The mitotic spindle mediates inheritance of the Golgi ribbon structure

The mammalian Golgi ribbon disassembles during mitosis and reforms in both daughter cells after division. Mitotic Golgi membranes concentrate around the spindle poles, suggesting that the spindle may control Golgi partitioning. To test this, cells were induced to divide asymmetrically with the entire spindle segregated into only one daughter cell. A ribbon reforms in the nucleated karyoplasts, whereas the Golgi stacks in the cytoplasts are scattered. However, the scattered Golgi stacks are polarized and transport cargo. Microinjection of Golgi extract together with tubulin or incorporation of spindle materials rescues Golgi ribbon formation. Therefore, the factors required for postmitotic Golgi ribbon assembly are transferred by the spindle, but the constituents of functional stacks are partitioned independently, suggesting that Golgi inheritance is regulated by two distinct mechanisms.     

Golgi complex fragmentation in G2/M transition: An organelle-based cell-cycle checkpoint

In mammalian cells, the Golgi complex is organized into a continuous membranous system known as the Golgi ribbon, which is formed by individual Golgi stacks that are laterally connected by tubular bridges. During mitosis, the Golgi ribbon undergoes extensive fragmentation through a multistage process that is required for its correct partitioning into the daughter cells. Importantly, inhibition of this Golgi disassembly results in cell-cycle arrest at the G2 stage, suggesting that accurate inheritance of the Golgi complex is monitored by a "Golgi mitotic checkpoint." Here, we discuss the mechanisms and regulation of the Golgi ribbon breakdown and briefly comment on how Golgi partitioning may inhibit G2/M transition.     

Accommodation of large cargo within Golgi cisternae

In mammalian cells, the Golgi complex has an elaborate structure consisting of stacked, flattened cisternal membranes collected into a ribbon in the center of the cell. Amazingly, the flattened cisternae can rapidly dilate to accommodate large cargo as it traffics through the organelle. The mechanism by which this occurs is unknown. Exocytosis of large cargo is essential for many physiological processes, including collagen and lipoprotein secretion, and defects in the process lead to disease. In addition, enveloped viruses that bud into the endoplasmic reticulum or Golgi complex must also be transported through Golgi cisternae for secretion from the infected cell. This review summarizes our understanding of intra-Golgi transport of large cargo, and outlines current questions open for experimentation.     

Unraveling the Golgi ribbon

The Golgi apparatus lies at the heart of the secretory pathway where it receives, modifies and sorts protein cargo to the proper intracellular or extracellular location. Although this secretory function is highly conserved throughout the eukaryotic kingdom, the structure of the Golgi complex is arranged very differently among species. In particular, Golgi membranes in vertebrate cells are integrated into a single compact entity termed the Golgi ribbon that is normally localized in the perinuclear area and in close vicinity to the centrosomes. This organization poses a challenge for cell division when the single Golgi ribbon needs to be partitioned into the two daughter cells. To ensure faithful inheritance in the progeny, the Golgi ribbon is divided in three consecutive steps in mitosis, namely disassembly, partitioning and reassembly. However, the structure of the Golgi ribbon is only present in higher animals and Golgi disassembly during mitosis is not ubiquitous in all organisms. Therefore, there must be unique reasons to build up the Golgi in this particular conformation and to preserve it over generations. In this review, we first highlight the diversity of the Golgi architecture in different organisms and revisit the concept of the Golgi ribbon. Following on, we discuss why the ribbon is needed and how it forms in vertebrate cells. Lastly, we conclude with likely purposes of mitotic ribbon disassembly and further propose mechanisms by which it regulates mitosis.     

