A glucose-starvation response regulates the diffusion of macromolecules
- University of California, Berkeley, United States
- ETH Zurich, Switzerland
- New York University School of Medicine, United States
- University of Rennes, France
Figures
Figure 1 with 2 supplements
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 …
Figure 1—figure supplement 1
Glucose starvation affects the mobility of nuclear and cytoplasmic objects.
(A) Individual log-log MSD plots of POA1 loci in non-starved (left) and starved (right) cells. (B) Individual log-log MSD plots of GFA1 mRNP particles in non-starved (left) and starved (right) …
Figure 1—figure supplement 2
Starvation confines macromolecular mobility.
(A) Log-log MSD plot of the URA3 locus during exponential growth and after acute starvation. (B) Log-log MSD plot of the GFA1 mRNP during exponential growth and quiescence (see 'Materials and …
Figure 2 with 1 supplement
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 …
Figure 2—figure supplement 1
Starvation confines the cytoskeleton-independent mobility of mRNPs and the cytoskeleton-influenced mobility of chromatin.
(A) Log-log MSD plot of the FBA1 mRNP after treatment as described in Figure 2C. (B) Log-log MSD plot of the URA3 locus after treatment as described in Figure 2C. (C) Log-log MSD plot of the pLacO …
Figure 3 with 1 supplement
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 …
Figure 3—figure supplement 1
Respiration maintains intracellular ATP concentrations after acute glucose starvation.
Log-growing wild-type (WT) and cbp2∆ yeast cells were acutely starved for glucose and back-diluted into media containing 2% dextrose (WT) or maintained in (–) glucose media. Intracellular ATP …
Figure 4 with 2 supplements
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., …
Figure 4—figure supplement 1
A drop in intracellular pH (pHi) titrates macromolecular mobility.
(A) Intracellular pH calibration curve (Orij et al., 2009) (see 'Materials and methods'). Error bars represent SD. (B) Log-log MSD plot of the URA3 locus. Cells were treated as described in Figure 4A.
Figure 4—figure supplement 2
Sodium azide induces a pleiotropic reduction in intracellular pH, which may explain the subsequent confinement of macromolecular mobility.
(A) Intracellular ATP concentrations after differing treatments with 0.02% sodium azide (‘wash’ versus ‘spike’) (see 'Materials and methods'). Intracellular ATP concentrations were determined as in F…
Figure 5 with 2 supplements
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 …
Figure 5—figure supplement 1
Histograms of cell volumes after various treatments.
(A) Cells were treated with K+Sorbate as in Figure 4 and cell volume measured as in Figure 5B. (B) Cells were starved of glucose in either low or high pH (5.0 or 7.4) and cell volume was measured.
Figure 5—figure supplement 2
Hyperosmotic shock increasingly confines macromolecular mobility without affecting intracellular ATP concentrations or intracellular pH.
(A) Intracellular ATP concentrations after treatment with either 0.4 M or 0.8 M NaCl. Cells were treated as described in Figure 5E and intracellular ATP concentrations were determined as in Figure 3A…
Figure 6 with 2 supplements
A conserved glucose starvation response alters the mechanical properties of the cytoplasm (Figure 6—figure supplements 1 and 2).
(A) Dry mass measurements of non-starved and starved yeast. Yeast cells in normal glucose, glucose deprivation, and raffinose growth conditions were lyophilized, and their cellular dry masses were …
Figure 6—figure supplement 1
Cell stiffness negatively correlates with cell size.
Both glucose-starved and non-starved budding yeast cells show an anti-correlation of cell stiffness with cell diameter. Linear fitting R2 value for non-starved cell data = 0.54; R2 value for starved …
Figure 6—figure supplement 2
The actin cytoskeleton is a major consumer of intracellular ATP.
Intracellular ATP concentrations of log-growing yeast were determined as in Figure 3A after treatment with latrunculin A or the solvent control DMSO. The zero minute time point was taken immediately …
Author response image 1
Comparison of acute glucose starvation and 2-deoxyglucose/antimycin A treatments.
(A) MSD curves for GFA1 mRNP particles in non-starved cells (blue), cells acutely starved of glucose (red), and cells acutely starved of glucose additionally treated with 20 mM 2-deoxyglucose (2DG) …
Videos
Video 1
Spheroplasted budding yeast shmoos.
https://doi.org/10.7554/eLife.09376.020
Video 2
Spheroplasted starved budding yeast shmoos.
https://doi.org/10.7554/eLife.09376.021
Video 3
Re-addition of glucose to spheroplasted starved budding yeast shmoos.
https://doi.org/10.7554/eLife.09376.022
Video 4
Spheroplasted fission yeast.
https://doi.org/10.7554/eLife.09376.023
Video 5
Spheroplasted starved fission yeast.
https://doi.org/10.7554/eLife.09376.024
Video 6
Re-addition of glucose to spheroplasted starved fission yeast.
https://doi.org/10.7554/eLife.09376.025Tables
Table 1
Effective diffusion coefficients (K; µm2/s) and anomalous diffusion exponents (α) for macromolecules in each condition.
