Journal: Journal of Fungi

Article Title: Specific Functional Features of the Cell Integrity MAP Kinase Pathway in the Dimorphic Fission Yeast Schizosaccharomyces japonicus

doi: 10.3390/jof7060482

Figure Lengend Snippet: Functional roles, localization, and activation pattern of the CIP MAPK Pmk1 in S. japonicus. ( A ) Upper : Exponentially growing S. japonicus wild-type and pmk1 Δ strains were incubated for 10 h in YES medium plus 0.6M KCl, and the percentage of unseptated, septated, and multiseptated cells (represented as mean ± SD from biological triplicates; number of total cells ≥ 200) was determined by fluorescence microscopy after calcofluor white staining. Lower : Representative images are shown; ( B ) Decimal dilutions of the indicated strains growing in YES medium were spotted onto YES plates supplemented with caspofungin (0.5–2 µg/mL) (upper panels), and 0.5 µg/mL FK506 plus 0.1, 0.15, or 0.2 M MgCl 2 (VIC assay; lower panels), and incubated for 3 days at 30 °C before being photographed. Representative experiments are shown; ( C ) S. japonicus and S. pombe wild-type cells expressing genomic Pmk1–GFP fusions were grown in YES medium to mid-log phase or treated for 6 h with 0.2 μM CPT ( S. japonicus ), and observed both by fluorescence and DIC microscopy after calcofluor white staining. Images were acquired as single medial plane images and are inverted for fluorescence; ( D ) S. pombe and S. japonicus wild-type strains expressing genomic Pmk1–GFP fusions were grown in YES medium to mid-log phase, and activated/total Pmk1 were detected with anti-phospho-p44/42 and anti-GFP antibodies, respectively. Anti-Cdc2 was used as a loading control. Relative units as mean ± SD (biological triplicates) for Pmk1 phosphorylation (anti-phospho-p44/42 blot) were determined with respect to the internal control (anti-GFP blot). **, p < 0.005; as calculated by unpaired Student’s t -test; ( E ) S. pombe and S. japonicus wild-type strains expressing genomic Pmk1–GFP fusions were grown in YES medium to mid-log phase, and treated with 0.6 M KCl, 1 µg/mL caspofungin, or recovered by filtration and resuspended in osmotically equilibrated medium lacking glucose for the indicated times. Activated/total Pmk1 was detected with anti-phospho-p44/42 and anti-GFP antibodies, respectively. Anti-Cdc2 was used as a loading control. Relative Pmk1 activity units as mean ± SD (biological duplicates) were determined as described in ( C ); ( F ) S. japonicus wild-type and sty1 Δ strains expressing a genomic Pmk1–GFP fusion were grown in YES medium to mid-log phase, and treated with 0.6 M KCl for the indicated times. Activated/total Pmk1 were detected with anti-phospho-p44/42 and anti-GFP antibodies, respectively. Relative Pmk1 activity units as mean ± SD (biological duplicates) were determined as above.

Article Snippet: Rabbit polyclonal anti-Cdk1/Cdc2 antibody (PSTAIR; Millipore) was used for loading control.

Techniques: Functional Assay, Activation Assay, Incubation, Fluorescence, Microscopy, Staining, Expressing, Filtration, Activity Assay

Journal: Journal of Fungi

Article Title: Specific Functional Features of the Cell Integrity MAP Kinase Pathway in the Dimorphic Fission Yeast Schizosaccharomyces japonicus

