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 cell biology

Article Title: Reversible amyloids of pyruvate kinase couple cell metabolism and stress granule disassembly

doi: 10.1038/s41556-021-00760-4

Figure Lengend Snippet: (A) Schematic representation of the domains of Cdc19. The four mutated residues located within the LCR in the Cdc19 irrev mutant are indicated (red circles). (B)-(C) Both Cdc19 WT and Cdc19 irrev form ThT- and Congo Red (CR)-positive aggregates upon heat shock in vitro . Purified Cdc19 WT , Cdc19 irrev and a non-aggregating Cdc19 mutant as negative control (Cdc19 ΔPEP ) were incubated with ThT (B) or CR (C) at 4 °C or after heat shock (42 °C, 10 min). Fluorescence emission was measured at 490 nm or 614 nm, respectively. Graphs show mean ± S.E.M. of three independent experiments (two-tailed t-test, ThT: P WT = 0.0000149, P irrev = 0.0091, CR: P WT = 0.0003, P irrev = 0.0128). (D) In vivo -formed Cdc19 WT and Cdc19 irrev aggregates are CR-positive. Cells expressing GFP-tagged Cdc19 WT or Cdc19 irrev were harvested when exponentially growing or after heat shock (42 °C, 30 min) and lysed. Cdc19-GFP was immobilized in a GFP-trap microfluidic chamber and stained with CR. GFP and CR signals were detected by fluorescence microscopy, and merged to visualize co-localization. Arrowheads indicate CR-positive Cdc19-GFP aggregates. Images are representative of three independent experiments. Scale bar: 10 μm. (E) Limited-Proteolysis Mass Spectrometry (LiP-MS) results indicate that Cdc19 WT and Cdc19 irrev undergo comparable structural transitions upon aggregation in vitro and in vivo . Purified soluble or aggregated (42 °C, 10 min) Cdc19 WT or Cdc19 irrev , as well as cell extracts obtained from cells expressing Cdc19 WT -GFP or Cdc19 irrev -GFP harvested during exponential growth or stationary phase (2 days) to induce aggregation were analysed by LiP-MS as described in the (n = 3 independent experiments). Peptides detected in soluble and aggregated Cdc19 WT and Cdc19 irrev are displayed in volcano plots, and upregulated (red) or downregulated (blue) conformation-sensitive peptides are highlighted. Conformation-specific LiP-MS-peptides detected in vitro and in vivo were mapped to the Cdc19 schematic drawing (green). (F) Intracellular ATP levels (mM) were determined in the indicated strains after heat shock (42 °C, 30 min) and recovery (30 °C, 60 min). Mean ± S.E.M. of n = 5 independent experiments is shown (two-tailed Mann-Whitney test, P = 0.0079). Source data for all graphical representations are found in .

Article Snippet: For time-lapse experiments, cells were loaded in microfluidic plates (CellASIC ONIX2, Merck Millipore) at 30 °C [ ].

Techniques: Mutagenesis, In Vitro, Purification, Negative Control, Incubation, Fluorescence, Two Tailed Test, In Vivo, Expressing, Staining, Microscopy, Mass Spectrometry, MANN-WHITNEY

Journal: Wellcome Open Research

Article Title: Cdc14 phosphatase directs centrosome re-duplication at the meiosis I to meiosis II transition in budding yeast

doi: 10.12688/wellcomeopenres.10507.2

Figure Lengend Snippet: Wild type (AM16065), cdc14-1 (AM16066), slk19Δ (AM16110) and spo12Δ (AM15985) cells carrying TUB1-GFP and PDS1-tdTomato were induced to sporulate, released from prophase I arrest and imaged at 10 min intervals for a total of 12 h in a microfluidics device. ( A ) Representative images are shown. The black arrow denotes Pds1 degradation, marking entry of cells into anaphase I. The white asterisk marks the time of spindle breakdown. ( B ) The time taken for complete spindle disassembly after Pds1 degradation was recorded for individual cells and plotted for a total of 67 wild type cells and 100 of each of cdc14-1 , slk19Δ and spo12Δ cells. Mean rates of spindle breakdown are shown (red line), with error bars representing standard deviation. The two-tailed Student’s t -test was used to calculate p values. ( C ) Inactivation of Cdc14 results in abnormal spindle behaviour. Cells were categorised based on spindle morphology as indicated in the legend.

Article Snippet: Live-cell meiotic movies were generated using CellASIC® ONIX Y04D Microfluidics plates (Merck Millipore).

