Hypoosmotic shock causes rapid cell swelling and surface expansion. (A and B) Wild-type cells expressing pHluorin-mCherry (SEY6210 containing pMB517) were grown in SD_-URA+1 M sorbitol, flushed into a microfluidics chamber, and shifted to a growth medium without sorbitol. Fluorescence microscopy pictures from cells before treatment and the same cells 2 min after hypoosmotic shock were used to measure changes in the maximal and minimal diameter of the ellipsoidal cells (A). The resulting calculated changes in surface area are shown in B. (C) Wild type, tcb2/3∆, ist2∆, and fps1∆ cells expressing pHluorin-mCherry (SEY6210, MTY18, MTY2, and LGY83 containing pMB517) were exposed in a microfluidics chamber to a hypoosmotic shock (1 M sorbitol > 0 M sorbitol), and the observed changes in morphology and pHluorin/mCherry fluorescence were used to determine the number of lysed cells (loss of pHluorin signal, indicating loss of PM integrity) and ruptured cells (loss of pHluorin and mCherry signal, indicating ruptured cell wall and PM). (D) Strains expressing Nce102-mCherry (AMY4, MTY81, MTY55, MTY52, MTY83, MTY47, and MTY68) were hypoosmotic-shocked in a microfluidics chamber and the cell surface change was determined based on changes of a specified diameter. For the EGTA treatment, the growth medium contained 10 mM EGTA. To deplete ATP, 5 mM NaN₃ and 5 mM NaF were added to the cells 5 min before treatment (WT -ATP). The graphs in A, B, and D are box-and-whisker plots, indicating the mean.