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Polarized focal adhesion kinase activity within a focal adhesion during cell migration

Nature Chemical Biology volume 19, pages 1458–1468 (2023)Cite this article

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Abstract

Focal adhesion kinase (FAK) relays integrin signaling from outside to inside cells and contributes to cell adhesion and motility. However, the spatiotemporal dynamics of FAK activity in single FAs is unclear due to the lack of a robust FAK reporter, which limits our understanding of these essential biological processes. Here we have engineered a genetically encoded FAK activity sensor, dubbed FAK–separation of phases-based activity reporter of kinase (SPARK), which visualizes endogenous FAK activity in living cells and vertebrates. Our work reveals temporal dynamics of FAK activity during FA turnover. Most importantly, our study unveils polarized FAK activity at the distal tip of newly formed single FAs in the leading edge of a migrating cell. By combining FAK–SPARK with DNA tension probes, we show that tensions applied to FAs precede FAK activation and that FAK activity is proportional to the strength of tension. These results suggest tension-induced polarized FAK activity in single FAs, advancing the mechanistic understanding of cell migration.

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Fig. 1: Phosphorylation-induced GFP phase separation-based FAK reporter visualizes the endogenous activity of FAK in living cells and vertebrates.
Fig. 2: FAK–SPARK detects FAK activation during cell spreading and visualizes FAK activity within single FAs after FA assembly.
Fig. 3: FAK is activated before the disassembly of FAs or during sliding and turnover.
Fig. 4: FAK activity is polarized at the distal tip of newly formed FAs in the leading edge.
Fig. 5: Polarization of FAK activity depends on FERM domain, integrin and actin cytoskeleton.
Fig. 6: FAK–SPARK droplet formation scales with integrin ligand Ttol.

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Acknowledgements

We would like to thank S. Narum for carrying out FLIM and analysis, W. Weiss for sharing cell lines, W. Degrado and D. Sheppard for sharing integrin inhibitors and antibodies and O. Weiner and D. Sheppard for constructive comments. Funding for this work was provided by NIH NIGMS R35GM131766 to X.S. and NIH NIGMS R01GM131099 and R01GM124472 to K.S.

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Authors and Affiliations

  1. Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA

    Xiaoquan Li & Xiaokun Shu

  2. Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA, USA

    Xiaoquan Li & Xiaokun Shu

  3. Department of Chemistry, Emory University, Atlanta, GA, USA

    Joseph Dale Combs III & Khalid Salaita

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Contributions

X.S. initiated the project. X.L. and X.S. designed the experiments and analyzed the data. X.L. conducted the experiments. D.C. and K.S. designed the tension experiments and analyzed the data. X.L., D.C., K.S. and X.S. wrote the paper.

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Correspondence to Xiaokun Shu.

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Extended data

Extended Data Fig. 1 FAK-SPARK forms droplets after removal of the FAK inhibitor.

Data are mean ± SEM (n = 3 biological replicates). Scale bar, 10 μm.

Source data

Extended Data Fig. 2 FAK-SPARK is applicable to various cancer cells in imaging FAK activity using a lentivirus expressing FAK-SPARK.

Data are mean ± SEM, n = 5 biological replicates, two-sided nonpaired t-test. p = 5.52 × 10−7 for MDA-MB-231, p = 0.00056 for Kelly, p = 0.0026 for MEF, p = 2.64 × 10−8 for SHEP, p = 0.0019 for SKNAS and p = 8.17 × 10−6 for U2OS). **: p value < 0.01. ***: p value < 0.001. Scale bar, 10 μm.

Source data

Extended Data Fig. 3 FAK-SPARK visualizes FAK activity with spatiotemporal resolution by targeting active FAK into specific locations.

a Fluorescence images showing that FAK-SPARK visualizes FAK activity in the centrosome when constitutively active FAK (CA-FAK) is fused to aurora A kinase that is located in the centrosome (left panels). FAK activity is absent in the centrosome without CA-FAK (right panels). b Fluorescence images showing that nucleus-located FAK-SPARK-NLS (nuclear localization signal) visualizes FAK activity in the nuclear HOTag1 condensates that are tagged with CA-FAK (left panels). FAK activity is absent the CA-FAK-absent HOTag1 condensates. a and b were repeated three times independently with similar results. (c) Cartoon showing experimental procedure of measuring FAK-SPARK temporal resolution. d Time-lapse images after addition of rapamycin. This experiment was repeated three times with similar results. e Normalized fluorescence along the dash line in (D). f Normalized SPARK signal over time. Scale bar, 5 μm (a, b, d).

Source data

Extended Data Fig. 4 FAK-SPARK expression does not perturb dynamics of focal adhesion assembly and disassembly.

a, b, Representative images of HeLa cells expressing mApple-paxillin + FAK-SPARK and mApple-paxillin + GFP, respectively. a and b were repeated for three times independently with similar results. c, d, Tracking of FAs using Focal Adhesion Analysis Server {Steenkiste:2021iv, Berginski:2013dj} in a & b. e, A typical trace of assembly and disassembly rate calculation. f, g, Assembly rate and disassembly rate in HeLa cells expressing FAK-SPARK or GFP. two-sided nonpaired t-test, p = 0.12 and 0.10 for f and g respectively. Data represent mean ± SEM (n indicates FA numbers, 305 and 298 for GFP and FAK-SAPRK in f, 331 for both GFP and FAK-SPARK in g). Data represent mean ± SEM. n.s., not significant. Scale= 10 μm.

