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Microcephalin and pericentrin regulate mitotic entry via centrosome-associated Chk1.

Tibelius A, Marhold J, Zentgraf H, Heilig CE, Neitzel H, Ducommun B, Rauch A, Ho AD, Bartek J, Krämer A - J. Cell Biol. (2009)

Bottom Line: Primary microcephaly, Seckel syndrome, and microcephalic osteodysplastic primordial dwarfism type II (MOPD II) are disorders exhibiting marked microcephaly, with small brain sizes reflecting reduced neuron production during fetal life.Although primary microcephaly can be caused by mutations in microcephalin (MCPH1), cells from patients with Seckel syndrome and MOPD II harbor mutations in ataxia telangiectasia and Rad3 related (ATR) or pericentrin (PCNT), leading to disturbed ATR signaling.In this study, we show that a lack of MCPH1 or PCNT results in a loss of Chk1 from centrosomes with subsequently deregulated activation of centrosomal cyclin B-Cdk1.

View Article: PubMed Central - PubMed

Affiliation: Clinical Cooperation Unit Molecular Hematology/Oncology, German Cancer Research Center, 69120 Heidelberg, Germany.

ABSTRACT
Primary microcephaly, Seckel syndrome, and microcephalic osteodysplastic primordial dwarfism type II (MOPD II) are disorders exhibiting marked microcephaly, with small brain sizes reflecting reduced neuron production during fetal life. Although primary microcephaly can be caused by mutations in microcephalin (MCPH1), cells from patients with Seckel syndrome and MOPD II harbor mutations in ataxia telangiectasia and Rad3 related (ATR) or pericentrin (PCNT), leading to disturbed ATR signaling. In this study, we show that a lack of MCPH1 or PCNT results in a loss of Chk1 from centrosomes with subsequently deregulated activation of centrosomal cyclin B-Cdk1.

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Related in: MedlinePlus

Down-regulation of MCPH1 induces premature entry into mitosis via depletion of centrosomal Chk1. (A and B) U2OS cells transfected with luciferase- (as control [siLUC]), MCPH1-, or PCNT-specific siRNA (A) as well as normal, MCPH1427insA, and PCNT3109G>T LBCs (B) were costained with propidium iodide and anti–P-S10–histone H3 and analyzed by fluorescence-activated cell sorting to quantify cells in mitosis. Statistical significance versus control by two-tailed Student's t test is as follows: (A) *, P = 0.02 (U2OS-siMCPH1); **, P = 0.001 (U2OS-siPCNT); (B) **, P = 0.009 (MCPH1427insA); P = 0.09 (PCNT3109G>T). (C) Parental U2OS cells or U2OS cells conditionally expressing wild-type (Chk1[WT]) or kinase-dead (Chk1[KD]) GFP-Chk1-PACT were transfected with MCPH1-specific siRNA and analyzed by fluorescence microscopy. The mean percentages of cells with PCC are indicated. Statistical significance versus Chk1(WT) by two-tailed Student's t test is as follows: ***, P = 0.0008 (U2OS); P = 0.0002 (Chk1[KD]). (D) Characterization of the PCC phenotype. U2OS cells were transfected with luciferase- or MCPH1-specific siRNA and immunostained with antibodies to cyclin B1 (top), lamin A (middle), and P-S10–histone H3 (pH3; bottom). DNA was counterstained with To-Pro 3. After transfection with MCPH1-specific siRNA, PCC cells have an intact nuclear membrane as judged by lamin A staining, are P-S10–histone H3 negative, and show no nuclear cyclin B1 accumulation, thereby demonstrating that they exhibit G2 rather than mitotic characteristics. Error bars represent the standard deviation after combining the results of three different experiments. Bars, 10 µm.
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fig5: Down-regulation of MCPH1 induces premature entry into mitosis via depletion of centrosomal Chk1. (A and B) U2OS cells transfected with luciferase- (as control [siLUC]), MCPH1-, or PCNT-specific siRNA (A) as well as normal, MCPH1427insA, and PCNT3109G>T LBCs (B) were costained with propidium iodide and anti–P-S10–histone H3 and analyzed by fluorescence-activated cell sorting to quantify cells in mitosis. Statistical significance versus control by two-tailed Student's t test is as follows: (A) *, P = 0.02 (U2OS-siMCPH1); **, P = 0.001 (U2OS-siPCNT); (B) **, P = 0.009 (MCPH1427insA); P = 0.09 (PCNT3109G>T). (C) Parental U2OS cells or U2OS cells conditionally expressing wild-type (Chk1[WT]) or kinase-dead (Chk1[KD]) GFP-Chk1-PACT were transfected with MCPH1-specific siRNA and analyzed by fluorescence microscopy. The mean percentages of cells with PCC are indicated. Statistical significance versus Chk1(WT) by two-tailed Student's t test is as follows: ***, P = 0.0008 (U2OS); P = 0.0002 (Chk1[KD]). (D) Characterization of the PCC phenotype. U2OS cells were transfected with luciferase- or MCPH1-specific siRNA and immunostained with antibodies to cyclin B1 (top), lamin A (middle), and P-S10–histone H3 (pH3; bottom). DNA was counterstained with To-Pro 3. After transfection with MCPH1-specific siRNA, PCC cells have an intact nuclear membrane as judged by lamin A staining, are P-S10–histone H3 negative, and show no nuclear cyclin B1 accumulation, thereby demonstrating that they exhibit G2 rather than mitotic characteristics. Error bars represent the standard deviation after combining the results of three different experiments. Bars, 10 µm.

Mentions: These findings suggest that MCPH1 or PCNT depletion–induced loss of Chk1 from centrosomes leads to reduced inhibitory phosphorylation of both centrosomal Cdc25B at S230 and Cdk1 at Y15 and therefore to premature entry into mitosis. In agreement with these findings, it has been reported recently that human and Drosophila melanogaster MCPH1-deficient cells have low levels of total P-Y15-Cdk1 in S and G2 phases, resulting in accelerated mitotic entry in MCPH1427insA LBCs and premature centrosome separation in MCPH1 mutant Drosophila embryos (Alderton et al., 2006; Brunk et al., 2007). To assay for the rates of cells in late G2 or mitosis, U2OS cells treated with mock-, MCPH1-, or PCNT-specific siRNAs were examined by flow cytometry measurement using P-S10–histone H3 as a mitotic marker. 72 h after MCPH1 or PCNT siRNA transfection, significantly more cells were in late G2 or mitosis (Fig. 5 A). Similar results were obtained when the cell cycle distribution of exponentially growing control, MCPH1427insA, and PCNT3109G>T LBCs was examined (Fig. 5 B and Fig. S3 A). In addition, transient transfection of MCPH1427insA LBCs with wild-type MCPH1-Flag led to a significant reduction of G2/mitotic cells from 1.28 ± 0.2% after mock transfection to 0.75 ± 0.1% 12 h after transfection with MCPH1-Flag (Fig. S3 B).


Microcephalin and pericentrin regulate mitotic entry via centrosome-associated Chk1.

Tibelius A, Marhold J, Zentgraf H, Heilig CE, Neitzel H, Ducommun B, Rauch A, Ho AD, Bartek J, Krämer A - J. Cell Biol. (2009)

Down-regulation of MCPH1 induces premature entry into mitosis via depletion of centrosomal Chk1. (A and B) U2OS cells transfected with luciferase- (as control [siLUC]), MCPH1-, or PCNT-specific siRNA (A) as well as normal, MCPH1427insA, and PCNT3109G>T LBCs (B) were costained with propidium iodide and anti–P-S10–histone H3 and analyzed by fluorescence-activated cell sorting to quantify cells in mitosis. Statistical significance versus control by two-tailed Student's t test is as follows: (A) *, P = 0.02 (U2OS-siMCPH1); **, P = 0.001 (U2OS-siPCNT); (B) **, P = 0.009 (MCPH1427insA); P = 0.09 (PCNT3109G>T). (C) Parental U2OS cells or U2OS cells conditionally expressing wild-type (Chk1[WT]) or kinase-dead (Chk1[KD]) GFP-Chk1-PACT were transfected with MCPH1-specific siRNA and analyzed by fluorescence microscopy. The mean percentages of cells with PCC are indicated. Statistical significance versus Chk1(WT) by two-tailed Student's t test is as follows: ***, P = 0.0008 (U2OS); P = 0.0002 (Chk1[KD]). (D) Characterization of the PCC phenotype. U2OS cells were transfected with luciferase- or MCPH1-specific siRNA and immunostained with antibodies to cyclin B1 (top), lamin A (middle), and P-S10–histone H3 (pH3; bottom). DNA was counterstained with To-Pro 3. After transfection with MCPH1-specific siRNA, PCC cells have an intact nuclear membrane as judged by lamin A staining, are P-S10–histone H3 negative, and show no nuclear cyclin B1 accumulation, thereby demonstrating that they exhibit G2 rather than mitotic characteristics. Error bars represent the standard deviation after combining the results of three different experiments. Bars, 10 µm.
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Related In: Results  -  Collection

