Fig. 7: PH-domain-mediated membrane recruitment and diffusion of kindlin-2 is crucial during cell spreading and mature FA formation.

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.