doi: 10.3390/jof7060482

Figure Lengend Snippet: Antagonistic control of Pmk1 activity by PKC orthologs Pck1 and Pck2 modulates S. japonicus hyphal differentiation. ( A ) S. japonicus strains of the indicated genotypes were grown in YES medium to mid-log phase, and activated Pmk1 were detected with anti-phospho-p44/42, whereas anti-Cdc2 was used as a loading control. Relative units as mean ± SD (biological triplicates) for Pmk1 phosphorylation (anti-phospho-p44/42 blot) were determined with respect to the internal control (anti-Cdc2 blot). ***, p < 0.001; as calculated by unpaired Student’s t -test; ( B ) S. japonicus strains of the indicated genotypes were grown in YES medium to mid-log phase, treated with either 0.6 M KCl (upper panels) or 1 µg/mL caspofungin (lower panels) for the indicated times, and activated Pmk1 was detected with anti-phospho-p44/42, whereas anti-Cdc2 was used as a loading control. Results from representative experiments are shown; ( C ) Exponentially growing S. japonicus cells of the indicated genotypes were inoculated at an initial cell density of 10 6 cells/mL in YES medium with 6% glucose and incubated for 0 and 6 h with 0.2 μM CPT. Cell length at each time point is represented as box and whisker plots. Data obtained after quantification of one experiment performed per triplicate ( n ≥ 400 cells/strain) is shown. ****, p < 0.0001; ns, not significant, as calculated by one-way ANOVA; ( D ) Representative images of strains analyzed in (C) were obtained by fluorescence microscopy after calcofluor white staining; ( E ) Exponentially growing S. japonicus wild-type cells (YES medium with 6% glucose) expressing a Pmk1–GFP fusion were treated with 0.2 μM CPT for the indicated times. Activated/total Pmk1 were detected with anti-phospho-p44/42 and anti-GFP antibodies, respectively. Anti-Cdc2 was used as a loading control. Relative units as mean ± SD (biological duplicates) for Pmk1 phosphorylation (anti-phospho-p44/42 blot) were determined with respect to the internal control (anti-GFP blot). ns, not significant, as calculated by unpaired Student’s t -test; ( F ) Left : S. japonicus strains of the indicated genotypes were grown in YES medium plus 0.2 μM CPT for 12 h, and the percentage of hyphae were quantified. Percentages are expressed as mean ± SD and correspond to biological duplicates ( n ≥ 200 cells/sample). Right : Representative images of strains were obtained by fluorescence microscopy after calcofluor white staining; ( G ) Left : cells from log-phase cultures of the indicated strains growing in YES medium (~2.10 6 cells in each case) were spotted on YEMA and RGE plates, incubated at 30 °C for 7 days, and then photographed. Right : the total area of mycelial expansion (expressed as relative units, RU) was measured for each strain genotype ( n ≥ 6) and is represented as scatter plot. *, p < 0.05; ****, p < 0.0001; ns, not significant, as calculated by one-way ANOVA.

Article Snippet: Rabbit polyclonal anti-Cdk1/Cdc2 antibody (PSTAIR; Millipore) was used for loading control.

Techniques: Activity Assay, Incubation, Whisker Assay, Fluorescence, Microscopy, Staining, Expressing

Journal: eLife

Article Title: Stress-activated MAPK signaling controls fission yeast actomyosin ring integrity by modulating formin For3 levels

doi: 10.7554/eLife.57951

Figure Lengend Snippet: ( A ) The stress activated MAPK pathway (SAPK) in S. pombe . Please see text for a detailed description of its main components and functions. ( B ) Decimal dilutions of strains of the indicated genotypes were spotted on YES and YES solid plates with a range of concentrations of LatA, incubated at 30°C for 3 days, and photographed. A representative experiment is shown. ( C ) Left panel: S. pombe wild type cells expressing a genomic Sty1-HA6his fusion were grown in YES medium to mid-log phase, and remained untreated (0), or treated with a range of concentrations of LatA for 1 hr. Activated/total Sty1 were detected with anti-phospho-p38 and anti-HA antibodies, respectively. Total Atf1 levels were detected with anti-Atf1 antibody. Anti-Cdc2 was used as a loading control. Right panel: Relative units as mean ± SD (biological triplicates) for Sty1 phosphorylation (anti-phospho-p38 blot) were determined with respect to the internal control (anti-HA blot). **, p<0.005; *, p<0.05; ns, not significant, as calculated by unpaired Student's t test. ( D ) S. pombe wild-type cells expressing a genomic Sty1-HA6his fusion were grown in YES medium to mid-log phase, and remained untreated (0), or treated with a range of concentrations of LatA for the indicated times. Activated/total Sty1 were detected with anti-phospho-p38 and anti-HA antibodies, respectively. ( E ) S. pombe wild type, mcs4∆ and Mcs4( D512N ) cells expressing a genomic Sty1-HA6his fusion were grown in YES medium to mid-log phase, and treated with 1 µM LatA for the indicated times. Activated/total Sty1 were detected with anti-phospho-p38 and anti-HA antibodies, respectively. A representative experiment is shown. ( F ) Decimal dilutions of strains of the indicated genotypes were spotted on YES and YES solid plates with a range of concentrations of LatA, incubated at 30°C for 3 days, and photographed. ( G ) S. pombe wild-type cells expressing a CRIB-3xGFP fusion were grown in YES medium to mid-log phase, and remained untreated (0) or treated with a range of concentrations of LatA for 30 and 60 min. Left panel: The percentage of cells at G1 and G2 that show dispersal of the CRIB-3xGFP fusion from the cell poles was estimated in each case by fluorescence microscopy, and is presented as mean ± SD (biological duplicates). nd: no dispersal from the cell poles is detected. Right panel: representative fluorescence micrographs of control and LatA-treated cells. Figure 1—source data 1. Values used for graphical representations and statistical analysis in .

