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FAK, talin and PIPKIγ regulate endocytosed integrin activation to polarize focal adhesion assembly

Nature Cell Biology volume 18, pages 491–503 (2016)Cite this article

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Abstract

Integrin endocytic recycling is critical for cell migration, yet how recycled integrins assemble into new adhesions is unclear. By synchronizing endocytic disassembly of focal adhesions (FAs), we find that recycled integrins reassemble FAs coincident with their return to the cell surface and dependent on Rab5 and Rab11. Unexpectedly, endocytosed integrins remained in an active but unliganded state in endosomes. FAK and Src kinases co-localized with endocytosed integrin and were critical for FA reassembly by regulating integrin activation and recycling, respectively. FAK sustained the active integrin conformation by maintaining talin association with Rab11 endosomes in a type I phosphatidylinositol phosphate kinase (PIPKIγ)-dependent manner. In migrating cells, endocytosed integrins reassembled FAs polarized towards the leading edge, and this polarization required FAK. These studies identify unanticipated roles for FA proteins in maintaining endocytosed integrin in an active conformation. We propose that the conformational memory of endocytosed integrin enhances polarized reassembly of FAs to enable directional cell migration.

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Figure 1: FA reassembly requires endocytic recycling.
Figure 2: FAK kinase activity is dispensable for FA disassembly but critical for FA reassembly.
Figure 3: FAK and SFKs differentially affect integrin activation and return to the cell surface.
Figure 4: FAK maintains integrin in an active state after FA disassembly and during endocytic recycling.
Figure 5: Active integrin and FN exhibit little co-localization in endocytic compartments following FA disassembly.
Figure 6: Talin maintains activity of endocytosed integrin in a FAK-dependent manner.
Figure 7: PIPKIγi2 modulates activity of endocytosed integrin.
Figure 8: Effects of inhibiting FAK and SFK in migrating NIH3T3 fibroblasts reflect their roles in regulating integrin activation and recycling.

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Acknowledgements

We thank G. DiPaolo and E. Marcantonio for suggestions and R. Horwitz (Allen Institute for Cell Science, USA), J. Goldenring (Vanderbilt University, USA), D. Calderwood (Yale University, USA), D. Schlaepfer (University of California, San Diego, USA) and R. Anderson (University of Wisconsin-Madison, USA) for reagents. This work was supported by NIH grants GM062939 and GM099481 to G.G.G.

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  1. Department of Pathology & Cell Biology, Columbia University, P&S 15-420, 630 West 168th Street, New York, New York 10032, USA

    Guilherme P. F. Nader, Ellen J. Ezratty & Gregg G. Gundersen

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Contributions

G.P.F.N. conducted and designed experiments, analysed and interpreted the data and wrote the manuscript. E.J.E. conducted initial experiments, interpreted the data and edited the manuscript. G.G.G. designed the experiments, analysed and interpreted the data and edited the manuscript.

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Correspondence to Gregg G. Gundersen.

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Integrated supplementary information

Supplementary Figure 1 FAs reassembled after NZ washout contain typical FA markers and are biased towards the cell periphery.

(a) Immunofluorescence images of FAK-pY397, paxillin and phalloidin staining (actin) of NIH3T3 fibroblasts after NZ treatment and washout for the indicated times. Bar, 10 μm. (b) Quantification of cell area before and after MT-induced FA disassembly. n = 25 cells pooled from 3 independent experiments. Box and whisker plots show median values by horizontal lines, 75% of values by boxes and 95% of the range by whiskers. (c, left panels) Immunofluorescence of paxillin-EGFP NIH3T3 fibroblast cell line showing a similar distribution of paxillin-EGFP and endogenous FAK-pY397. Bar, 15 μm. (c, right panels) Immunofluorescence of NIH3T3 fibroblasts stained for paxillin and vinculin showing that FAs are similar in size, number and distribution compared to the FAs in paxillin-EGFP NIH3T3 fibroblasts. Bar, 15 μm. (d) Representative immunoblots of paxillin levels in paxillin-EGFP NIH3T3 fibroblasts; 2 independent experiments. Note that paxillin-EGFP is expressed at levels comparable to endogenous paxillin. (e) Representative immunoblots of paxillin-pY118 and total paxillin in NIH3T3 fibroblast lysates after NZ washout in the presence of DMSO, 10 μM PF228 or 10 μM PP2. Tubulin, loading control; data represent 1 out of 2 independent experiments. (f) Quantification of cell migration velocity for parental NIH3T3 fibroblasts and paxillin-EGFP NIH3T3 fibroblasts. DIC images were taken at 5 min intervals for 12 h; n = 35 cells pooled from 3 independent experiments. (g) TIRF images from a movie of paxillin-EGFP NIH3T3 fibroblasts at 0, 60 and 100 min after NZ washout. Bar, 5 μm. (h) FA reassembly occurs preferentially at the cell periphery. Left: diagram illustrating the regions used to quantify the distribution of reassembled FAs: FAs located within 2 μm of the cell periphery were classified as peripheral; those outside the 2 μm range were classified as internal. Middle and right: quantification of peripheral FA area and number as defined in the diagram; n = 10 cells pooled from 2 independent experiments. Error bars (f,h) are mean ± SD. Student’s two-tailed unpaired t-test; P values are indicated; ns, not significant. See Supplementary Figure 9 for uncropped images of blots.

