs labeling (scale bar: 3 µm). Right: color-coded trajectories overlaid on FAs labeled by GFP-paxillin (greyscale) show the diffusion modes: free diffusion (green), confined diffusion (yellow) and immobilization (red). e Distributions of the diffusion coefficient D computed from the trajectories of β1-WT-mEos2 (black), β1-Y795A-mEos2 (pink), β1-Y783A-mEos2 (orange) obtained inside (left) and outside FAs (right), are shown in a logarithmic scale. The gray area including D values inferior to 0.011 µm².s −1 corresponds to immobilized proteins. Values represent the average of the distributions obtained from different cells. f Same as e , but with β3-WT-mEos2 (black), β3-Y759A-mEos2 (pink), β3-Y747A-mEos2 (orange). g Fraction of β1-WT and mutants undergoing free diffusion, confined diffusion or immobilization inside (left) and outside FAs (right) with or without manganese activation (Mn 2+ ). Values represent the average of the fractions obtained from different cells (error bars: SEM). h Same as g , but for β3-WT and mutants. Super-resolution PALM intensity image of β1-WT ( i ) and β1-Y795A ( j ) density in a FA (inset: labeled with GFP–paxillin) obtained from a sptPALM sequence. k Kymographs generated from a sptPALM time-lapse image along the length of a FA as shown by the dashed lines encompassing the FA in i and j . l Box plots displaying the median (notch) and mean (square) ± percentile (box: 25–75%, whiskers: 10–90%) of immobilization time inside FAs of β1-WT and mutants (left) and of β3-WT and mutants (right). a – h : results for β1-WT-mEos2 without (17 cells) and with Mn 2+ (15 cells), β1-Y795A-mEos2 without (22 cells) and with Mn 2+ (22 cells), β1-Y783A-mEos2 without (16 cells) and with Mn 2+ (20 cells), β3-mEos2 without (16 cells) and with Mn 2+ (13 cells), β3-Y759A-mEos2 without (20 cells) and with Mn 2+ (11 cells, 2 ind. exp.) and β3-Y747A-mEos2 without (16 cells) and with Mn 2+ (10 cells, 2 ind. exp.) correspond to pooled data from three independent experiments unless indicated. For (l), 6 (β1-Y795A, β1-Y783A, β3-WT), 5 (β3-Y759A, β3-Y747A), or 3 cells (β1-WT) corresponding to pooled data from three independent experiments were analysed. Where indicated, statistical significance was obtained using two‐tailed, non‐parametric Mann–Whitney rank sum test. Inside and outside FAs, the different conditions without Mn 2+ were compared to the corresponding β-integrin-WT condition; with Mn 2+ , each given condition was compared with the value obtained without Mn 2+ . Otherwise, a black line indicates which conditions were compared. The exact P values are indicated on the figure except when P < 0.0001. Source data are provided as a Source Data file.
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Left: Super‐resolution PALM intensity images of β1-WT-mEos2 ( a ), β1-Y795A-mEos2 ( b ), β3-WT-mEos2 ( c ), β3-Y759A-mEos2 ( d ) in MEFs obtained from sptPALM sequences (50 Hz, >80 s). Inset: low resolution image of GFP-paxillin, which was co-expressed for FAs labeling (scale bar: 3 µm). Right: color-coded trajectories overlaid on FAs labeled by GFP-paxillin (greyscale) show the diffusion modes: free diffusion (green), confined diffusion (yellow) and immobilization (red). e Distributions of the diffusion coefficient D computed from the trajectories of β1-WT-mEos2 (black), β1-Y795A-mEos2 (pink), β1-Y783A-mEos2 (orange) obtained inside (left) and outside FAs (right), are shown in a logarithmic scale. The gray area including D values inferior to 0.011 µm².s −1 corresponds to immobilized proteins. Values represent the average of the distributions obtained from different cells. f Same as e , but with β3-WT-mEos2 (black), β3-Y759A-mEos2 (pink), β3-Y747A-mEos2 (orange). g Fraction of β1-WT and mutants undergoing free diffusion, confined diffusion or immobilization inside (left) and outside FAs (right) with or without manganese activation (Mn 2+ ). Values represent the average of the fractions obtained from different cells (error bars: SEM). h Same as g , but for β3-WT and mutants. Super-resolution PALM intensity image of β1-WT ( i ) and β1-Y795A ( j ) density in a FA (inset: labeled with GFP–paxillin) obtained from a sptPALM sequence. k Kymographs generated from a sptPALM time-lapse image along the length of a FA as shown by the dashed lines encompassing the FA in i and j . l Box plots displaying the median (notch) and mean (square) ± percentile (box: 25–75%, whiskers: 10–90%) of immobilization time inside FAs of β1-WT and mutants (left) and of β3-WT and mutants (right). a – h : results for β1-WT-mEos2 without (17 cells) and with Mn 2+ (15 cells), β1-Y795A-mEos2 without (22 cells) and with Mn 2+ (22 cells), β1-Y783A-mEos2 without (16 cells) and with Mn 2+ (20 cells), β3-mEos2 without (16 cells) and with Mn 2+ (13 cells), β3-Y759A-mEos2 without (20 cells) and with Mn 2+ (11 cells, 2 ind. exp.) and β3-Y747A-mEos2 without (16 cells) and with Mn 2+ (10 cells, 2 ind. exp.) correspond to pooled data from three independent experiments unless indicated. For (l), 6 (β1-Y795A, β1-Y783A, β3-WT), 5 (β3-Y759A, β3-Y747A), or 3 cells (β1-WT) corresponding to pooled data from three independent experiments were analysed. Where indicated, statistical significance was obtained using two‐tailed, non‐parametric Mann–Whitney rank sum test. Inside and outside FAs, the different conditions without Mn 2+ were compared to the corresponding β-integrin-WT condition; with Mn 2+ , each given condition was compared with the value obtained without Mn 2+ . Otherwise, a black line indicates which conditions were compared. The exact P values are indicated on the figure except when P < 0.0001. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

