Integrin adhesion and force coupling are independently regulated by localized PtdIns(4,5)2 synthesis

Abstract

The 90-kDa isoform of the lipid kinase PIP kinase Type I γ (PIPKIγ) localizes to focal adhesions (FAs), where it provides a local source of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). Although PtdIns(4,5)P(2) regulates the function of several FA-associated molecules, the role of the FA-specific pool of PtdIns(4,5)P(2) is not known. We report that the genetic ablation of PIPKIγ specifically from FAs results in defective integrin-mediated adhesion and force coupling. Adhesion defects in cells deficient in FAPtdIns(4,5)P(2) synthesis are corrected within minutes while integrin-actin force coupling remains defective over a longer period. Talin and vinculin, but not kindlin, are less efficiently recruited to new adhesions in these cells. These data demonstrate that the specific depletion of PtdIns(4,5)P(2) from FAs temporally separates integrin-ligand binding from integrin-actin force coupling by regulating talin and vinculin recruitment. Furthermore, it suggests that force coupling relies heavily on locally generated PtdIns(4,5)P(2) rather than bulk membrane PtdIns(4,5)P(2).

Figure 1

PIPKIγ_i2 is the primary PtdIns(4,5)P₂-generating enzyme at FAs. (A) Western blot analysis of fibroblast protein lysates from WT and ΔE17 cells. In all, 50 μg of lysate was loaded to detect PIPKIγ_i2, 20 μg of lysate was loaded to detect PIPKIγ and talin, and 5 μg of lysate was loaded to detect actin. (B) Western blot analysis of cytoskeletal extract (CSK) from WT cells, and enriched FA fractions from WT and ΔE17 cells. (C) Kinase assays using equal protein amounts of CSK from WT cells, and enriched FA fractions from WT and ΔE17 cells, analysed by thin layer chromatography. The migration positions of PtdIns(4,5)P₂ (PIP₂) and an unidentified lipid contaminant from the FA preparation (asterisk) are identified. Ori, origin. Figure source data can be found in Supplementary data.

Figure 2

Talin and vinculin recruitment to new FAs is impaired in PIPKIγΔE17 fibroblasts. (A) TIRF time-lapse images were collected from the leading edge of GFP–talin-expressing WT or ΔE17 fibroblasts. The incorporation of talin into new adhesions (arrows) was monitored by quantifying the rate of increase in GFP epifluorescence. Heatmaps of pixel intensities from a representative example are shown. (B–E) Rate constants of incorporation were determined from the linear phase of GFP epifluorescence increase in cells expressing GFP-tagged (B) talin (n=11 WT, 9 ΔE17), (C) vinculin (n=7 WT, 7 ΔE17), (D) kindlin2 (n=6 WT, 7 ΔE17) and (E) paxillin (n=6 WT, 6 ΔE17). (F) Talin incorporation rate was calculated for ΔE17 cells expressing GFP (vector; 4 cells), kinase-dead GFP–PIPKIγ_i2 (PIPKIγ KD; 7 cells) or wild-type GFP–PIPKIγ_i2 (PIPKIγ; 6 cells). (G) The incorporation rates of the talin ‘unclasping’ mutants K318A/K320A (KK → AA) and E1770K were calculated in WT cells (n=6 KK → AA, 8 E1770K) and ΔE17 cells (n=9 KK → AA, 6 E1770K). (H) The incorporation rate of talinK274E was compared with the incorporation rate of WT talin in WT (n=6 WT, 5 K274E) and ΔE17 cells (n=5 WT, 5 K274E). All data are mean±s.e.m. Mann–Whitney tests were used to establish statistical significance in (B–H); NS=not significant. Scale bar in (A)=1 μm.

Figure 3

Initial adhesion is reduced in PIPKIγΔE17 fibroblasts but rate of bond accumulation is unchanged. (A) Plate-and-wash assay performed with WT (filled circles) and ΔE17 fibroblasts (open circles) plated onto 2 μg/ml FN-coated plastic in the presence of 1 mM Mg²⁺. Experiments including 100 μM cilengitide are depicted by triangles. Data are mean±s.e.m. for three independent experiments. Exponential rise-to-max best-fit lines were used to determine kinetic parameters. (B) Plate-and-wash assay as in (A) but performed in the presence of 1 mM Mn²⁺. (C) Cells were attached to a tipless cantilever and brought into contact with a 2 μg/ml FN-coated surface for 30 s. To probe for the contribution of α5β1 and αvβ3 to adhesion, an α5β1 function blocking antibody or 100 nM cilengitide was used, respectively. Data are mean±s.e.m. of 10 cells per condition. (D, E) A spinning disc assay was used to analyse relative numbers of adhesive bonds. (D) Typical data obtained from this assay following a 30-min incubation and 5-min spin. Shear stress increases linearly as a function of the radial distance from the centre (depicted by the diagonal line). The profile of cell detachment follows a sigmoid curve (WT cells=open circles; ΔE17 cells=open squares). The τ50 value, or the mean shear stress required for cell detachment, is shown. (E) Spinning disc data for WT cells (closed circles) and ΔE17 cells (open circles) plated onto 3 μg/ml FN-coated coverslips for the indicated time periods. Data are mean±s.e.m. for three independent experiments, conducted in duplicate. Exponential rise to max curves were used to determine kinetic parameters. ^*P<0.05; ^**P<0.005; ^***P<0.0005.

