A pH-driven transition of the cytoplasm from a fluid- to a solid-like state promotes entry into dormancy
- Max Planck Institute of Molecular Cell Biology and Genetics, Germany
- Max Planck Institute for the Physics of Complex Systems, Germany
- Technische Universität Dresden, Germany
Figures
Figure 1 with 3 supplements
Reduced mobility of organelles and foreign tracer particles under energy depletion conditions.
(A) Representative trajectories of a range of different organelle markers tracked under control conditions (●, cells in log-phase, upper panel) and upon energy depletion (▲, lower panel). Organelle …
Figure 1—figure supplement 1
GFP-µNS particles form discrete particles in the yeast cytoplasm (left).
These particles explore the cytoplasm over time (middle and right).
Figure 1—figure supplement 2
GFP-µNS particles show size-dependent MSD over short (left) and long (right) observation times.
The fluorescence intensity of particles was used as a proxy for particle size.
Figure 1—figure supplement 3
Same data as in Figure 1—figure supplement 2, shown as log-log plots (left).
The generalized diffusion constant (right) was obtained from the time-averaged MSD of individual trajectories at the lag time of =2 s divided by , where =0.88 is the power-law exponent of the …
Figure 2 with 1 supplement
Energy depletion causes a drop in cytosolic pH, which may explain reduced particle mobility.
(A) MSD of GFP-µNS particles in untreated cells (control), cells treated with 100 μM latrunculin A (LatA) and energy-depleted cells. (B) MSD of GFP-µNS particles in untreated cells (control), cells …
Figure 2—figure supplement 1
Yeast cells expressing the pH sensor pHluorin2 in the cytoplasm (left panel) were used to generate a pH calibration curve (right panel), as described in materials and methods and reported previously (Brett et al., 2005).
https://doi.org/10.7554/eLife.09347.009
Figure 3
Acidification of the cytosol reduces particle mobility.
(A) The cytosolic pH of yeast cells exposed to phosphate buffers of different pH containing 2 mM 2,4-dinitrophenol (DNP) and 2% glucose was measured over time in a microfluidic flow chamber (left). …
Figure 4 with 6 supplements
Characterization of the acidified and energy-depleted cytoplasm from a particle perspective.
(A) The time- and ensemble-averaged MSD are shown as a function of lag-time, together with the fitted power-law scalings as dashed lines. We obtained the following power-law exponents (DNP …
Figure 4—figure supplement 1
Top: The power spectral density (PSD) of particle displacements is shown as a function of frequency .
The dashed lines correspond to a power law scaling of the PSD as with where is the exponent in the power-law scaling of the corresponding MSD (see Figure 4A). Bottom: The PSD after randomly …
Figure 4—figure supplement 2
The cumulative distribution functions (CDFs) of particle displacements for some representative trajectories in all considered conditions (log-phase, energy-depleted, and pH-adjusted to pH 6.0 and pH 7.4).
The dashed lines are fits to CDFs of Gaussian distributions. The inset shows the values of the non-Gaussian parameter (defined in the Materials and methods section) for each individual trajectory in …
Figure 4—figure supplement 3
Left: The probability density functions (PDFs) of particle displacements for all conditions (log-phase, energy-depleted, and pH-adjusted to pH 6.0 and pH 7.4) and at two different lag times after rescaling the displacements both by and the generalized diffusion constant .
The dashed line is the PDF of the Gaussian distribution with the unit variance. Right: The unscaled PDFs of particles’ displacements for log phase and energy depleted cells for two lag times of 0.2 …
Figure 4—figure supplement 4
The directional correlation function, defined as , where is the lag-time, is an integer, is the change in particle position between time and and denotes the length of the vector , for all conditions (log-phase, energy depleted, and pH adjusted to pH 6.0 and pH 7.4) as a function of for lag-time ms.
https://doi.org/10.7554/eLife.09347.017
Figure 4—figure supplement 5
Correlations of subsequent displacements (defined as , where and are the displacement vectors at two consecutive time intervals) were calculated from trajectories recorded in cells adjusted to pH 7.4 and pH 6.0, respectively, and plotted as a function of the initial displacement length .
The lag-time (time used to calculate the displacement) is 200 ms. The linear slope b quantifies the negative correlations of subsequent particle displacements and its magnitude increases from to upon acidification. The cytosolic pH was adjusted by treating cells with phosphate buffers of pH 6.0 and pH 7.4, respectively, containing 2 mM DNP and 2% glucose.
Figure 4—figure supplement 6
Time-averaged (solid lines) and ensemble-averaged (symbols) MSDs of particles in control and energy-depleted cells, respectively (left panel) Time-averaged (solid lines) and ensemble-averaged (symbols) MSDs of particles in cells adjusted to pH 6.0 and pH 7.4 with DNP, respectively (right panel).