doi: 10.1038/s41467-021-23372-w

Figure Lengend Snippet: Left: Super‐resolution PALM intensity images of β1-WT-mEos2 ( a ), β1-Y795A-mEos2 ( b ), β3-WT-mEos2 ( c ), β3-Y759A-mEos2 ( d ) in MEFs obtained from sptPALM sequences (50 Hz, >80 s). Inset: low resolution image of GFP-paxillin, which was co-expressed for FAs labeling (scale bar: 3 µm). Right: color-coded trajectories overlaid on FAs labeled by GFP-paxillin (greyscale) show the diffusion modes: free diffusion (green), confined diffusion (yellow) and immobilization (red). e Distributions of the diffusion coefficient D computed from the trajectories of β1-WT-mEos2 (black), β1-Y795A-mEos2 (pink), β1-Y783A-mEos2 (orange) obtained inside (left) and outside FAs (right), are shown in a logarithmic scale. The gray area including D values inferior to 0.011 µm².s −1 corresponds to immobilized proteins. Values represent the average of the distributions obtained from different cells. f Same as e , but with β3-WT-mEos2 (black), β3-Y759A-mEos2 (pink), β3-Y747A-mEos2 (orange). g Fraction of β1-WT and mutants undergoing free diffusion, confined diffusion or immobilization inside (left) and outside FAs (right) with or without manganese activation (Mn 2+ ). Values represent the average of the fractions obtained from different cells (error bars: SEM). h Same as g , but for β3-WT and mutants. Super-resolution PALM intensity image of β1-WT ( i ) and β1-Y795A ( j ) density in a FA (inset: labeled with GFP–paxillin) obtained from a sptPALM sequence. k Kymographs generated from a sptPALM time-lapse image along the length of a FA as shown by the dashed lines encompassing the FA in i and j . l Box plots displaying the median (notch) and mean (square) ± percentile (box: 25–75%, whiskers: 10–90%) of immobilization time inside FAs of β1-WT and mutants (left) and of β3-WT and mutants (right). a – h : results for β1-WT-mEos2 without (17 cells) and with Mn 2+ (15 cells), β1-Y795A-mEos2 without (22 cells) and with Mn 2+ (22 cells), β1-Y783A-mEos2 without (16 cells) and with Mn 2+ (20 cells), β3-mEos2 without (16 cells) and with Mn 2+ (13 cells), β3-Y759A-mEos2 without (20 cells) and with Mn 2+ (11 cells, 2 ind. exp.) and β3-Y747A-mEos2 without (16 cells) and with Mn 2+ (10 cells, 2 ind. exp.) correspond to pooled data from three independent experiments unless indicated. For (l), 6 (β1-Y795A, β1-Y783A, β3-WT), 5 (β3-Y759A, β3-Y747A), or 3 cells (β1-WT) corresponding to pooled data from three independent experiments were analysed. Where indicated, statistical significance was obtained using two‐tailed, non‐parametric Mann–Whitney rank sum test. Inside and outside FAs, the different conditions without Mn 2+ were compared to the corresponding β-integrin-WT condition; with Mn 2+ , each given condition was compared with the value obtained without Mn 2+ . Otherwise, a black line indicates which conditions were compared. The exact P values are indicated on the figure except when P < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Beta1-integrin-Y795A-mEos2 construct (human, vector: pFB) was kindly provided by C. Albiges-Rizo (IAB, U. Grenoble Alpes). mEos2-paxillin construct (chicken, promotor: CMV, vector: pmEos2) was obtained from Addgene (Plasmid #57409).

Techniques: Labeling, Diffusion-based Assay, Activation Assay, Sequencing, Generated, Two Tailed Test, MANN-WHITNEY

