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• W2983642713 abstract "Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract The organization and biophysical properties of the cytosol implicitly govern molecular interactions within cells. However, little is known about mechanisms by which cells regulate cytosolic properties and intracellular diffusion rates. Here, we demonstrate that the intracellular environment of budding yeast undertakes a startling transition upon glucose starvation in which macromolecular mobility is dramatically restricted, reducing the movement of both chromatin in the nucleus and mRNPs in the cytoplasm. This confinement cannot be explained by an ATP decrease or the physiological drop in intracellular pH. Rather, our results suggest that the regulation of diffusional mobility is induced by a reduction in cell volume and subsequent increase in molecular crowding which severely alters the biophysical properties of the intracellular environment. A similar response can be observed in fission yeast and bacteria. This reveals a novel mechanism by which cells globally alter their properties to establish a unique homeostasis during starvation. https://doi.org/10.7554/eLife.09376.001 eLife digest Most organisms live in unpredictable environments, which can often lead to nutrient shortages and other conditions that limit their ability to grow. To survive in these harsh conditions, many organisms adopt a dormant state in which their metabolism slows down to conserve vital energy. When the environmental conditions improve, the organisms can return to their normal state and continue to grow. The interior of cells is known as the cytoplasm. It is very crowded and contains many molecules and compartments that carry out a variety of vital processes. The cytoplasm has long been considered to be fluid-like in nature, but recent evidence suggests that in bacterial cells it can solidify to resemble a glass-like material under certain conditions. When cells experience stress they stop dividing and alter their metabolism. However, it was not clear whether cells also alter their physical properties in response to changes in the environment. Now, Joyner et al. starve yeast cells of sugar and track the movements of two large molecules called mRNPs and chromatin. Chromatin is found in a cell compartment known as the nucleus, while mRNPs are found in the cytoplasm. The experiments show that during starvation, both molecules are less able to move around in their respective areas of the cell. This appears to be due to water loss from the cells, which causes the cells to become smaller and leads to the interior of the cell becoming more crowded. Joyner et al. also observed a similar response in bacteria. Furthermore, Joyner et al. suggest that the changes in physical properties are critical for cells to survive the stress caused by starvation. A separate study by Munder et al. found that when cells become dormant the cytoplasm becomes more acidic, which causes many proteins to bind to each other and form large clumps. Together, the findings of the studies suggest that the interior of cells can undergo a transition from a fluid-like to a more solid-like state to protect the cells from damage when energy is in short supply. The next challenge is to understand the molecular mechanisms that cause the physical properties of the cells to change under different conditions. https://doi.org/10.7554/eLife.09376.002 Introduction Eukaryotic cells expend a significant amount of energy to establish and maintain a high degree of intracellular organization in order to regulate and orchestrate their complex metabolism. This includes the active enrichment of macromolecules into organelles, which in turn allows for the separation of biochemical pathways and enhances their efficiency by increasing local concentrations of enzymes and metabolites. Similarly, active transport by motor proteins on the cytoskeleton enables large eukaryotic cells to overcome the limits of Brownian motion in which the diffusion time scales with the square of the distance. Nevertheless, many cellular pathways and biochemical interactions are dependent on Brownian diffusion. Much research has