Limits...
Abnormal centrosomal structure and duplication in Cep135-deficient vertebrate cells.

Inanç B, Pütz M, Lalor P, Dockery P, Kuriyama R, Gergely F, Morrison CG - Mol. Biol. Cell (2013)

Bottom Line: DT40 cells that lack Cep135 are viable and show no major defects in centrosome composition or function, although we note a small decrease in centriole numbers and a concomitant increase in the frequency of monopolar spindles.Centrosome amplification after hydroxyurea treatment increases significantly in Cep135-deficient cells, suggesting an inhibitory role for the protein in centrosome reduplication during S-phase delay.We propose that Cep135 is required for the structural integrity of centrioles in proliferating vertebrate cells, a role that also limits centrosome amplification in S-phase-arrested cells.

View Article: PubMed Central - PubMed

Affiliation: Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland.

ABSTRACT
Centrosomes are key microtubule-organizing centers that contain a pair of centrioles, conserved cylindrical, microtubule-based structures. Centrosome duplication occurs once per cell cycle and relies on templated centriole assembly. In many animal cells this process starts with the formation of a radially symmetrical cartwheel structure. The centrosomal protein Cep135 localizes to this cartwheel, but its role in vertebrates is not well understood. Here we examine the involvement of Cep135 in centriole function by disrupting the Cep135 gene in the DT40 chicken B-cell line. DT40 cells that lack Cep135 are viable and show no major defects in centrosome composition or function, although we note a small decrease in centriole numbers and a concomitant increase in the frequency of monopolar spindles. Furthermore, electron microscopy reveals an atypical structure in the lumen of Cep135-deficient centrioles. Centrosome amplification after hydroxyurea treatment increases significantly in Cep135-deficient cells, suggesting an inhibitory role for the protein in centrosome reduplication during S-phase delay. We propose that Cep135 is required for the structural integrity of centrioles in proliferating vertebrate cells, a role that also limits centrosome amplification in S-phase-arrested cells.

Show MeSH

Related in: MedlinePlus

Cep135  cells are viable and show no proliferative defect. (A) Proliferation analysis of Cep135 KO DT40 cells. Cells were seeded at 105/ml, and cell numbers were counted over 72 h. Data show mean ± SD of three separate experiments. (B) Quantitative cell cycle analysis of asynchronous cells of the indicated genotype stained for incorporated BrdU and total DNA levels. The G1 (bottom left), S (top), and G2/M (bottom right) gates are indicated in boxes, and the numbers refer to the percentage of cells detected in each of the gates averaged from three separate experiments. (C) Mitotic indices for wild type and two Cep135 KO clones were determined by M-phase marker phosphorylated histone H3 (PH3). The numbers refer to the percentage of cells in M phase averaged from three separate experiments. (D) Duration of mitosis in wild-type and Cep135 KO cells. Graph shows the mean of time in mitosis and the mean of time from the beginning of prometaphase to the end metaphase and the mean of time from the beginning of anaphase to the end of telophase. Each data point represents one cell. Data show mean ± SD for the 50 individual cells analyzed in three different experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3756917&req=5

Figure 3: Cep135 cells are viable and show no proliferative defect. (A) Proliferation analysis of Cep135 KO DT40 cells. Cells were seeded at 105/ml, and cell numbers were counted over 72 h. Data show mean ± SD of three separate experiments. (B) Quantitative cell cycle analysis of asynchronous cells of the indicated genotype stained for incorporated BrdU and total DNA levels. The G1 (bottom left), S (top), and G2/M (bottom right) gates are indicated in boxes, and the numbers refer to the percentage of cells detected in each of the gates averaged from three separate experiments. (C) Mitotic indices for wild type and two Cep135 KO clones were determined by M-phase marker phosphorylated histone H3 (PH3). The numbers refer to the percentage of cells in M phase averaged from three separate experiments. (D) Duration of mitosis in wild-type and Cep135 KO cells. Graph shows the mean of time in mitosis and the mean of time from the beginning of prometaphase to the end metaphase and the mean of time from the beginning of anaphase to the end of telophase. Each data point represents one cell. Data show mean ± SD for the 50 individual cells analyzed in three different experiments.