Left: Super‐resolution PALM intensity images of mEos2‐kindlin-2 ( a ) and talin-1-tdEos ( b ) in MEFs obtained from a sptPALM sequence (50 Hz, >80 s). Inset: low resolution image of GFP-paxillin, which was co-expressed for FAs labeling (scale bar: 3 µm). Right: color-coded trajectories overlaid on FAs labeled by GFP-paxillin (greyscale) show the diffusion modes: free diffusion (green), confined diffusion (yellow) and immobilization (red). c Distributions of the diffusion coefficient D computed from the trajectories of talin-1-tdEos (yellow) and mEos2‐kindlin-2 (blue) obtained inside (left) and outside FAs (right), are shown in a logarithmic scale. The gray area including D values inferior to 0.011 µm².s −1 corresponds to immobilized proteins. Values represent the average of the distributions obtained from different cells. d Fraction of mEos2‐kindlin-2 (blue) and talin-1-tdEos (yellow) undergoing free diffusion, confined diffusion or immobilization inside (left) and outside FAs (right). Values represent the average of the fractions obtained from different cells (error bars: SEM). e Box plots displaying the median (notch) and mean (square) ± percentile (25–75%) of diffusion coefficients corresponding to the free diffusion trajectories inside (left) and outside FAs (right). Super-resolution PALM intensity image of mEos2-kindlin-2 ( f ) and talin-1-tdEos ( g ) density in a FA (inset: labeled with GFP–paxillin) obtained from a sptPALM sequence. h Kymographs generated from a sptPALM time-lapse image along the length of a FA as shown by the dashed lines encompassing the FA in f and g . i Box plots displaying the median (notch) and mean (square) ± percentile (box: 25–75%, whiskers: 10–90%) of immobilization time inside FAs for kindlin-2 and talin-1. j Distribution of rearward speed (mean ± s.d. for cells) for kindlin-2 and talin-1. k Fraction of rearward speed equal or superior to 2 nm.s −1 (mean ± s.d. for cells) for kindlin-2 and talin-1. Corresponding data points are overlaid on the bar chart as dot plots. Results for mEos2‐kindlin-2 ( a – e : 13 cells; f – k : 1127 immobilization events in 12 cells) correspond to pooled data from three independent experiments. Results for β1-WT-mEos2 (dark gray) and β3-WT-mEos2 (light gray) corresponds to data shown in Fig. and Supplementary Fig. . Results for talin-1-tdEos ( a – e : 8 cells; f – k : 584 immobilization events in 7 cells) correspond to pooled data from two independent experiments. Where indicated, statistical significance was obtained using two‐tailed, non‐parametric Mann–Whitney rank sum test. Inside and outside FAs, the different conditions were compared to the corresponding mEos2‐kindlin-2 condition Otherwise, a black line indicates which conditions were compared. The exact P values are indicated on the figure except when P < 0.0001. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

doi: 10.1038/s41467-021-23372-w

Figure Lengend Snippet: Left: Super‐resolution PALM intensity images of mEos2‐kindlin-2 ( a ) and talin-1-tdEos ( b ) in MEFs obtained from a sptPALM sequence (50 Hz, >80 s). Inset: low resolution image of GFP-paxillin, which was co-expressed for FAs labeling (scale bar: 3 µm). Right: color-coded trajectories overlaid on FAs labeled by GFP-paxillin (greyscale) show the diffusion modes: free diffusion (green), confined diffusion (yellow) and immobilization (red). c Distributions of the diffusion coefficient D computed from the trajectories of talin-1-tdEos (yellow) and mEos2‐kindlin-2 (blue) obtained inside (left) and outside FAs (right), are shown in a logarithmic scale. The gray area including D values inferior to 0.011 µm².s −1 corresponds to immobilized proteins. Values represent the average of the distributions obtained from different cells. d Fraction of mEos2‐kindlin-2 (blue) and talin-1-tdEos (yellow) undergoing free diffusion, confined diffusion or immobilization inside (left) and outside FAs (right). Values represent the average of the fractions obtained from different cells (error bars: SEM). e Box plots displaying the median (notch) and mean (square) ± percentile (25–75%) of diffusion coefficients corresponding to the free diffusion trajectories inside (left) and outside FAs (right). Super-resolution PALM intensity image of mEos2-kindlin-2 ( f ) and talin-1-tdEos ( g ) density in a FA (inset: labeled with GFP–paxillin) obtained from a sptPALM sequence. h Kymographs generated from a sptPALM time-lapse image along the length of a FA as shown by the dashed lines encompassing the FA in f and g . i Box plots displaying the median (notch) and mean (square) ± percentile (box: 25–75%, whiskers: 10–90%) of immobilization time inside FAs for kindlin-2 and talin-1. j Distribution of rearward speed (mean ± s.d. for cells) for kindlin-2 and talin-1. k Fraction of rearward speed equal or superior to 2 nm.s −1 (mean ± s.d. for cells) for kindlin-2 and talin-1. Corresponding data points are overlaid on the bar chart as dot plots. Results for mEos2‐kindlin-2 ( a – e : 13 cells; f – k : 1127 immobilization events in 12 cells) correspond to pooled data from three independent experiments. Results for β1-WT-mEos2 (dark gray) and β3-WT-mEos2 (light gray) corresponds to data shown in Fig. and Supplementary Fig. . Results for talin-1-tdEos ( a – e : 8 cells; f – k : 584 immobilization events in 7 cells) correspond to pooled data from two independent experiments. Where indicated, statistical significance was obtained using two‐tailed, non‐parametric Mann–Whitney rank sum test. Inside and outside FAs, the different conditions were compared to the corresponding mEos2‐kindlin-2 condition Otherwise, a black line indicates which conditions were compared. The exact P values are indicated on the figure except when P < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Beta1-integrin-Y795A-mEos2 construct (human, vector: pFB) was kindly provided by C. Albiges-Rizo (IAB, U. Grenoble Alpes). mEos2-paxillin construct (chicken, promotor: CMV, vector: pmEos2) was obtained from Addgene (Plasmid #57409).