Golgi apparatus partitioning during cell division

This review discusses the mitotic segregation of the Golgi apparatus. The results from classical biochemical and morphological studies have suggested that in mammalian cells this organelle remains distinct during mitosis, although highly fragmented through the formation of mitotic Golgi clusters of small tubules and vesicles. Shedding of free Golgi-derived vesicles would consume Golgi clusters and disperse this organelle throughout the cytoplasm. Vesicles could be partitioned in a stochastic and passive way between the two daughter cells and act as a template for the reassembly of this key organelle. This model has recently been modified by results obtained using GFP- or HRP-tagged Golgi resident enzymes, live cell imaging and electron microscopy. Results obtained with these techniques show that the mitotic Golgi clusters are stable entities throughout mitosis that partition in a microtubule spindle-dependent fashion. Furthermore, a newer model proposes that at the onset of mitosis, the Golgi apparatus completely loses its identity and is reabsorbed into the endoplasmic reticulum. This suggests that the partitioning of the Golgi apparatus is entirely dependent on the partitioning of the endoplasmic reticulum. We critically discuss both models and summarize what is known about the molecular mechanisms underlying the Golgi disassembly and reassembly during and after mitosis. We will also review how the study of the Golgi apparatus during mitosis in other organisms can answer current questions and perhaps reveal novel mechanisms.     

Active ADP-ribosylation factor-1 (ARF1) is required for mitotic Golgi fragmentation

In mammalian cells the Golgi apparatus undergoes an extensive disassembly process at the onset of mitosis that is believed to facilitate equal partitioning of this organelle into the two daughter cells. However, the underlying mechanisms for this fragmentation process are so far unclear. Here we have investigated the role of the ADP-ribosylation factor-1 (ARF1) in this process to determine whether Golgi fragmentation in mitosis is mediated by vesicle budding. ARF1 is a small GTPase that is required for COPI vesicle formation from the Golgi membranes. Treatment of Golgi membranes with mitotic cytosol or with purified coatomer together with wild type ARF1 or its constitutive active form, but not the inactive mutant, converted the Golgi membranes into COPI vesicles. ARF1-depleted mitotic cytosol failed to fragment Golgi membranes. ARF1 is associated with Golgi vesicles generated in vitro and with vesicles in mitotic cells. In addition, microinjection of constitutive active ARF1 did not affect mitotic Golgi fragmentation or cell progression through mitosis. Our results show that ARF1 is active during mitosis and that this activity is required for mitotic Golgi fragmentation.     

Mena–GRASP65 interaction couples actin polymerization to Golgi ribbon linking

In mammalian cells, the Golgi reassembly stacking protein 65 (GRASP65) has been implicated in both Golgi stacking and ribbon linking by forming trans-oligomers through the N-terminal GRASP domain. Because the GRASP domain is globular and relatively small, but the gaps between stacks are large and heterogeneous, it remains puzzling how GRASP65 physically links Golgi stacks into a ribbon. To explore the possibility that other proteins may help GRASP65 in ribbon linking, we used biochemical methods and identified the actin elongation factor Mena as a novel GRASP65-binding protein. Mena is recruited onto the Golgi membranes through interaction with GRASP65. Depleting Mena or disrupting actin polymerization resulted in Golgi fragmentation. In cells, Mena and actin were required for Golgi ribbon formation after nocodazole washout; in vitro, Mena and microfilaments enhanced GRASP65 oligomerization and Golgi membrane fusion. Thus Mena interacts with GRASP65 to promote local actin polymerization, which facilitates Golgi ribbon linking.     

The coiled-coil membrane protein golgin-84 is a novel rab effector required for Golgi ribbon formation

Fragmentation of the mammalian Golgi apparatus during mitosis requires the phosphorylation of a specific subset of Golgi-associated proteins. We have used a biochemical approach to characterize these proteins and report here the identification of golgin-84 as a novel mitotic target. Using cryoelectron microscopy we could localize golgin-84 to the cis-Golgi network and found that it is enriched on tubules emanating from the lateral edges of, and often connecting, Golgi stacks. Golgin-84 binds to active rab1 but not cis-Golgi matrix proteins. Overexpression or depletion of golgin-84 results in fragmentation of the Golgi ribbon. Strikingly, the Golgi ribbon is converted into mini-stacks constituting only approximately 25% of the volume of a normal Golgi apparatus upon golgin-84 depletion. These mini-stacks are able to carry out protein transport, though with reduced efficiency compared with a normal Golgi apparatus. Our results suggest that golgin-84 plays a key role in the assembly and maintenance of the Golgi ribbon in mammalian cells.     

