Piezo1 deficiency abrogates integrin-dependent motility in human T cells which is mediated through redistribution of Piezo1 at the leading edge in response to chemokine stimulation.

A. MSD versus time calculated for GFP+ (potential Piezo1-knockdown cells) and GFP- (potential control cells) in GFP plasmid and Piezo1 siRNA co-transfected human CD4+ T lymphocytes, that were allowed to migrate in presence of recombinant CCL19 on ICAM1-coated dishes. B. Representative tracks of GFP+ and GFP- CD4+ T lymphocytes. Comparisons of C. % GFP+ cells after 72 hours of nucleofection D & E. 3D transwell migration assay of siRNA-transfected primary human CD4+ T lymphocytes (D) and Jurkat T cells (E), respectively. Data representative of at least 3 independent experiments. F. Representative confocal images of Piezo1 distribution in fixed untreated and CCL19-treated CD4+ T lymphocytes. 63X oil magnification. G. Comparison between Piezo1 polarity index calculated for fixed, stained, human CD4+ T lymphocytes with or without 0.5µg/ml CCL19 treatment for 20-30 minutes. N > 510 random cells, each. H. Piezo1 polarity index calculated for Jurkat cells, expressing mCherry-tagged Piezo1 during live-cell tracking in the presence of recombinant SDF1α. Each dot represents polarity index of each cell, averaged over all the time-frames. I. Representative time kinetics of particle image velocimetry (PIV) analysis of Piezo1-mcherry transfected Jurkat cells, allowed to move on ICAM-coated dishes in the presence of recombinant SDF1α. Top panel: No chemokine. Bottom Panel: SDF1α.

Piezo1 redistribution in migrating T cells follow increased membrane tension in the leading edge A.

Schematic illustration of interference reflection microscopy imaging setup. B. Probability distribution of amplitude of temporal fluctuations (SDtime, left) and tension (right) before and after addition of chemokines. C. Temporal trajectory of tension of cells before and after addition of chemokine with ** indicating significant difference from control. D. Representative IRM images of Jurkat cells (top), corresponding tension maps (middle) and R2 maps (bottom), before and after chemokine treatment. E Correlation between normalized Piezo intensity, tension magnitudes with time. F. Scatter plot to show correlation between normalized pixel count of Piezo1 and normalized tension. Piezo1 intensity at specific time-points were correlated with tension magnitudes at the preceding timepoints. Normalization has been done such that the maximum value is set to 1 and others are accordingly scaled. G. Colour-coded Piezo1-GFP intensity map (left), epifluorescence image (middle), and tension map (right) of representative Piezo1-GFP expressing Jurkat cell, after 30 minutes of 0.1µg/ml of SDF1α treatment.

Focal adhesion following chemokine receptor activation does not depend on Piezo1

A. Representative confocal images of human CD4+ T lymphocytes, fixed and stained for phospho-FAK (pFAK) under untreated and CCL19-treated conditions. B. Increased polarity of pFAK upon chemokine stimulation of CD4+ T lymphocytes as compared to unstimulated controls. N > 120 cells, each. C. Representative confocal images of pFAK distribution in control and Piezo1 siRNA-transfected CD4+ T lymphocytes stimulated with chemokine. D. Comparison of pFAK polarity in CCL19-stimulated control and Piezo1-knockdown CD4+ T lymphocytes. n > 450 random cells, each. E. Representative confocal images of paxillin distribution in untreated versus CCL19-stimulated CD4+ T lymphocytes. F. Increased paxillin polarity in response to CCL19 stimulation as compared to untreated CD4+ T lymphocytes. Untreated n > 70, CCL19-treated n > 150. G. Representative confocal images of immunostained paxillin in chemokine-stimulated, control and Piezo1-knockdown CD4+ T lymphocytes. H. Comparison of paxillin polarity in CCL19-stimulated control and Piezo1-knockdown CD4+ T lymphocytes. n > 550 random cells, each. I. Representative confocal images of stained Piezo1 in CD4+ T cells stimulated with CCL19 in the presence or absence of FAK inhibitor 14. J. Effect of FAK inhibition on Piezo1 polarity in CD4+ T lymphocytes stimulated with recombinant CCL19 versus untreated cells. n > 600 random cells, each. Data represented from at least 3 independent experiments.