Techniques: Standard Deviation, Two Tailed Test

Journal: Wellcome Open Research

Article Title: Cdc14 phosphatase directs centrosome re-duplication at the meiosis I to meiosis II transition in budding yeast

doi: 10.12688/wellcomeopenres.10507.2

Figure Lengend Snippet: ( A ) Sporulating CDC14-GFP SPC42-tdTomato cells (AM11517) were imaged in a microfluidics chamber at 15 min intervals for a total of 12 h. Example of a cell in which Cdc14-GFP localizes asymmetrically to one SPB during anaphase I (arrows). ( B and C ) Wild type cells as in ( A ) were released from a prophase I arrest and imaged at 30 min intervals on agarose pads. ( B ) The ratio of Cdc14-GFP/Spc42-tdTomato signal per SPB is shown with error bars representing standard error. Cells were classified into different cell cycle stages (metaphase I, anaphase I, metaphase II, anaphase II) by scoring the number of SPBs, the distance between them and Cdc14 nucleolar sequestration. The two-tailed Student’s t- test was used to calculate p values (* p<0.001) n= 50 cells. ( C ) Co-localization of Cdc14-GFP with SPBs was scored in the indicated cell cycle stages. ( D and E ) Cdc14 localises prematurely to SPBs in the absence of Cdc55. pCLB2-3HA-CDC55 cells carrying CDC14-GFP and SPC42-tdTomato (AM13123) were induced to sporulate, released from pGAL-NDT80 block and imaged 3h later on agarose pads. Cdc14 localisation was classified into three categories: sequestered in nucleolus (1), partially released from nucleolus (2) and completely released from nucleolus (3). ( D ) Example images of with numbered arrows showing examples of each category. ( E ) Co-localisation of Cdc14 with SPBs was scored in 100 cells of each category.

Article Snippet: Live-cell meiotic movies were generated using CellASIC® ONIX Y04D Microfluidics plates (Merck Millipore).

Techniques: Two Tailed Test, Blocking Assay

Journal: Wellcome Open Research

Article Title: Cdc14 phosphatase directs centrosome re-duplication at the meiosis I to meiosis II transition in budding yeast

doi: 10.12688/wellcomeopenres.10507.2

Figure Lengend Snippet: ( A ) Wild type (AM13989) cells containing SPC42-tdTomato were induced to sporulate, released from pGAL-NDT80 block and imaged at 15 minute intervals for a total of 12 hours in a microfluidics chamber. Representative images of wild type cells are shown. ( B ) SPBs are duplicated at meiotic prophase. Wild type cdc14-1 (AM16163), and pCLB2-3HA-CDC55 (AM15984) cells containing SPC42-tdTomato and pGAL-NDT80 and were resuspended in sporulation medium in the absence of β-oestradiol and imaged immediately (t=0) or after 3 and 6h as they progress into the prophase I arrest due to the absence of Ndt80. Individual SPB foci were quantified and mean total SPB fluorescence intensity (F i ) per cell was plotted, with error bars (obscured by the markers) representing standard error, n=100 per timepoint. ( C – E ) SPB fluorescence was quantified as in B from movies of live cells after release from the prophase I arrest by addition of β-oestradiol as in ( A ). The first time point at which a cell contained 2 Spc42-tdTomato foci was defined as 1h and SPB fluorescence in the preceding 4 (1 focus) and following 4 time points was quantified (n=10 cells). Note that at the 1.5h time point 7 wild type cells carried 2 Spc42-tdTomato foci, while 3 wild type cells carried 4 Spc42-tdTomato foci. ( F and G ) LFQ Proteomic analysis of SPB composition and environment in wild type and cdc14-1 cells. Strains used were AM1835 (no tag), AM11444 ( SPC42-3FLAG ), AM9459 ( cdc14-1 ) and AM11443 ( cdc14-1 SPC42-3FLAG ) After 4 h, cells were harvested and SPBs purified by anti-FLAG immunoprecipitation. Peptides were generated by in-gel trypsin digestion and LC-MS data sets for 3 biological replicas was analysed using MaxQuant software. ( F ) Example silver stained SDS-PAGE showing Spc42-3FLAG immunoprecipitates of one of three biological replicas used in LFQ proteomic analysis. BSA standards were used to estimate protein concentration. ( G ) Statistical analysis of relative LFQ intensity output was carried out using Perseus. Volcano plot shows –log of P values versus ratio of wild type/c dc14-1 for all 254 proteins in >5 columns. No significant change in composition and environment was observed between wild type and cdc14-1 SPBs. (FDR = 0.05, s0 = 1). Proteins of interest are highlighted as follows: red = SPB components; blue = Cdc14; green = Bfa1/Bub2. See also https://osf.io/g5cmh/ .

Article Snippet: Live-cell meiotic movies were generated using CellASIC® ONIX Y04D Microfluidics plates (Merck Millipore).

Techniques: Blocking Assay, Fluorescence, Purification, Immunoprecipitation, Generated, Liquid Chromatography with Mass Spectroscopy, Software, Staining, SDS Page, Protein Concentration