Article Snippet: Total Sty1 was detected in S. pombe extracts with mouse monoclonal anti-HA antibody (RRID: AB_514505 ; 12CA5, Roche Molecular Biochemicals), whereas rabbit polyclonal anti-Cdk1/Cdc2 (PSTAIR) (RRID: AB_310302 ; Millipore) was used as loading control in S. japonicus extracts.

Techniques: Incubation, Expressing, Fluorescence, Microscopy

Journal: eLife

Article Title: Stress-activated MAPK signaling controls fission yeast actomyosin ring integrity by modulating formin For3 levels

doi: 10.7554/eLife.57951

Figure Lengend Snippet: Total extracts from growing cultures of wild-type, sty1 Δ, and wis1DD strains were resolved by SDS-PAGE, and total Act1 levels were detected by incubation with anti-actin antibody. Anti-Cdc2 was used as a loading control. Right panel: results are shown as relative fold expression (mean ± SD) from three biological repeats. ns, not significant, as calculated by unpaired Student´s t test.

Article Snippet: Total Sty1 was detected in S. pombe extracts with mouse monoclonal anti-HA antibody (RRID: AB_514505 ; 12CA5, Roche Molecular Biochemicals), whereas rabbit polyclonal anti-Cdk1/Cdc2 (PSTAIR) (RRID: AB_310302 ; Millipore) was used as loading control in S. japonicus extracts.

Techniques: SDS Page, Incubation, Expressing

Journal: eLife

Article Title: Stress-activated MAPK signaling controls fission yeast actomyosin ring integrity by modulating formin For3 levels

doi: 10.7554/eLife.57951

Figure Lengend Snippet: Wild-type cells expressing a Cdc12-3HA genomic fusion were grown in YES medium to mid-log phase, and remained untreated or treated with the indicated concentrations of LatA for 1 hr. Total cell extracts were resolved by SDS-PAGE in the presence of 15 µM PhosTag (upper panel), or conventional SDS-PAGE (middle panel), and Cdc12 levels were detected by incubation with anti-HA HRP-conjugated antibody. Anti-Cdc2 (lower panel) was used as a loading control. Representative experiments are shown.

Article Snippet: Total Sty1 was detected in S. pombe extracts with mouse monoclonal anti-HA antibody (RRID: AB_514505 ; 12CA5, Roche Molecular Biochemicals), whereas rabbit polyclonal anti-Cdk1/Cdc2 (PSTAIR) (RRID: AB_310302 ; Millipore) was used as loading control in S. japonicus extracts.

Techniques: Expressing, SDS Page, Incubation

Journal: eLife

Article Title: Stress-activated MAPK signaling controls fission yeast actomyosin ring integrity by modulating formin For3 levels

doi: 10.7554/eLife.57951

Figure Lengend Snippet: Wild-type, sty1 Δ, and wis1DD strains expressing a Cdc12-3HA genomic fusion were grown in YES medium to mid-log phase, and remained untreated (0), or treated with 0.2 µM LatA for 2 hr. Total cell extracts were resolved by SDS-PAGE in the presence of 15 µM PhosTag (upper panel), or conventional SDS-PAGE (middle panel), and Cdc12 levels were detected by incubation with anti-HA HRP-conjugated antibody. Anti-Cdc2 (lower panel) was used as a loading control. Representative experiments are shown.

Article Snippet: Total Sty1 was detected in S. pombe extracts with mouse monoclonal anti-HA antibody (RRID: AB_514505 ; 12CA5, Roche Molecular Biochemicals), whereas rabbit polyclonal anti-Cdk1/Cdc2 (PSTAIR) (RRID: AB_310302 ; Millipore) was used as loading control in S. japonicus extracts.