Membrane recruitment of LFA1 on chemokine receptor activation disrupts with Piezo1 deficiency.

A. Representative confocal images of fixed, immunostained Piezo1 and LFA1 in unstimulated and CCL19-stimulated CD4+ T lymphocytes. B. Increased LFA1 polarity in response to recombinant CCL19 stimulation in CD4+ T cells. n > 295 random cells, each. C. Manders’ co-localisation analysis of Piezo1 and LFA1 in untreated and CCL19-treated CD4+ T lymphocytes. D. Comparison of LFA1 polarity in chemokine-treated control and Piezo1 knockdown CD4+ T lymphocytes. E. Representative confocal stained images of LFA1 polarity of CCL19-treated control and Piezo1-knockdown CD4+ T lymphocytes. n > 490 random cells, each. F. Representative confocal images of phospho-Akt (pAkt) and Akt distribution upon chemokine treatment of control and Piezo1-knockdown cells. G & H. Quantitative analyses of pAkt (M) and Akt (N) polarity in control and Piezo1-knockdown cells after CCL19-treatment. n > 580 random cells, each. Data represented from at least 3 independent experiments.

Local Ca2+ mobilization with Piezo1 redistribution upon chemokine stimulation.

A. Representative TIRF image of a Jurkat Cell stained with Fluo3AM and transiently expressing with Piezo1 mCherry. Lower panel: zoomed in image and overlap of binary images of objects detected from Calcium and Piezo channels as well as simulated randomly placed punctas having similar total (in the zoomed in section) and average area as that of the Calcium punctas detected in the real images. Overlap RGB image shows the overlap between Real Calcium (Red) and piezo puncta (Green), overlap puncta (Yellow), simulated calcium puncta (blue). B. Comparison between percentage calcium colocalization of simulated data and real data. * Denote p value < 0.05 calculated using Mann Whitney U test. N cell= 52 (13 cells x 4 time points). NROIS= 52. C. Representative confocal images of LFA1 and actin distribution in CCL19-stimulated CD4+ T lymphocytes with or without inhibition of calpain. 100μM of PD15606 was added to the cells 1 hour prior to addition of chemokine. Quantitative comparisons of D. LFA1 and E. actin polarity upon chemokine stimulation, in the presence or absence of calpain pre-inhibition. N > 290 random cells, each. Data represented is of at least 3 independent experiments.

Piezo1 deficiency disrupts F-actin retrograde flow in T cells despite chemokine receptor activation.

A. Representative fixed confocal images of actin and Piezo1 distribution in CD4+ T lymphocytes after 30 mins of 0.5µg/ml recombinant CCL19 treatment. B. Quantitative comparison of actin polarity in control and Piezo1-knockdown CD4+ T lymphocytes cells after 30 mins of chemokine treatment. n > 300 random cells, each. C. Representative fixed confocal images of actin distribution in chemokine-treated, control and Piezo1-knockdown CD4+ T lymphocytes. Front-back (F/B) of Piezo1 and actin-GFP in SDF1α treated (D) and untreated (E) Jurkat cells co-expressing actin-GFP and Piezo1-mCherry. F. A snapshot of time-lapse imaging of Piezo1-mCherry and actin-GFP expressing Jurkat cell treated with 0.1µg/ml of SDF1α. Image is maximum Z-projection of the cell at 63X/1.40 magnification. Data represented is of at least 3 independent experiments.

The mechanistic model depicting involvement of Piezo1 mechanosensing in leading edge events in a migrating T cell.

Proposed model suggests chemokine receptor activation in human T cells lead to focal adhesion kinase activation and focal adhesion formation. Focal adhesions lead to localized increase in membrane tension at the leading edge plasma membrane which leads to Piezo1 recruitment and activation. Piezo1 activation leads to calpain activation which potentially drives further cytoskeletal consolidation to recruit integrin LFA1. LFA1 recruitment and activation lead to phosphorylation of AKT and downstream signaling eventually driving the retrograde actin flow in migrating human T cells.