Yeast cells actively tune their membranes to phase separate at temperatures that scale with growth temperatures.

Membranes of vacuoles, the lysosomal organelles of Saccharomyces cerevisiae (budding yeast), undergo extraordinary changes during the cell's normal growth cycle. The cycle begins with a stage of rapid cell growth. Then, as glucose becomes scarce, growth slows, and vacuole membranes phase separate into micrometer-scale domains of two liquid phases. Recent studies suggest that these domains promote yeast survival by organizing membrane proteins that play key roles in a central signaling pathway conserved among eukaryotes (TORC1). An outstanding question in the field has been whether cells regulate phase transitions in response to new physical conditions and how this occurs. Here, we measure transition temperatures and find that after an increase of roughly 15 °C, vacuole membranes appear uniform, independent of growth temperature. Moreover, populations of cells grown at a single temperature regulate this transition to occur over a surprisingly narrow temperature range. Remarkably, the transition temperature scales linearly with the growth temperature, demonstrating that the cells physiologically adapt to maintain proximity to the transition. Next, we ask how yeast adjust their membranes to achieve phase separation. We isolate vacuoles from yeast during the rapid stage of growth, when their membranes do not natively exhibit domains. Ergosterol is the major sterol in yeast. We find that domains appear when ergosterol is depleted, contradicting the prevalent assumption that increases in sterol concentration generally cause membrane phase separation in vivo, but in agreement with previous studies using artificial and cell-derived membranes.

Remodeling of yeast vacuole membrane lipidomes from the log (one phase) to stationary stage (two phases).

Upon nutrient limitation, budding yeast of Saccharomyces cerevisiae shift from fast growth (the log stage) to quiescence (the stationary stage). This shift is accompanied by liquid-liquid phase separation in the membrane of the vacuole, an endosomal organelle. Recent work indicates that the resulting micrometer-scale domains in vacuole membranes enable yeast to survive periods of stress. An outstanding question is which molecular changes might cause this membrane phase separation. Here, we conduct lipidomics of vacuole membranes in both the log and stationary stages. Isolation of pure vacuole membranes is challenging in the stationary stage, when lipid droplets are in close contact with vacuoles. Immuno-isolation has previously been shown to successfully purify log-stage vacuole membranes with high organelle specificity, but it was not previously possible to immuno-isolate stationary-stage vacuole membranes. Here, we develop Mam3 as a bait protein for vacuole immuno-isolation, and demonstrate low contamination by non-vacuolar membranes. We find that stationary-stage vacuole membranes contain surprisingly high fractions of phosphatidylcholine lipids (∼40%), roughly twice as much as log-stage membranes. Moreover, in the stationary stage, these lipids have higher melting temperatures, due to longer and more saturated acyl chains. Another surprise is that no significant change in sterol content is observed. These lipidomic changes, which are largely reflected on the whole-cell level, fit within the predominant view that phase separation in membranes requires at least three types of molecules to be present: lipids with high melting temperatures, lipids with low melting temperatures, and sterols.

Colony context and size-dependent compensation mechanisms give rise to variations in nuclear growth trajectories.

To investigate the fundamental question of how cellular variations arise across spatiotemporal scales in a population of identical healthy cells, we focused on nuclear growth in hiPS cell colonies as a model system. We generated a 3D timelapse dataset of thousands of nuclei over multiple days, and developed open-source tools for image and data analysis and an interactive timelapse viewer for exploring quantitative features of nuclear size and shape. We performed a data-driven analysis of nuclear growth variations across timescales. We found that individual nuclear volume growth trajectories arise from short timescale variations attributable to their spatiotemporal context within the colony. We identified a strikingly time-invariant volume compensation relationship between nuclear growth duration and starting volume across the population. Notably, we discovered that inheritance plays a crucial role in determining these two key nuclear growth features while other growth features are determined by their spatiotemporal context and are not inherited.