A striking variation in electron density of the vacuolar lumen was observed with electron microscopy. In logarithmically growing non-stressed yeast cells, vacuoles are often very electron dense. After heat shock, the vacuoles were often electron translucent and sometimes contained a grainy texture. Vacuoles were therefore classified into four major categories according to their appearance: dense, grainy, medium or translucent (Fig. 3A). The electron density of vacuoles fluctuated strongly throughout the time course in each experiment, showing lower electron densities when exposed to stress and a recovery to darkly stained vacuoles at 90 min (Fig. 3B).

When comparing vacuoles of dividing cells, daughter cells had either a higher or equal vacuolar electron density than their mother (Fig. 3C,D, n=53 dividing cells). A previous study showed that upon cell division in yeast, the daughter cell has a more acidic vacuole than its older mother cell (Hughes and Gottschling, 2012). We thus hypothesised that lower electron density in vacuoles could be due to a deacidification of that organelle. The effect of vacuolar pH on the electron density of the samples was investigated using cells from a vma2Δ strain (Fig. 3E). Vma2 is a subunit of the V1 domain of the vacuolar H+ ATPase and its deletion causes an increased vacuolar pH compared with its usual pH of 5-6.5 (Li and Kane, 2009), this high pH also reduces vacuolar protease activity (Nakamura et al., 1997). As opposed to wild-type cells, almost all vacuoles in vma2Δ cells displayed the electron-translucent phenotype, demonstrating that increased pH leads to altered vacuolar staining (Fig. 3E, n=181 wild-type and 274 vma2Δ cells). To determine whether vacuoles deacidify during heat shock, we used the pH-sensitive vacuolar probe BCECF-AM, which displays increased fluorescence at increased pH (Plant et al., 1999; Hughes and Gottschling, 2012). As a positive deacidification control, BCECF-AM staining also confirmed the expected elevated pH in the vma2Δ mutant (Fig. 3F,G). Vacuoles exposed to 45 min heat shock fluoresced brightly, compared with those of cells grown at 30°C, confirming that heat shock causes elevated internal pH in vacuoles (Fig. 3F,G). Furthermore, cells of the deletion mutant prb1Δ, where the gene PRB1 encoding a vacuolar protease is deleted, showed more electron-dense vacuoles than wild type (Fig. 3E, n=150). In fluorescence microscopy images of cells stained with quinacrine, an indicator for acidity, vacuoles had higher fluorescence than wild-type cells (Fig. 3H,I). Accordingly, prb1Δ cells stained using BCECF-AM had lower fluorescence than wild type (Fig. S1B). Therefore, using our electron microscopy sample preparation protocol, changes in vacuolar pH can potentially be observed as altered electron density of the vacuolar lumen. To summarise, vacuoles are affected by heat shock in several ways: a decrease in number, an increase in size and increased luminal pH.