Journal: Molecular Biology of the Cell

Article Title: Transient activation of fission yeast AMPK is required for cell proliferation during osmotic stress

doi: 10.1091/mbc.E17-04-0235

Figure Lengend Snippet: Transient activation of Ssp2 by hyperosmotic stress. (A) Western blot showing levels of Ssp2-pT189 in response to the indicated environmental stresses. Treatments were for 15 min. We used α-myc as a loading control for total Ssp2. For α-Ssp2-pT189, asterisks denote background bands, and arrowheads mark the Ssp2-pT189 band. (B) Western blot showing activation kinetics of Ssp2-pT189 in response to 1 M KCl. We used 0.1% glucose treatment as a control for Ssp2-pT189 induction and α-myc as a loading control for total Ssp2. (C) Western blot showing activation kinetics of Ssp2-pT189 in response to 1.2 M sorbitol. We used 0.1% glucose treatment as a control for Ssp2-pT189 induction and α-myc as a loading control for total Ssp2. (D) Western blot showing activation kinetics of Ssp2-pT189 in response to 1 M NaCl. We used 1 M KCl treatment as a control for Ssp2-pT189 induction and α-myc as a loading control for total Ssp2. (E) Localization of Scr1-mEGFP with control EMM4S treatment or EMM4S plus 1 M KCl treatment using a microfluidics device. Images are single focal planes. Preswitch indicates image taken before switch to control or 1 M KCl medium. Scale bar, 8 μm. (F) Quantification of Scr1-mEGFP nuclear localization from microfluidics-based movies as in E. More than 50 cells/condition. Mean ± SD based on three individual biological replicates.

Article Snippet: For and , cells were imaged using a CellASIC ONIX Y04C Microfluidics Plate in conjunction with the ONIX microfluidic perfusion platform (CellASIC).

Techniques: Activation Assay, Western Blot, Control

Journal: Molecular Biology of the Cell

Article Title: Transient activation of fission yeast AMPK is required for cell proliferation during osmotic stress

doi: 10.1091/mbc.E17-04-0235

Figure Lengend Snippet: ssp2 ∆ mutants arrest growth and division during osmotic stress. (A) Differential interference contrast (DIC) images of selected time points for wild-type or ssp2∆ cells growing in a microfluidics device before and after exposure to 1 M KCl. Yellow triangles indicate ssp2∆ cells; unmarked cells are wild type. Time is indicated in hours:minutes. (B) Quantification of total cell number for wild-type vs. ssp2∆ strains after shift to 1 M KCl. Cells were imaged in time lapse using microfluidics, as in A. Cells were manually counted from each time frame, and only cells that were present in the imaging field throughout the entire experiment were counted.

Article Snippet: For and , cells were imaged using a CellASIC ONIX Y04C Microfluidics Plate in conjunction with the ONIX microfluidic perfusion platform (CellASIC).

Techniques: Imaging

Journal: Nature Metabolism

Article Title: Cell cycle-linked vacuolar pH dynamics regulate amino acid homeostasis and cell growth

doi: 10.1038/s42255-023-00872-1

Figure Lengend Snippet: a , Fluorescence intensity fold-change of SEP (blue squares) and v-SEP (red squares) from cells permeabilized in situ with digitonin and exposed to solutions of different pH. Fluorescence was measured with a spectrophotometer. The expected vacuolar pH range is boxed in magenta. b , A schematic of the ratiometric pH-sensitive reporter composed of the acid sensitive v-SEP fluorophore and the acid insensitive mCherry fluorophore targeted to the vacuole by the N-terminal fusion of the first 50 amino acids of carboxypeptidase Y (CPY leader). The micrograph shows both fluorophores localized to the vacuole in a single cell. c , Time-series data show the ratio of vacuolar v-SEP intensity to mCherry intensity over time for two representative cells grown in medium containing glucose and amino acids (SDC) and imaged every 2 min for 1,000 min in microfluidics chambers (CellASIC). Dashed lines indicate the timing of bud emergence. d , The 1,417 single cell ratiometric fluorescence traces of v-SEP/mCherry in cells grown in custom-built microfluidics devices in the presence of SDC. Fluorescence data are aligned to the first peak of vacuolar pH alkalinization. e , Single frames of timelapse data show brightfield and the ratiometric signal of vacuolar accumulated BCECF. Absolute pH values are calculated from in situ generated standard curves. A red arrow highlights the peak of a vacuolar alkalinization event and the change in refractive index in the brightfield panel. f , Absolute pH changes calculated from ratiometric BCECF imaging plotted over time for two representative cells. g , Peak pH and minimum pH observed over 129 cell cycle events from 27 different cells. Average values are denoted by horizontal red bars. h , The difference between peak and minimum pH values within 129 discrete cell cycle events from 27 cells. Average value is denoted by a horizontal red bar. i , A schematic model shows vacuolar pH changes as wild-type (WT) cells progress through the cell cycle. In unbudded cells (left), vacuolar pH is more acidic and as cells grow (right), the organelle becomes less acidic.

