Limits...
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.

Show MeSH

Related in: MedlinePlus

Loss of Chk1 from centrosomes induces activation of centrosome-associated Cdc25B and Cdk1. (A, C, and D) Normal, MCPH1427insA, and PCNT3109G>T LBCs were synchronized in G1/early S phase by a mimosine block, released for 8 h to reach G2 phase, and subsequently costained with rabbit anti–P-S230-Cdc25B (green) and mouse anti–γ-tubulin (red) antibodies (A), rabbit anti–P-Y15-Cdk1 (green) and mouse anti–γ-tubulin (red) antibodies (C), or mouse anti-Cdk1 (red) and rabbit anti–γ-tubulin (green) antibodies (D) and analyzed by confocal microscopy. (B) Loss of Chk1 from centrosomes induces activation of centrosome-associated Cdc25B. The mean percentages of cells with centrosomal colocalization of γ-tubulin and P-S230-Cdc25B are indicated. Statistical significance versus control (LBC) by two-tailed Student's t test is as follows: ***, P = 0.0006 (MCPH1427insA); **, P = 0.006 (PCNT3109G>T). (E) Loss of Chk1 from centrosomes induces activation of centrosome-associated Cdk1. The mean percentages of cells with centrosomal colocalization of γ-tubulin and total Cdk1 (light gray bars) or P-Y15-Cdk1 (dark gray bars) are indicated. Statistical significance versus control (LBC) by two-tailed Student's t test is as follows: *, P = 0.013 (MCPH1427insA); P = 0.019 (PCNT3109G>T). (F) Reduced levels of P-S230-Cdc25B and P-Y15-Cdk1 in isolated centrosome preparations from MCPH1427insA and PCNT3109G>T LBCs. Immunoblots were performed on three sucrose centrifugation fractions of centrosome preparations (left) and whole cell lysates (right) as an input control from control (LBC), MCPH1427insA, and PCNT3109G>T LBCs using antibodies against P-S230-Cdc25B, P-Y15-Cdk1, Cdk1, and, for comparison, Nek2 as a loading control and Mcm7 to exclude nuclear contamination. For whole cell lysates, antibodies to Cdc25B, Cdk1, and actin were included. Arrowheads point to centrosomes, which are shown enlarged in insets. Error bars represent the standard deviation after combining the results of three different experiments. Bars, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2712957&req=5

fig4: Loss of Chk1 from centrosomes induces activation of centrosome-associated Cdc25B and Cdk1. (A, C, and D) Normal, MCPH1427insA, and PCNT3109G>T LBCs were synchronized in G1/early S phase by a mimosine block, released for 8 h to reach G2 phase, and subsequently costained with rabbit anti–P-S230-Cdc25B (green) and mouse anti–γ-tubulin (red) antibodies (A), rabbit anti–P-Y15-Cdk1 (green) and mouse anti–γ-tubulin (red) antibodies (C), or mouse anti-Cdk1 (red) and rabbit anti–γ-tubulin (green) antibodies (D) and analyzed by confocal microscopy. (B) Loss of Chk1 from centrosomes induces activation of centrosome-associated Cdc25B. The mean percentages of cells with centrosomal colocalization of γ-tubulin and P-S230-Cdc25B are indicated. Statistical significance versus control (LBC) by two-tailed Student's t test is as follows: ***, P = 0.0006 (MCPH1427insA); **, P = 0.006 (PCNT3109G>T). (E) Loss of Chk1 from centrosomes induces activation of centrosome-associated Cdk1. The mean percentages of cells with centrosomal colocalization of γ-tubulin and total Cdk1 (light gray bars) or P-Y15-Cdk1 (dark gray bars) are indicated. Statistical significance versus control (LBC) by two-tailed Student's t test is as follows: *, P = 0.013 (MCPH1427insA); P = 0.019 (PCNT3109G>T). (F) Reduced levels of P-S230-Cdc25B and P-Y15-Cdk1 in isolated centrosome preparations from MCPH1427insA and PCNT3109G>T LBCs. Immunoblots were performed on three sucrose centrifugation fractions of centrosome preparations (left) and whole cell lysates (right) as an input control from control (LBC), MCPH1427insA, and PCNT3109G>T LBCs using antibodies against P-S230-Cdc25B, P-Y15-Cdk1, Cdk1, and, for comparison, Nek2 as a loading control and Mcm7 to exclude nuclear contamination. For whole cell lysates, antibodies to Cdc25B, Cdk1, and actin were included. Arrowheads point to centrosomes, which are shown enlarged in insets. Error bars represent the standard deviation after combining the results of three different experiments. Bars, 10 µm.