Mentions: We next analyzed the proliferative properties of Cep135 KO cells. As shown in Figure 3A, Cep135-deficient cells proliferated at a rate indistinguishable from that of wild-type cells. Flow cytometry analysis of the Cep135-targeted cells showed no difference from wild type with respect to the proportions of the population undergoing DNA replication (Figure 3B) or mitosis (Figure 3C). To extend our analysis of mitosis in Cep135-deficient cells, we measured the duration of mitosis by live cell imaging using wild-type and Cep135-deficient cells that stably expressed histone H2B-RFP (Dodson et al., 2007). The mean time taken from chromosome condensation to decondensation was found to be 42.9 min in both wild-type cells and Cep135-deficient cells (Figure 3D), indicating that mitotic progression is normal in cells that lack Cep135. Although the data presented in this figure were obtained from clones generated with construct A, those that resulted from targeting with construct B showed similar proliferative characteristics with no significant difference from wild-type cells (data not shown). These results indicate that full-length Cep135 is dispensable for cell viability and normal cell cycle progression in DT40 cells. Given the degree to which Cep135 is evolutionarily conserved, we had expected a stronger phenotype, so an obvious issue is that of potential redundancy. Extensive BLAST searching of the chicken genome and EST databases has not provided any evidence for a duplication of Cep135. In a detailed bioinformatic analysis of Cep135, Carvalho-Santos et al. (2010) found that testis-specific protein 10 (Tsga10) shared high levels of sequence similarity with Cep135 within a specific, conserved region. We confirmed that Tsga10 was expressed in DT40 cells (Supplemental Figure S1C). To test whether Tsga10 might serve redundant functions with Cep135, we disrupted Tsga10 in Cep135-knockout cells, using the strategy shown in Supplemental Figure S1, A and B, and confirmed the loss of Tsga10 expression (Supplemental Figure S1C). We found no significant alteration of spindle integrity in Cep135/Tsga10 knockouts (Supplemental Figure S1D) and no effect on centriole number in monastrol-induced monopoles (Supplemental Figure S1E). From these data, we conclude that Cep135 is nonredundant in DT40 cells.


Abnormal centrosomal structure and duplication in Cep135-deficient vertebrate cells.

Inanç B, Pütz M, Lalor P, Dockery P, Kuriyama R, Gergely F, Morrison CG - Mol. Biol. Cell (2013)

Cep135  cells are viable and show no proliferative defect. (A) Proliferation analysis of Cep135 KO DT40 cells. Cells were seeded at 105/ml, and cell numbers were counted over 72 h. Data show mean ± SD of three separate experiments. (B) Quantitative cell cycle analysis of asynchronous cells of the indicated genotype stained for incorporated BrdU and total DNA levels. The G1 (bottom left), S (top), and G2/M (bottom right) gates are indicated in boxes, and the numbers refer to the percentage of cells detected in each of the gates averaged from three separate experiments. (C) Mitotic indices for wild type and two Cep135 KO clones were determined by M-phase marker phosphorylated histone H3 (PH3). The numbers refer to the percentage of cells in M phase averaged from three separate experiments. (D) Duration of mitosis in wild-type and Cep135 KO cells. Graph shows the mean of time in mitosis and the mean of time from the beginning of prometaphase to the end metaphase and the mean of time from the beginning of anaphase to the end of telophase. Each data point represents one cell. Data show mean ± SD for the 50 individual cells analyzed in three different experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3756917&req=5

