Mitotic Golgi Disassembly Research Articles

Ran pathway-independent regulation of mitotic Golgi disassembly by Importin-\u03b1

To facilitate proper mitotic cell partitioning, the Golgi disassembles by suppressing vesicle fusion. However, the underlying mechanism has not been characterized previously. Here, we report a Ran pathway-independent attenuation mechanism that allows Importin-α (a nuclear transport factor) to suppress the vesicle fusion mediated by p115 (a vesicular tethering factor) and is required for mitotic Golgi disassembly. We demonstrate that Importin-α directly competes with p115 for interaction with the Golgi protein GM130. This interaction, promoted by a phosphate moiety on GM130, is independent of Importin-β and Ran. A GM130 K34A mutant, in which the Importin-α-GM130 interaction is specifically disrupted, exhibited abundant Golgi puncta during metaphase. Importantly, a mutant showing enhanced p115-GM130 interaction presented proliferative defects and G2/M arrest, demonstrating that Importin-α-GM130 binding modulates the Golgi disassembly that governs mitotic progression. Our findings illuminate that the Ran and kinase-phosphatase pathways regulate multiple aspects of mitosis coordinated by Importin-α (e.g. spindle assembly, Golgi disassembly).

Mitotic Golgi disassembly is required for bipolar spindle formation and mitotic progression

During mitosis, the mammalian Golgi vesiculates and, upon partitioning, reassembles in each daughter cell; however, it is not clear whether the disassembly process per se is important for partitioning or is merely an outcome of mitotic entry. Here, we show that Golgi vesiculation is required for progression to metaphase. To prevent Golgi disassembly, we expressed HRP linked to a Golgi-resident protein and acutely triggered the polymerization of 3,3'-diaminobenzidine (DAB) in the Golgi lumen. The DAB polymer does not affect interphase cell viability, but inhibits Golgi fragmentation by nocodazole and brefeldin A and also halts cells in early mitosis. The arrest is Golgi specific and does not occur when DAB is polymerized in the endosomes. Cells with a DAB polymer in the Golgi enter mitosis normally but arrest with an intact Golgi clustered at a monopolar spindle and an active spindle assembly checkpoint (SAC). Mitotic progression is restored upon centrosome depletion by the Polo-like kinase 4 inhibitor, centrinone, indicating that the link between the Golgi and the centrosomes must be dissolved to reach metaphase. These results demonstrate that Golgi disassembly is required for mitotic progression because failure to vesiculate the Golgi activates the canonical SAC. This requirement suggests that cells actively monitor Golgi integrity in mitosis.

Mechanisms and Regulation of the Mitotic Inheritance of the Golgi Complex.

In mammalian cells, the Golgi complex is structured in the form of a continuous membranous system composed of stacks connected by tubular bridges: the “Golgi ribbon.” At the onset of mitosis, the Golgi complex undergoes a multi-step fragmentation process that is required for its correct partition into the dividing cells. Importantly, inhibition of Golgi disassembly results in cell-cycle arrest at the G2 stage, which indicates that accurate inheritance of the Golgi complex is monitored by a “Golgi mitotic checkpoint.” Moreover, mitotic Golgi disassembly correlates with the release of a set of Golgi-localized proteins that acquire specific functions during mitosis, such as mitotic spindle formation and regulation of the spindle checkpoint. Most of these events are regulated by small GTPases of the Arf and Rab families. Here, we review recent studies that are revealing the fundamental mechanisms, the molecular players, and the biological significance of mitotic inheritance of the Golgi complex in mammalian cells. We also briefly comment on how Golgi partitioning is coordinated with mitotic progression.

