Limits...
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

Microtubules in the wild-type strain treated with CIPC.(A) GFP-tagged α-tubulin (GFP-Atb2) as a microtubule marker and Sad1 tagged with mCherry (Sad1-mCherry) as an SPB marker were expressed from their native promoters. The cells were grown at 30°C and treated with 260 μM or 300 μM CIPC for 5 hours in EMM medium. DMSO was used as a solvent. Misoriented MTs are marked with arrowheads. The bar indicates 5 μm. (B) The wild-type strain in metaphase was observed in the presence of CIPC. The bar indicates 5 μm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4359140&req=5

pone.0120109.g001: Microtubules in the wild-type strain treated with CIPC.(A) GFP-tagged α-tubulin (GFP-Atb2) as a microtubule marker and Sad1 tagged with mCherry (Sad1-mCherry) as an SPB marker were expressed from their native promoters. The cells were grown at 30°C and treated with 260 μM or 300 μM CIPC for 5 hours in EMM medium. DMSO was used as a solvent. Misoriented MTs are marked with arrowheads. The bar indicates 5 μm. (B) The wild-type strain in metaphase was observed in the presence of CIPC. The bar indicates 5 μm.

Mentions: It was previously reported that isopropyl N-3-chlorophenylcarbamate (CIPC) induces abnormal multipolar spindle formation in higher eukaryotes [31]. We therefore examined the effect of the drug on fission yeast, focusing on the morphology of microtubules (MTs). Throughout this study we employed strains expressing α-tubulin tagged with GFP from the native nda3 promoter integrated at the lys1 locus or the native atb2 promoter at the native locus for visualization of MTs (GFP-Atb2) [43–45]. Because the survival rate of the wild-type cells is more than 90% at a concentration of 250 μM of CIPC and drops to nearly 0% at 300 μM [34], we first observed cells incubated in the presence of the drug at a semi-lethal concentration (260 μM) for 5 hours. As shown in Fig. 1A, the MTs in the cells treated with the drug were misoriented. For a statistical analysis, we measured the angle (θ) between the long cell axis and each MT. The results (Fig. 2B) showed that although the angles between the cell axis and most of the MTs (approximately 70%) in the control cells were less than 10°, they were much larger in the cells treated with the drug at 260 μM. We next examined wild-type cells treated with the drug at a lethal concentration (300 μM) for 5 hours. Despite the increase in the dose of CIPC, we did not note an apparent difference in the MT morphology (Figs. 1A and 2B). Under both conditions, the mitotic spindle and SPB (spindle pole body), a structure equivalent to the centrosome, were morphologically normal (Fig. 1B). We also observed living cells by time-lapse microscopy and found cell cycle progression was not disturbed during mitosis (S1 Fig).


Fission yeast Scp3 potentially maintains microtubule orientation through bundling.

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

Microtubules in the wild-type strain treated with CIPC.(A) GFP-tagged α-tubulin (GFP-Atb2) as a microtubule marker and Sad1 tagged with mCherry (Sad1-mCherry) as an SPB marker were expressed from their native promoters. The cells were grown at 30°C and treated with 260 μM or 300 μM CIPC for 5 hours in EMM medium. DMSO was used as a solvent. Misoriented MTs are marked with arrowheads. The bar indicates 5 μm. (B) The wild-type strain in metaphase was observed in the presence of CIPC. The bar indicates 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0120109.g001: Microtubules in the wild-type strain treated with CIPC.(A) GFP-tagged α-tubulin (GFP-Atb2) as a microtubule marker and Sad1 tagged with mCherry (Sad1-mCherry) as an SPB marker were expressed from their native promoters. The cells were grown at 30°C and treated with 260 μM or 300 μM CIPC for 5 hours in EMM medium. DMSO was used as a solvent. Misoriented MTs are marked with arrowheads. The bar indicates 5 μm. (B) The wild-type strain in metaphase was observed in the presence of CIPC. The bar indicates 5 μm.
Mentions: It was previously reported that isopropyl N-3-chlorophenylcarbamate (CIPC) induces abnormal multipolar spindle formation in higher eukaryotes [31]. We therefore examined the effect of the drug on fission yeast, focusing on the morphology of microtubules (MTs). Throughout this study we employed strains expressing α-tubulin tagged with GFP from the native nda3 promoter integrated at the lys1 locus or the native atb2 promoter at the native locus for visualization of MTs (GFP-Atb2) [43–45]. Because the survival rate of the wild-type cells is more than 90% at a concentration of 250 μM of CIPC and drops to nearly 0% at 300 μM [34], we first observed cells incubated in the presence of the drug at a semi-lethal concentration (260 μM) for 5 hours. As shown in Fig. 1A, the MTs in the cells treated with the drug were misoriented. For a statistical analysis, we measured the angle (θ) between the long cell axis and each MT. The results (Fig. 2B) showed that although the angles between the cell axis and most of the MTs (approximately 70%) in the control cells were less than 10°, they were much larger in the cells treated with the drug at 260 μM. We next examined wild-type cells treated with the drug at a lethal concentration (300 μM) for 5 hours. Despite the increase in the dose of CIPC, we did not note an apparent difference in the MT morphology (Figs. 1A and 2B). Under both conditions, the mitotic spindle and SPB (spindle pole body), a structure equivalent to the centrosome, were morphologically normal (Fig. 1B). We also observed living cells by time-lapse microscopy and found cell cycle progression was not disturbed during mitosis (S1 Fig).

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