Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Nov 11;8(45):eabo1461.
doi: 10.1126/sciadv.abo1461. Epub 2022 Nov 9.

Force- and cell state-dependent recruitment of Piezo1 drives focal adhesion dynamics and calcium entry

Mingxi Yao  1   2   3 , Ajay Tijore  3   4 , Delfine Cheng  5 , Jinyuan Vero Li  5 , Anushya Hariharan  3 , Boris Martinac  5 , Guy Tran Van Nhieu  6   7 , Charles D Cox  5 , Michael Sheetz  3   8   9
Affiliations

Force- and cell state-dependent recruitment of Piezo1 drives focal adhesion dynamics and calcium entry

Mingxi Yao et al. Sci Adv. .

Abstract

Mechanosensing is an integral part of many physiological processes including stem cell differentiation, fibrosis, and cancer progression. Two major mechanosensing systems-focal adhesions and mechanosensitive ion channels-can convert mechanical features of the microenvironment into biochemical signals. We report here unexpectedly that the mechanosensitive calcium-permeable channel Piezo1, previously perceived to be diffusive on plasma membranes, binds to matrix adhesions in a force-dependent manner, promoting cell spreading, adhesion dynamics, and calcium entry in normal but not in most cancer cells tested except some glioblastoma lines. A linker domain in Piezo1 is needed for binding to adhesions, and overexpression of the domain blocks Piezo1 binding to adhesions, decreasing adhesion size and cell spread area. Thus, we suggest that Piezo1 is a previously unidentified component of focal adhesions in nontransformed cells that catalyzes adhesion maturation and growth through force-dependent calcium signaling, but this function is absent in most cancer cells.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.. Piezo1 is stably recruited to maturing adhesions on fibronectin surfaces.
(A) Time lapse of cell spreading of HFF cells seeded on fibronectin-coated glass surface. The cells are transiently expressing Piezo1-mRuby3 and paxillin-BFP. Scale bars, 10 μm. (B) Localization of Piezo1 spreading on fibronectin surface during polarization and under stable condition. Scale bars, 10 μm (main figure) and 5 μm (zoom). (C) Adhesion enrichment of Piezo1 at 1, 2, and 4 hours after cell seeding. The adhesion enrichment was defined as the average intesity ratio of Piezo1 in the adhesions and background. ** denotes p-value < 0.01, *** denotes p-value < 0.001. (D) Cell-spreading area and aspect ratio 1, 2, and 4 hours after cell seeding. The P values were calculated using two-sample t test. (E) Left: Live TIRF images of HFF cells transiently transfected with Piezo1-GFP and paxillin-mapple on fibronectin-coated surfaces. The adhesion region marked by a white square was bleached, and snapshots of fluorescent recovery for Piezo1 and talin are shown in the zoom in figure on bottom. Scale bars, 5 μm. Right: The FRAP recovery curve of the bleached region (means ± SD). (F) The fitted mobile fraction and recovery rates from the FRAP measurements. More than five independent cells and 20 adhesions were picked for each condition. The bracket after the fitting value denote 95% confidence interval of the fitting parameters. In the FRAP experiment, Piezo1 is either coexpressed with Integrin β3 or paxillin.
Fig. 2.
Fig. 2.. Piezo1 localization in HFF cells is controlled by contractility.
(A) Top: Distribution of Piezo1-mRuby3 in HFF cells 15 min after treatment with 30 μM Y-compound and 30 min after washout. Experiments were repeated on >5 cells with similar results. Bottom: Representative Piezo1 localization and Piezo1 adhesion enrichment quantification in control HFF cells and HFF cells expressing constitutively active RhoA V14 mutant. Scale bars, 10 μm. ** denotes p-value < 0.01, *** denotes p-value < 0.001. (B) Time-lapse image of HFF cells transfected with Piezo1-mRuby3, integrin β3–Emerald, and IpaA-GFP with the addition of 30 μM Y-27632. Experiments were repeated on >3 cells with similar results. (C) Fluorescence image of Piezo1-GFP in COS-7 cells with/without overexpressing myosin IIA–mCherry. WT, wild type.
Fig. 3.
Fig. 3.. Piezo1 recruitment of adhesions precede integrin β3 disassembly.
(A to C) Time lapse of HFF cells cotransfected with Piezo1-mRuby3 and mEmerald–integrin β3 spreading on fibronectin surfaces. (A) A spreading cell 1 hour after seeding. (B) A polarized cell after overnight seeding. (C) Time lapse of HFF cells transfected with Piezo1-mRuby3 and mEmerald–integrin β1 spreading on fibronectin surfaces overnight. The normalized intensity profile of adhesions highlighted by the red line is shown below the figure. (D) Kymograph of Piezo1 and integrin β3 dynamics of the focal adhesion marked by red line in (B). (E) Kymograph of Piezo1 and integrin β1 dynamics of the focal adhesion marked in red line in (C).
Fig. 4.
