Figure 5. The kindlin/paxillin complex induces FAK signaling and cell spreading.

(A) FAK and EGFR activation after seeding serum-starved Kind^Ctr, Tln^Ko and Kind^Ko cells on PLL or FN and treating them with or without EGF and Mn²⁺. (B) Immunofluorescence staining of activated (Tyr-397 phosphorylated) FAK and F-actin in cells seeded on FN and treated with Mn²⁺ for 30 min (FAKGFP indicates exogenous expression of FAKGFP fusion protein). (C) FAK and Akt activation in Kind^Ko cells stably transduced with K2GFP or K2ΔPHGFP either seeded on FN or kept in suspension. GFP indicates similar expression of transduced GFP-tagged constructs. GAPDH levels served to control loading. (D) Levels of phosphorylated signaling mediators downstream of FAK in Mn²⁺-treated, serum-starved or EGF-treated Kind^Ctr, Tln^Ko and Kind^Ko cells. GAPDH levels served to control loading. (E) Quantification of lamellipodia formation of FN-seeded Tln^Ko and Kind^Ko cells treated with Mn²⁺ and either DMSO or the FAK inhibitor PF-228 (n=3 independent repeats; >100 cells/condition; error bars indicate standard error of the mean; significances are given in comparison to the DMSO control). (F) FAK activity in Tln^Ko and Kind^Ko cells stably transduced with FAKGFP (n=3 independent experiments). (G) Quantification of lamellipodia formation in Tln^Ko and Kind^Ko cells stably transduced with FAKGFP (n=3 independent experiments; significances are given in comparison to untreated control; error bars indicate standard error of the mean). Bar, 10 µm. DMSO, dimethyl sulfoxide; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FAK, focal adhesion kinase; FAKGFP, green fluorescent protein-tagged FAK; FN, fibronectin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; PLL, poly-L-lysine.

DOI: http://dx.doi.org/10.7554/eLife.10130.023

Figure 5—figure supplement 1. FAK phosphorylation in Tln^Ctr, Tln^Ko, Tln^Ko+T1V, Kind^Ctr, Kind^Ko and Kind^Ko+K2GFP cells.

(A) Densitometric quantification of western blot signals of lysates from untreated, EGF- and Mn²⁺-treated Kind^Ctr, Tln^Ko and Kind^Ko cells seeded either on FN or PLL and probed with anti-Tyr-397 phosphorylated FAK (pY397-FAK) antibodies (n=3 independent repeats; significances are calculated with respect to PLL adherent cells; error bars indicate standard error of the mean). (B,C) Western blotting of indicated signaling proteins in untreated, EGF- and Mn²⁺-treated Tln^Ctr, Tln^Ko and Tln^Ko cells re-expressing Venus-tagged talin-1 (Tln^Ko+T1V) (B), and Kind^Ctr, Kind^Ko and Kind^Ko cells re-expressing GFP-tagged kindlin-2 (Kind^Ko+K2GFP) (C) seeded either on FN or PLL. EGF, epidermal growth factor; FAK, focal adhesion kinase; K2GFP, green fluorescent protein-tagged kindlin-2; FN, fibronectin; GFP, green fluorescent protein; PLL, poly-L-lysine; T1V, Venus-tagged full length talin-1.

DOI: http://dx.doi.org/10.7554/eLife.10130.024

Figure 5—figure supplement 2. Kindlin-2 forms a ternary complex with paxillin and FAK.

GFP-IP in lysates of K2GFP-, K2ΔPHGFP- or GFP-reconstituted Kind^Ko cells overexpressing Myc-tagged FAK (FAK-Myc) and Cherry-tagged paxillin (PxnCH). K2GFP but not K2ΔPHGFP forms a ternary complex with paxillin and FAK. FAK, focal adhesion kinase; GFP, green fluorescent protein; IP, immunoprecipitation; K2GFP, green fluorescent protein-tagged kindlin-2.

DOI: http://dx.doi.org/10.7554/eLife.10130.025

Figure 5—figure supplement 3. Activity of signaling mediators downstream of FAK in Tln^Ctr, Tln^Ko, Tln^Ko+T1V, Kind^Ctr, Kind^Ko and Kind^Ko+K2GFP cells.

(A,B) Western blotting of indicated signaling proteins in untreated (first three lanes), EGF- and Mn²⁺-treated Tln^Ctr, Tln^Ko and Tln^Ko cells re-expressing Venus-tagged talin-1 (Tln^Ko+T1V) (A), and Kind^Ctr, Kind^Ko and Kind^Ko cells re-expressing GFP-tagged kindlin-2 (Kind^Ko+K2GFP) (B) seeded on FN. EGF, epidermal growth factor; FN, fibronectin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; K2GFP, green fluorescent protein-tagged kindlin-2; T1V, Venus-tagged full length talin-1.

DOI: http://dx.doi.org/10.7554/eLife.10130.026

Figure 5—figure supplement 4. Cell spreading of FAK overexpressing Tln^Ko and Kind^Ko cells.

(A,B) Cell spreading area of Tln^Ko (A) and Kind^Ko cells (B) seeded on PLL, FN or on FN after FAKGFP overexpression measured by image quantification (n=3; independent repeats are pooled; >100 cells/condition and repeat; resulting areas are shown as binning histograms; significances are calculated between non-transfected cells and FAKGFP expressing cells plated on FN). FAK, focal adhesion kinase; FAKGFP, green fluorescent protein-tagged FAK; FN, fibronectin; GFP, green fluorescent protein; PLL, poly-L-lysine.

DOI: http://dx.doi.org/10.7554/eLife.10130.027