Monoubiquitination Regulates Golgi Membrane Dynamics In The Cell Cycle

Partitioning of the Golgi membrane into daughter cells during mammalian cell division occurs through a unique disassembly and reassembly process. Several converging lines of evidence have suggested that monoubiquitination plays an essential role in the regulation of post‐mitotic Golgi membrane fusion. Monoubiquitination, as a regulatory signal, occurs during mitotic Golgi disassembly and is required for subsequent Golgi reassembly. The AAA ATPase p97 and its adapter protein p47 are involved in membrane fusion during post‐mitotic Golgi reassembly. The p97/p47 complex binds to monoubiquitin through the UBA domain of p47, and this interaction is required for p97‐mediated Golgi membrane fusion. Proteasome activity is not involved in either Golgi disassembly or reassembly. We have identified a Golgi‐localized ubiquitin E3 ligase HACE1 which is involved in mitotic Golgi disassembly; reduced HACE1 activity is human cancer such as Wilms' tumor results in fragmented Golgi. We have also discovered the p97/p47 binding protein VCIP135 as a deubiquitinating enzyme whose activity is required for post‐mitotic Golgi reassembly. VCIP135 activity is regulated by phosphorylation in the cell cycle; it is inactivated by phosphorylation in metaphase and activated upon dephosphorylation in telophase. Recently, we have identified the substrates on the Golgi membranes whose ubiquitination and deubiquitination are regulated by HACE1 and VCIP135 in the cell cycle. These data suggest that cycles of addition and removal of ubiquitin to and from substrates regulate Golgi membrane dynamics during cell division.

AMPK phosphorylates GBF1 for mitotic Golgi disassembly

In mammalian cells, the Golgi apparatus undergoes extensive fragmentation during mitosis; this is required not only for the partitioning of the complex but also for the process of mitosis. However, the molecular mechanism underlying the mitotic fragmentation of the Golgi is far from clear. Here, we show that AMP-activated protein kinase (AMPK) is phosphorylated and activated when cells enter mitosis. Activated AMPK phosphorylates GBF1, a guanine nucleotide exchange factor (GEF) for Arf-GTPases, disassociating GBF1 from the Golgi membrane and abolishing the action of GBF1 as an Arf1-GEF. We further demonstrate that the phosphorylation of AMPK and GBF1 is essential for Golgi disassembly and subsequent mitosis entry. These data suggest that AMPK-GBF1-Arf1 signaling is involved in the regulation of Golgi fragmentation during mitosis.

Sequential phosphorylation of GRASP65 during mitotic Golgi disassembly

SummaryGRASP65 phosphorylation during mitosis and dephosphorylation after mitosis are required for Golgi disassembly and reassembly during the cell cycle. At least eight phosphorylation sites on GRASP65 have been identified, but whether they are modified in a coordinated fashion during mitosis is so far unknown. In this study, we raised phospho-specific antibodies that recognize phosphorylated T220/T224, S277 and S376 residues of GRASP65, respectively. Biochemical analysis showed that cdc2 phosphorylates all three sites, while plk1 enhances the phosphorylation. Microscopic studies using these antibodies for double and triple labeling demonstrate sequential phosphorylation and dephosphorylation during the cell cycle. S277 and S376 are phosphorylated from late G2 phase through metaphase until telophase when the new Golgi is reassembled. T220/224 is not modified until prophase, but is highly modified from prometaphase to anaphase. In metaphase, phospho-T220/224 signal localizes on both Golgi haze and mitotic Golgi clusters that represent dispersed Golgi vesicles and Golgi remnants, respectively, while phospho-S277 and S376 labeling is more concentrated on mitotic Golgi clusters. Expression of a phosphorylation-resistant GRASP65 mutant T220A/T224A inhibited mitotic Golgi fragmentation to a much larger extent than the expression of the S277A and S376A mutants. In cytokinesis, T220/224 dephosphorylation occurs prior to that of S277, but after S376. This study provides evidence that GRASP65 is sequentially phosphorylated and dephosphorylated during mitosis at different sites to orchestrate Golgi disassembly and reassembly during cell division, with phosphorylation of the T220/224 site being most critical in the process.

The ubiquitin ligase HACE1 regulates Golgi membrane dynamics during the cell cycle

Partitioning of the Golgi membrane into daughter cells during mammalian cell division occurs through a unique disassembly and reassembly process that is regulated by ubiquitination. However, the identity of the ubiquitin ligase is unknown. Here we show that the Homologous to the E6-AP Carboxyl Terminus (HECT) domain containing ubiquitin ligase HACE1 is targeted to the Golgi membrane through interactions with Rab proteins. The ubiquitin ligase activity of HACE1 in mitotic Golgi disassembly is required for subsequent postmitotic Golgi membrane fusion. Depletion of HACE1 using small interfering RNAs or expression of an inactive HACE1 mutant protein in cells impaired postmitotic Golgi membrane fusion. The identification of HACE1 as a Golgi-localized ubiquitin ligase provides evidence that ubiquitin has a critical role in Golgi biogenesis during the cell cycle.