Fig. 4.. Piezo1 localizes to the adhesions of nontransformed cells but is diffusive in transformed cells.
(A) Immunostaining of Piezo1 localization in paired normal and transformed cell lines with similar origin. (B) Quantification of Piezo1 adhesion enrichment for cells in (A). P values were calculated using two-sample t test. (C) FRAP recovery curve of Piezo1 in HFF cells and transformed HT1080 and MDA-MB-231 cells. The error bar denotes 95% confidence interval of the mean. (D) Quantification of adhesion enrichment of Piezo1 across various normal and transformed cancer cell lines. Error bars denote SEM. There were statistically significant differences (P < 0.001) between HFF/HMEC and all the cancer cells. ** denotes p-value < 0.01, *** denotes p-value < 0.001.
Fig. 5.
Fig. 5.. Piezo1 localization affects adhesion maturation and correlates with transformation states of cells.
(A) TIRF image of fixed-cell staining of nontransformed HFF cell line and transformed fibrosarcoma HT1080 with/without Piezo1 knockdown (KD) after overnight spreading on fibronectin surfaces. Scale bars, 20 μm. (B) Box plots of the cell-spreading areas, cell aspect ratio, and mean adhesion sizes of the cells in (A). Box and error bar denote the SEM and SD of the measurements. The significance value was calculated using two-sample t tests. (C) Western blots of Piezo1 level in control siRNA and piezo1 knock-down cells. ** denotes p-value < 0.01, *** denotes p-value < 0.001. NS, not significant.
Fig. 6.
Fig. 6.. Normal and transformed cells have distinct adhesion calcium signals.
(A) Domain Illustration of the novel adhesion targeting calcium sensor based on jGCaMP7s from Janelia. (B) Left: TIRF image montages of representative HFF cells (with/without Piezo1 knockdown) and HT1080 and MDA-MB-231 cells transfected with paxillin calcium sensor. The cells were seeded on fibronectin surfaces overnight before imaging. Normalized ratio represents the ratio image between jGCaMP7s and mScarlet-I, which is a measure of local Ca2+ concentrations. Right: Calcium level fluctuations over time in the tracked adhesions of the cell presented in the left panel; each line in the image denotes a single adhesion. The interval of measurement is 30 s. FA, Focal Adhesions. (C) Quantification of average calcium ratio in tracked adhesions over 60 min for different cell types. A.U., arbitrary units. ** denotes p-value < 0.01, *** denotes p-value < 0.001.
Fig. 7.
Fig. 7.. The linker domain of Piezo1 is linked to adhesion recruitment and alters cell morphology in HFF cells.
(A) Domain map of Piezo1 and the long-linker construct. The residue number is based on mouse Piezo1 structures. (B) Immunostaining of HFF and HT1080 cells transfected with mNeongreen control vector or mNeongreen-labeled Piezo1 linker construct. The cells were seeded on fibronectin surface overnight and stained with paxillin and Piezo1. (C) Box plot of cell-spreading area and cell aspect ratio of positively transfected cells. Box and error bar present SEM and SD, respectively. P values were calculated using two-sample t test. ** denotes p-value < 0.01, *** denotes p-value < 0.001, NS, not significant.
Fig. 8.
Fig. 8.. Model of Piezo1’s regulation in normal and transformed cells.
(Top) In normal cells, Piezo1 is recruited to maturing focal adhesions in a force-dependent manner, where it regulates both the growth and turnover of focal adhesions. (Bottom) In transformed cells, Piezo1’s localization is diffusive, and it no longer regulates adhesion morphology, which might contribute to the misregulated calcium signaling observed in cancer cells including stretch-dependent apoptosis (16).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Geiger B., Spatz J. P., Bershadsky A. D., Environmental sensing through focal adhesions. Nat. Rev. Mol. Cell Biol. 10, 21–33 (2009). - PubMed
    1. Vogel V., Sheetz M., Local force and geometry sensing regulate cell functions. Nat. Rev. Mol. Cell Biol. 7, 265–275 (2006). - PubMed
    1. Jain R. K., Martin J. D., Stylianopoulos T., The role of mechanical forces in tumor growth and therapy. Annu. Rev. Biomed. Eng. 16, 321–346 (2014). - PMC - PubMed
    1. Li J., Hou B., Tumova S., Muraki K., Bruns A., Ludlow M. J., Sedo A., Hyman A. J., McKeown L., Young R. S., Yuldasheva N. Y., Majeed Y., Wilson L. A., Rode B., Bailey M. A., Kim H. R., Fu Z., Carter D. A. L. L., Bilton J., Imrie H., Ajuh P., Dear T. N., Cubbon R. M., Kearney M. T., Prasad K. R., Evans P. C., Ainscough J. F. X. X., Beech D. J., Piezo1 integration of vascular architecture with physiological force. Nature 515, 279–282 (2014). - PMC - PubMed
    1. Bershadsky A. D., Balaban N. Q., Geiger B., Adhesion-dependent cell mechanosensitivity. Annu. Rev. Cell Dev. Biol. 19, 677–695 (2003). - PubMed