Article Snippet: Numbers inset below bar graphs are the median period of vacuolar pH oscillations. b , Cells grown in SDC imaged every 2 min for 1,000 min in microfluidics chambers (CellASIC) and treated in the device with rapamycin (vertical red bar). v-SEP intensity over time is shown for two representative cells. c , Cells grown in SDC imaged every 2 min for 1,000 min in microfluidics chambers (CellASIC) and treated in the device with ConcA (vertical red bar). v-SEP intensity over time is shown for two representative cells.

Techniques: Fluorescence, In Situ, Spectrophotometry, Generated, Refractive Index, Imaging

Journal: Nature Metabolism

Article Title: Cell cycle-linked vacuolar pH dynamics regulate amino acid homeostasis and cell growth

doi: 10.1038/s42255-023-00872-1

Figure Lengend Snippet: a , A schematic of the PSD pipeline to transform v-SEP intensity time-series data from cells grown in SDC (top) or YNBD (bottom) to amplitude versus frequency plots. Dominant amplitude is proportional to the magnitude of oscillatory pH changes over the imaging period (oscillatory vacuolar pH amplitude). b , Oscillatory vacuolar pH amplitude was obtained using the PSD pipeline for 339 cells grown in SDC (+amino acids (AA) +glucose), 133 cells grown in SCEG (+AA, +ethanol/glycerol) and 321 cells grown in YNBD (−AA, +glucose). Median vacuolar pH amplitude is displayed as box-and-whisker plots with whiskers calculated as 1.5 × the interquartile ranges, median values indicated by the horizontal red bar and P values are calculated by a two-sided Dunn’s test with Holm adjustment. Numbers inset below bar graphs are the median period of vacuolar pH oscillations. c , Cells grown in YNBD imaged every 2 min for 1,000 min in microfluidics chambers (CellASIC). v-SEP intensity over time is shown for two representative cells. Vertical dashed lines indicate the timing of bud emergence. d , Oscillatory vacuolar pH amplitude from cells grown in the presence of individual amino acid addbacks. Median vacuolar pH amplitude is displayed as box-and-whisker plots with whiskers calculated as 1.5 × the interquartile ranges, median values indicated by the horizontal black bar. Note, the y axis is fixed at 30,000 AU. Cell number used in the analysis and periodicity of vacuolar pH oscillations in minutes for each condition: Lys ( n = 255, 111 ± 3 min), His ( n = 132, 110 ± 5 min), Gly ( n = 92, 104 ± 10 min), Pro ( n = 26, 82 ± 18 min), Gln ( n = 64, 96 ± 11 min), Ser ( n = 60, 95 ± 8 min), Glu ( n = 90, 87 ± 10 min), Thr ( n = 54, 91 ± 13 min), Arg ( n = 280, 110 ± 3 min), Val ( n = 56, 106 ± 10 min), Asn ( n = 163, 105 ± 3 min), Asp ( n = 102, 100 ± 10 min), Ile ( n = 420, 98 ± 2 min), Trp ( n = 121, 112 ± 4 min), Leu ( n = 356, 104 ± 2 min), Ala ( n = 157, 97 ± 3 min), Phe ( n = 429, 90 ± 2 min) and Tyr ( n = 334, 101 ± 2 min). e , Cells grown in SCEG imaged every 2 min for 1,000 min in microfluidics chambers (CellASIC). v-SEP intensity over time is shown for two representative cells. Dashed lines indicate the timing of bud emergence. See Source Data for additional curves.

