Fig. 2: Microgeometry alters PIEZO1 mediated Ca²⁺ mobilization at the tongue of the aspirated RBC.

a–c Human RBCs ∆F_max–∆p curve under aspiration by micropipette with different geometries. Ca²⁺ fold changes relative to the resting state in the aspirated RBCs were quantified along the pressure ranges from ∆p = −5 to −40 mmHg. When the tip angle θ is fixed, larger diameters upshifted the ∆F_max–∆p curve, indicating an overall more rapid PIEZO1 activity, while larger tip angle from θ = 0° (a), 5° (b), and 10° (c) resulted a stronger upshift effect on the curve. All results were measured from n ≥ 56 cells within 3 independent experiments and presented as mean ± s.e.m. d Detailed Ca²⁺ intensity changes comparison amongst different geometries when ∆p = −40 mmHg. Although increasing tip diameter d caused an enhanced Ca²⁺ intensity change due to aspiration, larger tip angle θ had a more significant enhancement to the Ca²⁺ mobilization. e Schematic illustration of the larger tip angle θ impact on local PIEZO1 activities. We defined the cell part outside the micropipette as the body (brown circled) with its Ca²⁺ intensity, F_body, while the cell part being aspirated into the micropipette as the tongue (red circled), namely F_tongue. The ratio between F_tongue,max and F_body,max illustrates the spatial difference of the Ca²⁺ mobilization in the cell. f, g Representative normalized Ca²⁺ intensity of F_body (brown) and F_tongue (red) when the RBC was aspirated. The F_body,max and F_tongue,max are indicated by the horizontal dash line in each representative trace, respectively. h Ca²⁺ mobilization mapping is neck angle mediated, of which a higher ratio represents more Ca²⁺ influx happens at the tongue of the aspirated cell (black, θ = 10°) or a lower ratio represents Ca²⁺ influx was preferrable to happen at the cell body (light gray, θ = 0°). The number of cells (n value) analyzed in each group was indicated above the bars (d, h). Data are presented as box plots with medium, minima, and maxima and analyzed by Welch’s ANOVA. e is created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en.