Techniques: Expressing, SDS Page, Incubation

Journal: eLife

Article Title: Stress-activated MAPK signaling controls fission yeast actomyosin ring integrity by modulating formin For3 levels

doi: 10.7554/eLife.57951

Figure Lengend Snippet: ( A ) Extracts from S. pombe growing cells expressing a genomic For3-3GFP fusion were treated with lambda phosphatase in the presence/absence of specific phosphatase inhibitor. Total and phosphorylated For3 levels (arrows) were determined by immunobloting with anti-GFP antibody. Anti-Cdc2 was used as a loading control. Results from a representative experiment are shown. ( B ) Total extracts from growing cultures of wild-type, sty1 Δ, pyp1 Δ, and wis1DD strains expressing a For3-3GFP genomic fusion were resolved by SDS-PAGE, and total For3 levels were detected by incubation with anti-GFP antibody. Anti-Cdc2 was used as a loading control. Lower panel: quantification of western blot experiments. Relative For3 levels are represented as mean ± SD (biological triplicates). * , p<0.05, as calculated by unpaired Student's t test with respect to the wild type. ( C ) Sty1-T97A (as) cells expressing a For3-3GFP genomic fusion were grown to mid-log phase and remained untreated (MetOH; solvent control), or treated with 10 µM 3BrBPP1 at the indicated times. Total extracts were resolved by SDS-PAGE, and For3 levels were detected by incubation with anti-GFP antibody. Anti-Cdc2 was used as a loading control. Lower panel: quantification of western blot experiments. Relative For3 levels are represented as mean ± SD (biological duplicates). * * , p<0.005; ns, not significant, as calculated by unpaired Student's t test. ( D ) Upper panel: Representative maximum-projection image of mixed wild type (For3-3GFP, Hht1-RFP; representative cells are marked with asterisks) and sty1∆ (For3-3GFP; representative cells are marked with point arrows) cells growing to mid-log phase observed by fluorescence microscopy. Lower panels: intensity plots of For3-3GFP and Pmk1-GFP fusions (shown as arbitrary fluorescence units) were generated from line scans across the equatorial region of both wild-type and sty1∆ cells (n> 28) with early septum. Individual (dotted lines) and average scans (solid lines) are shown in each case. ( E ) mRNA levels of for3 + gene were measured by qPCR from total RNA extracted from cell samples corresponding to S. pombe wild type cells growing exponentially in YES medium that remained untreated (0), or treated with the indicated concentrations of LatA for 1 hr. Results are shown as relative fold expression (mean ± SD) from three biological repeats. **** , p<0.0001; **, p<0.005; ns, not significant, as calculated by unpaired Student's t test with respect to the wild type. ( F ) S. pombe wild-type, wis1DD and pyp1 Δ cells expressing genomic Sty1-HA6his and For3-3GFP fusions were grown in YES medium to mid-log phase, and remained untreated (0), or treated with the indicated concentrations of LatA for 2 hr. Activated/total Sty1 were detected with anti-phospho-p38 and anti-HA antibodies, respectively. Total For3 levels were detected with anti-GFP antibody. Anti-Cdc2 was used as a loading control. Lower panel: quantification of Western blot experiments. Relative For3 levels are represented as mean ± SD (biological duplicates). * ** , p<0.001; **, p<0.005; *, p<0.05, as calculated by unpaired Student's t test. ( G ) Exponentially growing cdc2-asM17 cells expressing a For3-3GFP fusion were treated with 1 µM 3-MB-PP1 for 3 hr to hold the cycle at G2, released from the arrest for 10 min after ATP-analogue washout, and treated with 0.2 µM LatA for the indicated times. Total For3 levels were detected with anti-GFP antibody. Anti-Cdc2 was used as a loading control. Lower panel: quantification of Western blot experiments. Relative For3 levels are represented as mean ± SD (biological duplicates). * , p<0.05, as calculated by unpaired Student's t test. ( H ) Wild type and Act1-LQ (LatA-insensitive mutant) cells expressing a For3-3GFP genomic fusion were grown to mid-log phase and remained untreated (0), or treated with 0.15 µM LatA for 2, 4, or 6 hr. Total extracts were resolved by SDS-PAGE, and For3 levels were detected by incubation with anti-GFP antibody. Anti-Cdc2 was used as a loading control. Results from a representative experiment are shown. ( I ) Wild-type, sty1 Δ, pyp1 Δ, and wis1DD cells expressing a For3-3GFP genomic fusion were grown to mid-log phase and remained untreated (0), or treated with 0.15 µM LatA for 4 hr. Total extracts were resolved by SDS-PAGE, and For3 levels were detected by incubation with anti-GFP antibody. Anti-Cdc2 was used as a loading control. Results from a representative experiment are shown. Figure 5—source data 1. Values used for graphical representations and statistical analysis in .

