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Golgi partitioning controls mitotic entry through Aurora-A kinase.

Persico A, Cervigni RI, Barretta ML, Corda D, Colanzi A - Mol. Biol. Cell (2010)

Bottom Line: We show that a block of Golgi partitioning impairs centrosome recruitment and activation of Aurora-A, which results in the G2 block of cell cycle progression.Overexpression of Aurora-A overrides this cell cycle block, indicating that Aurora-A is a major effector of the Golgi checkpoint.Our findings provide the basis for further understanding of the signaling pathways that coordinate organelle inheritance and cell duplication.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro, Chieti, Italy.

ABSTRACT
At the onset of mitosis, the Golgi complex undergoes a multistep fragmentation process that is required for its correct partitioning into the daughter cells. Inhibition of this Golgi fragmentation results in cell cycle arrest at the G2 stage, suggesting that correct inheritance of the Golgi complex is monitored by a "Golgi mitotic checkpoint." However, the molecular basis of this G2 block is not known. Here, we show that the G2-specific Golgi fragmentation stage is concomitant with centrosome recruitment and activation of the mitotic kinase Aurora-A, an essential regulator for entry into mitosis. We show that a block of Golgi partitioning impairs centrosome recruitment and activation of Aurora-A, which results in the G2 block of cell cycle progression. Overexpression of Aurora-A overrides this cell cycle block, indicating that Aurora-A is a major effector of the Golgi checkpoint. Our findings provide the basis for further understanding of the signaling pathways that coordinate organelle inheritance and cell duplication.

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Block of Golgi fragmentation inhibits Aur-A recruitment through a novel mechanism. HeLa cells were grown on coverslips and arrested in S phase by using the double-thymidine block. (A–C) Four hours after S phase block release, the cells were either left in growth medium (nontreated) or treated with 9 μM RO-3306 (a Cdk1 inhibitor) and fixed and processed for immunofluorescence under confocal microscopy or immunoblotting at the indicated times after thymidine removal. Cells were labeled with Hoechst 33342 to determine the mitotic indices up to 12 h after S phase release (A) and with antibodies against T288-phosphorylated Aur-A to determine the relative percentages of pT288 Aur-A–positive cells calculated according to the nontreated cells fixed up to 12 h after S phase release (B). A similar set of samples was processed for immunoblotting to reveal total Aur-A and active Aur-A (PT288) (C). (D–F) Cells were either nonmicroinjected (D and E) or microinjected 1 h after thymidine washout with recombinant GST (GST-inj; 8 mg/ml) or recombinant GRASP65 (GR65-inj, 8–10 mg/ml; E and F), and with FITC-conjugated dextran as microinjection marker (E and F). Eight hours after S phase block release, the nonmicroinjected cells were either left in growth medium (D and E, −) or treated with 30 μM IPA3 (D and E, a Pak inhibitor) and fixed 12 h after the S-phase block release for immunofluorescence under confocal microscopy with antibodies against T288-phosphorylated Aur-A (D), against T423-phosphorylated Pak (E), or against Plk1 (F). The relative percentages of PT288 Aur-A-positive (D), T423 Pak-positive (E) were calculated according to the relevant cells treated with vehicle (−). The relative percentages of Plk1-positive cells (F) were calculated according the noninjected cells on the same coverslip. Quantification data are means ± SD from at least two independent experiments, each carried out in duplicate. More than 200 cells were microinjected for each condition.
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Figure 5: Block of Golgi fragmentation inhibits Aur-A recruitment through a novel mechanism. HeLa cells were grown on coverslips and arrested in S phase by using the double-thymidine block. (A–C) Four hours after S phase block release, the cells were either left in growth medium (nontreated) or treated with 9 μM RO-3306 (a Cdk1 inhibitor) and fixed and processed for immunofluorescence under confocal microscopy or immunoblotting at the indicated times after thymidine removal. Cells were labeled with Hoechst 33342 to determine the mitotic indices up to 12 h after S phase release (A) and with antibodies against T288-phosphorylated Aur-A to determine the relative percentages of pT288 Aur-A–positive cells calculated according to the nontreated cells fixed up to 12 h after S phase release (B). A similar set of samples was processed for immunoblotting to reveal total Aur-A and active Aur-A (PT288) (C). (D–F) Cells were either nonmicroinjected (D and E) or microinjected 1 h after thymidine washout with recombinant GST (GST-inj; 8 mg/ml) or recombinant GRASP65 (GR65-inj, 8–10 mg/ml; E and F), and with FITC-conjugated dextran as microinjection marker (E and F). Eight hours after S phase block release, the nonmicroinjected cells were either left in growth medium (D and E, −) or treated with 30 μM IPA3 (D and E, a Pak inhibitor) and fixed 12 h after the S-phase block release for immunofluorescence under confocal microscopy with antibodies against T288-phosphorylated Aur-A (D), against T423-phosphorylated Pak (E), or against Plk1 (F). The relative percentages of PT288 Aur-A-positive (D), T423 Pak-positive (E) were calculated according to the relevant cells treated with vehicle (−). The relative percentages of Plk1-positive cells (F) were calculated according the noninjected cells on the same coverslip. Quantification data are means ± SD from at least two independent experiments, each carried out in duplicate. More than 200 cells were microinjected for each condition.