Golgi Biogenesis

The Golgi is an essential membrane-bound organelle in the secretary pathway of eukaryotic cells. In mammalian cells, the Golgi stacks are integrated into a continuous perinuclear ribbon, which poses a challenge for the daughter cells to inherit this membrane organelle during cell division. To facilitate proper partitioning, the mammalian Golgi ribbon is disassembled into vesicles in early mitosis. Following segregation into the daughter cells, a functional Golgi is reformed. Here we summarize our current understanding of the molecular mechanisms that control the mitotic Golgi disassembly and postmitotic reassembly cycle in mammalian cells.

The Role of GRASP65 in Golgi Cisternal Stacking and Cell Cycle Progression

In vitro assays identified the Golgi peripheral protein GRASP65 as a Golgi stacking factor that links adjacent Golgi cisternae by forming mitotically regulated trans-oligomers. These conclusions, however, require further confirmation in the cell. In this study, we showed that the first 112 amino acids at the N-terminus (including the first PDZ domain, PDZ1) of the protein are sufficient for oligomerization. Systematic electron microscopic analysis showed that the expression of non-regulatable GRASP65 mutants in HeLa cells enhanced Golgi stacking in interphase and inhibited Golgi fragmentation during mitosis. Depletion of GRASP65 by small interference RNA (siRNA) reduced the number of cisternae in the Golgi stacks; this reduction was rescued by expressing exogenous GRASP65. These results provided evidence and a molecular mechanism by which GRASP65 stacks Golgi cisternal membranes. Further experiments revealed that inhibition of mitotic Golgi disassembly by expressing non-regulatable GRASP65 mutants did not affect equal partitioning of the Golgi membranes into the daughter cells. However, it delayed mitotic entry and suppressed cell growth; this effect was diminished by dispersing the Golgi apparatus with Brefeldin A treatment prior to mitosis, suggesting that Golgi disassembly at the onset of mitosis plays a role in cell cycle progression.

Molecular Mechanism of Mitotic Golgi Disassembly and Reassembly Revealed by a Defined Reconstitution Assay

In mammalian cells, flat Golgi cisternae closely arrange together to form stacks. During mitosis, the stacked structure undergoes a continuous fragmentation process. The generated mitotic Golgi fragments are distributed into the daughter cells, where they are reassembled into new Golgi stacks. In this study, an in vitro assay has been developed using purified proteins and Golgi membranes to reconstitute the Golgi disassembly and reassembly processes. This technique provides a useful tool to delineate the mechanisms underlying the morphological change. There are two processes during Golgi disassembly: unstacking and vesiculation. Unstacking is mediated by two mitotic kinases, cdc2 and plk, which phosphorylate the Golgi stacking protein GRASP65 and thus disrupt the oligomer of this protein. Vesiculation is mediated by the COPI budding machinery ARF1 and the coatomer complex. When treated with a combination of purified kinases, ARF1 and coatomer, the Golgi membranes were completely fragmented into vesicles. After mitosis, there are also two processes in Golgi reassembly: formation of single cisternae by membrane fusion, and restacking. Cisternal membrane fusion requires two AAA ATPases, p97 and NSF (N-ethylmaleimide-sensitive fusion protein), each of which functions together with specific adaptor proteins. Restacking of the newly formed Golgi cisternae requires dephosphorylation of Golgi stacking proteins by the protein phosphatase PP2A. This systematic study revealed the minimal machinery that controls the mitotic Golgi disassembly and reassembly processes.

Dispersal of Golgi matrix proteins during mitotic Golgi disassembly.

During mitosis, the mammalian Golgi disassembles into numerous vesicles and larger membrane structures referred to as clusters or remnants. Following mitosis, the vesicles and clusters reassemble to form an intact Golgi in each daughter cell. One model of Golgi biogenesis states that Golgi matrix proteins remain assembled in mitotic clusters and then serve as a template for Golgi reassembly. To test this idea, we performed a 3D-computational analysis of mitotic cells to determine the extent to which these proteins remain in mitotic clusters. As a control we used brefeldin A-induced Golgi disassembly which causes dispersal of Golgi enzymes, but leaves matrix proteins in remnant structures. Unlike brefeldin A-treated cells, in which matrix proteins were clearly sorted from non-matrix proteins, we observed extensive dispersal of matrix proteins in metaphase cells with no evidence of differential sorting of these proteins from other Golgi proteins. The extensive disassembly of matrix proteins argues against their participation in a stable template and supports a self-assembly mode of Golgi biogenesis.

A role for Arf1 in mitotic Golgi disassembly, chromosome segregation, and cytokinesis.

In mitosis, chromosome, cytoskeleton, and organelle dynamics must be coordinated for successful cell division. Here, we present evidence for a role for Arf1, a small GTPase associated with the Golgi apparatus, in the orchestration of mitotic Golgi breakdown, chromosome segregation, and cytokinesis. We show that early in mitosis Arf1 becomes inactive and dissociates from Golgi membranes. This is followed by the dispersal of numerous Arf1-dependent peripheral Golgi proteins and subsequent Golgi disassembly. If Arf1 is kept in an active state by treatment with the small molecule H89 or expression of its GTP-locked form, intact Golgi membranes with bound peripheral proteins persist throughout mitosis. These cells enter mitosis but exhibit gross defects in chromosome segregation and cytokinetic furrow ingression. These findings suggest that mitotic Golgi disassembly depends on Arf1 inactivation and is used by the cell to disperse numerous peripheral Golgi proteins for coordinating the behavior of Golgi membranes, chromosomes, and cytoskeleton during mitosis.

In Search of an Essential Step during Mitotic Golgi Disassembly and Inheritance

In Search of an Essential Step during Mitotic Golgi Disassembly and Inheritance

Mitotic phosphorylation of Golgi reassembly stacking protein 55 by mitogen-activated protein kinase ERK2.

The role of the mitogen-activated protein kinase kinase (MKK)/extracellular-activated protein kinase (ERK) pathway in mitotic Golgi disassembly is controversial, in part because Golgi-localized targets have not been identified. We observed that Golgi reassembly stacking protein 55 (GRASP55) was phosphorylated in mitotic cells and extracts, generating a mitosis-specific phospho-epitope recognized by the MPM2 mAb. This phosphorylation was prevented by mutation of ERK consensus sites in GRASP55. GRASP55 mitotic phosphorylation was significantly reduced, both in vitro and in vivo, by treatment with U0126, a potent and specific inhibitor of MKK and thus ERK activation. Furthermore, ERK2 directly phosphorylated GRASP55 on the same residues that generated the MPM2 phospho-epitope. These results are the first demonstration of GRASP55 mitotic phosphorylation and indicate that the MKK/ERK pathway directly phosphorylates the Golgi during mitosis.

Binding of the Vesicle Docking Protein p115 to Golgi Membranes Is Inhibited under Mitotic Conditions

The vesicle docking protein p115 showed saturable, high affinity binding to interphase Golgi membranes. The affinity of binding was up to 20-fold lower using membranes preincubated with mitotic cytosol. In contrast, binding was not affected by mitotic pretreatment of p115. The reduction in p115 binding was mediated by phosphorylation, could be induced by a cyclin-dependent kinase, and was fully reversible. A shift of p115 from membranes to cytosol was also found after fractionating mitotic cells. The functional significance of the decreased binding was addressed by in vitro mitotic incubations which disassemble Golgi cisternae, predominantly producing transport vesicles. The addition of excess p115 decreased loss of membrane from cisternae, indicating that p115's action is limiting while transport vesicles accumulate. The cessation of intra-Golgi traffic in mitosis has been hypothesized to result from an inhibition of membrane fusion while budding of transport vesicles continues. This process also contributes to mitotic Golgi disassembly. Our results imply that there is a mitotic modification to Golgi membranes leading to a reduction in the affinity of the p115 receptor. Reduced p115 binding may play a part in the inhibition of membrane fusion by preventing prior vesicle docking.