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Fission yeast Scp3 potentially maintains microtubule orientation through bundling.

Ozaki K, Chikashige Y, Hiraoka Y, Matsumoto T - PLoS ONE (2015)

Bottom Line: In this study, we investigated the function of Scp3 along with the effect of CIPC in the fission yeast Schizosaccharomyces pombe.Functional analysis suggested that Scp3 functions independently from Ase1, a protein largely required for the bundling of the mitotic spindle.These results suggested that multiple systems are independently involved to ensure microtubule orientation by bundling in fission yeast.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Biostudies, Kyoto University, Kyoto, Kyoto, Japan.

ABSTRACT
Microtubules play important roles in organelle transport, the maintenance of cell polarity and chromosome segregation and generally form bundles during these processes. The fission yeast gene scp3+ was identified as a multicopy suppressor of the cps3-81 mutant, which is hypersensitive to isopropyl N-3-chlorophenylcarbamate (CIPC), a poison that induces abnormal multipolar spindle formation in higher eukaryotes. In this study, we investigated the function of Scp3 along with the effect of CIPC in the fission yeast Schizosaccharomyces pombe. Microscopic observation revealed that treatment with CIPC, cps3-81 mutation and scp3+ gene deletion disturbed the orientation of microtubules in interphase cells. Overexpression of scp3+ suppressed the abnormal orientation of microtubules by promoting bundling. Functional analysis suggested that Scp3 functions independently from Ase1, a protein largely required for the bundling of the mitotic spindle. A strain lacking the ase1+ gene was more sensitive to CIPC, with the drug affecting the integrity of the mitotic spindle, indicating that CIPC has a mitotic target that has a role redundant with Ase1. These results suggested that multiple systems are independently involved to ensure microtubule orientation by bundling in fission yeast.

No MeSH data available.


Related in: MedlinePlus

Localization of Scp3.(A) Each strain was grown on EMM medium with or without thiamine at 30°C for 5 days. (B) Microtubules in interphase or mitosis were observed in each strain. The cells were incubated in the absence of thiamine for 20 hours at 30°C. The bars indicate 5 μm. (C) Quantitative of normal mitotic spindle. (D) Cells expressing Scp3 tagged with GFP from the native promoter (left) or the promoter of pREP81 (right) were grown in EMM medium at 30°C. For induction of the scp3+ gene, the cells were incubated in the absence of thiamine for 20 hours. Sad1-mCherry was used as a maker of SPB. The bars indicate 5 μm.
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pone.0120109.g004: Localization of Scp3.(A) Each strain was grown on EMM medium with or without thiamine at 30°C for 5 days. (B) Microtubules in interphase or mitosis were observed in each strain. The cells were incubated in the absence of thiamine for 20 hours at 30°C. The bars indicate 5 μm. (C) Quantitative of normal mitotic spindle. (D) Cells expressing Scp3 tagged with GFP from the native promoter (left) or the promoter of pREP81 (right) were grown in EMM medium at 30°C. For induction of the scp3+ gene, the cells were incubated in the absence of thiamine for 20 hours. Sad1-mCherry was used as a maker of SPB. The bars indicate 5 μm.

Mentions: The scp3+ gene was previously identified as a multicopy suppressor of the cps3–81 mutant, which is hypersensitive to CIPC [46]. The gene encodes a protein of 583 amino acids, with a predicted molecular weight of 62.8 kDa and two zinc finger motifs starting at the 41st and 70th residues, respectively. As the scp3+ gene was physically mapped on chromosome I, it is not allelic to the cps3+ gene, which was mapped on chromosome III. We first confirmed that the cps3–81 mutation is suppressed by the ectopic expression of the scp3+ gene. cps3–81 mutant cells ectopically expressing the scp3+ gene from the nmt promoter of the pREP81 plasmid were able to grow on a medium containing CIPC at a concentration of 260 μM. Furthermore, MT observation indicated that scp3+ overexpression corrected the abnormal morphology of MTs in the mutant (Fig. 2A and 2B). The ectopic expression of the scp3+ gene from the nmt promoter of pREP81 also overcame the effect of the drug in wild-type cells (Fig. 2A and 2B). When scp3+ expression was driven by the pREP1 promoter for a higher induction, growth retardation resulted in the wild-type strain (Fig. 4A). Microscopic observation indicated fewer interphase MTs and also aberrant mitotic spindles (Fig. 4B and 4C), suggesting that Scp3 promotes the bundling of MTs.