Mentions: Because many signaling networks are composed of elements that are functionally connected by feedback loops, it was important to determine whether reduction of Aur-A recruitment to and activation at the centrosome is a secondary consequence of a block of the essential mitotic complex cycB-Cdk1. Indeed, although according to some reports Aur-A activation is independent of Cdk1 activity (Hirota et al., 2003), other reports have proposed that Aur-A is activated downstream of cycB-Cdk1 (Marumoto et al., 2002). HeLa cells were subjected to the double-thymidine synchronization protocol, and 4 h after thymidine washout they were incubated with 9 μM RO-3306 (RO), a specific inhibitor of Cdk1 kinase (Vassilev, 2006). The cells were fixed at various times after thymidine washout and stained with Hoechst to determine the mitotic index and with antibodies against the active T288-phosphorylated Aur-A. A similar set of samples was treated for immunoblotting, to reveal the levels of total and active Aur-A. Despite a complete block of entry into mitosis in the RO-treated cells (Figure 5A), activation of Aur-A at the centrosome was not affected by RO addition (Figure 5B). Conversely, this treatment strongly reduced the global levels of active Aur-A (Figure 5C). Thus, through this single-cell analysis and in our experimental setting, early activation of Aur-A at the centrosome (i.e., during the G2 phase) is independent of cycB-Cdk1. This centrosome pool of active Aur-A represents a minimal yet functionally significant fraction that is below the detection limits of Western blotting. On the contrary, the global levels of active Aur-A become detectable by Western blotting only during the mitotic phase, and they were influenced by a positive feedback loop that includes cycB-Cdk1. More importantly, these data show that Golgi-dependent inhibition of Aur-A activation cannot be mediated by a signaling pathway that primarily affects cycB-Cdk1.


Golgi partitioning controls mitotic entry through Aurora-A kinase.

Persico A, Cervigni RI, Barretta ML, Corda D, Colanzi A - Mol. Biol. Cell (2010)