| Condition | POA1 Locus | pLacO Plasmid | URA3 Locus | GFA1 mRNP | FBA1 mRNP | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| K | α | K | α | K | α | K | α | K | α | |
| (+) Glucose | 0.0057 | 0.69 | 0.0067 | 0.78 | 0.0076 | 0.65 | 0.0420 | 0.83 | 0.0501 | 0.85 |
| (-) Glucose | 0.0023 | 0.64 | 0.0021 | 0.80 | 0.0022 | 0.73 | 0.0131 | 0.77 | 0.0139 | 0.77 |
| (-) Glucose pH 7.4 | 0.0015 | 0.65 | -- | -- | -- | -- | 0.0120 | 0.75 | -- | -- |
| DMSO | 0.0059 | 0.56 | 0.0046 | 0.70 | 0.0060 | 0.61 | 0.0491 | 0.83 | 0.0541 | 0.83 |
| Nocodazole | 0.0040 | 0.48 | 0.0025 | 0.57 | 0.0046 | 0.51 | 0.0364 | 0.85 | 0.0397 | 0.85 |
| Latrunculin A | 0.0038 | 0.50 | 0.0024 | 0.63 | 0.0038 | 0.55 | 0.0476 | 0.82 | 0.0550 | 0.81 |
| Nocodazole + LatA | 0.0028 | 0.48 | 0.0014 | 0.49 | 0.0026 | 0.52 | 0.0303 | 0.81 | 0.0367 | 0.82 |
| 2 mM K+Sorbate | 0.0056 | 0.73 | 0.0051 | 0.80 | 0.0059 | 0.68 | 0.0402 | 0.82 | 0.0296 | 0.80 |
| 4 mM K+Sorbate | 0.0050 | 0.75 | 0.0044 | 0.72 | 0.0048 | 0.71 | 0.0406 | 0.78 | 0.0242 | 0.79 |
| 6 mM K+Sorbate | 0.0039 | 0.70 | 0.0018 | 0.66 | 0.0027 | 0.66 | 0.0378 | 0.76 | 0.0270 | 0.78 |
| 8 mM K+Sorbate | 0.0023 | 0.64 | 0.0012 | 0.61 | 0.0014 | 0.61 | 0.0340 | 0.76 | 0.0164 | 0.77 |
| 0.4 M NaCl | 0.0030 | 0.69 | 0.0026 | 0.75 | -- | -- | 0.0129 | 0.83 | 0.0146 | 0.85 |
| 0.6 M NaCl | 0.0012 | 0.60 | 0.0011 | 0.58 | -- | -- | 0.0047 | 0.83 | 0.0057 | 0.84 |
| 0.8 M NaCl | 0.0009 | 0.63 | 0.0011 | 0.63 | -- | -- | 0.0013 | 0.67 | 0.0016 | 0.77 |
| Quiescence | -- | -- | -- | -- | -- | -- | 0.0004 | 0.29 | 0.0015 | 0.68 |
| 0.02% Azide (Wash) | 0.0037 | 0.74 | -- | -- | -- | -- | 0.0293 | 0.82 | -- | -- |
| 0.02% Azide (Spike) | 0.0012 | 0.67 | -- | -- | -- | -- | 0.0155 | 0.81 | -- | -- |
Table 2
Yeast strains used in this study.
| Strain | Genotype | Source |
|---|---|---|
| KWY165 | W303; MATa ura3-1 leu2-3 his3-11,15 trp1-1 ade2-1 | This Study |
| KWY1622 | W303; MATα ybr022w::256LacO::LEU2 his3::LacI-GFP::HIS3 trp1::dsRED-HDEL::TRP1 | Green et al., 2012 |
| KWY3541 | KWY 1622, cbp2∆::KANMX | This Study |
| KWY3538 | W303; MATα his3::LacI-GFP::HIS3 trp1::dsRED-HDEL::TRP1 256LacO::URA3 | This Study |
| KWY2848 | W303; MATα his3::LacI-GFP::HIS3 trp1::dsRED-HDEL::TRP1 yel021w::128LacO::URA3 | This Study |
| KWY4586 | W303; MATa ybr022w::112TetO::URA3 yfr023w::256LacO::LEU2 his3::LacI-GFP_TetR-3XmCherry::HIS3 | This Study |
| KWY970 | KWY 165, ura3::pHIS-GFP-TUB1::URA3 | This Study |
| KWY3661 | W303; MATa pHluorin::URA3 | This Study |
| KWY4796 | W303; MATα trp1::dsRED-HDEL::TRP1 leu2::TetR-GFP::LEU2 | This Study |
| KWY4736 | W303 MATa/S288c MATα NDC1/ndc1::NDC1-tdTomato::KANMX GFA1/gfa1::GFA1-24PP7 3xYFP-PP7-CFP::HIS3 | This Study |
| KWY4737 | W303 MATa/S288c MATα NDC1/ndc1::NDC1-tdTomato::KANMX FBA1/fba1::FBA1-24PP7 3xYFP-PP7-CFP::HIS3 | This Study |
| KWY5112 | W303; MATa ura3-1 leu2-3 his3-11,15 trp1-1 ade2-1 VPH1::VPH1-mCherry::KANMX leu2::GFP::CaURA3 | This Study |
| KWY6241 | CAF13 (S. pombe wildtype) | This Study |
| yYB5978 | S288c; MATa his3∆1 leu2∆0 ura3∆0 met15∆0 LYS2 ADE2 TRP1 bar1::kanMX | Caudron and Barral, 2013 |
Table 3
Plasmids used in this study.
| Plasmid | Description | Source |
|---|---|---|
| pKW2734 | pHMX-256LacO (CEN, URA3) | This Study |
| pKW2957 | pYES2-PACT1-pHluorin (CEN, URA3) | Orij et al., 2009 |
| pKW544 | MET25pro-PP7-CP-3xYFP (CEN, HIS3) | This Study |