Article Snippet: Numbers inset below bar graphs are the median period of vacuolar pH oscillations. b , Cells grown in SDC imaged every 2 min for 1,000 min in microfluidics chambers (CellASIC) and treated in the device with rapamycin (vertical red bar). v-SEP intensity over time is shown for two representative cells. c , Cells grown in SDC imaged every 2 min for 1,000 min in microfluidics chambers (CellASIC) and treated in the device with ConcA (vertical red bar). v-SEP intensity over time is shown for two representative cells.

Techniques: Imaging, Whisker Assay

Journal: Nature Metabolism

Article Title: Cell cycle-linked vacuolar pH dynamics regulate amino acid homeostasis and cell growth

doi: 10.1038/s42255-023-00872-1

Figure Lengend Snippet: a , Oscillatory vacuolar pH amplitude of wild-type cells and cells lacking the SPS components, Ptr3 (288 cells), Ssy1 (447 cells) and Ssy5 (399 cells), grown in SDC. Median vacuolar pH amplitude is displayed as box-and-whisker plots with whiskers calculated as 1.5 × the interquartile ranges, median values indicated by the horizontal red bar and P values are calculated by a two-sided Dunn’s test with Holm adjustment. Numbers inset below bar graphs are the median period of vacuolar pH oscillations. b , Cells grown in SDC imaged every 2 min for 1,000 min in microfluidics chambers (CellASIC) and treated in the device with rapamycin (vertical red bar). v-SEP intensity over time is shown for two representative cells. c , Cells grown in SDC imaged every 2 min for 1,000 min in microfluidics chambers (CellASIC) and treated in the device with ConcA (vertical red bar). v-SEP intensity over time is shown for two representative cells. Dashed lines indicate the timing of bud emergence. d , Oscillatory vacuolar pH amplitude of WT cells and cells lacking the Fab1 components, Vac7 (177 cells), Vac14 (287 cells), Atg18 (200 cells), Fab1 (21 cells) and Fig4 (206 cells), grown in SDC. Median vacuolar pH amplitude is displayed as box-and-whisker plots with whiskers calculated as 1.5 × the interquartile ranges, median values indicated by the horizontal red bar and P values are calculated by a two-sided Dunn’s test with Holm adjustment. Numbers inset below bar graphs are the median period of vacuolar pH oscillations. e , Oscillatory vacuolar pH amplitude of wild-type cells and cells lacking the CDK5 homolog Pho85 (201 cells) and associated cyclins Pho80 (240 cells), Pcl6 (315 cells), Pcl7 (277 cells) and the Pcl6 and Pcl7 double mutant (206 cells). Cells were grown in SDC. Median vacuolar pH amplitude is displayed as box-and-whisker plots with whiskers calculated as 1.5 × the interquartile ranges, median values indicated by the horizontal red bar and P values are calculated by a two-sided Dunn’s test with Holm adjustment. Numbers inset below bar graphs are the median period of vacuolar pH oscillations. f , Oscillatory vacuolar pH amplitude of WT cells and cells lacking seven putative Pho85 phospho-sites in Vac7 ( vac7-7A , 228 cells) and 21 putative phospho-sites in Fab1 ( fab1-21A , 168 cells) and the double mutant ( vac7-7A fab1-21A , 257 cells). Cells were grown in SDC. Median vacuolar pH amplitude is displayed as box-and-whisker plots with whiskers calculated as 1.5 × the interquartile ranges, median values indicated by the horizontal red bar and P values are calculated by a two-sided Dunn’s test with Holm adjustment. Numbers inset below bar graphs are the median period of vacuolar pH oscillations. See Source Data for additional curves.

Article Snippet: Numbers inset below bar graphs are the median period of vacuolar pH oscillations. b , Cells grown in SDC imaged every 2 min for 1,000 min in microfluidics chambers (CellASIC) and treated in the device with rapamycin (vertical red bar). v-SEP intensity over time is shown for two representative cells. c , Cells grown in SDC imaged every 2 min for 1,000 min in microfluidics chambers (CellASIC) and treated in the device with ConcA (vertical red bar). v-SEP intensity over time is shown for two representative cells.

