Hypoosmotic-induced spheroplast expansion results in cER loss. (A) Spheroplasted wild-type cells expressing ER-pHluorin and Nce102-mCherry (AMY4 containing MRV66) were flushed into a microfluidics chamber in the presence of 0.4 M sorbitol and then switched to a medium containing 0.1 M sorbitol. The arrow indicates an enlarged eisosome that is commonly observed in spheroplasts. (B) Analysis of time-lapse imaging of spheroplast swelling (AMY4 containing MRV66). The corresponding movie can be found in Video 1 (video is 3× sped up). (C) Quantification of the surface increase observed with hypoosmotic treated wild-type and mutant spheroplasts (AMY4, MTY52, and MTY47 containing MRV66). (D) Quantification of the number of cells that lysed (loss of PM integrity) during hypoosmotic-induced cell expansion (0.4 M sorbitol > 0.1 M sorbitol). (E) Example of a tcb2/3∆ spheroplast (MTY52 containing MRV66) that lysed during hypoosmotic shock. (F) Five examples of wild-type cells expressing ER-pHluorin as an ER marker, before and after the hypoosmotic shock (0.4 M > 0.1 M sorbitol). The microscopy pictures show the surface of the cell, which visualizes cER. (G) Quantification of the change in cER surface caused by hypoosmotic spheroplast expansion. The graphs in C and G are box-and-whisker plots, indicating the mean.