Figure 4

FA formation and cell spreading are delayed in the absence of local PtdIns(4,5)P₂ synthesis. (A) Immunofluorescence images of WT and ΔE17 fibroblasts expressing GFP–talin (green) and immunostained for paxillin (red) after 20, 40 or 60 min of spreading on 5 μg/ml FN-coated glass coverslips. Boxed areas in the upper panels are magnified in the lower panels. (B) Quantification of the cell area occupied by FAs in 25 cells of each genotype. Values are mean±s.d. A Mann–Whitney test was used to establish statistical significance. (C) FA lengths measured from cells immunostained for paxillin were measured and binned into 0.5 μm increments to obtain a comparative distribution of FA length in WT (black bars, 1128 FAs from 10 cells) and ΔE17 (grey bars 1006 FAs from 10 cells) cells. (D) Representative bright field images of WT and ΔE17 fibroblasts plated onto plastic dishes coated with the indicated concentrations of FN for 1 h, or overnight in the absence of serum. (E) Temporal analysis of spreading behaviour for the cells plated on 2 μg/ml fibronectin. Squares=wild type; triangles=ΔE17. Values are mean±s.e.m. for 15 cells of each genotype.

Figure 5

Integrin bond tensioning is reduced in the absence of localized PtdIns(4,5)P₂ synthesis. (A) Cells were plated onto 5 μg/ml FN-coated plastic for the incubated times and integrins were crosslinked with the extracellular crosslinker DTSSP. The amount of crosslinked β1-integrin from WT (closed circles) and ΔE17 cells (open circles) was quantified from western blots and best-fit curves were fitted to a first order exponential function as described in Materials and methods. Data are mean±s.e.m. for three experiments. (B, C) Contraction of collagen gels was used to assess force transduction to the extracellular environment. Plugs of a collagen and FN mixture containing 3 × 10⁵ cells were photographed at defined time intervals. (B) A representative experiment and (C) the mean±s.e.m. of four independent experiments are shown. Exponential decay curves were used to determine kinetic parameters. (D) Strain energy heatmaps of wild-type (WT) and ΔE17 cells plated onto FN-coated polyacrylamide supports (E=12.8 kPa). Bright field and traction field images are overlaid to show the position of the cell. (E) The product of the strain energy normalized for cell area is presented. (F) Cells were plated for 6 h or kept in suspension for 1 h and myosin light chain (MLC) phosphorylation was assessed by glycerol-urea PAGE. The migration positions of MLC and phospho-MLC (pMLC) are indicated. Figure source data can be found in Supplementary data.

Figure 6

Enhanced phase contrast and fluorescence microscopy reveals an adhesion-actin coupling defect in PIPKIγΔE17 cells. (A) A single frame from a time-lapse series of WT cells. The black line marks the region from where a kymograph was generated. (B) The kymograph obtained from the wild-type cell depicted in (A). Red lines are examples of regions used to measure the distance and angle of the dark bands corresponding to retrograde actin flow. (C) A single frame from a time-lapse series of ΔE17 cells, showing the region from which the kymograph was derived (black line). (D) The kymograph obtained from the cell depicted in (C). Red lines are example regions used to calculate the length and angle of bands resulting from retrograde actin flow. (E, F) Lengths and angles of 50 actin flow lines from 9 cells of each genotype were used to calculate the rate of retrograde actin flow (E) and depth of penetration of the fast actin flow (defined as lamellipodial width) (F). Plots are mean±s.d. (G, H) GFP–talin-expressing WT (G) and ΔE17 cells (H) were immunostained for cortactin (blue) and localization of talin (green) relative to the lamellipodium was examined. Arrowheads denote adhesions that terminate at the lamella–lamellipodium border. Arrows in (H) highlight regions where the GFP–talin signal overlaps with the lamellipodium in ΔE17 cells.

Figure 7

Migration analysis of wild-type and ΔE17 cells. (A) Calculated migration speeds from the closure of a wounded cell monolayer (Scratch; mean±s.d. from four scratches for each genotype) or the manual tracking of sparsely seeded cells (mean±s.d. of 70 cells for each genotype). (B) Individual tracks of all cells from the manual tracking assay, plotted using the chemotaxis and migration plugin for ImageJ (Ibidi, Martinsried, DE). (C) Migration towards an EGF or PDGF gradient using a modified Boyden chamber transwell assay, using the indicated concentrations of EGF or 25 ng/ml PDGF. The migration index is the fold increase of migration across the filter in response to growth factor compared with 1% BSA. Data are mean±s.e.m. of three experiments.