Supplementary Figure 2 FA reassembly involves endocytic recycling.

(a) Recycling of biotinylated integrin following FA disassembly. NIH3T3 fibroblasts were treated with NZ for 3 h and then surface labeled with biotin at 4 °C. NZ was washed out and biotinylated proteins were allowed to internalize for 60 min. Biotin was removed from receptors remaining at the cell surface by reduction with MesNa at 4 °C. Cells were then rewarmed to 37 °C for the times indicated followed by a second reduction. Cells were lysed and integrin-biotinylation was determined by capture-ELISA using microtiter wells coated with α5 integrin antibody; data represent the mean of n = 6 independent experiments. [ok?](b) Representative immunoblots of α5 integrin in NIH3T3 fibroblasts lysates after NZ washout. Vinculin, loading control; data represent 1 out of 3 independent experiments. (c) Immunofluorescence images of α5 integrin-GFP, Rab11 and GM-130 (Golgi) in NIH3T3 fibroblasts expressing α5 integrin-GFP and fixed at 60 min of NZ washout. Arrows indicate the Rab11 ERC; arrowhead indicates the Golgi. Note colocalization of α5 integrin-GFP with the Rab11 ERC, but not the Golgi. Bar, 5 μm. (d) Immunofluorescence images of paxillin-pY118, GM-130 (Golgi) and MTs in NIH3T3 fibroblasts after NZ washout for the indicated times in the presence of either 4 μg/ml brefeldin A or 0.1% ethanol (vehicle). Bar, 15 μm. (e) Immunofluorescence images of vinculin and Rab11 in NIH3T3 fibroblasts expressing Rab11 WT-EGFP or Rab11 S25N-EGFP fixed at 120 min of NZ washout. Arrows indicate transfected cells. Note reassembly of FAs in cells expressing Rab11 WT but not dominant negative Rab11 S25N. Bar, 20 μm. (f) Immunofluorescence image of zyxin and phalloidin actin staining of a Rab5 S34N-EGFP-transfected NIH3T3 fibroblast (arrow) 120 min after NZ washout. Bar, 15 μm. (g) Quantification of FA disassembly and reassembly in NIH3T3 fibroblasts transfected with the indicated EGFP-tagged Rab constructs; n = 3 independent experiments; 150 cells analysed per condition in each experiment. (h) Representative immunoblots of Rab11a/b in NIH3T3 fibroblasts treated with non-coding (NC) or Rab11a/b siRNAs. Tubulin, loading control. Data represent 1 out of 3 independent experiments. Error bars are (a) SEM and (g) mean ± SD. Student’s two-tailed unpaired t-test; P values are indicated. See Supplementary Figure 9 for uncropped images of blots.

Supplementary Figure 3 FAK and SFK inhibitors inhibit FA reassembly and do so in a reversible fashion.

