Summary and interpretation of our observations. (A) Microfluidics-based analysis of yeast hypoosmotic shock. Time 0 marks the time point at which we observed the beginning of the cell surface increase (blue line). Based on this observed swelling, we predict that the turgor pressure increased shortly before (black line). In wild type, the cytoplasmic calcium increase followed ∼4 s after cell expansion started (yellow line) and another 4 s later the cytoplasmic pH showed a small drop (green line). In contrast, tcb2/3∆ cells exhibited a calcium spike and a dramatic pH drop ∼8 s after cell expansion started. Furthermore, the increased cell size did not remain and when the turgor pressure dropped the mutant cells lost some of their surface expansion. (B) Model of the tricalbin-mediated transport of cER membrane to the PM. At cER sheets, the tricalbins form a ring structure that deforms the membrane into ER peaks. High PM fluidity, caused either by high tension or heat-shock, opens the calcium channel Cch1 (step 1). The local influx of calcium (step 2) causes the tricalbins to shorten the distance between the cER and the PM (step 3), thereby bringing the fusogenic ER peak into close proximity to the high-fluidity PM. As a result, the membranes fuse, which allows rapid delivery of ER lipids and the GPI-anchored protein Crh2 to the cell surface (step 4). The tricalbin ring acts as a diffusion barrier for transmembrane proteins and thus prevents the delivery of Ist2 to the cell surface. High-calcium concentrations activate the chloride channel Ist2, which after fusion becomes part of the PM. The resulting secretion of chloride ions (step 5) lowers the membrane potential and as a consequence increases the calcium influx via Cch1 at other fusion sites. Finally, the fusion pore collapses, leaving behind a cER with a reduced lipid/protein ratio and thus a changed morphology.