Techniques: Whisker Assay, Mutagenesis

Journal: bioRxiv

Article Title: Starvation induces shrinkage of the bacterial cytoplasm

doi: 10.1101/2020.12.06.413849

Figure Lengend Snippet: A) Representative images of phase-contrast, cytoplasmic GFP, and periplasmic mCherry images of KC1193 cells after transitioning from LB to M9 salts for 4 h. Shrinkage of the cytoplasm was evident by the bright periplasmic mCherry signal. B) The relative cytoplasmic area of KC1193 cells (as measured by the GFP signal relative to the total cell area with GFP or mCherry signal) plateaued only after multiple washes. n > 100 cells for each condition, and p -values were calculated from two-tailed Student’s t -tests. C) After shifting from LB to M9 salts in a microfluidic chamber, the cytoplasmic area of KC1193 cells continued to decrease for several hours. D) The rate of shrinkage was faster in cells transitioned from LB to M9 salts compared to transitioning from M9 glucose to M9 salts. Data points are mean ± standard error of mean (sem) for n>100 cells in each condition. E) Cytoplasmic shrinkage occurs predominantly at the new pole. After pulse-chase labeling with Alexa488-WGA to label the cell periphery, the pole with separation between the cytoplasm and cell wall was almost always at the unlabeled (new) cell pole. Inset: a typical cell with shrinkage in the new (dark) pole. F) Quantitative phase imaging demonstrated that starved cells have higher dry-mass density than exponentially growing cells, yet the density is still lower compared to stationary-phase cells (n > 200 cells for each condition). G) The total dry mass of starved cells was similar to that of exponentially growing cells. Inset: typical quantitative phase images for each condition, with the white arrow highlighting the pole with shrinkage. Scale bar: 1 µm. H) Proteomics measurements highlighted classes of proteins involved in translation and DNA replication with significantly lower abundance in starved cells ( p < 0.01, FDR < 0.05 with bootstrapping). Colored circles represent data from individual proteins. White circles are mean values across all proteins in the class and error bars are 1.5 times the interquartile range.

Article Snippet: Overnight cultures were diluted 100-fold into 1 mL of fresh LB and incubated for 2 h with shaking at 37 °C, excepting experiments in minimal medium, for which overnight cultures were diluted into M9+0.4% glucose and incubated for 4 h. Cells were imaged in B04A microfluidic perfusion plates (CellASIC Corp.) and medium was exchanged using the ONIX microfluidic platform (CellASIC Corp.).

Techniques: Two Tailed Test, Pulse Chase, Labeling, Imaging

Journal: bioRxiv

Article Title: Starvation induces shrinkage of the bacterial cytoplasm

doi: 10.1101/2020.12.06.413849

Figure Lengend Snippet: A) Time-lapse images of KC1193 cells grown in fresh LB in a microfluidic flow cell for 10 min, then switched to spent LB for 100 min, then switched back to fresh LB. The osmolalities of the fresh and spent LB were equalized. The periplasmic area has expanded after 20 min in spent LB, and gradually shrinks after the resumption of growth in fresh LB. B) Quantification of single-cell growth rate and periplasmic area revealed that growth dropped to zero over ∼10 min after switching to spent LB. The periplasmic area only started to increase after the growth rate reached zero, and increased steadily over the 100 min in spent LB. After the switch back to fresh LB, growth rate immediately started to increase, gradually reaching pre-spent LB levels after >60 min. The resolution of the periplasmic area was coincident with the growth-rate increase. Data points are mean ± sem with n >150 cells.

Article Snippet: Overnight cultures were diluted 100-fold into 1 mL of fresh LB and incubated for 2 h with shaking at 37 °C, excepting experiments in minimal medium, for which overnight cultures were diluted into M9+0.4% glucose and incubated for 4 h. Cells were imaged in B04A microfluidic perfusion plates (CellASIC Corp.) and medium was exchanged using the ONIX microfluidic platform (CellASIC Corp.).

Techniques: