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The kinetochore protein Kis1/Eic1/Mis19 ensures the integrity of mitotic spindles through maintenance of kinetochore factors Mis6/CENP-I and CENP-A.

Hirai H, Arai K, Kariyazono R, Yamamoto M, Sato M - PLoS ONE (2014)

Bottom Line: The kis1 mutant frequently failed to assemble a normal bipolar spindle.We also found that the inner kinetochore proteins Mis6/CENP-I and Cnp1/CENP-A were delocalized from kinetochores in the kis1 cells and that kinetochore-microtubule attachment was defective.We conclude that Kis1 is required for inner kinetochore organization, through which Kis1 ensures kinetochore-microtubule attachment and spindle integrity.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cytoskeletal Logistics, Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Technology, Waseda University, TWIns, Tokyo, Japan.

ABSTRACT
Microtubules play multiple roles in a wide range of cellular phenomena, including cell polarity establishment and chromosome segregation. A number of microtubule regulators have been identified, including microtubule-associated proteins and kinases, and knowledge of these factors has contributed to our molecular understanding of microtubule regulation of each relevant cellular process. The known regulators, however, are insufficient to explain how those processes are linked to one another, underscoring the need to identify additional regulators. To find such novel mechanisms and microtubule regulators, we performed a screen that combined genetics and microscopy for fission yeast mutants defective in microtubule organization. We isolated approximately 900 mutants showing defects in either microtubule organization or the nuclear envelope, and these mutants were classified into 12 categories. We particularly focused on one mutant, kis1, which displayed spindle defects in early mitosis. The kis1 mutant frequently failed to assemble a normal bipolar spindle. The responsible gene encoded a kinetochore protein, Mis19 (also known as Eic1), which localized to the interface of kinetochores and spindle poles. We also found that the inner kinetochore proteins Mis6/CENP-I and Cnp1/CENP-A were delocalized from kinetochores in the kis1 cells and that kinetochore-microtubule attachment was defective. Another mutant, mis6, also displayed similar spindle defects. We conclude that Kis1 is required for inner kinetochore organization, through which Kis1 ensures kinetochore-microtubule attachment and spindle integrity. Thus, we propose an unexpected relationship between inner kinetochore organization and spindle integrity.

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kis1 encodes an essential protein.(A, B) Identification of kis1. (A) A multicopy suppressor plasmid of kis1-1 contained an insert with the indicated region of chromosome II. (B) Ten-fold serial dilution assays on SD medium without leucine at 25 or 36°C. The kis1-1 mutants harbor plasmids which express indicated genes (mmm1, SPBC27B12.02 and erg32) or empty vector (empty). Plasmids in 1, 3, and 6 were derived from pREP1, whereas plasmids in 2, 4, 5, and 7 were derived from pREP41. The promoter in pREP1 is stronger than in pREP41. (C) Domain structure of Kis1. The asterisk indicates the mutation site of the kis1-1 mutant (R65C). Gray boxes indicate predicted coiled-coil regions. (D) Alignment of Kis1/Mis19 with predicted orthologs in yeast. Identical residues are boxed in black. Similar residues are shaded in gray. Spom, Schizosaccharomyces pombe; Scry, S. cryophilus; Soct, S. octosporus; Sjap, S. japonicus; Scer, Saccharomyces cerevisiae. The arrow indicates the mutation site of kis1-1. (E, F) Kis1/Mis19 is essential for growth. (E) Diploid cells heterozygous for kis1 (kis1+/kis1::kan) were sporulated and individual spores in each ascus were dissected on the nonselective medium (N/S). Two viable and two non-viable segregation patterns were obtained. Viable colonies were sensitive to kanamycin (G418) without exception, meaning that those are colonies of kis1+ haploid cells. (F) Growth of WT and kis1-1 cells at 36°C. (G) kis1-1 displays unequal chromosome segregation. Left: Centromeres of chromosome II were visualized with GFP (cen2-GFP: green), DAPI (red), and differential interference contrast (DIC). Cells were grown at 36°C for 6 h. Right: Population of cells with unequal segregation of cen2-GFP. n>200.
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pone-0111905-g004: kis1 encodes an essential protein.(A, B) Identification of kis1. (A) A multicopy suppressor plasmid of kis1-1 contained an insert with the indicated region of chromosome II. (B) Ten-fold serial dilution assays on SD medium without leucine at 25 or 36°C. The kis1-1 mutants harbor plasmids which express indicated genes (mmm1, SPBC27B12.02 and erg32) or empty vector (empty). Plasmids in 1, 3, and 6 were derived from pREP1, whereas plasmids in 2, 4, 5, and 7 were derived from pREP41. The promoter in pREP1 is stronger than in pREP41. (C) Domain structure of Kis1. The asterisk indicates the mutation site of the kis1-1 mutant (R65C). Gray boxes indicate predicted coiled-coil regions. (D) Alignment of Kis1/Mis19 with predicted orthologs in yeast. Identical residues are boxed in black. Similar residues are shaded in gray. Spom, Schizosaccharomyces pombe; Scry, S. cryophilus; Soct, S. octosporus; Sjap, S. japonicus; Scer, Saccharomyces cerevisiae. The arrow indicates the mutation site of kis1-1. (E, F) Kis1/Mis19 is essential for growth. (E) Diploid cells heterozygous for kis1 (kis1+/kis1::kan) were sporulated and individual spores in each ascus were dissected on the nonselective medium (N/S). Two viable and two non-viable segregation patterns were obtained. Viable colonies were sensitive to kanamycin (G418) without exception, meaning that those are colonies of kis1+ haploid cells. (F) Growth of WT and kis1-1 cells at 36°C. (G) kis1-1 displays unequal chromosome segregation. Left: Centromeres of chromosome II were visualized with GFP (cen2-GFP: green), DAPI (red), and differential interference contrast (DIC). Cells were grown at 36°C for 6 h. Right: Population of cells with unequal segregation of cen2-GFP. n>200.

Mentions: To identify the gene responsible for the phenotype of kis1-1, we introduced the S. pombe genomic DNA library into kis1-1 cells to search for clones that suppress the temperature sensitivity, and we isolated a DNA clone containing coding sequences of chromosome 2, i.e., mmm1, SPBC27B12.02, and erg32 (Figure 4A). Subsequent complementation assays were performed with DNA clones containing one of the three coding sequences. SPBC27B12.02 was the only gene of the three to complement the temperature sensitivity (Figure 4B). This gene was recently identified in two other studies [68], [69], in which it was named mis19 and eic1. According to the S. pombe gene database PomBase [70], the registered gene name is mis19, and thus hereafter we refer to it as kis1/mis19.


The kinetochore protein Kis1/Eic1/Mis19 ensures the integrity of mitotic spindles through maintenance of kinetochore factors Mis6/CENP-I and CENP-A.

Hirai H, Arai K, Kariyazono R, Yamamoto M, Sato M - PLoS ONE (2014)

kis1 encodes an essential protein.(A, B) Identification of kis1. (A) A multicopy suppressor plasmid of kis1-1 contained an insert with the indicated region of chromosome II. (B) Ten-fold serial dilution assays on SD medium without leucine at 25 or 36°C. The kis1-1 mutants harbor plasmids which express indicated genes (mmm1, SPBC27B12.02 and erg32) or empty vector (empty). Plasmids in 1, 3, and 6 were derived from pREP1, whereas plasmids in 2, 4, 5, and 7 were derived from pREP41. The promoter in pREP1 is stronger than in pREP41. (C) Domain structure of Kis1. The asterisk indicates the mutation site of the kis1-1 mutant (R65C). Gray boxes indicate predicted coiled-coil regions. (D) Alignment of Kis1/Mis19 with predicted orthologs in yeast. Identical residues are boxed in black. Similar residues are shaded in gray. Spom, Schizosaccharomyces pombe; Scry, S. cryophilus; Soct, S. octosporus; Sjap, S. japonicus; Scer, Saccharomyces cerevisiae. The arrow indicates the mutation site of kis1-1. (E, F) Kis1/Mis19 is essential for growth. (E) Diploid cells heterozygous for kis1 (kis1+/kis1::kan) were sporulated and individual spores in each ascus were dissected on the nonselective medium (N/S). Two viable and two non-viable segregation patterns were obtained. Viable colonies were sensitive to kanamycin (G418) without exception, meaning that those are colonies of kis1+ haploid cells. (F) Growth of WT and kis1-1 cells at 36°C. (G) kis1-1 displays unequal chromosome segregation. Left: Centromeres of chromosome II were visualized with GFP (cen2-GFP: green), DAPI (red), and differential interference contrast (DIC). Cells were grown at 36°C for 6 h. Right: Population of cells with unequal segregation of cen2-GFP. n>200.
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Related In: Results  -  Collection

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Show All Figures
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pone-0111905-g004: kis1 encodes an essential protein.(A, B) Identification of kis1. (A) A multicopy suppressor plasmid of kis1-1 contained an insert with the indicated region of chromosome II. (B) Ten-fold serial dilution assays on SD medium without leucine at 25 or 36°C. The kis1-1 mutants harbor plasmids which express indicated genes (mmm1, SPBC27B12.02 and erg32) or empty vector (empty). Plasmids in 1, 3, and 6 were derived from pREP1, whereas plasmids in 2, 4, 5, and 7 were derived from pREP41. The promoter in pREP1 is stronger than in pREP41. (C) Domain structure of Kis1. The asterisk indicates the mutation site of the kis1-1 mutant (R65C). Gray boxes indicate predicted coiled-coil regions. (D) Alignment of Kis1/Mis19 with predicted orthologs in yeast. Identical residues are boxed in black. Similar residues are shaded in gray. Spom, Schizosaccharomyces pombe; Scry, S. cryophilus; Soct, S. octosporus; Sjap, S. japonicus; Scer, Saccharomyces cerevisiae. The arrow indicates the mutation site of kis1-1. (E, F) Kis1/Mis19 is essential for growth. (E) Diploid cells heterozygous for kis1 (kis1+/kis1::kan) were sporulated and individual spores in each ascus were dissected on the nonselective medium (N/S). Two viable and two non-viable segregation patterns were obtained. Viable colonies were sensitive to kanamycin (G418) without exception, meaning that those are colonies of kis1+ haploid cells. (F) Growth of WT and kis1-1 cells at 36°C. (G) kis1-1 displays unequal chromosome segregation. Left: Centromeres of chromosome II were visualized with GFP (cen2-GFP: green), DAPI (red), and differential interference contrast (DIC). Cells were grown at 36°C for 6 h. Right: Population of cells with unequal segregation of cen2-GFP. n>200.
Mentions: To identify the gene responsible for the phenotype of kis1-1, we introduced the S. pombe genomic DNA library into kis1-1 cells to search for clones that suppress the temperature sensitivity, and we isolated a DNA clone containing coding sequences of chromosome 2, i.e., mmm1, SPBC27B12.02, and erg32 (Figure 4A). Subsequent complementation assays were performed with DNA clones containing one of the three coding sequences. SPBC27B12.02 was the only gene of the three to complement the temperature sensitivity (Figure 4B). This gene was recently identified in two other studies [68], [69], in which it was named mis19 and eic1. According to the S. pombe gene database PomBase [70], the registered gene name is mis19, and thus hereafter we refer to it as kis1/mis19.

Bottom Line: The kis1 mutant frequently failed to assemble a normal bipolar spindle.We also found that the inner kinetochore proteins Mis6/CENP-I and Cnp1/CENP-A were delocalized from kinetochores in the kis1 cells and that kinetochore-microtubule attachment was defective.We conclude that Kis1 is required for inner kinetochore organization, through which Kis1 ensures kinetochore-microtubule attachment and spindle integrity.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cytoskeletal Logistics, Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Technology, Waseda University, TWIns, Tokyo, Japan.

ABSTRACT
Microtubules play multiple roles in a wide range of cellular phenomena, including cell polarity establishment and chromosome segregation. A number of microtubule regulators have been identified, including microtubule-associated proteins and kinases, and knowledge of these factors has contributed to our molecular understanding of microtubule regulation of each relevant cellular process. The known regulators, however, are insufficient to explain how those processes are linked to one another, underscoring the need to identify additional regulators. To find such novel mechanisms and microtubule regulators, we performed a screen that combined genetics and microscopy for fission yeast mutants defective in microtubule organization. We isolated approximately 900 mutants showing defects in either microtubule organization or the nuclear envelope, and these mutants were classified into 12 categories. We particularly focused on one mutant, kis1, which displayed spindle defects in early mitosis. The kis1 mutant frequently failed to assemble a normal bipolar spindle. The responsible gene encoded a kinetochore protein, Mis19 (also known as Eic1), which localized to the interface of kinetochores and spindle poles. We also found that the inner kinetochore proteins Mis6/CENP-I and Cnp1/CENP-A were delocalized from kinetochores in the kis1 cells and that kinetochore-microtubule attachment was defective. Another mutant, mis6, also displayed similar spindle defects. We conclude that Kis1 is required for inner kinetochore organization, through which Kis1 ensures kinetochore-microtubule attachment and spindle integrity. Thus, we propose an unexpected relationship between inner kinetochore organization and spindle integrity.

Show MeSH