therefore been performed to characterize diffusional processes that occur within eukaryotic cells. These studies have revealed that the very complex and extremely crowded cytosol of eukaryotic cells cannot be viewed as an ideal liquid but, instead, can be modeled as a polymer gel (Luby-Phelps, 2000; Clegg, 1984; Knull and Minton, 1996) or soft colloidal glass (Fabry et al., 2001; Mandadapu et al., 2008; Luby-Phelps, 2000). Still, a comprehensive understanding of the biophysical properties of eukaryotic cells is lacking, and it remains poorly understood how these characteristics influence macromolecular movement. Furthermore, little is known about the biological determinants of intracellular diffusion including whether cells functionally regulate their diffusional properties in response to changes in growth conditions or the environment. A particularly well-studied model of intracellular movement is the motion of chromatin in the nucleus. Various groups have analyzed chromatin mobility, but a coherent mechanistic understanding of this process has yet to emerge (reviewed in Hübner and Spector, 2010). For example, it was shown that chromosomes wiggle in a manner consistent with constrained diffusion and it was proposed that chromosome movement results from Brownian motion rather than from active motility (Marshall et al., 1997). In contrast, other studies have suggested that chromatin movement is ATP dependent (Heun et al., 2001), and it was hypothesized that chromatin movement is driven by a multitude of ATP-dependent processes along the length of the chromosome (Neumann et al., 2012). Similarly, the role of the cytoskeleton in chromatin movement has remained controversial. For example, both microtubule-dependent and -independent movement was reported (Heun et al., 2001, Marshall et al., 1997), and several recent studies have also implicated the actin cytoskeleton in chromatin mobility (Chuang et al., 2006; Koszul et al., 2008; Spagnol and Dahl, 2014; Spichal et al., 2016). To better characterize the diffusional properties of eukaryotic cells, we have analyzed here the movement of chromatin and mRNPs in budding yeast under changing growth conditions. Our results demonstrate that cells dramatically change their biophysical properties in response to glucose starvation, which causes a confinement of macromolecules and affects the mechanical properties of the cell. This effect cannot be explained by changes in ATP levels or pH but can be induced by a loss of cell volume without any change in cell mass. This response seems to be conserved as bacteria similarly restrict macromolecular mobility in response to starvation (Parry et al., 2014), and we show here that starvation also induces a volume loss in bacterial cells and severely affects the rigidity of fission yeast cells. Our results suggest a novel mechanism by which cells regulate their biophysical properties in order to adapt to environmental stress. Results Glucose starvation limits macromolecular mobility in the nucleus and cytoplasm To examine the effects of changes in growth conditions on nuclear chromatin dynamics in budding yeast, we analyzed the movement of various gene loci. LacO repeats were integrated at the POA1 locus on chromosome II, the URA3 locus on chromosome V, and on a centromeric plasmid (pLacO). Co-expression of LacI-GFP allowed us to visualize these three loci and track their mobility over minute-long sequences. Whereas several changes in growth conditions, including growth in different carbon sources or nitrogen starvation, had no obvious effect on chromatin mobility (data not shown), acute glucose starvation induced a dramatic cessation of chromatin movement (Figure 1A). This suggests that chromatin mobility is regulated by the presence of glucose. Figure 1 with 2 supplements see all Download asset Open asset Acute glucose starvation confines macromolecular mobility in the nucleus and cytoplasm (Figure 1—figure supplement 1). (A) Minute-long trajectories of the POA1 locus from both (+) glucose (blue) and (–) glucose (red) conditions projected on bright field images. Log-growing cells in (+) glucose were acutely starved for glucose, (–) glucose, for 30 min minutes prior to imaging. Scale bar: 