Article Snippet: Total Sty1 was detected in S. pombe extracts with mouse monoclonal anti-HA antibody (RRID: AB_514505 ; 12CA5, Roche Molecular Biochemicals), whereas rabbit polyclonal anti-Cdk1/Cdc2 (PSTAIR) (RRID: AB_310302 ; Millipore) was used as loading control in S. japonicus extracts.

Techniques: Expressing, Western Blot, SDS Page, Incubation, Fluorescence, Microscopy, Generated, Mutagenesis

Journal: eLife

Article Title: Stress-activated MAPK signaling controls fission yeast actomyosin ring integrity by modulating formin For3 levels

doi: 10.7554/eLife.57951

Figure Lengend Snippet: cdc10-129 (G1-phase arrest), cdc25-22 (G2-phase arrest), and nda3-km311 (M-phase arrest) cells expressing a genomic For3-3GFP fusion were incubated at either 25°C (asynchronous cultures), 36.5°C for 3.5 hr ( cdc10-129 and cdc25-22 backgrounds) and 25 or 18°C for 7 hr ( nda3-km311 background). Total For3 levels were determined by immunobloting with anti-GFP antibody. Anti-Cdc2 was used as a loading control. Relative For3 levels are represented as mean ± SD (biological duplicates). ns, not significant, as calculated by unpaired Student's t test.

Article Snippet: Total Sty1 was detected in S. pombe extracts with mouse monoclonal anti-HA antibody (RRID: AB_514505 ; 12CA5, Roche Molecular Biochemicals), whereas rabbit polyclonal anti-Cdk1/Cdc2 (PSTAIR) (RRID: AB_310302 ; Millipore) was used as loading control in S. japonicus extracts.

Techniques: Expressing, Incubation, Western Blot

Journal: eLife

Article Title: Stress-activated MAPK signaling controls fission yeast actomyosin ring integrity by modulating formin For3 levels

doi: 10.7554/eLife.57951

Figure Lengend Snippet: S. pombe wild-type and sty1 Δ cells expressing a genomic For3-3GFP fusion were grown in YES medium to mid-log phase, and incubated at 40°C, or treated with 0.6 M KCl or 1 mM H 2 O 2 for the indicated times. Total For3 levels were detected with anti-GFP antibody. Anti-Cdc2 was used as a loading control. Results from representative experiments are shown.

Article Snippet: Total Sty1 was detected in S. pombe extracts with mouse monoclonal anti-HA antibody (RRID: AB_514505 ; 12CA5, Roche Molecular Biochemicals), whereas rabbit polyclonal anti-Cdk1/Cdc2 (PSTAIR) (RRID: AB_310302 ; Millipore) was used as loading control in S. japonicus extracts.

Techniques: Expressing, Incubation

Journal: eLife

Article Title: Stress-activated MAPK signaling controls fission yeast actomyosin ring integrity by modulating formin For3 levels

doi: 10.7554/eLife.57951

Figure Lengend Snippet: S. pombe wild-type and sty1 Δ cells expressing a genomic For3-3GFP fusion were grown in YES medium to mid-log phase, and incubated with 100 µg/ml LatA for the indicated times. Total For3 levels were detected with anti-GFP antibody. Anti-Cdc2 was used as a loading control. Relative For3 levels are represented as mean ± SD (biological duplicates) with respect to the zero time.

Article Snippet: Total Sty1 was detected in S. pombe extracts with mouse monoclonal anti-HA antibody (RRID: AB_514505 ; 12CA5, Roche Molecular Biochemicals), whereas rabbit polyclonal anti-Cdk1/Cdc2 (PSTAIR) (RRID: AB_310302 ; Millipore) was used as loading control in S. japonicus extracts.

Techniques: Expressing, Incubation

Journal: eLife

Article Title: Stress-activated MAPK signaling controls fission yeast actomyosin ring integrity by modulating formin For3 levels

doi: 10.7554/eLife.57951

Figure Lengend Snippet:

Article Snippet: Total Sty1 was detected in S. pombe extracts with mouse monoclonal anti-HA antibody (RRID: AB_514505 ; 12CA5, Roche Molecular Biochemicals), whereas rabbit polyclonal anti-Cdk1/Cdc2 (PSTAIR) (RRID: AB_310302 ; Millipore) was used as loading control in S. japonicus extracts.

Techniques: SYBR Green Assay, Western Blot, Software