Mentions: Centrosome-associated Chk1 prevents premature activation of cyclin B–Cdk1 and thereby mitotic entry by inhibitory phosphorylation of centrosomal Cdc25B at S230 with consecutive inhibition of P-Y15-Cdk1 dephosphorylation (Krämer et al., 2004; Schmitt et al., 2006). To determine whether down-regulation of MCPH1 or PCNT has an impact on the phosphorylation status of the centrosomal fractions of Cdc25B and Cdk1, primary MCPH1427insA and PCNT3109G>T LBCs as well as control LBCs were synchronized in G1/early S phase by a mimosine block and released for 8 h to reach G2 phase. Subsequently, cells were coimmunostained with antibodies to P-S230-Cdc25B or P-Y15-Cdk1 and γ-tubulin. Although centrosomes were decorated with anti–P-S230-Cdc25B in 65.7 ± 5.1% in control LBCs, only 14.0 ± 2.6% and 36.7 ± 1.5% of MCPH1427insA and PCNT3109G>T LBCs harbored centrosomes that stained positive for P-S230-Cdc25B (Fig. 4, A and B). Similarly, in contrast to control LBCs (27.3 ± 5.1%), only 5.7 ± 1.5% and 10.0 ± 1.7% of MCPH1427insA and PCNT3109G>T LBCs, respectively, harbored centrosomes with discernible P-Y15-Cdk1 decoration, despite almost all centrosomes stained positive for total Cdk1 (Fig. 4, C–E).


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)

Loss of Chk1 from centrosomes induces activation of centrosome-associated Cdc25B and Cdk1. (A, C, and D) Normal, MCPH1427insA, and PCNT3109G>T LBCs were synchronized in G1/early S phase by a mimosine block, released for 8 h to reach G2 phase, and subsequently costained with rabbit anti–P-S230-Cdc25B (green) and mouse anti–γ-tubulin (red) antibodies (A), rabbit anti–P-Y15-Cdk1 (green) and mouse anti–γ-tubulin (red) antibodies (C), or mouse anti-Cdk1 (red) and rabbit anti–γ-tubulin (green) antibodies (D) and analyzed by confocal microscopy. (B) Loss of Chk1 from centrosomes induces activation of centrosome-associated Cdc25B. The mean percentages of cells with centrosomal colocalization of γ-tubulin and P-S230-Cdc25B are indicated. Statistical significance versus control (LBC) by two-tailed Student's t test is as follows: ***, P = 0.0006 (MCPH1427insA); **, P = 0.006 (PCNT3109G>T). (E) Loss of Chk1 from centrosomes induces activation of centrosome-associated Cdk1. The mean percentages of cells with centrosomal colocalization of γ-tubulin and total Cdk1 (light gray bars) or P-Y15-Cdk1 (dark gray bars) are indicated. Statistical significance versus control (LBC) by two-tailed Student's t test is as follows: *, P = 0.013 (MCPH1427insA); P = 0.019 (PCNT3109G>T). (F) Reduced levels of P-S230-Cdc25B and P-Y15-Cdk1 in isolated centrosome preparations from MCPH1427insA and PCNT3109G>T LBCs. Immunoblots were performed on three sucrose centrifugation fractions of centrosome preparations (left) and whole cell lysates (right) as an input control from control (LBC), MCPH1427insA, and PCNT3109G>T LBCs using antibodies against P-S230-Cdc25B, P-Y15-Cdk1, Cdk1, and, for comparison, Nek2 as a loading control and Mcm7 to exclude nuclear contamination. For whole cell lysates, antibodies to Cdc25B, Cdk1, and actin were included. Arrowheads point to centrosomes, which are shown enlarged in insets. Error bars represent the standard deviation after combining the results of three different experiments. Bars, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2712957&req=5

