A glucose-starvation response regulates the diffusion of macromolecules

  1. Ryan P Joyner
  2. Jeffrey H Tang
  3. Jonne Helenius
  4. Elisa Dultz
  5. Christiane Brune
  6. Liam J Holt
  7. Sebastien Huet
  8. Daniel J Müller
  9. Karsten Weis  Is a corresponding author
  1. University of California, Berkeley, United States
  2. ETH Zurich, Switzerland
  3. New York University School of Medicine, United States
  4. University of Rennes, France
7 figures, 6 videos and 3 tables

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 …

https://doi.org/10.7554/eLife.09376.003
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) …

https://doi.org/10.7554/eLife.09376.004
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 …

https://doi.org/10.7554/eLife.09376.005
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 …

https://doi.org/10.7554/eLife.09376.007
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 …

https://doi.org/10.7554/eLife.09376.008
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 …

https://doi.org/10.7554/eLife.09376.009
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 …

https://doi.org/10.7554/eLife.09376.010
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., …

https://doi.org/10.7554/eLife.09376.011
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.

https://doi.org/10.7554/eLife.09376.012
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…

https://doi.org/10.7554/eLife.09376.013
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 …

https://doi.org/10.7554/eLife.09376.014
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.

https://doi.org/10.7554/eLife.09376.015
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

https://doi.org/10.7554/eLife.09376.016
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 …

https://doi.org/10.7554/eLife.09376.017
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 …

https://doi.org/10.7554/eLife.09376.018
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 …

https://doi.org/10.7554/eLife.09376.019
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) …

https://doi.org/10.7554/eLife.09376.028

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.09376.025

Tables

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 mRNP
KαKαKαKαKα
(+) 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----
DMSO0.00590.560.00460.700.00600.610.04910.830.05410.83
Nocodazole0.00400.480.00250.570.00460.510.03640.850.03970.85
Latrunculin A0.00380.500.00240.630.00380.550.04760.820.05500.81
Nocodazole + LatA0.00280.480.00140.490.00260.520.03030.810.03670.82
2 mM K+Sorbate0.00560.730.00510.800.00590.680.04020.820.02960.80
4 mM K+Sorbate0.00500.750.00440.720.00480.710.04060.780.02420.79
6 mM K+Sorbate0.00390.700.00180.660.00270.660.03780.760.02700.78
8 mM K+Sorbate0.00230.640.00120.610.00140.610.03400.760.01640.77
0.4 M NaCl0.00300.690.00260.75----0.01290.830.01460.85
0.6 M NaCl0.00120.600.00110.58----0.00470.830.00570.84
0.8 M NaCl0.00090.630.00110.63----0.00130.670.00160.77
Quiescence------------0.00040.290.00150.68
0.02% Azide (Wash)0.00370.74--------0.02930.82----
0.02% Azide (Spike)0.00120.67--------0.01550.81----
Table 2

Yeast strains used in this study.

https://doi.org/10.7554/eLife.09376.026
StrainGenotypeSource
KWY165W303; MATa ura3-1 leu2-3 his3-11,15 trp1-1 ade2-1This Study
KWY1622W303; MATα ybr022w::256LacO::LEU2 his3::LacI-GFP::HIS3 trp1::dsRED-HDEL::TRP1Green et al., 2012
KWY3541KWY 1622, cbp2∆::KANMXThis Study
KWY3538W303; MATα his3::LacI-GFP::HIS3 trp1::dsRED-HDEL::TRP1 256LacO::URA3This Study
KWY2848W303; MATα his3::LacI-GFP::HIS3 trp1::dsRED-HDEL::TRP1 yel021w::128LacO::URA3This Study
KWY4586W303; MATa ybr022w::112TetO::URA3 yfr023w::256LacO::LEU2 his3::LacI-GFP_TetR-3XmCherry::HIS3This Study
KWY970KWY 165, ura3::pHIS-GFP-TUB1::URA3This Study
KWY3661W303; MATa pHluorin::URA3This Study
KWY4796W303; MATα trp1::dsRED-HDEL::TRP1 leu2::TetR-GFP::LEU2This Study
KWY4736W303 MATa/S288c MATα NDC1/ndc1::NDC1-tdTomato::KANMX GFA1/gfa1::GFA1-24PP7 3xYFP-PP7-CFP::HIS3This Study
KWY4737W303 MATa/S288c MATα NDC1/ndc1::NDC1-tdTomato::KANMX FBA1/fba1::FBA1-24PP7 3xYFP-PP7-CFP::HIS3This Study
KWY5112W303; MATa ura3-1 leu2-3 his3-11,15 trp1-1 ade2-1 VPH1::VPH1-mCherry::KANMX leu2::GFP::CaURA3This Study
KWY6241CAF13 (S. pombe wildtype)This Study
yYB5978S288c; MATa his3∆1 leu2∆0 ura3∆0 met15∆0 LYS2 ADE2 TRP1 bar1::kanMXCaudron and Barral, 2013
Table 3

Plasmids used in this study.

https://doi.org/10.7554/eLife.09376.027
PlasmidDescriptionSource
pKW2734pHMX-256LacO (CEN, URA3)This Study
pKW2957pYES2-PACT1-pHluorin (CEN, URA3)Orij et al., 2009
pKW544MET25pro-PP7-CP-3xYFP (CEN, HIS3)This Study

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