Block of Golgi fragmentation inhibits Aur-A recruitment through a novel mechanism. HeLa cells were grown on coverslips and arrested in S phase by using the double-thymidine block. (A–C) Four hours after S phase block release, the cells were either left in growth medium (nontreated) or treated with 9 μM RO-3306 (a Cdk1 inhibitor) and fixed and processed for immunofluorescence under confocal microscopy or immunoblotting at the indicated times after thymidine removal. Cells were labeled with Hoechst 33342 to determine the mitotic indices up to 12 h after S phase release (A) and with antibodies against T288-phosphorylated Aur-A to determine the relative percentages of pT288 Aur-A–positive cells calculated according to the nontreated cells fixed up to 12 h after S phase release (B). A similar set of samples was processed for immunoblotting to reveal total Aur-A and active Aur-A (PT288) (C). (D–F) Cells were either nonmicroinjected (D and E) or microinjected 1 h after thymidine washout with recombinant GST (GST-inj; 8 mg/ml) or recombinant GRASP65 (GR65-inj, 8–10 mg/ml; E and F), and with FITC-conjugated dextran as microinjection marker (E and F). Eight hours after S phase block release, the nonmicroinjected cells were either left in growth medium (D and E, −) or treated with 30 μM IPA3 (D and E, a Pak inhibitor) and fixed 12 h after the S-phase block release for immunofluorescence under confocal microscopy with antibodies against T288-phosphorylated Aur-A (D), against T423-phosphorylated Pak (E), or against Plk1 (F). The relative percentages of PT288 Aur-A-positive (D), T423 Pak-positive (E) were calculated according to the relevant cells treated with vehicle (−). The relative percentages of Plk1-positive cells (F) were calculated according the noninjected cells on the same coverslip. Quantification data are means ± SD from at least two independent experiments, each carried out in duplicate. More than 200 cells were microinjected for each condition.
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Figure 5: Block of Golgi fragmentation inhibits Aur-A recruitment through a novel mechanism. HeLa cells were grown on coverslips and arrested in S phase by using the double-thymidine block. (A–C) Four hours after S phase block release, the cells were either left in growth medium (nontreated) or treated with 9 μM RO-3306 (a Cdk1 inhibitor) and fixed and processed for immunofluorescence under confocal microscopy or immunoblotting at the indicated times after thymidine removal. Cells were labeled with Hoechst 33342 to determine the mitotic indices up to 12 h after S phase release (A) and with antibodies against T288-phosphorylated Aur-A to determine the relative percentages of pT288 Aur-A–positive cells calculated according to the nontreated cells fixed up to 12 h after S phase release (B). A similar set of samples was processed for immunoblotting to reveal total Aur-A and active Aur-A (PT288) (C). (D–F) Cells were either nonmicroinjected (D and E) or microinjected 1 h after thymidine washout with recombinant GST (GST-inj; 8 mg/ml) or recombinant GRASP65 (GR65-inj, 8–10 mg/ml; E and F), and with FITC-conjugated dextran as microinjection marker (E and F). Eight hours after S phase block release, the nonmicroinjected cells were either left in growth medium (D and E, −) or treated with 30 μM IPA3 (D and E, a Pak inhibitor) and fixed 12 h after the S-phase block release for immunofluorescence under confocal microscopy with antibodies against T288-phosphorylated Aur-A (D), against T423-phosphorylated Pak (E), or against Plk1 (F). The relative percentages of PT288 Aur-A-positive (D), T423 Pak-positive (E) were calculated according to the relevant cells treated with vehicle (−). The relative percentages of Plk1-positive cells (F) were calculated according the noninjected cells on the same coverslip. Quantification data are means ± SD from at least two independent experiments, each carried out in duplicate. More than 200 cells were microinjected for each condition.
Mentions: Because many signaling networks are composed of elements that are functionally connected by feedback loops, it was important to determine whether reduction of Aur-A recruitment to and activation at the centrosome is a secondary consequence of a block of the essential mitotic complex cycB-Cdk1. Indeed, although according to some reports Aur-A activation is independent of Cdk1 activity (Hirota et al., 2003), other reports have proposed that Aur-A is activated downstream of cycB-Cdk1 (Marumoto et al., 2002). HeLa cells were subjected to the double-thymidine synchronization protocol, and 4 h after thymidine washout they were incubated with 9 μM RO-3306 (RO), a specific inhibitor of Cdk1 kinase (Vassilev, 2006). The cells were fixed at various times after thymidine washout and stained with Hoechst to determine the mitotic index and with antibodies against the active T288-phosphorylated Aur-A. A similar set of samples was treated for immunoblotting, to reveal the levels of total and active Aur-A. Despite a complete block of entry into mitosis in the RO-treated cells (Figure 5A), activation of Aur-A at the centrosome was not affected by RO addition (Figure 5B). Conversely, this treatment strongly reduced the global levels of active Aur-A (Figure 5C). Thus, through this single-cell analysis and in our experimental setting, early activation of Aur-A at the centrosome (i.e., during the G2 phase) is independent of cycB-Cdk1. This centrosome pool of active Aur-A represents a minimal yet functionally significant fraction that is below the detection limits of Western blotting. On the contrary, the global levels of active Aur-A become detectable by Western blotting only during the mitotic phase, and they were influenced by a positive feedback loop that includes cycB-Cdk1. More importantly, these data show that Golgi-dependent inhibition of Aur-A activation cannot be mediated by a signaling pathway that primarily affects cycB-Cdk1.

Bottom Line: We show that a block of Golgi partitioning impairs centrosome recruitment and activation of Aurora-A, which results in the G2 block of cell cycle progression.Overexpression of Aurora-A overrides this cell cycle block, indicating that Aurora-A is a major effector of the Golgi checkpoint.Our findings provide the basis for further understanding of the signaling pathways that coordinate organelle inheritance and cell duplication.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro, Chieti, Italy.

ABSTRACT
At the onset of mitosis, the Golgi complex undergoes a multistep fragmentation process that is required for its correct partitioning into the daughter cells. Inhibition of this Golgi fragmentation results in cell cycle arrest at the G2 stage, suggesting that correct inheritance of the Golgi complex is monitored by a "Golgi mitotic checkpoint." However, the molecular basis of this G2 block is not known. Here, we show that the G2-specific Golgi fragmentation stage is concomitant with centrosome recruitment and activation of the mitotic kinase Aurora-A, an essential regulator for entry into mitosis. We show that a block of Golgi partitioning impairs centrosome recruitment and activation of Aurora-A, which results in the G2 block of cell cycle progression. Overexpression of Aurora-A overrides this cell cycle block, indicating that Aurora-A is a major effector of the Golgi checkpoint. Our findings provide the basis for further understanding of the signaling pathways that coordinate organelle inheritance and cell duplication.

Show MeSH
Related in: MedlinePlus