Figure 3: Cep135 cells are viable and show no proliferative defect. (A) Proliferation analysis of Cep135 KO DT40 cells. Cells were seeded at 105/ml, and cell numbers were counted over 72 h. Data show mean ± SD of three separate experiments. (B) Quantitative cell cycle analysis of asynchronous cells of the indicated genotype stained for incorporated BrdU and total DNA levels. The G1 (bottom left), S (top), and G2/M (bottom right) gates are indicated in boxes, and the numbers refer to the percentage of cells detected in each of the gates averaged from three separate experiments. (C) Mitotic indices for wild type and two Cep135 KO clones were determined by M-phase marker phosphorylated histone H3 (PH3). The numbers refer to the percentage of cells in M phase averaged from three separate experiments. (D) Duration of mitosis in wild-type and Cep135 KO cells. Graph shows the mean of time in mitosis and the mean of time from the beginning of prometaphase to the end metaphase and the mean of time from the beginning of anaphase to the end of telophase. Each data point represents one cell. Data show mean ± SD for the 50 individual cells analyzed in three different experiments.
Mentions: We next analyzed the proliferative properties of Cep135 KO cells. As shown in Figure 3A, Cep135-deficient cells proliferated at a rate indistinguishable from that of wild-type cells. Flow cytometry analysis of the Cep135-targeted cells showed no difference from wild type with respect to the proportions of the population undergoing DNA replication (Figure 3B) or mitosis (Figure 3C). To extend our analysis of mitosis in Cep135-deficient cells, we measured the duration of mitosis by live cell imaging using wild-type and Cep135-deficient cells that stably expressed histone H2B-RFP (Dodson et al., 2007). The mean time taken from chromosome condensation to decondensation was found to be 42.9 min in both wild-type cells and Cep135-deficient cells (Figure 3D), indicating that mitotic progression is normal in cells that lack Cep135. Although the data presented in this figure were obtained from clones generated with construct A, those that resulted from targeting with construct B showed similar proliferative characteristics with no significant difference from wild-type cells (data not shown). These results indicate that full-length Cep135 is dispensable for cell viability and normal cell cycle progression in DT40 cells. Given the degree to which Cep135 is evolutionarily conserved, we had expected a stronger phenotype, so an obvious issue is that of potential redundancy. Extensive BLAST searching of the chicken genome and EST databases has not provided any evidence for a duplication of Cep135. In a detailed bioinformatic analysis of Cep135, Carvalho-Santos et al. (2010) found that testis-specific protein 10 (Tsga10) shared high levels of sequence similarity with Cep135 within a specific, conserved region. We confirmed that Tsga10 was expressed in DT40 cells (Supplemental Figure S1C). To test whether Tsga10 might serve redundant functions with Cep135, we disrupted Tsga10 in Cep135-knockout cells, using the strategy shown in Supplemental Figure S1, A and B, and confirmed the loss of Tsga10 expression (Supplemental Figure S1C). We found no significant alteration of spindle integrity in Cep135/Tsga10 knockouts (Supplemental Figure S1D) and no effect on centriole number in monastrol-induced monopoles (Supplemental Figure S1E). From these data, we conclude that Cep135 is nonredundant in DT40 cells.

Bottom Line: DT40 cells that lack Cep135 are viable and show no major defects in centrosome composition or function, although we note a small decrease in centriole numbers and a concomitant increase in the frequency of monopolar spindles.Centrosome amplification after hydroxyurea treatment increases significantly in Cep135-deficient cells, suggesting an inhibitory role for the protein in centrosome reduplication during S-phase delay.We propose that Cep135 is required for the structural integrity of centrioles in proliferating vertebrate cells, a role that also limits centrosome amplification in S-phase-arrested cells.

View Article: PubMed Central - PubMed

Affiliation: Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland.

ABSTRACT
Centrosomes are key microtubule-organizing centers that contain a pair of centrioles, conserved cylindrical, microtubule-based structures. Centrosome duplication occurs once per cell cycle and relies on templated centriole assembly. In many animal cells this process starts with the formation of a radially symmetrical cartwheel structure. The centrosomal protein Cep135 localizes to this cartwheel, but its role in vertebrates is not well understood. Here we examine the involvement of Cep135 in centriole function by disrupting the Cep135 gene in the DT40 chicken B-cell line. DT40 cells that lack Cep135 are viable and show no major defects in centrosome composition or function, although we note a small decrease in centriole numbers and a concomitant increase in the frequency of monopolar spindles. Furthermore, electron microscopy reveals an atypical structure in the lumen of Cep135-deficient centrioles. Centrosome amplification after hydroxyurea treatment increases significantly in Cep135-deficient cells, suggesting an inhibitory role for the protein in centrosome reduplication during S-phase delay. We propose that Cep135 is required for the structural integrity of centrioles in proliferating vertebrate cells, a role that also limits centrosome amplification in S-phase-arrested cells.

Show MeSH
Related in: MedlinePlus