4 µm. (B) Mean square displacement (MSD) curves for POA1 mobility. Upper panel: individual MSDs were averaged into an aggregate MSD for each condition. Error bars represent standard error of the mean (SEM). Lower panel: log-log MSD plot of the same data. (C) Log-log MSD plot of the pLacO plasmid during exponential growth and after acute glucose starvation. (D) Minute-long trajectories of GFA1 mRNPs from both (+) glucose (blue) and (–) glucose (red) conditions projected on bright field images. (E) Mean square displacement (MSD) curves for GFA1 mRNP mobility. Upper panel: individual MSDs were averaged into an aggregate MSD for each condition. Error bars represent SEM. Lower panel: log-log MSD plot of the same data. (F) Log-log MSD plot of the FBA1 mRNP during exponential growth and after acute glucose starvation. Dashed gray lines represent a slope of one to guide the eye. https://doi.org/10.7554/eLife.09376.003 To quantify the dramatic changes in chromatin mobility, we calculated ensemble-averaged mean square displacements (MSDs) for the chromatin loci (n = 183–1172 trajectories each) (Figure 1B and C; Figure 1—figure supplement 1A; Figure 1—figure supplement 2A). These plots express the magnitude of diffusion for a given particle, quantifying the average displacement per unit time and are used to compute their effective diffusion coefficients (Qian et al., 1991). We find that the confinement of chromatin upon glucose starvation (Figure 1B and C; Figure 1—figure supplement 2) leads to an approximately three-fold reduction of the apparent diffusion coefficient (K): for instance, KPOA1 decreased from 5.7 x 10–3 µm2/s to 2.3 x 10–3 µm2/s upon starvation (Table 1). The change in mobility at this time scale was not caused by a change in the anomaly of the diffusion process as the anomalous diffusion exponent (α), which is given by the slope of the curves in the MSD log-log plot, is not affected (see also Table 1). Table 1 Effective diffusion coefficients (K; µm2/s) and anomalous diffusion exponents (α) for macromolecules in each condition. https://doi.org/10.7554/eLife.09376.006 Condition POA1 LocuspLacO Plasmid URA3 Locus GFA1 mRNP FBA1 mRNPKαKαKαKαKα(+) Glucose0.00570.690.00670.780.00760.650.04200.830.05010.85(-) Glucose0.00230.640.00210.800.00220.730.01310.770.01390.77(-) Glucose pH 7.40.00150.65--------0.01200.75----DMSO0.00590.560.00460.700.00600.610.04910.830.05410.83Nocodazole0.00400.480.00250.570.00460.510.03640.850.03970.85Latrunculin A0.00380.500.00240.630.00380.550.04760.820.05500.81Nocodazole + LatA0.00280.480.00140.490.00260.520.03030.810.03670.822 mM K+Sorbate0.00560.730.00510.800.00590.680.04020.820.02960.804 mM K+Sorbate0.00500.750.00440.720.00480.710.04060.780.02420.796 mM K+Sorbate0.00390.700.00180.660.00270.660.03780.760.02700.788 mM K+Sorbate0.00230.640.00120.610.00140.610.03400.760.01640.770.4 M NaCl0.00300.690.00260.75----0.01290.830.01460.850.6 M NaCl0.00120.600.00110.58----0.00470.830.00570.840.8 M NaCl0.00090.630.00110.63----0.00130.670.00160.77Quiescence------------0.00040.290.00150.680.02% Azide (Wash)0.00370.74--------0.02930.82----0.02% Azide (Spike)0.00120.67--------0.01550.81---- To analyze whether glucose starvation uniquely affects chromatin dynamics in the nucleus, or whether it also influences the mobility of other macromolecules, we imaged the movement of cytoplasmic mRNPs, which can be conveniently tracked as single particles (Shav-Tal et al., 2004). 24-PP7 stem-loops were integrated into the 3’ UTR of GFA1 and FBA1, essential genes involved in distinct processes (Lagorce et al., 2002; Schwelberger et al., 1989), and the movement of individual mRNPs was examined upon co-expression of the coat-binding protein, CP-PP7-3xYFP. Cumulative track projections revealed substantially higher mobility for mRNPs than chromosomal loci in glucose, which is expected given the significantly smaller size of mRNPs compared to chromosome fibers (Figure 1D) (Thompson et al. 2010; Zarnack and Feldbrügge, 2007). Yet, upon glucose starvation, GFA1 and FBA1 mRNPs also exhibited a dramatic reduction in their mobility (Figure 1E and F; Figure 1—figure supplement 1B). Removal of glucose led to a three- to four-fold decrease in the diffusion coefficient