Techniques: Sequencing, Labeling, Diffusion-based Assay, Generated, Two Tailed Test, MANN-WHITNEY

Left: Super‐resolution PALM intensity images of mEos2-kindlin-2-WT in Mn 2+ -stimulated MEFs ( a ) and mEos2-kindlin-2-QW614/615AA in MEFs ( b ) obtained from a sptPALM sequence (50 Hz, >80 s). Inset: low resolution image of GFP-paxillin, which was co-expressed for FAs labeling (scale bar: 3 µm). Right: color-coded trajectories overlaid on FAs labeled by GFP-paxillin (greyscale) show the diffusion modes: free diffusion (green), confined diffusion (yellow) and immobilization (red). c Distributions of the diffusion coefficient D computed from the trajectories of mEos2-kindlin-2-WT (blue), mEos2-kindlin-2-WT in Mn 2+ -stimulated MEFs (black), mEos2-kindlin-2-QW614/615AA (red) obtained inside (left) and outside FAs (right), are shown in a logarithmic scale. The gray area including D values inferior to 0.011 µm².s −1 corresponds to immobilized proteins. Values represent the average of the distributions obtained from different cells. d Fraction of proteins undergoing free diffusion, confined diffusion or immobilization inside (left) and outside FAs (right) for mEos2-kindlin-2-WT, mEos2-kindlin-2-WT in Mn 2+ -stimulated MEFs, mEos2-kindlin-2-QW614/615AA (same color-code). Values represent the average of the fractions obtained from several cells (error bars: SEM). e Box plots displaying the median (notch) and mean (square) ± percentile (25–75%) of diffusion coefficients corresponding to the free diffusion trajectories inside (left) and outside FAs (right). Results for mEos2-kindlin-2-WT (17 cells) and mEos2-kindlin-2- QW614/615AA (33 cells) correspond to pooled data from five independent experiments. Results for mEos2-kindlin-2-WT + integrin activation by Mn 2+ (17 cells) correspond to pooled data from four independent experiments. Where indicated, statistical significance was obtained using two‐tailed, non‐parametric Mann–Whitney rank sum test. Inside and outside FAs, the different conditions were compared to the corresponding mEos2-kindlin-2-WT condition. The exact P values are indicated on the figure except when P < 0.0001. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

doi: 10.1038/s41467-021-23372-w

Figure Lengend Snippet: Left: Super‐resolution PALM intensity images of mEos2-kindlin-2-WT in Mn 2+ -stimulated MEFs ( a ) and mEos2-kindlin-2-QW614/615AA in MEFs ( b ) obtained from a sptPALM sequence (50 Hz, >80 s). Inset: low resolution image of GFP-paxillin, which was co-expressed for FAs labeling (scale bar: 3 µm). Right: color-coded trajectories overlaid on FAs labeled by GFP-paxillin (greyscale) show the diffusion modes: free diffusion (green), confined diffusion (yellow) and immobilization (red). c Distributions of the diffusion coefficient D computed from the trajectories of mEos2-kindlin-2-WT (blue), mEos2-kindlin-2-WT in Mn 2+ -stimulated MEFs (black), mEos2-kindlin-2-QW614/615AA (red) obtained inside (left) and outside FAs (right), are shown in a logarithmic scale. The gray area including D values inferior to 0.011 µm².s −1 corresponds to immobilized proteins. Values represent the average of the distributions obtained from different cells. d Fraction of proteins undergoing free diffusion, confined diffusion or immobilization inside (left) and outside FAs (right) for mEos2-kindlin-2-WT, mEos2-kindlin-2-WT in Mn 2+ -stimulated MEFs, mEos2-kindlin-2-QW614/615AA (same color-code). Values represent the average of the fractions obtained from several cells (error bars: SEM). e Box plots displaying the median (notch) and mean (square) ± percentile (25–75%) of diffusion coefficients corresponding to the free diffusion trajectories inside (left) and outside FAs (right). Results for mEos2-kindlin-2-WT (17 cells) and mEos2-kindlin-2- QW614/615AA (33 cells) correspond to pooled data from five independent experiments. Results for mEos2-kindlin-2-WT + integrin activation by Mn 2+ (17 cells) correspond to pooled data from four independent experiments. Where indicated, statistical significance was obtained using two‐tailed, non‐parametric Mann–Whitney rank sum test. Inside and outside FAs, the different conditions were compared to the corresponding mEos2-kindlin-2-WT condition. The exact P values are indicated on the figure except when P < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Beta1-integrin-Y795A-mEos2 construct (human, vector: pFB) was kindly provided by C. Albiges-Rizo (IAB, U. Grenoble Alpes). mEos2-paxillin construct (chicken, promotor: CMV, vector: pmEos2) was obtained from Addgene (Plasmid #57409).