(a) Immunofluorescence images of FAK, vinculin and MTs in NIH3T3 fibroblasts at the indicated times after NZ washout in the presence of vehicle (DMSO) or PF228 (1 or 5 μM). Note that even low concentrations of PF228 still inhibit focal adhesion reassembly without affecting focal adhesion disassembly. Bar, 10 μm. (b) Immunofluorescence images of vinculin, paxillin and MTs in NIH3T3 fibroblasts at indicated times after NZ washout in the presence of DMSO or 2 μM FAK inhibitor-I. Note that FAK inhibitor-I does not affect FA disassembly (60 min NZ washout) but prevents reassembly of FAs (120 min NZ washout). Bar, 15 μm. (c) Immunofluorescence images of vinculin and MTs in NIH3T3 after NZ washout in the presence of DMSO or 10 μM PF228 or 10 μM PP2. After 120 min of NZ washout, kinase inhibitors were washed out and cells were fixed at the indicated times of “release”. Bar, 15 μm (d) Quantification of FA reassembly following drug release of cells treated as in “c”; n = 3 independent experiments; 200 cells analysed per condition in each experiment. Error bars are mean ± SD; ns, not significant. (e) Immunofluorescence images of src and Rab11-EGFP in NIH3T3 fibroblasts expressing Rab11-EGFP fixed at 60 min after NZ washout. Arrows indicate src colocalizing with Rab11 in the ERC. Bar, 15 μm.

Supplementary Figure 4 FA reassembly is rescued by Mn2+ in FAK−/− cells transfected with FAK kinase mutants but not in Rab11-depleted cells.

(a) Immunofluorescence images of vinculin in NIH3T3 fibroblasts treated with non-coding (NC) or Rab11a/b siRNAs and fixed at 120 min after NZ washout. MnCl2 (100 μM) was added 90 min after NZ washout. Bar, 15 μm. (b) Quantification of FA reassembly of cells treated as in “a”; n = 3 independent experiments; 150 cells analysed per condition in each experiment. (c) Immunofluorescence images of FAK (WT or different mutants) and vinculin in FAK-/- cells expressing FAK-WT-GFP, FAK-Y397F-GFP or FAK-K454R-HA and fixed at the indicated times after NZ washout. MnCl2 (100 μM) was added to the cells 60 min after NZ washout. Arrows indicate transfected cells. Bar, 15 μm. (d) Quantification of FA disassembly and reassembly in transfected FAK-/- cells at the indicated times after NZ washout as in “c”; n = 3 independent experiments; 100 cells analysed per condition in each experiment. Error bars are mean ± SD. Student’s two-tailed unpaired t-test; P values are indicated; ns, not significant.

Supplementary Figure 5 FAK inhibitor blocks FA reassembly without interfering with FA disassembly in HT1080 fibrosarcoma cells.

(a) Immunofluorescence images of vinculin, paxillin-pY118 and MTs in HT1080 fibrosarcoma cells fixed at the indicated times after NZ washout in the presence of DMSO or 10 μM PF228. Bar, 25 μm. (b) Quantification of FA disassembly (60 min NZ washout) and reassembly (120 min NZ washout) in HT1080 cells treated with DMSO or PF228 as in “a”. n = 3 independent experiments, 200 cells analysed per condition in each experiment. Bars are SD. (c) Immunofluorescence images of active β1 integrin (12G10 antibody) and FAK-pY397 in HT1080 cells fixed 60 min after NZ washout. Arrows indicate colocalization of active integrin and FAK-pY397 signals. Bar, 10 μm. (d) Immunofluorescence images of FAK-pY397 in HT1080 cells fixed 60 min after NZ washout in the presence of DMSO or 80 μM dynasore. Bar, 15 μm. (e) Immunofluorescence images of FAK-pY397 and Rab11 in HT1080 cells fixed 60 min after NZ washout in the presence of DMSO or 80 μM dynasore. Bar, 15 μm. (f) Quantification of FA disassembly in HT1080 cells fixed 60 min after NZ washout in the presence of DMSO or 80 μM dynasore; n = 60 cells analysed per condition, pooled from 2 independent experiments. Box and whisker plots represent values as defined in Supplementary Fig. 1b legend. Student’s two-tailed unpaired t-test; P values are indicated; ns, not significant.

Supplementary Figure 6 An antibody to a second epitope on active β1 integrin detects endocytosed integrin in HT1080 fibrosarcoma cells and SFKs are not critical for maintaining integrin activation in the ERC.

(a) Immunofluorescence images of active β1 integrin (HUTS-4 antibody), total β1 integrin (K20 antibody) and Rab11 in HT1080 cells fixed at 60 min after NZ washout. Boxed regions are enlarged in the images on the right. Bar, 10 μm. (b) Immunofluorescence images of active β1 integrin (12G10 antibody), total β1 integrin (K20 antibody) and Rab11 in HT1080 cells fixed at 60 min after NZ washout in the presence of DMSO or 10 μM PP2. Boxed regions are enlarged in the images on the right. Bar, 15 μm. (c) Quantification of fluorescence intensity of active β1 integrin measured in the Rab11 ERC of HT1080 cells 60 min after NZ washout in DMSO or 10 μM PP2. n = 3 independent experiments, 30 cells analysed per condition in each experiment. Box and whisker plots represent values as defined in Supplementary Fig. 1b legend; ns, not significant.