Grey areas indicate +/- SEM of the time-averaged MSD.
Figure 5 with 9 supplements
Mechanical characterization of acidified and energy-depleted cells.
(A) The apparent elastic modulus of S. cerevisiae spheroplasts (without rigid cell walls) at pH 7.4 (E = 636 ± 16 Pa (mean ± SEM); N = 249) and pH 6.0 (E = 1459 ± 59 Pa; N = 257) was measured by …
Figure 5—figure supplement 1
(Left) The apparent elastic moduli of S. cerevisiae spheroplasts, incubated in phosphate buffers of different pH in the presence and absence of DNP, were determined by AFM-based indentation.
Independent measurements were repeated at least four times, with a total of N = 242–409 cells, for each condition. Cells were significantly stiffer at pH 6.0 (E = 1459 ± 59 Pa; mean ± SEM) than at …
Figure 5—figure supplement 2
The viscosity of the spheroplasted cells, extracted from the AFM-based indentation-retraction curves, decreased from η = 90 ± 16 Pa s (mean ± SEM; N = 31) at pH 7.4 to η = 70 ± 14 Pa s (N = 23) at pH 6.0.
Boxes show the 25th, 50th (the median), and 75th percentiles, whiskers the spread of the data (and a few outliers). Asterisk * indicates significance level p<0.05 (Mann-Whitney-test).
Figure 5—figure supplement 3
The volume of pH-adjusted (left panel) and sorbitol-treated (right panel) yeast cells was measured with an imaging-based method (see materials and methods).
The cytosolic pH was adjusted by treating cells with phosphate buffers of indicated pH containing 2 mM DNP and 2% glucose. Sorbitol treatment was done in synthetic complete medium. The term …
Figure 5—figure supplement 4
Both low pH and sorbitol treatment cause a reduction in particle mobility.
MSD of GFP-µNS particles in cells exposed to increasing concentrations of sorbitol and in cells adjusted to low pH. The cytosolic pH was adjusted by treating cells with phosphate buffers of pH 5.5 …
Figure 5—figure supplement 5
The diffusivity of a mCherry-GFP fusion protein was measured with fluorescence recovery after photobleaching (FRAP) in cells exposed to different concentrations of sorbitol (left panel), cells adjusted to different cytosolic pH (middle panel), and in energy-depleted cells (right panel).
Sorbitol treatment was done in synthetic complete medium containing the indicated amount of sorbitol. The cytosolic pH was adjusted by treating cells with phosphate buffers of pH 5.5, pH 6.0 and pH …
Figure 5—figure supplement 6
Energy-depleted S. pombe cells retain a rod-like shape in the absence of a cell wall.
Left: Energy-depleted S. pombe cells before (top) and after (bottom) cell wall removal. BF: Bright field image. Calcofluor white staining confirms the presence of a cell wall before and absence of a …
Figure 5—figure supplement 7
Cell wall digesting enzymes work equally well in buffers of different pH.
Top: Still images of cells incubated with and without (Ctrl) cell wall removing enzyme mix in buffers of indicated pH. No sorbitol was added in order to allow for cell lysis upon cell wall removal. …
Figure 5—figure supplement 8
Energy-depleted S. pombe cells were imaged by time-lapse bright field microscopy after addition of the cell wall removing enzyme mix.
Still images show multiple events (yellow arrows) in which the cell body separates from the cell wall sheath and maintains its rod-like shape. Also see corresponding Video 6.
Figure 5—figure supplement 9
Low pH adjusted, rod-shaped S. pombe spheroplasts round up when exposed to glucose-containing medium.
Prior to imaging the spheroplast was kept in phosphate buffer of pH 6.0 containing 1 M sorbitol and 2 mM DNP and did not round up for several hours. At time point 0 min the buffer was replaced with …
Figure 6 with 2 supplements
Acidification of the cytosol causes widespread assembly of cytoplasmic proteins.
(A) The isoelectric points and the molecular weight of all yeast proteins were computed from their primary amino acid sequence and plotted as a virtual 2D gel. The green line indicates optimal …
Figure 6—figure supplement 1
Response of 68 cytoplasmic proteins to a drop in cytosolic pH and to treatment with 1 M sorbitol.
Representative images of proteins that respond with assembly formation to low pH were also tested for their response to 1 M sorbitol (left). The percentage of cells showing protein assemblies at …
Figure 6—figure supplement 2
Different subunits of the hetero-pentameric eIF2B complex colocalize in the same filamentous structures, suggesting that these protein retain their native-like structure when they form higher-order assemblies.
Cells were energy-depleted in phosphate buffer of pH 6.0 for 3 hours prior to imaging.
Figure 7
Acidification of the cytosol promotes survival under energy depletion conditions.
(A) Growth assay of S. cerevisiae cells that were energy-depleted with 20 mM 2-DG and 10 µM antimycin A in growth medium adjusted to pH 7.0 and 6.0, respectively. (B) Similar to A, but cells were …
Author response image 1
Summary of experiments showing that the cell wall was completely removed in our spheroplasting procedure.
(A) A rod-shaped spheroplasted fission yeast cell rounds up when media is added. (B) Acidified control and spheroplasted cells were treated with calcofluor white to stain the cell wall. Note the …
Author response image 2
Comparison of the power spectral density of displacements for trajectories of different length (control conditions).
The black line corresponds to the data set presented in the paper and the blue line is the new data for 227 trajectories that are 10 min long and acquired with 5 s resolution. As we see from this …
Author response image 3
AFM based indentation measurements of the apparent elastic modulus of cells with and without cell wall.
In both cases, the cells were indented up to a maximum force of 2 nN and the resulting force-distance curves were analyzed with the Hertz model as described in the material and Methods section. Note …
Author response image 4
The cell size of S. cerevisiae cells in response to low pH adjustment was measured over time with an RT-DC setup.
Cells were loaded into the microfluidic chip of the RT-DC device immediately after exposure to phosphate buffer of pH 6 containing 2 mM DNP and 2% glucose. The median diameter of more than 530 cells …
Author response image 5
Viscosity of S. cerevisiae spheroplasts at pH 7.4 and pH 6.0 measured by AFM nano-indentation experiments and analyzed according to Rebelo et al.
The viscosity reduces from η= 90 ± 16 Pa s (mean ± SEM; N = 31; median is 81 Pa s) at pH 7.4 to η = 70 ± 14 Pa s (N = 23; median is 59 Pa s) at pH 6.0.
Author response image 6
Normalized velocity autocorrelation function plotted for different lag times τ from 30 ms to 500 ms, which are used to define the velocity from the displacement data (the lag time change is color-coded from blue to red in steps of 10 ms).
Non-vanishing negative correlations indicate that they are not caused by a localization error. Left and right panels show log phase and energy depleted cells data respectively.
Videos
Video 1
Time-lapse microscopy of GFP-µNS particles moving in the S. cerevisiae cytoplasm.
To illustrate how particles explore the yeast cytoplasm over time, the fluorescence channel and the reference bright field channel were merged.
Video 2
Brightfield time-lapse microscopy of S. cerevisiae cells growing in a microfluidic flow chamber.
Cells were exposed to buffers of different pH containing 2 mM DNP as indicated.
Video 3
Fluorescence time-lapse microscopy of GFP-µNS expressing S. cerevisiae cells growing in a microfluidic flow chamber.
Cells were repeatedly exposed to buffers of different pH containing 2 mM DNP as indicated.
Video 4
Brightfield time-lapse microscopy of S. pombe cells during cell wall removal.
Cells were imaged in EMM5 medium containing glucose (control, left panel) or in EMM5 medium without glucose containing 20 mM 2-desoxyglucose (2DG) and 10 µM antimycin A (right panel). Cell wall …
Video 5
Brightfield time-lapse microscopy of S. pombe cells during cell wall removal.
Cells were imaged in phosphate buffer of pH 5.5 (right panel) and pH 7.4 (left) containing 2 mM DNP and 2% glucose. Cell wall removing enzyme mix was added after 30 min of imaging. Buffers contained …
Video 6
Brightfield time-lapse microscopy of energy-depleted S. pombe cells during enzymatic cell wall removal.
Cells were energy-depleted in EMM5 medium (pH 6.0) without glucose supplemented with 20 mM 2-deoxyglucose and 10 μM antimycin A for 2 hr before imaging. Imaging was then done in cell wall removal …
Video 7
Brightfield time-lapse microscopy of a S. pombe spheroplast.
The spheroplast was generated prior to imaging in phosphate buffer of pH 6.0 containing 1 M sorbitol, 2 mM DNP and cell wall digesting enzyme mix and kept in this cell wall removal buffer for …
Video 8
Fluorescence time-lapse microscopy of Gcn3-GFP (left side) and GFP-µNS (right side) expressing yeast cells growing in a microfluidic flow chamber.
Cells were exposed to a phosphate buffer of pH 5.5 containing 2 mM DNP and 2% glucose as indicated.
Additional files
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Supplementary file 1
List of strains used in this study.
- https://doi.org/10.7554/eLife.09347.039
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Supplementary file 2
List of plasmids used in this study.
- https://doi.org/10.7554/eLife.09347.040
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Supplementary file 3
Table with osmolality values of media and buffers.
- https://doi.org/10.7554/eLife.09347.041