Source data

Extended Data Fig. 5 FAK-SPARK expression does not perturb cell migration during wound healing using wound scratch assay.

Wound was induced in HeLa cells expressing FAK-SPARK using a scratcher. a, b, Representative images of HeLa cells expressing FAK-SPARK or GFP, respectively. c, d, Typical images showing wound healing right after scratching (day 0), one day (day 1) and two days (day 2) after scratching. The red line marks cell boundary. a, b, c and d were repeated three times independently with similar results. e, Quantification of wound healing, n = 3 biological replicates. two-sided nonpaired t-test, p = 0.65 and 0.46 for Day 1 and Day 2. Data represent mean ± SEM. n.s., not significant. Scale bar, 20 μm for A & B and 200 μm for c & d.

Source data

Extended Data Fig. 6 Integrin-ECM ligand tension precedes FAK activity.

a Diagram of 19 pN DNA-based MTFM probes and FAK-SPARK activation mechanism. b Representative cell RICM (gray) and fluorescence micrographs of 19 pN hairpin tension(red), IFP2-Paxillin(magenta), and FAK-SPARK(green) at t = 0.0 min. Scale bar, 5 μm. c Overlayed kymographs, yellow line in d, of tension, FAK-SPARK, and paxillin channels. Yellow arrows denote the point of tension and paxillin recruitment and FAK-SPARK droplet formation respectively. d Individual fluorescence micrographs of yellow inset in b at different timepoints, yellow dotted line denotes the linear ROI used for kymograph analysis in (c). Scale bar, 2 μm. e Plot of normalized maximum fluorescence over time derived from the kymograph, with threshold points used to derive the time delay denoted with black circles f Histogram of time delay measurements yielding 21.0 ± 9.0 min average delay between 19 pN integrin tension and FAK-SPARK droplet formation. n = 19 focal adhesions from 4 cells on 4 different surfaces (4 biological replicates).

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–24, Tables 1 and 2, Videos 1–15, References and Supplementary Fig. 9A (uncropped data of blots).

Reporting Summary

Supplementary Video 1

FAK–SPARK achieves a spatiotemporal resolution in visualizing FAK activity in living cells. HOTag1–NLS–mKO3–Frb (blue), FAK–SPARK–NLS (green) and FKBP–IFP2–CA–FAK (red). Images were taken every 30 s per frame.

Supplementary Video 2

FAK activation at the leading of a spreading cell. FAK–SPARK (green) and mApple–paxillin (red). Images were taken every 30 s per frame.

Supplementary Video 3

FAK activity in a living cell. FAK–SPARK (green) and mApple–paxillin (red). Images were taken every 30 s per frame.

Supplementary Video 4

An ROI showing FAK activity in a living cell. FAK–SPARK (green) and mApple–paxillin (red). Images were taken every 30 s per frame.

Supplementary Video 5

FAK activation during assembly of a single FA in cells. FAK–SPARK (green) and mApple–paxillin (red). Images were taken every 30 s per frame.

Supplementary Video 6

FAK activation before disassembly of FAs in cells. FAK–SPARK (green) and mApple–paxillin (red). Images were taken every 30 s per frame.

Supplementary Video 7

Disassembly of a single FA following FAK activation. FAK–SPARK (green) and mApple–paxillin (red). Images were taken every 30 s per frame.

Supplementary Video 8

FAK activation during sliding of single FAs. FAK–SPARK (green) and mApple–paxillin (red). Images were taken every 30 s per frame.

Supplementary Video 9

FAK activation during the turnover of FAs in cells. FAK–SPARK (green) and mApple–paxillin (red). Images were taken every 30 s per frame.

Supplementary Video 10

An ROI showing FAK activation during the turnover of FAs in cells. FAK–SPARK (green) and mApple–paxillin (red). Images were taken every 30 s per frame.

Supplementary Video 11

Polarized FAK activity at the distal tip of newly formed single FAs in a migrating cell. FAK–SPARK (green) and mApple–paxillin (red). Images were taken every 30 s per frame.

Supplementary Video 12

No distal polarization of FAK activity when CA–FAK is fused to paxillin and targeted to FAs in cells with endogenous FAK knocked down by shRNA against FAK. FAK–SPARK (green) and mApple–paxillin (red). Images were taken every 30 s per frame.

Supplementary Video 13

Nineteen piconewton hairpin integrin tension, IFP2–paxillin and TIRF-imaged FAK–SPARK droplets generated at regions experiencing 19 pN tension and enriched in paxillin.

Supplementary Video 14

TGT rupture (red), IFP2–paxillin (blue) and FAK–SPARK droplets (green), generated overtime on 12 pN TGTs.

Supplementary Video 15

TGT rupture (red), IFP2–paxillin (blue) and FAK–SPARK droplets (green), generated overtime on 56 pN TGTs.

Supplementary Data

Supporting data for Supplementary Figs. 1, 3, 5–9 and 22–24.

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Li, X., Combs, J.D., Salaita, K. et al. Polarized focal adhesion kinase activity within a focal adhesion during cell migration. Nat Chem Biol 19, 1458–1468 (2023). https://doi.org/10.1038/s41589-023-01353-y

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