The Golgin Tether Giantin Regulates the Secretory Pathway by Controlling Stack Organization within Golgi Apparatus

Golgins are coiled-coil proteins that play a key role in the regulation of Golgi architecture and function. Giantin, the largest golgin in mammals, forms a complex with p115, rab1, GM130, and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), thereby facilitating vesicle tethering and fusion processes around the Golgi apparatus. Treatment with the microtubule destabilizing drug nocodazole transforms the Golgi ribbon into individual Golgi stacks. Here we show that siRNA-mediated depletion of giantin resulted in more dispersed Golgi stacks after nocodazole treatment than by control treatment, without changing the average cisternal length. Furthermore, depletion of giantin caused an increase in cargo transport that was associated with altered cell surface protein glycosylation. Drosophila S2 cells are known to have dispersed Golgi stacks and no giantin homolog. The exogenous expression of mammalian giantin cDNA in S2 cells resulted in clustered Golgi stacks, similar to the Golgi ribbon in mammalian cells. These results suggest that the spatial organization of the Golgi ribbon is mediated by giantin, which also plays a role in cargo transport and sugar modifications.     

The Golgi ribbon structure facilitates anterograde transport of large cargoes

In mammalian cells, individual Golgi stacks fuse laterally to form the characteristic perinuclear ribbon structure. Yet the purpose of this remarkable structure has been an enigma. We report that breaking down the ribbon of mammalian cells strongly inhibits intra-Golgi transport of large cargoes without altering the rate of transport of smaller cargoes. In addition, insect cells that naturally harbor dispersed Golgi stacks have limited capacity to transport artificial oversized cargoes. These results imply that the ribbon structure is an essential requirement for transport of large cargoes in mammalian cells, and we suggest that this is because it enables the dilated rims of cisternae (containing the aggregates) to move across the stack as they transfer among adjacent stacks within the ribbon structure.     

Caspase-mediated cleavage of the stacking protein GRASP65 is required for Golgi fragmentation during apoptosis

The mammalian Golgi complex is comprised of a ribbon of stacked cisternal membranes often located in the pericentriolar region of the cell. Here, we report that during apoptosis the Golgi ribbon is fragmented into dispersed clusters of tubulo-vesicular membranes. We have found that fragmentation is caspase dependent and identified GRASP65 (Golgi reassembly and stacking protein of 65 kD) as a novel caspase substrate. GRASP65 is cleaved specifically by caspase-3 at conserved sites in its membrane distal COOH terminus at an early stage of the execution phase. Expression of a caspase-resistant form of GRASP65 partially preserved cisternal stacking and inhibited breakdown of the Golgi ribbon in apoptotic cells. Our results suggest that GRASP65 is an important structural component required for maintenance of Golgi apparatus integrity.     

Inheritance of the endoplasmic reticulum and Golgi apparatus

Yeast and mammalian cells use a variety of different mechanisms to ensure that the endoplasmic reticulum and Golgi apparatus are inherited by both daughter cells on cell division. In yeast, endoplasmic reticulum inheritance involves both active microtubule and passive actin-based mechanisms, while the Golgi is transported into the forming daughter cell by an active actin-based mechanism. Animal cells actively partition the endoplasmic reticulum and Golgi apparatus, but association with the mitotic spindle-rather than the actin cytoskeleton-appears to be the mechanism     

The localization and phosphorylation of p47 are important for Golgi disassembly-assembly during the cell cycle

In mammalian cells, the Golgi apparatus is disassembled at the onset of mitosis and reassembled at the end of mitosis. This disassembly-reassembly is generally believed to be essential for the equal partitioning of Golgi into two daughter cells. For Golgi disassembly, membrane fusion, which is mediated by NSF and p97, needs to be blocked. For the NSF pathway, the tethering of p115-GM130 is disrupted by the mitotic phosphorylation of GM130, resulting in the inhibition of NSF-mediated fusion. In contrast, the p97/p47 pathway does not require p115-GM130 tethering, and its mitotic inhibitory mechanism has been unclear. Now, we have found that p47, which mainly localizes to the nucleus during interphase, is phosphorylated on Serine-140 by Cdc2 at mitosis. The phosphorylated p47 does not bind to Golgi membranes. An in vitro assay shows that this phosphorylation is required for Golgi disassembly. Microinjection of p47(S140A), which is unable to be phosphorylated, allows the cell to keep Golgi stacks during mitosis and has no effect on the equal partitioning of Golgi into two daughter cells, suggesting that Golgi fragmentation-dispersion may not be obligatory for equal partitioning even in mammalian cells.     