of both GFA1 (KGFA1reduced from 4.2 x 10–2 µm2/s to 1.3 x 10–2 µm2/s) and FBA1 (KFBA1reduced from 5.0 x 10–2 µm2/s to 1.4 x 10-2 µm2/s) mRNPs without affecting the anomalous exponent (α). These values are similar to the relative change that we observed in the diffusion of chromatin (Table 1). Depletion of glucose by growth into quiescence also confined the mobility of mRNPs; therefore, the effects we observe are not a consequence of our cell washes (Figure 1—figure supplement 2B and C). The decrease in mobility of both chromosomal loci and mRNP particles suggests that glucose starvation causes a confinement of macromolecules in the nucleus as well as the cytoplasm. Starvation arrests cytoskeletal dynamics, which constrains chromatin mobility but has little effect on mRNPs To begin to understand the nature and mechanism of the starvation-induced reduction in chromatin and mRNP mobility, we first focused on the cytoskeleton. In eukaryotes, the movement of many macromolecules is directly influenced by the dynamics of the cytoskeleton. For instance, mRNPs can be actively transported by motor proteins along the cytoskeleton and both the actin cytoskeleton and microtubules were reported to influence chromatin dynamics (Thompson et al., 2010; Heun et al., 2001; Koszul et al., 2008; Spagnol and Dahl, 2014; Spichal et al., 2016). Interestingly, actin filaments were also shown to rapidly depolymerize upon starvation (Uesono et al., 2004; Sagot et al., 2006). We therefore wanted to explore whether the starvation-induced confinement of macromolecular mobility resulted from changes in cytoskeletal dynamics. Indeed, our starvation condition lead to ablation of actin filaments in nearly all cells (Figure 2A) and a nearly four-fold reduction of microtubule elongation events in G1 cells as visualized by Tub1-GFP (Figure 2B). Figure 2 with 1 supplement see all Download asset Open asset Starvation confines both cytoskeleton-independent macromolecular mobility and mobility influenced by the cytoskeleton (Figure 2—figure supplement 1). (A) Quantification of filamentous actin during logarithmic growth, (+) glucose, and after acute starvation, (–) glucose. Cells were fixed and stained with phalloidin. Z-stack projections were then processed and cells were classified based on the presence or absence of filamentous actin. Error bars are standard error for three biological replicates (n = 406–709 cells per replicate). (B) Average number of microtubule projections per G1 cell during logarithmic growth, (+) glucose, and after acute starvation, (–) glucose. Error bars are standard deviation (SD) of the mean from three biological replicates (n = 10–1510-15 cells per replicate). The p-value for a two-tailed t-test for unpaired values assuming equal variance is shown. (C) Log-log MSD plot of the POA1 locus after treatment with nocodazole and/or latrunculin-A (LatA) for 20 min prior to imaging. (D) Log-log MSD plot of the GFA1 mRNP after treatment as described in (C). Dashed gray lines represent a slope of one to guide the eye. https://doi.org/10.7554/eLife.09376.007 If the changes in the mobility of chromatin or mRNPs upon glucose withdrawal were due to the reduction of cytoskeletal dynamics, we would predict that drug-induced inhibition of actin and/or microtubule polymerization mimics the starvation response. In order to test this hypothesis, we treated cells with the actin depolymerizer, latrunculin A (LatA), and/or the microtubule depolymerizing drug, nocodazole (Ayscough et al., 1997; Jacobs et al., 1988). Indeed, treatment with either drug reduced chromatin movement, although the effect was insufficient to mimic the confinement of chromatin during starvation (Figure 2C; Figure 2—figure supplement 1). Concurrent treatments with LatA and nocodazole reduced chromatin mobility in an additive manner to substantially reduce chromatin mobility (Figure 2C; Figure 2—figure supplement 1). Moreover, log-log MSD plots indicate that the effect of inhibiting the cytoskeleton is most apparent at longer time intervals and may augment the anomalous behavior of chromatin motion (αPOA1-DMSO = 0.56 versus αPOA1-Noc+LatA = 0.48; Figure 2C; Figure 2—figure supplement 1; Table 1). The actin and microtubule