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fig5: Down-regulation of MCPH1 induces premature entry into mitosis via depletion of centrosomal Chk1. (A and B) U2OS cells transfected with luciferase- (as control [siLUC]), MCPH1-, or PCNT-specific siRNA (A) as well as normal, MCPH1427insA, and PCNT3109G>T LBCs (B) were costained with propidium iodide and anti–P-S10–histone H3 and analyzed by fluorescence-activated cell sorting to quantify cells in mitosis. Statistical significance versus control by two-tailed Student's t test is as follows: (A) *, P = 0.02 (U2OS-siMCPH1); **, P = 0.001 (U2OS-siPCNT); (B) **, P = 0.009 (MCPH1427insA); P = 0.09 (PCNT3109G>T). (C) Parental U2OS cells or U2OS cells conditionally expressing wild-type (Chk1[WT]) or kinase-dead (Chk1[KD]) GFP-Chk1-PACT were transfected with MCPH1-specific siRNA and analyzed by fluorescence microscopy. The mean percentages of cells with PCC are indicated. Statistical significance versus Chk1(WT) by two-tailed Student's t test is as follows: ***, P = 0.0008 (U2OS); P = 0.0002 (Chk1[KD]). (D) Characterization of the PCC phenotype. U2OS cells were transfected with luciferase- or MCPH1-specific siRNA and immunostained with antibodies to cyclin B1 (top), lamin A (middle), and P-S10–histone H3 (pH3; bottom). DNA was counterstained with To-Pro 3. After transfection with MCPH1-specific siRNA, PCC cells have an intact nuclear membrane as judged by lamin A staining, are P-S10–histone H3 negative, and show no nuclear cyclin B1 accumulation, thereby demonstrating that they exhibit G2 rather than mitotic characteristics. Error bars represent the standard deviation after combining the results of three different experiments. Bars, 10 µm.
Mentions: These findings suggest that MCPH1 or PCNT depletion–induced loss of Chk1 from centrosomes leads to reduced inhibitory phosphorylation of both centrosomal Cdc25B at S230 and Cdk1 at Y15 and therefore to premature entry into mitosis. In agreement with these findings, it has been reported recently that human and Drosophila melanogaster MCPH1-deficient cells have low levels of total P-Y15-Cdk1 in S and G2 phases, resulting in accelerated mitotic entry in MCPH1427insA LBCs and premature centrosome separation in MCPH1 mutant Drosophila embryos (Alderton et al., 2006; Brunk et al., 2007). To assay for the rates of cells in late G2 or mitosis, U2OS cells treated with mock-, MCPH1-, or PCNT-specific siRNAs were examined by flow cytometry measurement using P-S10–histone H3 as a mitotic marker. 72 h after MCPH1 or PCNT siRNA transfection, significantly more cells were in late G2 or mitosis (Fig. 5 A). Similar results were obtained when the cell cycle distribution of exponentially growing control, MCPH1427insA, and PCNT3109G>T LBCs was examined (Fig. 5 B and Fig. S3 A). In addition, transient transfection of MCPH1427insA LBCs with wild-type MCPH1-Flag led to a significant reduction of G2/mitotic cells from 1.28 ± 0.2% after mock transfection to 0.75 ± 0.1% 12 h after transfection with MCPH1-Flag (Fig. S3 B).

Bottom Line: Primary microcephaly, Seckel syndrome, and microcephalic osteodysplastic primordial dwarfism type II (MOPD II) are disorders exhibiting marked microcephaly, with small brain sizes reflecting reduced neuron production during fetal life.Although primary microcephaly can be caused by mutations in microcephalin (MCPH1), cells from patients with Seckel syndrome and MOPD II harbor mutations in ataxia telangiectasia and Rad3 related (ATR) or pericentrin (PCNT), leading to disturbed ATR signaling.In this study, we show that a lack of MCPH1 or PCNT results in a loss of Chk1 from centrosomes with subsequently deregulated activation of centrosomal cyclin B-Cdk1.

View Article: PubMed Central - PubMed

Affiliation: Clinical Cooperation Unit Molecular Hematology/Oncology, German Cancer Research Center, 69120 Heidelberg, Germany.

ABSTRACT
Primary microcephaly, Seckel syndrome, and microcephalic osteodysplastic primordial dwarfism type II (MOPD II) are disorders exhibiting marked microcephaly, with small brain sizes reflecting reduced neuron production during fetal life. Although primary microcephaly can be caused by mutations in microcephalin (MCPH1), cells from patients with Seckel syndrome and MOPD II harbor mutations in ataxia telangiectasia and Rad3 related (ATR) or pericentrin (PCNT), leading to disturbed ATR signaling. In this study, we show that a lack of MCPH1 or PCNT results in a loss of Chk1 from centrosomes with subsequently deregulated activation of centrosomal cyclin B-Cdk1.

Show MeSH
Related in: MedlinePlus