Crn7 interacts with AP-1 and is required for the maintenance of Golgi morphology and protein export from the Golgi

Crn7 is a novel cytosolic mammalian WD-repeat protein of unknown function that associates with Golgi membranes. Here, we demonstrate that Crn7 knockdown by small interfering RNA results in dramatic changes in the Golgi morphology and function. First, the Golgi ribbon is disorganized in Crn7 KD cells. Second, the Golgi export of several marker proteins including VSV envelope G glycoprotein is greatly reduced but not the retrograde protein import into the Golgi complex. We further establish that Crn7 co-precipitates with clathrin adaptor AP-1 but is not required for AP-1 targeting to Golgi membranes. We identify tyrosine 288-based motif as part of a canonical YXXPhi sorting signal and a major mu1-adaptin binding site in vitro. This study provides the first insight into the function of mammalian Crn7 protein in the Golgi complex.     

An Ordered Inheritance Strategy for the Golgi Apparatus: Visualization of Mitotic Disassembly Reveals a Role for the Mitotic Spindle

During mitosis, the ribbon of the Golgi apparatus is transformed into dispersed tubulo-vesicular membranes, proposed to facilitate stochastic inheritance of this low copy number organelle at cytokinesis. Here, we have analyzed the mitotic disassembly of the Golgi apparatus in living cells and provide evidence that inheritance is accomplished through an ordered partitioning mechanism. Using a Sar1p dominant inhibitor of cargo exit from the endoplasmic reticulum (ER), we found that the disassembly of the Golgi observed during mitosis or microtubule disruption did not appear to involve retrograde transport of Golgi residents to the ER and subsequent reorganization of Golgi membrane fragments at ER exit sites, as has been suggested. Instead, direct visualization of a green fluorescent protein (GFP)-tagged Golgi resident through mitosis showed that the Golgi ribbon slowly reorganized into 1–3-μm fragments during G2/early prophase. A second stage of fragmentation occurred coincident with nuclear envelope breakdown and was accompanied by the bulk of mitotic Golgi redistribution. By metaphase, mitotic Golgi dynamics appeared to cease. Surprisingly, the disassembly of mitotic Golgi fragments was not a random event, but involved the reorganization of mitotic Golgi by microtubules, suggesting that analogous to chromosomes, the Golgi apparatus uses the mitotic spindle to ensure more accurate partitioning during cytokinesis.     

The biogenesis of the Golgi ribbon: the roles of membrane input from the ER and of GM130

The Golgi complex in mammalian cells forms a continuous ribbon of interconnected stacks of flat cisternae. We show here that this distinctive architecture reflects and requires the continuous input of membranes from the endoplasmic reticulum (ER), in the form of pleiomorphic ER-to-Golgi carriers (EGCs). An important step in the biogenesis of the Golgi ribbon is the complete incorporation of the EGCs into the stacks. This requires the Golgi-matrix protein GM130, which continuously cycles between the cis-Golgi compartments and the EGCs. On acquiring GM130, the EGCs undergo homotypic tethering and fusion, maturing into larger and more homogeneous membrane units that appear primed for incorporation into the Golgi stacks. In the absence of GM130, this process is impaired and the EGCs remain as distinct entities. This induces the accumulation of tubulovesicular membranes, the shortening of the cisternae, and the breakdown of the Golgi ribbon. Under these conditions, however, secretory cargo can still be delivered to the Golgi complex, although this occurs less efficiently, and apparently through transient and/or limited continuities between the EGCs and the Golgi cisternae.     