Fission yeast Scp3 potentially maintains microtubule orientation through bundling.

Ozaki K, Chikashige Y, Hiraoka Y, Matsumoto T - PLoS ONE (2015)

Localization of Scp3.(A) Each strain was grown on EMM medium with or without thiamine at 30°C for 5 days. (B) Microtubules in interphase or mitosis were observed in each strain. The cells were incubated in the absence of thiamine for 20 hours at 30°C. The bars indicate 5 μm. (C) Quantitative of normal mitotic spindle. (D) Cells expressing Scp3 tagged with GFP from the native promoter (left) or the promoter of pREP81 (right) were grown in EMM medium at 30°C. For induction of the scp3+ gene, the cells were incubated in the absence of thiamine for 20 hours. Sad1-mCherry was used as a maker of SPB. The bars indicate 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4359140&req=5

pone.0120109.g004: Localization of Scp3.(A) Each strain was grown on EMM medium with or without thiamine at 30°C for 5 days. (B) Microtubules in interphase or mitosis were observed in each strain. The cells were incubated in the absence of thiamine for 20 hours at 30°C. The bars indicate 5 μm. (C) Quantitative of normal mitotic spindle. (D) Cells expressing Scp3 tagged with GFP from the native promoter (left) or the promoter of pREP81 (right) were grown in EMM medium at 30°C. For induction of the scp3+ gene, the cells were incubated in the absence of thiamine for 20 hours. Sad1-mCherry was used as a maker of SPB. The bars indicate 5 μm.
Mentions: The scp3+ gene was previously identified as a multicopy suppressor of the cps3–81 mutant, which is hypersensitive to CIPC [46]. The gene encodes a protein of 583 amino acids, with a predicted molecular weight of 62.8 kDa and two zinc finger motifs starting at the 41st and 70th residues, respectively. As the scp3+ gene was physically mapped on chromosome I, it is not allelic to the cps3+ gene, which was mapped on chromosome III. We first confirmed that the cps3–81 mutation is suppressed by the ectopic expression of the scp3+ gene. cps3–81 mutant cells ectopically expressing the scp3+ gene from the nmt promoter of the pREP81 plasmid were able to grow on a medium containing CIPC at a concentration of 260 μM. Furthermore, MT observation indicated that scp3+ overexpression corrected the abnormal morphology of MTs in the mutant (Fig. 2A and 2B). The ectopic expression of the scp3+ gene from the nmt promoter of pREP81 also overcame the effect of the drug in wild-type cells (Fig. 2A and 2B). When scp3+ expression was driven by the pREP1 promoter for a higher induction, growth retardation resulted in the wild-type strain (Fig. 4A). Microscopic observation indicated fewer interphase MTs and also aberrant mitotic spindles (Fig. 4B and 4C), suggesting that Scp3 promotes the bundling of MTs.

Bottom Line: In this study, we investigated the function of Scp3 along with the effect of CIPC in the fission yeast Schizosaccharomyces pombe.Functional analysis suggested that Scp3 functions independently from Ase1, a protein largely required for the bundling of the mitotic spindle.These results suggested that multiple systems are independently involved to ensure microtubule orientation by bundling in fission yeast.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Biostudies, Kyoto University, Kyoto, Kyoto, Japan.

ABSTRACT
Microtubules play important roles in organelle transport, the maintenance of cell polarity and chromosome segregation and generally form bundles during these processes. The fission yeast gene scp3+ was identified as a multicopy suppressor of the cps3-81 mutant, which is hypersensitive to isopropyl N-3-chlorophenylcarbamate (CIPC), a poison that induces abnormal multipolar spindle formation in higher eukaryotes. In this study, we investigated the function of Scp3 along with the effect of CIPC in the fission yeast Schizosaccharomyces pombe. Microscopic observation revealed that treatment with CIPC, cps3-81 mutation and scp3+ gene deletion disturbed the orientation of microtubules in interphase cells. Overexpression of scp3+ suppressed the abnormal orientation of microtubules by promoting bundling. Functional analysis suggested that Scp3 functions independently from Ase1, a protein largely required for the bundling of the mitotic spindle. A strain lacking the ase1+ gene was more sensitive to CIPC, with the drug affecting the integrity of the mitotic spindle, indicating that CIPC has a mitotic target that has a role redundant with Ase1. These results suggested that multiple systems are independently involved to ensure microtubule orientation by bundling in fission yeast.

No MeSH data available.


Related in: MedlinePlus