Fig 2: N-glycosylation of Piezo1 in a split construct.a Diagram depicting the human split Piezo1 protein. b Representative blot showing the split human Piezo1 protein and the split human Piezo1 protein with the double cap mutant N2294Q and N2331Q with and without PNGaseF treatment. c Diagram illustrating where glycans are likely to be located. d Electrophysiological recordings of Piezo1−/− HEK293T cells expressing human split Piezo1 and the double cap mutant N2294Q and N2331Q in the cell-attached configuration in response to negative pressure applied using a high-speed pressure-clamp (red). e Quantification of peak current elicited per patch. f Representative blot showing the C-terminal portion of the split human Piezo1 protein and the mutants N2294Q, N2331Q and N2294Q/N2331Q. Note that the single mutants run at the same size but the double mutant is smaller. *p < 0.05 determined by Mann–Whitney U-test. −ve represents an un-transfected control.

Fig 3: 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).

Fig 4: 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 5: N-linked and glycosylation of Piezo1 in a heterologous expression system and in native cell types.a A representative western blot of hPiezo1-1591-GFP and Piezo1-C-GFP expressed in HEK293 cells. b A representative western blot illustrating cleavage of the upper FG (fully glycosylated) band of the Piezo1 doublet in the presence of the enzyme PNGaseF and a mix of O-linked and N-linked deglycosylase (CG—core-glycosylated, UG—unglycosylated). c Schematic illustration of PNGaseF mediated cleavage of N-linked glycans at the molecular level. d Representative western blot showing the effect of PNGaseF digestion on Kv11.1-HA and e Kv11.1FLAG protein expressed in HEK293 cells. The red arrow denotes a previously identified PNGaseF resistant Kv11.1 component. f Representative western blot comparing the effect of PNGaseF digestion of mouse Piezo1-TdTomato from red blood cells (RBCs) and lung tissue. g Comparison of western blot of human Piezo1-1591-mCherry expressed in HEK293 cells with mouse Piezo1-TdTomato from mouse lung tissue (different exposure denoted by dashed white line). h Reduced intensity, of representative blot shown in g to illustrate the reduction in size of the lower CG band in addition to the loss of the upper FG band. i Schematic representation of where N-glycans are added and the site of action of the fungal metabolite brefeldin A. j Representative western blot of brefeldin A treatment (0–16 h) on human Piezo1-GFP expressed in HEK293 cells. k Raw electrophysiological traces from cell-attached patches of brefeldin A treated Piezo1−/− HEK293T expressing Piezo1-GFP recorded at −65 mV. l Quantification of peak Piezo1 currents per patch elicited by negative pressure pulses in the presence of brefeldin A for escalating durations. Data expressed as mean ± SEM *p < 0.05 determined by Kruskal–Wallis test with Dunn’s post-hoc test.