Organelle size scaling of the budding yeast vacuole is tuned by membrane trafficking rates

Abstract

Organelles serve as biochemical reactors in the cell, and often display characteristic scaling trends with cell size, suggesting mechanisms that coordinate their sizes. In this study, we measure the vacuole-cell size scaling trends in budding yeast using optical microscopy and a novel, to our knowledge, image analysis algorithm. Vacuole volume and surface area both show characteristic scaling trends with respect to cell size that are consistent among different strains. Rapamycin treatment was found to increase vacuole-cell size scaling trends for both volume and surface area. Unexpectedly, these increases did not depend on macroautophagy, as similar increases in vacuole size were observed in the autophagy deficient mutants atg1Δ and atg5Δ. Rather, rapamycin appears to act on vacuole size by inhibiting retrograde membrane trafficking, as the atg18Δ mutant, which is defective in retrograde trafficking, shows similar vacuole size scaling to rapamycin-treated cells and is itself insensitive to rapamycin treatment. Disruption of anterograde membrane trafficking in the apl5Δ mutant leads to complementary changes in vacuole size scaling. These quantitative results lead to a simple model for vacuole size scaling based on proportionality between cell growth rates and vacuole growth rates.

Figure 1

Vacuole size extraction and vacuole size scaling in WT strains. (A) Cells (brightfield) and vacuoles (VPH1-GFP) were imaged using a spinning disk confocal microscope. (B) Z-stacks were processed using a computational algorithm to reconstruct surfaces for each vacuole. (C) Cell and vacuole sizes measurements were fit with power-law scaling trends, which can be compared between different mutants and conditions. (D) Vacuole volume-cell volume scaling and (E) Vacuole surface area-cell volume scaling trends for W303A and BY4741 background strains.

Figure 2

Vacuole size and size ratios in W303A WT. (A) Vacuole volume- and (B) Vacuole surface area-to-cell volume scaling plots. (C) Vacuole volume-to-cell size ratios and (D) Vacuole surface area-to-cell size ratios plotted against cell volume. Plots contain data for total (mother+bud) cells (blue), individual mothers (red), and individual buds (green). In (C) and (D), lines indicate least squares linear regressions to the data with listed Pearson correlation r values.

Figure 3

Rapamycin induces larger vacuole sizes independent of autophagy. (A) Yeast cells (grayscale) and vacuoles (green) growing in log phase (left) tend to become enlarged and more fused after incubation with 0.2 μg/mL rapamycin for 4 h (right). (B) Vacuole volume- and (C) vacuole surface area-to-cell size scaling trends are similar in W303A and in atg1Δ and atg5Δ autophagy mutants. Lines indicate power-law fits to the data, and Pearson’s r values are listed. Note that for all strains, both vacuole volume and surface area scaling trends are increased in rapamycin-treated cells compared to untreated cells.

Figure 4

Retrograde and anterograde membrane trafficking affect vacuole size. Comparison of vacuole size-to-cell volume scaling trends between WT and atg18Δ ((A) image, (B) volume, and (C) surface area) or between WT and apl5Δ ((D) image, (E) volume, and (F) surface area). Lines indicate power-law fits to the data, and Pearson’s r values are listed. Without rapamycin, atg18Δ and apl5Δ show larger and smaller vacuole size scaling trends, respectively. In the presence of rapamycin, atg18Δ mutants show vacuole size scaling trends similar to WT yeast, whereas apl5Δ vacuoles still show smaller size scaling trends. (G) Based on these findings, we propose this interaction network in which rapamycin affects vacuole surface area primarily through effects on retrograde traffic and vacuole volume through effects on vacuole fusion.