Fig. 4: Micropipette tip angle influences the accumulation of F-actin at the cell neck.

a Confocal snapshots of a HEK293T WT cell aspirated by the micropipette. The transmitted channel (1^st row) was used to visualize the aspiration, while Ca²⁺ mobilization (2^nd row) and F-actin dynamics (3^rd and 4^th row, cyan) were monitored using a separate scanner unit in the FV3000 microscope, respectively. The zoom in F-actin channel demonstrated the accumulation event at the cell neck region (dash line outlined) during aspiration. The merged fluorescence image (5^th row) showed that F-actin moved to the neck and accumulated during the aspiration, and co-localized with the dividend between low signal cell body and high signal cell tongue (t = 6.7 s). b Ca²⁺ fold changes of aspirated HEK293T with different tip angles θ has a consistent trend with human RBC. The suppressed response in PIEZO1-KO HEK293T (HEK293T KO) and amplified response in PIEZO1-OE HEK293T (HEK293T OE) validated that the Ca²⁺ mobilization in HEK293T cell was PIEZO1 mediated. The number of data points (n number) is marked above the box and data are presented as box plots with medium, maxima, and minima, and analyzed by Welch’s ANOVA test. c F-actin accumulation divided the high PIEZO1 activity tongue from the body. Average intensity profile of calcium (magenta) and F-actin (cyan) along the symmetric axis was plotted.