Techniques: Sequencing, Labeling, Diffusion-based Assay, Activation Assay, Two Tailed Test, MANN-WHITNEY

Left: Super‐resolution PALM intensity images of mEos2‐kindlin-2-ΔPH ( a ) and mEos2‐PH-domain of kindlin-2-WT ( b ) in MEFs obtained from a sptPALM sequence (50 Hz, >80 s). Inset: low resolution image of GFP-paxillin, which was co-expressed for FAs labeling (scale bar: 3 µm). Right: color-coded trajectories overlaid on FAs labeled by GFP-paxillin (greyscale) show the diffusion modes: free diffusion (green), confined diffusion (yellow) and immobilization (red). c Distributions of the diffusion coefficient D computed from the trajectories of mEos2‐kindlin-2-WT (blue), mEos2‐kindlin-2-ΔPH (orange) and mEos2‐PH-domain of kindlin-2-WT (black) obtained inside (left) and outside FAs (right), are shown in a logarithmic scale. The gray area including D values inferior to 0.011 µm².s −1 corresponds to immobilized proteins. Values represent the average of the distributions obtained from different cells. d Fraction of proteins undergoing free diffusion, confined diffusion or immobilization inside (left) and outside FAs (right) for mEos2‐kindlin-2-WT, mEos2‐kindlin-2-ΔPH, and mEos2‐PH-domain of kindlin-2-WT (same color-code). Values represent the average of the fractions obtained from different cells (error bars: SEM). e Box plots displaying the median (notch) and mean (square) ± percentile (25–75%) of diffusion coefficients corresponding to the free diffusion trajectories inside (left) and outside FAs (right). Results of sptPALM (in a – e ) for mEos2‐kindlin-2-WT (15 cells), mEos2‐kindlin-2-ΔPH (28 cells) and mEos2‐PH-domain of kindlin-2-WT (18 cells) correspond to pooled data from three independent experiments. f Fraction of proteins recruited at the membrane defined as the ratio of the membrane-level fluorescence signal (TIRF) to the total fluorescence signal of the cell (epifluorescence). Results of membrane fraction (in f ) for mEos2‐kindlin-2-WT (28 cells), mEos2‐kindlin-2-ΔPH (27 cells) correspond to pooled data from two independent experiments, whereas results for β3-integrin-mEos2 (17 cells), cytosolic mEos2 (13 cells), mEos2-kindlin-2-QW (15 cells), mEos2-kindlin-2-K390A (15 cells) and mEos2-kindlin-2-L357A (15 cells) correspond to one experiment. Where indicated, statistical significance was obtained using two‐tailed, non‐parametric Mann–Whitney rank sum test. Inside and outside FAs, the different conditions were compared to the corresponding mEos2‐kindlin-2-WT condition. Otherwise, a black line indicates which conditions were compared. The exact P values are indicated on the figure except when P < 0.0001. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

doi: 10.1038/s41467-021-23372-w

Figure Lengend Snippet: Left: Super‐resolution PALM intensity images of mEos2‐kindlin-2-ΔPH ( a ) and mEos2‐PH-domain of kindlin-2-WT ( b ) in MEFs obtained from a sptPALM sequence (50 Hz, >80 s). Inset: low resolution image of GFP-paxillin, which was co-expressed for FAs labeling (scale bar: 3 µm). Right: color-coded trajectories overlaid on FAs labeled by GFP-paxillin (greyscale) show the diffusion modes: free diffusion (green), confined diffusion (yellow) and immobilization (red). c Distributions of the diffusion coefficient D computed from the trajectories of mEos2‐kindlin-2-WT (blue), mEos2‐kindlin-2-ΔPH (orange) and mEos2‐PH-domain of kindlin-2-WT (black) obtained inside (left) and outside FAs (right), are shown in a logarithmic scale. The gray area including D values inferior to 0.011 µm².s −1 corresponds to immobilized proteins. Values represent the average of the distributions obtained from different cells. d Fraction of proteins undergoing free diffusion, confined diffusion or immobilization inside (left) and outside FAs (right) for mEos2‐kindlin-2-WT, mEos2‐kindlin-2-ΔPH, and mEos2‐PH-domain of kindlin-2-WT (same color-code). Values represent the average of the fractions obtained from different cells (error bars: SEM). e Box plots displaying the median (notch) and mean (square) ± percentile (25–75%) of diffusion coefficients corresponding to the free diffusion trajectories inside (left) and outside FAs (right). Results of sptPALM (in a – e ) for mEos2‐kindlin-2-WT (15 cells), mEos2‐kindlin-2-ΔPH (28 cells) and mEos2‐PH-domain of kindlin-2-WT (18 cells) correspond to pooled data from three independent experiments. f Fraction of proteins recruited at the membrane defined as the ratio of the membrane-level fluorescence signal (TIRF) to the total fluorescence signal of the cell (epifluorescence). Results of membrane fraction (in f ) for mEos2‐kindlin-2-WT (28 cells), mEos2‐kindlin-2-ΔPH (27 cells) correspond to pooled data from two independent experiments, whereas results for β3-integrin-mEos2 (17 cells), cytosolic mEos2 (13 cells), mEos2-kindlin-2-QW (15 cells), mEos2-kindlin-2-K390A (15 cells) and mEos2-kindlin-2-L357A (15 cells) correspond to one experiment. Where indicated, statistical significance was obtained using two‐tailed, non‐parametric Mann–Whitney rank sum test. Inside and outside FAs, the different conditions were compared to the corresponding mEos2‐kindlin-2-WT condition. Otherwise, a black line indicates which conditions were compared. The exact P values are indicated on the figure except when P < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Beta1-integrin-Y795A-mEos2 construct (human, vector: pFB) was kindly provided by C. Albiges-Rizo (IAB, U. Grenoble Alpes). mEos2-paxillin construct (chicken, promotor: CMV, vector: pmEos2) was obtained from Addgene (Plasmid #57409).