Mitosis controls the Golgi and the Golgi controls mitosis

In mammals, the Golgi complex is structured in the form of a continuous membranous system composed of up to 100 stacks connected by tubular bridges, the 'Golgi ribbon'. During mitosis, the Golgi undergoes extensive fragmentation through a multistage process that allows its correct partitioning and inheritance by daughter cells. Strikingly, this Golgi fragmentation is required not only for inheritance but also for mitotic entrance itself, since its block results in the arrest of the cell cycle in G2. This is called the 'Golgi mitotic checkpoint'. Recent studies have identified the severing of the ribbon into its constituent stacks during early G2 as the precise stage of Golgi fragmentation that controls mitotic entry. This opens new ways to elucidate the mechanism of the Golgi checkpoint.     

Golgi Ribbon Unlinking: An Organelle-Based G2/M Checkpoint

Cell cycle checkpoints have mostly been associated with signaling mechanisms monitoring the genetic material for abnormalities and inaccuracy in partitioning, such as DNA lesions or failure in chromosome attachment to kinetochores. However, it becomes more evident that other checkpoints are turned on by cytoplasmic events, such as the cytoskeleton organisation and the organelle structure. Here, we summarize recent evidence strongly suggesting that the integrity of the Golgi ribbon, and more precisely the tubules interconnecting the Golgi stacks to form this ribbon, at late G2/early prophase, is linked to a Golgi-related G2/M checkpoint. A number of kinases phosphorylating a so far small subset of Golgi-localized proteins have been shown to promote the G2-specific Golgi ribbon unlinking allowing the cell to enter mitosis. When these kinases are inactivated or when the substrates cannot be phosphorylated, Golgi unlinking is prevented and the cells are blocked or delayed in G2 phase.     

Actin- and microtubule-dependent regulation of Golgi morphology by FHDC1

The Golgi apparatus is the central hub of intracellular trafficking and consists of tethered stacks of cis, medial, and trans cisternae. In mammalian cells, these cisternae are stitched together as a perinuclear Golgi ribbon, which is required for the establishment of cell polarity and normal subcellular organization. We previously identified FHDC1 (also known as INF1) as a unique microtubule-binding member of the formin family of cytoskeletal-remodeling proteins. We show here that endogenous FHDC1 regulates Golgi ribbon formation and has an apparent preferential association with the Golgi-derived microtubule network. Knockdown of FHDC1 expression results in defective Golgi assembly and suggests a role for FHDC1 in maintenance of the Golgi-derived microtubule network. Similarly, overexpression of FHDC1 induces dispersion of the Golgi ribbon into functional ministacks. This effect is independent of centrosome-derived microtubules and instead likely requires the interaction between the FHDC1 microtubule-binding domain and the Golgi-derived microtubule network. These effects also depend on the interaction between the FHDC1 FH2 domain and the actin cytoskeleton. Thus our results suggest that the coordination of actin and microtubule dynamics by FHDC1 is required for normal Golgi ribbon formation.     

Disconnecting the Golgi ribbon from the centrosome prevents directional cell migration and ciliogenesis

Mammalian cells exhibit a frequent pericentrosomal Golgi ribbon organization. In this paper, we show that two AKAP450 N-terminal fragments, both containing the Golgi-binding GM130-interacting domain of AKAP450, dissociated endogenous AKAP450 from the Golgi and inhibited microtubule (MT) nucleation at the Golgi without interfering with centrosomal activity. These two fragments had, however, strikingly different effects on both Golgi apparatus (GA) integrity and positioning, whereas the short fragment induced GA circularization and ribbon fragmentation, the large construct that encompasses an additional p150glued/MT-binding domain induced separation of the Golgi ribbon from the centrosome. These distinct phenotypes arose by specific interference of each fragment with either Golgi-dependent or centrosome-dependent stages of Golgi assembly. We could thus demonstrate that breaking the polarity axis by perturbing GA positioning has a more dramatic effect on directional cell migration than disrupting the Golgi ribbon. Both features, however, were required for ciliogenesis. We thus identified AKAP450 as a key determinant of pericentrosomal Golgi ribbon integrity, positioning, and function in mammalian cells.