Organelle Size Scaling of the Budding Yeast Vacuole by Relative Growth and Inheritance

Abstract

It has long been noted that larger animals have larger organs compared to smaller animals of the same species, a phenomenon termed scaling [1]. Julian Huxley proposed an appealingly simple model of "relative growth"-in which an organ and the whole body grow with their own intrinsic rates [2]-that was invoked to explain scaling in organs from fiddler crab claws to human brains. Because organ size is regulated by complex, unpredictable pathways [3], it remains unclear whether scaling requires feedback mechanisms to regulate organ growth in response to organ or body size. The molecular pathways governing organelle biogenesis are simpler than organogenesis, and therefore organelle size scaling in the cell provides a more tractable case for testing Huxley's model. We ask the question: is it possible for organelle size scaling to arise if organelle growth is independent of organelle or cell size? Using the yeast vacuole as a model, we tested whether mutants defective in vacuole inheritance, vac8Δ and vac17Δ, tune vacuole biogenesis in response to perturbations in vacuole size. In vac8Δ/vac17Δ, vacuole scaling increases with the replicative age of the cell. Furthermore, vac8Δ/vac17Δ cells continued generating vacuole at roughly constant rates even when they had significantly larger vacuoles compared to wild-type. With support from computational modeling, these results suggest there is no feedback between vacuole biogenesis rates and vacuole or cell size. Rather, size scaling is determined by the relative growth rates of the vacuole and the cell, thus representing a cellular version of Huxley's model.

Figure 1. Vacuole inheritance defect causes divergence in vacuole-to-cell size scaling

(A) Representative images of wild-type and vac8Δ cells (brightfield) and vacuoles (Vph1p-GFP). In each image, the cells are arranged in a frequently-observed pattern where a 2+ generation mother, M, has produced a 1^st generation daughter, D, in the previous cell cycle, and is currently producing a new bud, B. (B) Vacuole volume and (C) vacuole surface area vs. cell volume scaling plots for wild-type (blue) and vac8Δ (red) cells. Wild-type and vac8Δ scaling plots are significantly different from one-another for both volume and surface area (p<0.001, comparison of regression lines.) See also Figure S1.

Figure 2. Vacuole size scaling defects in inheritance mutants increase with replicative age

(A) Wild-type vacuole surface area; (B) wild-type vacuole volume; (C) vac8Δ vacuole surface area; and (D) vac8Δ vacuole volume vs. cell volume scaling plots were separated according to generational age. Lines represent best linear fits (solid, p-value<0.05; dashed, p-value>0.05). (F) Vacuole surface area- and (G) vacuole volume-to-cell volume ratios were averaged for cells binned by replicative age. Lines represent best linear fits, and error bars represent standard deviations. vac8Δ cells (red) show a much steeper increase in scaling ratio than wild-type (blue). See also Figure S2.

Figure 3. Wild-type and vac8Δ vacuoles grow at similar rates regardless of the vacuole-to-cell size scaling ratio

(A) Total cell (mother cell and its bud) and total vacuole sizes were measured at 10min intervals for 1^st generation mothers undergoing their first G1 (blue shading) and budding (red shading) followed by their second cell cycle (no shading). Timecourse plots (B)–(K) show average values at each timepoint with error bars depicting standard deviations. (B) Wild-type (n=10) and (C) vac8Δ (n=10) average cell volume plotted against time. (D) Wild-type and (E) vac8Δ average vacuole surface area plots. (F) Wild-type and (G) vac8Δ average vacuole volume plots. In (D) & (F), the cyan line corresponds to the sum of vacuole sizes in the now 2^nd generation mother and her newborn 1^st generation daughter. This represents the cumulative amount of vacuole growth. Vacuole surface area-to-cell volume ratios were calculated averaged for each timepoint for (H) wild-type and (I) vac8Δ cells. Vacuole volume-to-cell volume ratios were calculated and averaged for each timepoint for (J) wild-type and (K) vac8Δ cells. See also Figures S3 and S4.

Figure 4. Model of cell growth, vacuole growth, and vacuole inheritance

(A) Illustration of the simulation of cell and vacuole growth. In G1, a mother cell and its vacuole grow. The mother enters the cell cycle in which growth is restricted to the bud. When the bud reaches a certain size, division can occur, producing two mothers that enter another iteration of the cell cycle. (B) Using constant cell and vacuole growth rates produces populations that are similar to wild-type, and increasing variance in these rates leads to decreasing R²-coefficients in the population scaling plots. (C) Vacuole growth can be localized to the bud (left, wild-type), mother (vac8Δ, right), or randomly distributed between both cells (middle). Changes to inheritance lead to divergence in the scaling plots with respect to replicative age. See also Table S1.