cytoskeleton could either act on chromatin itself or affect chromatin mobility indirectly by influencing overall nuclear motion. To differentiate between these two possibilities, we tracked the distance between two labelled chromosomal loci (POA1, chromosome II and PES4, chromosome VI) over time (Figure 2—figure supplement 1). This approach allows for the exclusive quantification of intranuclear movements because translational changes in the positioning of the nucleus affect both loci identically and thus, do not influence intranuclear distance measurements (Marshall et al., 1997). This two-locus analysis confirmed the LatA and nocodazole-induced reduction in chromatin mobility as the interchromosomal distance between the two loci still decreased considerably with simultaneous drug treatment (Figure 2—figure supplement 1). Of note, however, this result cannot differentiate between cytoplasmic or nuclear cytoskeletal dynamics in modulating chromatin mobility (e.g. via deformations of the nucleus). In conclusion, actin and microtubule dynamics independently contribute to the mobility of yeast chromatin. In contrast to chromatin mobility, mRNP mobility was only moderately affected by perturbation of cytoskeletal dynamics (Figure 2D; Figure 2—figure supplement 1) suggesting that the mobility of the GFA1 and FBA1 mRNPs is largely independent of the cytoskeleton. Overall, our results show that glucose starvation restricts cytoskeleton-independent mobility as well as the mobility of macromolecules influenced by the cytoskeleton. Reduction of ATP is insufficient to explain the macromolecular confinement Our results so far could be explained by two alternative models: 1) starvation impacts macromolecular diffusion through multiple, distinct mechanisms, or 2) a singular, starvation-induced pathway restricts the mobility of macromolecules, and leads to both the collapse of cytoskeletal dynamics and the restriction of mRNP mobility. The acute withdrawal of glucose in fermenting yeast cells is expected to have dramatic consequences on cellular physiology. For example, the cellular ATP concentration drops (Ashe et al., 2000) and the intracellular pH decreases in starved cells (Orij et al., 2009). We therefore tested whether these global changes in cellular physiology lead to the observed changes in macromolecular mobility. First, we investigated the changes in intracellular ATP concentration during starvation. Upon glucose starvation, the ATP concentration rapidly decreased by ~70%. Remarkably, after this initial drop, ATP levels were relatively stable at ~30% of the initial concentration for the remainder of the experiment (Figure 3A). Of note, the maintenance of this reduced ATP level required oxidative phosphorylation as the cellular ATP concentration quickly dropped to nearly undetectable levels when cells deficient in mitochondrial function were starved (cbp2∆; Shaw and Lewin, 1997) (Figure 3—figure supplement 1). In contrast, when cells washed in sugar-free medium were back-diluted into media containing glucose, ATP levels recovered to the initial concentration within 30. Thus, glucose starvation induces a rapid ~70% reduction of intracellular ATP levels in agreement with previously published results (Özalp et al., 2010). Figure 3 with 1 supplement see all Download asset Open asset A ~70% reduction of intracellular ATP is insufficient to replicate the macromolecular confinement of glucose starvation (Figure 3—figure supplement 1). (A) Intracellular ATP concentrations of acutely glucose-starved yeast were back-diluted into media containing 2% dextrose (n = 2 experiments), 2% dextrose + 0.02% azide (n = 2 experiments), or maintained in (–) glucose media (n = 3 experiments). Intracellular ATP concentrations were determined using a luciferase-based ATP assay (Ashe et al., 2000) and normalized to pre-treatment levels. The zero min time point was taken immediately after back-dilution into the described media. Error bars represent SD. (B) Log-log MSD plot of the POA1 locus. Cells were treated with azide as described in (A). The azide treatment fails to replicate the confinement of glucose-starved cells from Figure 1B. (C) Log-log MSD plot of the GFA1 mRNP. Cells were