Supplementary Figure 7 Talin is required for FA reassembly in NIH3T3 fibroblasts and localization of PIPKIγi1 in HT1080 cells.

(a) Representative immunoblots of lysates from NIH3T3 fibroblasts treated with non-coding (NC) or different talin1 siRNAs. Tubulin is a loading control. Data represent 1 out or 3 independent experiments. (b) Immunofluorescence images of paxillin-pY31 and MTs in NIH3T3 fibroblasts transfected with NC or talin1 siRNA and fixed at the indicated times after NZ washout. Bar, 15 μm. (c) Quantification of FA disassembly and reassembly after NZ washout from NIH3T3 fibroblasts treated with non-coding (NC) or talin siRNAs. n = 4 independent experiments, 150 cells analysed per condition in each experiment. Error bars are mean ± SD. (d) Immunofluorescence images of talin and Rab11 in NIH3T3 fibroblasts 60 min after NZ washout. Bar, 15 μm. (e,f) Immunofluorescence images of PIPKIγi1-GFP and vinculin in HT1080 cells before (e) and after (f) NZ washout. Cells were transduced with PIPKIγi1-GFP using retrovirus. Note that PIPKIγi1 does not localize to FAs before NZ washout or to the Rab11 ERC after NZ washout. Bar, 20 μm. Student’s two-tailed unpaired t-test P values are provided. See Supplementary Figure 9 for uncropped images of blots.

Supplementary Figure 8 PIPKIγ is required for FA reassembly and migrating cells accumulate active integrin in the Rab11 ERC in a FAK-dependent manner.

(a) Immunofluorescence images of paxillin-pY31 and MTs in HT1080 cells treated with a noncoding (NC) or a smart pool mixture of PIPKIγ siRNAs and fixed at the indicated times after NZ washout. Bar, 15 μm. (b) Quantification of FA disassembly and reassembly after NZ washout in HT1080 cells. n = 5 independent experiments; 150 cells analysed per condition in each experiment. (c) Representative immunoblots of lysates from HT1080 cells treated with a noncoding (NC) or a smart pool mixture of PIPKIγ siRNAs. Tubulin, loading control. Data represent 1 out of 3 independent experiments. (d) Immunofluorescence images of paxillin in NIH3T3 fibroblasts seeded on FN-coated cover slips for 4 h at low confluency and then allowed to migrate after treatment with vehicle (DMSO) or FAK inhibitor (PF228) for the indicated times. Two examples for each condition are shown. Note reduced FAs and lack of new focal adhesion in cells treated with PF228. Bar, 15 μm. (e) Immunofluorescence images of active β1 integrin, total β1 integrin and Rab11 in wounded monolayers of HT1080 cells treated with SFKs inhibitor alone (PP2 2h) or treated with PP2 for 2 h followed by addition of FAK inhibitor for another 2 h (PP2 2h + PF228 2h). Arrows point to active β1 integrin accumulation in the Rab11 ERC upon treatment with PP2. Note lack of active integrin staining in the Rab11 ERC after treatment with both PP2 and PF228. Bar, 20 μm. (f) Quantification of active β1 integrin signal (top) or total β1 integrin signal (bottom) in the Rab11 ERC of cells located at wounded monolayers; n = 60 cells analysed per condition, pooled from 2 independent experiments,. Box and whisker plots (f) represent values as defined in Supplementary Fig. 1b legend. Error bars (b) are mean ± SD. Student’s two-tailed unpaired t-test; P values are indicated; ns, not significant. See Supplementary Figure 9 for uncropped images of blots.

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Time-lapse movie showing FA disassembly and reassembly.

NIH3T3 fibroblasts stably expressing paxillin-EGFP were imaged by TIRF microscopy after NZ treatment for 3 h followed by NZ washout to stimulate FA disassembly and reassembly. Images were captured every 40 sec. (AVI 62450 kb)

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Nader, G., Ezratty, E. & Gundersen, G. FAK, talin and PIPKIγ regulate endocytosed integrin activation to polarize focal adhesion assembly. Nat Cell Biol 18, 491–503 (2016). https://doi.org/10.1038/ncb3333

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