Techniques: Sequencing, Labeling, Diffusion-based Assay, Fluorescence, Two Tailed Test, MANN-WHITNEY

Top panel: Epifluorescence images of RFP-paxillin showing the localization of FAs. Bottom panel: 3D super-resolution images by DONALD displaying the axial localization of kindlin-2-WT ( a ), kindlin-2-QW ( b ), kindlin-2-ΔPH ( c ), paxillin ( d ) and kindlin-2-ΔPH-CAAX ( e ); scale bar, 3 µm. For each pixel, the average axial localization of detected single molecules is color-coded. f Distributions of the single molecule axial localizations of kindlin-2-WT (blue) and paxillin (brown) inside (top) and outside FAs (bottom). Values represent the average of the distributions obtained from different cells. Positions of occurrence maxima (z peak ) were estimated by Gaussian fitting. g Same as f , but with kindlin-2-WT (blue), kindlin-2-QW (red), kindlin-2-ΔPH (orange) and kindlin-2-ΔPH-CAAX (green). h Box plots displaying the median (notch) and mean (square) ± percentile (25–75%) of single molecule localizations inside (top) and outside FAs (bottom). Micrographs ( a – h ) correspond to a single experiment, but are representative of kindlin-2-WT: 6 cells, kindlin-2-QW: 9 cells, kindlin-2-ΔPH: 7 cells, paxillin: 6 cells, kindlin-2-ΔPH-CAAX: 7 cells. Where indicated, statistical significance was obtained using two‐tailed, non‐parametric Mann–Whitney rank sum test. The different conditions were compared to the mEos2‐kindlin-2-WT condition. The exact P values are indicated on the figure except when P < 0.0001. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

doi: 10.1038/s41467-021-23372-w

Figure Lengend Snippet: Top panel: Epifluorescence images of RFP-paxillin showing the localization of FAs. Bottom panel: 3D super-resolution images by DONALD displaying the axial localization of kindlin-2-WT ( a ), kindlin-2-QW ( b ), kindlin-2-ΔPH ( c ), paxillin ( d ) and kindlin-2-ΔPH-CAAX ( e ); scale bar, 3 µm. For each pixel, the average axial localization of detected single molecules is color-coded. f Distributions of the single molecule axial localizations of kindlin-2-WT (blue) and paxillin (brown) inside (top) and outside FAs (bottom). Values represent the average of the distributions obtained from different cells. Positions of occurrence maxima (z peak ) were estimated by Gaussian fitting. g Same as f , but with kindlin-2-WT (blue), kindlin-2-QW (red), kindlin-2-ΔPH (orange) and kindlin-2-ΔPH-CAAX (green). h Box plots displaying the median (notch) and mean (square) ± percentile (25–75%) of single molecule localizations inside (top) and outside FAs (bottom). Micrographs ( a – h ) correspond to a single experiment, but are representative of kindlin-2-WT: 6 cells, kindlin-2-QW: 9 cells, kindlin-2-ΔPH: 7 cells, paxillin: 6 cells, kindlin-2-ΔPH-CAAX: 7 cells. Where indicated, statistical significance was obtained using two‐tailed, non‐parametric Mann–Whitney rank sum test. The different conditions were compared to the mEos2‐kindlin-2-WT condition. The exact P values are indicated on the figure except when P < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Beta1-integrin-Y795A-mEos2 construct (human, vector: pFB) was kindly provided by C. Albiges-Rizo (IAB, U. Grenoble Alpes). mEos2-paxillin construct (chicken, promotor: CMV, vector: pmEos2) was obtained from Addgene (Plasmid #57409).

Techniques: Two Tailed Test, MANN-WHITNEY

Top panel: TIRF images of photoconverted mEos2 coupled to kindlin-2-WT ( a ), kindlin-2-QW ( b ), kindlin-2-ΔPH ( c ), kindlin-2-ΔPH-CAAX ( d ), or photoconverted mEos2 alone ( e ). Bottom panel: TIRF images of GFP-paxillin showing the localization of FAs. Scale bar, 2 µm. f Enrichment inside FAs defined as the ratio of the average fluorescence intensity inside FAs to average fluorescence intensity outside FAs. Black bars represent medians and interquartile ranges. Displayed results correspond to pooled data from three independent experiments (kindlin-2-WT: 36 cells, kindlin-2-QW: 24 cells, kindlin-2-ΔPH: 27 cells, kindlin-2-ΔPH-CAAX: 50 cells, kindlin-2-L357A: 34 cells, kindlin-2-K390A: 30 cells, mEos2: 38 cells). Where indicated, statistical significance was obtained using two‐tailed, non‐parametric Mann–Whitney rank sum test. The different conditions were compared to the mEos2‐kindlin-2-WT condition. Otherwise, a black line indicates which conditions were compared. The exact P values are indicated on the figure except when P < 0.0001. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