treated with azide as described in (A). The azide treatment fails to replicate the confinement of glucose-starved cells from Figure 1E. https://doi.org/10.7554/eLife.09376.009 To determine whether a ~70% reduction in intracellular ATP levels is sufficient to confine cytoskeleton-independent macromolecular mobility as well as mobility influenced by the cytoskeleton, we reproduced this decrease in ATP concentrations in the presence of glucose. This was achieved by back-diluting washed cells into glucose media containing 0.02% sodium azide (NaN3), an inhibitor of oxidative phosphorylation (Figure 3A). After 0.02% azide treatment, ATP levels dropped to the same level as in glucose-starved cells, but the mobility of chromatin and mRNPs did not decrease to the same degree as in starvation (Figure 3B and C). We therefore conclude that a ~70% reduction of intracellular ATP, as observed in glucose-depleted cells, is insufficient to fully explain the confinement of macromolecular mobility during glucose starvation. Reduction of intracellular pH titrates macromolecular mobility but cannot explain starvation-induced macromolecular confinement Next, we investigated the contribution of intracellular pH to macromolecular confinement upon starvation. The intracellular pH (pHi) of budding yeast was reported to decrease from pH ~7.3 to pH ~6.4 in a time frame consistent with the observed confinement of macromolecular mobility upon glucose deprivation (Orij et al., 2009; Young et al., 2010). Using the pH-sensitive GFP analog, pHluorin, as a pHi biosensor (Miesenböck et al., 1998), we confirmed in our experimental conditions that glucose starvation causes a reduction in pHi from ~7.4 to ~6.4 (Figure 4A). We then manipulated intracellular proton concentrations by the addition of the weak acid potassium sorbate (K+Sorbate). K+Sorbate traverses the plasma membrane, releases a proton, and reduces pHi in a concentration-dependent manner (Bracey et al., 1998; Piper et al., 2001). Varying the extracellular concentration of K+Sorbate from 0 mM to 8 mM enabled us to titrate pHi from pH 7.4 to pH 5.9, thus covering more than the pHi range observed in cells grown either in (+) glucose or (–) glucose conditions, with little effect on intracellular ATP levels (Figure 4A and B). Remarkably, exposing cells to increasing concentrations of K+Sorbate induced macromolecular confinement, and both chromatin and mRNP mobility could be titrated with decreasing pHi (Figure 4C–F; Figure 4—figure supplement 1). Figure 4 with 2 supplements see all Download asset Open asset A drop in intracellular pH (pHi) can reduce macromolecular mobility but cannot explain the confinement observed in glucose-starved cells (Figure 4—figure supplements 1 and 2). (A) Boxplots of the pHi of cells acutely starved for glucose or treated with varying concentrations of potassium sorbate (K+Sorbate). Log-growing yeast cells expressing phluorin (Miesenböck et al., 1998) were acutely starved of glucose or treated with K+Sorbate for 30 min before ratiometric imaging. Intracellular pH was then estimated from a calibration curve (see 'Materials and methods'; Figure 4—figure supplement 1). Values from three biological replicates were pooled and compiled into boxplots. Whiskers represent the minimum and maximum respectively. (B) Intracellular ATP concentrations after treatment with either 2 mM or 8 mM K+Sorbate. Intracellular ATP concentrations were determined as in Figure 3A. The zero minute time point was taken immediately after each treatment. Error bars represent SD (n =2). (C) Log-log MSD plot of the POA1 locus after treatment with K+Sorbate as described in (A). (D) Log-log MSD plot of the GFA1 mRNP after treatment as described in (A). E) Log-log MSD plot of the pLacO plasmid after treatment as described in (A). (F) Log-log MSD plot of the FBA1 mRNP after treatment as described in (A). (G) Log-log MSD plot of the POA1 locus after acute glucose starvation into starvation media (SC) adjusted to pH 7.4. (H) Log-log MSD plot of the GFA1 mRNP after treatment as described in (G). https://doi.org/10.7554/eLife.09376.011 Importantly, however, treatment with the highest concentration of K+Sorbate (8 mM) was necessary to fully recapitulate