fig4: Loss of Chk1 from centrosomes induces activation of centrosome-associated Cdc25B and Cdk1. (A, C, and D) Normal, MCPH1427insA, and PCNT3109G>T LBCs were synchronized in G1/early S phase by a mimosine block, released for 8 h to reach G2 phase, and subsequently costained with rabbit anti–P-S230-Cdc25B (green) and mouse anti–γ-tubulin (red) antibodies (A), rabbit anti–P-Y15-Cdk1 (green) and mouse anti–γ-tubulin (red) antibodies (C), or mouse anti-Cdk1 (red) and rabbit anti–γ-tubulin (green) antibodies (D) and analyzed by confocal microscopy. (B) Loss of Chk1 from centrosomes induces activation of centrosome-associated Cdc25B. The mean percentages of cells with centrosomal colocalization of γ-tubulin and P-S230-Cdc25B are indicated. Statistical significance versus control (LBC) by two-tailed Student's t test is as follows: ***, P = 0.0006 (MCPH1427insA); **, P = 0.006 (PCNT3109G>T). (E) Loss of Chk1 from centrosomes induces activation of centrosome-associated Cdk1. The mean percentages of cells with centrosomal colocalization of γ-tubulin and total Cdk1 (light gray bars) or P-Y15-Cdk1 (dark gray bars) are indicated. Statistical significance versus control (LBC) by two-tailed Student's t test is as follows: *, P = 0.013 (MCPH1427insA); P = 0.019 (PCNT3109G>T). (F) Reduced levels of P-S230-Cdc25B and P-Y15-Cdk1 in isolated centrosome preparations from MCPH1427insA and PCNT3109G>T LBCs. Immunoblots were performed on three sucrose centrifugation fractions of centrosome preparations (left) and whole cell lysates (right) as an input control from control (LBC), MCPH1427insA, and PCNT3109G>T LBCs using antibodies against P-S230-Cdc25B, P-Y15-Cdk1, Cdk1, and, for comparison, Nek2 as a loading control and Mcm7 to exclude nuclear contamination. For whole cell lysates, antibodies to Cdc25B, Cdk1, and actin were included. Arrowheads point to centrosomes, which are shown enlarged in insets. Error bars represent the standard deviation after combining the results of three different experiments. Bars, 10 µm.
Mentions: Centrosome-associated Chk1 prevents premature activation of cyclin B–Cdk1 and thereby mitotic entry by inhibitory phosphorylation of centrosomal Cdc25B at S230 with consecutive inhibition of P-Y15-Cdk1 dephosphorylation (Krämer et al., 2004; Schmitt et al., 2006). To determine whether down-regulation of MCPH1 or PCNT has an impact on the phosphorylation status of the centrosomal fractions of Cdc25B and Cdk1, primary MCPH1427insA and PCNT3109G>T LBCs as well as control LBCs were synchronized in G1/early S phase by a mimosine block and released for 8 h to reach G2 phase. Subsequently, cells were coimmunostained with antibodies to P-S230-Cdc25B or P-Y15-Cdk1 and γ-tubulin. Although centrosomes were decorated with anti–P-S230-Cdc25B in 65.7 ± 5.1% in control LBCs, only 14.0 ± 2.6% and 36.7 ± 1.5% of MCPH1427insA and PCNT3109G>T LBCs harbored centrosomes that stained positive for P-S230-Cdc25B (Fig. 4, A and B). Similarly, in contrast to control LBCs (27.3 ± 5.1%), only 5.7 ± 1.5% and 10.0 ± 1.7% of MCPH1427insA and PCNT3109G>T LBCs, respectively, harbored centrosomes with discernible P-Y15-Cdk1 decoration, despite almost all centrosomes stained positive for total Cdk1 (Fig. 4, C–E).

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