doi: 10.1038/s41467-021-23372-w

Figure Lengend Snippet: Top panel: TIRF images of photoconverted mEos2 coupled to kindlin-2-WT ( a ), kindlin-2-QW ( b ), kindlin-2-ΔPH ( c ), kindlin-2-ΔPH-CAAX ( d ), or photoconverted mEos2 alone ( e ). Bottom panel: TIRF images of GFP-paxillin showing the localization of FAs. Scale bar, 2 µm. f Enrichment inside FAs defined as the ratio of the average fluorescence intensity inside FAs to average fluorescence intensity outside FAs. Black bars represent medians and interquartile ranges. Displayed results correspond to pooled data from three independent experiments (kindlin-2-WT: 36 cells, kindlin-2-QW: 24 cells, kindlin-2-ΔPH: 27 cells, kindlin-2-ΔPH-CAAX: 50 cells, kindlin-2-L357A: 34 cells, kindlin-2-K390A: 30 cells, mEos2: 38 cells). Where indicated, statistical significance was obtained using two‐tailed, non‐parametric Mann–Whitney rank sum test. The different conditions were compared to the mEos2‐kindlin-2-WT condition. Otherwise, a black line indicates which conditions were compared. The exact P values are indicated on the figure except when P < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Beta1-integrin-Y795A-mEos2 construct (human, vector: pFB) was kindly provided by C. Albiges-Rizo (IAB, U. Grenoble Alpes). mEos2-paxillin construct (chicken, promotor: CMV, vector: pmEos2) was obtained from Addgene (Plasmid #57409).

Techniques: Fluorescence, Two Tailed Test, MANN-WHITNEY

a Phase contrast images showing cell spreading of Kind Ko (kindlin-1, kindlin-2 knock-out) cells after 4 h on fibronectin. Two days before the experiment, the cells were transfected to induce the expression of GFP-paxillin (leftmost), or GFP-paxillin along with mEos2-kindlin-2-WT (middle left), mEos2-kindlin-2-ΔPH (middle right), or mEos2-kindlin-2-ΔPH-CAAX (rightmost). Images representative of 3 independent experiments. b Top: Phase contrast images showing the morphological features of cells classified as non-spread, partially spread, or spread. Bottom: Relative fraction of non-spread, partially spread, and spread Kind Ko cells 2 days after re-expression of kindlin-2-WT or mutated variants and 4 h after seeding on fibronectin. Values represent the average of fractions from 3 independent experiments (error bars: SEM). Results for GFP-paxillin (141 cells), GFP-paxillin + mEos2-kindlin-2-WT (312 cells), GFP-paxillin + mEos2-kindlin-2-ΔPH (311 cells), GFP-paxillin + mEos2-kindlin-2-ΔPH-CAAX (309 cells), GFP-paxillin + mEos2-kindlin-2-QW614/615AA (284 cells), GFP-paxillin + mEos2-kindlin-2-WT-CAAX (289 cells) correspond to pooled data from three independent experiments. Between 13 and 41 fields of view (20x objective) per condition and per experiment were used to quantify cell spreading, except for the cells expressing GFP-paxillin alone (negative control: between 8 and 17 fields per experience). Between 84 and 107 cells per condition and per experiment were included (except for the GFP-paxillin condition: between 31 and 58 cells per experiment). c TIRF images of GFP-paxillin showing adhesion sites of Kind Ko cells 2 days after re-expression of mEos2-kindlin-2-WT (top) and mEos2-kindlin-2-ΔPH-CAAX (bottom) and 4 h after seeding on fibronectin. For each image, the upper-right panel shows the outlined region at higher magnification. Quantification of number of adhesions per cell ( d ) and total cell area ( e ) of Kind Ko cells (4 h after seeding on fibronectin) re-expressing for 2 days mEos2-kindlin-2-WT (light blue), mEos2-kindlin-2-ΔPH (orange), mEos2-kindlin-2-ΔPH-CAAX (green), mEos2-kindlin-2-QW614/615AA (red) or mEos2-kindlin-2-WT-CAAX (dark blue). Each point in the distribution represents the value obtained from a single cell. FAs were drawn manually and cell boundaries were determined by manually setting a threshold on the pixel intensity values using the TIRF GFP-paxillin images as shown in (c). Black bars represent medians and interquartile ranges. GFP-paxillin + mEos2-kindlin-2-WT: 84 cells; GFP-paxillin + mEos2-kindlin-2-ΔPH: 74 cells; GFP-paxillin + mEos2-kindlin-2-ΔPH-CAAX: 62 cells; GFP-paxillin + mEos2-kindlin-2-QW614/615AA: 30 cells; GFP-paxillin + mEos2-kindlin-2-WT-CAAX: 63 cells. The results correspond to pooled data from three independent experiments with at least 8 cells per experiment. Where indicated, statistical significance was obtained using two‐tailed, non‐parametric Mann–Whitney rank sum test. The exact P values are indicated on the figure except when P < 0.0001. Source data are provided as a Source Data file. f Schematic model of integrin activation by kindlin and talin in mature FAs. Integrin and kindlin enter independently in FAs by membrane free diffusion whereas talin and paxillin reach FAs by a cytosolic free diffusion. In a FA, integrins display cycles of free-diffusion and immobilizations triggered by binding to kindlin and talin. The probability of freely diffusing integrin, kindlin and talin to meet simultaneously with the correct orientation is extremely low. Our results favor a model where the encounter of freely diffusing integrin and kindlin at the plasma membrane triggers the formation of an immobile integrin-kindlin complex (i). This complex could constrain the integrin β-tail orientation and favor the binding of talin to the proximal NPxY motif, leading to the formation of a transient tripartite integrin-kindlin-talin complex (ii). Then, kindlin could intermittently dissociate (iii) and re-associate (ii) with the longer-lived integrin-talin complex. The role of transient tripartite integrin-kindlin-talin complex, could extend the duration of integrin/talin interactions. Dissociation of both talin and kindlin will end this cycle of integrin immobilization before its next encounter with kindlin.