the starvation-induced confinement of both chromatin and mRNPs (Figure 4C–F). Under this condition, pHi dropped to 5.9, which is below the physiologically observed pHi of 6.4 in glucose-starved cells (Figure 4A). When pHi was lowered to 6.4 (by the addition of 2 mM K+Sorbate), the movement of both chromatin and mRNPs was only moderately reduced (Figure 4C–F). Furthermore, starvation in (–) glucose media adjusted to pH 7.4, which prevents a drop in intracellular pH (Dechant et al., 2010), failed to inhibit the confinement of chromatin and mRNPs upon glucose starvation (Figure 4G and H). Thus, the starvation-induced reduction of pHi is neither sufficient nor necessary and cannot fully explain the observed confinement of macromolecular mobility. Starvation induces a reduction in cellular volume In the course of our experiments, we found that the nuclear volume decreased upon glucose withdrawal (Figure 5A). Since nuclear volume and cellular volume are generally tightly linked (Neumann and Nurse, 2007; Jorgenesen et al., 2002), we utilized a Coulter Counter to examine whether cell size changes upon glucose starvation. Indeed, the median cell volume decreased from 101.6 fL (σ = 54.6) in glucose to 86.1 fL (σ = 45.6) in starved cells, corresponding to a volume reduction of ~15% (Figure 5B). In addition, we observed that the yeast vacuole, an organelle involved in various processes including protein degradation and metabolite storage, swelled in size under glucose starvation conditions (Figure 5C). In non-starved cells, the vacuole-to-cell volume ratio was 0.25 ± 0.02, whereas for starved cells this ratio increased to 0.40 ± 0.01 (mean ± standard error for three independent experiments) (Figure 5D). In combination, this vacuolar volume expansion together with the decrease in total cell and nuclear volume, reduces the cytoplasmic space that is available for diffusing molecules upon glucose starvation by ~30% ('Materials and methods'). Figure 5 with 2 supplements see all Download asset Open asset Starvation induces a constriction in cell size and an expansion in vacuolar volume (Figure 5—figure supplements 1 and 2). (A) Nuclear volume after acute glucose starvation. Histograms of nuclear volumes measured by reconstruction from three-dimensional image stacks using Imaris. The p-value resulting from a two-tailed t-test on the average volume in each condition (unpaired values assuming equal variance) is p<0.001. (B) Histograms of cell volumes of log-growing and acutely starved yeast cells. Log-growing cells, (+) glucose, were acutely starved of glucose, (–) glucose, and cell volume was measured using a Beckman Coulter Multisizer 3 (see 'Materials and methods'). Approximately 50,000 cells were measured for each condition. (C) Cytoplasmic free GFP (green) and vacuolar membrane protein Vph1-mCherry (magenta) fluorescence images. Scale bar: 10 μm. (D) Quantification of vacuole-to-cell volume ratio. Error bars represent standard errors about the mean (N = 3 independent experiments with > 55 cells per experiment). (E) Log-log MSD plot of the POA1 locus after treatment with increasing concentrations of NaCl. Cells were imaged approximately 10 min after hyperosmotic shock. (F) Log-log MSD plot of the GFA1 mRNP after treatment as described in (E). https://doi.org/10.7554/eLife.09376.014 A reduction in cellular volume is sufficient to confine macromolecular mobility We hypothesized that such a large reduction of the accessible cellular volume may be sufficient to induce the observed macromolecular confinement, for example, by an increase in macromolecular crowding or changes in cellular viscosity (Luby-Phelps, 2000). If the hypothesis was correct that a decrease of cell volume was the singular response that confines macromolecular mobility, two predictions could be made: (1) lowering the pHi by the addition of K+Sorbate might induce a decrease in cellular volume since high concentrations of potassium sorbate can replicate the confinement of" @default.
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• W2983642713 title "Author response: A glucose-starvation response regulates the diffusion of macromolecules" @default.
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