Journal: Nature Communications

Article Title: Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions

doi: 10.1038/s41467-021-23372-w

Figure Lengend Snippet: a Phase contrast images showing cell spreading of Kind Ko (kindlin-1, kindlin-2 knock-out) cells after 4 h on fibronectin. Two days before the experiment, the cells were transfected to induce the expression of GFP-paxillin (leftmost), or GFP-paxillin along with mEos2-kindlin-2-WT (middle left), mEos2-kindlin-2-ΔPH (middle right), or mEos2-kindlin-2-ΔPH-CAAX (rightmost). Images representative of 3 independent experiments. b Top: Phase contrast images showing the morphological features of cells classified as non-spread, partially spread, or spread. Bottom: Relative fraction of non-spread, partially spread, and spread Kind Ko cells 2 days after re-expression of kindlin-2-WT or mutated variants and 4 h after seeding on fibronectin. Values represent the average of fractions from 3 independent experiments (error bars: SEM). Results for GFP-paxillin (141 cells), GFP-paxillin + mEos2-kindlin-2-WT (312 cells), GFP-paxillin + mEos2-kindlin-2-ΔPH (311 cells), GFP-paxillin + mEos2-kindlin-2-ΔPH-CAAX (309 cells), GFP-paxillin + mEos2-kindlin-2-QW614/615AA (284 cells), GFP-paxillin + mEos2-kindlin-2-WT-CAAX (289 cells) correspond to pooled data from three independent experiments. Between 13 and 41 fields of view (20x objective) per condition and per experiment were used to quantify cell spreading, except for the cells expressing GFP-paxillin alone (negative control: between 8 and 17 fields per experience). Between 84 and 107 cells per condition and per experiment were included (except for the GFP-paxillin condition: between 31 and 58 cells per experiment). c TIRF images of GFP-paxillin showing adhesion sites of Kind Ko cells 2 days after re-expression of mEos2-kindlin-2-WT (top) and mEos2-kindlin-2-ΔPH-CAAX (bottom) and 4 h after seeding on fibronectin. For each image, the upper-right panel shows the outlined region at higher magnification. Quantification of number of adhesions per cell ( d ) and total cell area ( e ) of Kind Ko cells (4 h after seeding on fibronectin) re-expressing for 2 days mEos2-kindlin-2-WT (light blue), mEos2-kindlin-2-ΔPH (orange), mEos2-kindlin-2-ΔPH-CAAX (green), mEos2-kindlin-2-QW614/615AA (red) or mEos2-kindlin-2-WT-CAAX (dark blue). Each point in the distribution represents the value obtained from a single cell. FAs were drawn manually and cell boundaries were determined by manually setting a threshold on the pixel intensity values using the TIRF GFP-paxillin images as shown in (c). Black bars represent medians and interquartile ranges. GFP-paxillin + mEos2-kindlin-2-WT: 84 cells; GFP-paxillin + mEos2-kindlin-2-ΔPH: 74 cells; GFP-paxillin + mEos2-kindlin-2-ΔPH-CAAX: 62 cells; GFP-paxillin + mEos2-kindlin-2-QW614/615AA: 30 cells; GFP-paxillin + mEos2-kindlin-2-WT-CAAX: 63 cells. The results correspond to pooled data from three independent experiments with at least 8 cells per experiment. Where indicated, statistical significance was obtained using two‐tailed, non‐parametric Mann–Whitney rank sum test. The exact P values are indicated on the figure except when P < 0.0001. Source data are provided as a Source Data file. f Schematic model of integrin activation by kindlin and talin in mature FAs. Integrin and kindlin enter independently in FAs by membrane free diffusion whereas talin and paxillin reach FAs by a cytosolic free diffusion. In a FA, integrins display cycles of free-diffusion and immobilizations triggered by binding to kindlin and talin. The probability of freely diffusing integrin, kindlin and talin to meet simultaneously with the correct orientation is extremely low. Our results favor a model where the encounter of freely diffusing integrin and kindlin at the plasma membrane triggers the formation of an immobile integrin-kindlin complex (i). This complex could constrain the integrin β-tail orientation and favor the binding of talin to the proximal NPxY motif, leading to the formation of a transient tripartite integrin-kindlin-talin complex (ii). Then, kindlin could intermittently dissociate (iii) and re-associate (ii) with the longer-lived integrin-talin complex. The role of transient tripartite integrin-kindlin-talin complex, could extend the duration of integrin/talin interactions. Dissociation of both talin and kindlin will end this cycle of integrin immobilization before its next encounter with kindlin.

Article Snippet: Beta1-integrin-Y795A-mEos2 construct (human, vector: pFB) was kindly provided by C. Albiges-Rizo (IAB, U. Grenoble Alpes). mEos2-paxillin construct (chicken, promotor: CMV, vector: pmEos2) was obtained from Addgene (Plasmid #57409).

Techniques: Knock-Out, Transfection, Expressing, Negative Control, Two Tailed Test, MANN-WHITNEY, Activation Assay, Diffusion-based Assay, Binding Assay