The Golgi mitotic checkpoint is controlled by BARS-dependent fission of the Golgi ribbon into separate stacks in G2
2007; Springer Nature; Volume: 26; Issue: 10 Linguagem: Inglês
10.1038/sj.emboj.7601686
ISSN1460-2075
AutoresAntonino Colanzi, Cristina Hidalgo-Carcedo, Angela Persico, Claudia Cericola, Gabriele Turacchio, Matteo Bonazzi, Alberto Luini, Daniela Corda,
Tópico(s)Photoreceptor and optogenetics research
ResumoArticle12 April 2007free access The Golgi mitotic checkpoint is controlled by BARS-dependent fission of the Golgi ribbon into separate stacks in G2 Antonino Colanzi Antonino Colanzi Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro, Chieti, Italy Search for more papers by this author Cristina Hidalgo Carcedo Cristina Hidalgo CarcedoPresent address: Tumour Cell Biology Laboratory, Cancer Research UK London Research Institute, London WC2A 3PX, UK. Search for more papers by this author Angela Persico Angela Persico Search for more papers by this author Claudia Cericola Claudia Cericola Search for more papers by this author Gabriele Turacchio Gabriele Turacchio Search for more papers by this author Matteo Bonazzi Matteo Bonazzi Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro, Chieti, Italy Search for more papers by this author Alberto Luini Alberto Luini Search for more papers by this author Daniela Corda Corresponding Author Daniela Corda Search for more papers by this author Antonino Colanzi Antonino Colanzi Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro, Chieti, Italy Search for more papers by this author Cristina Hidalgo Carcedo Cristina Hidalgo CarcedoPresent address: Tumour Cell Biology Laboratory, Cancer Research UK London Research Institute, London WC2A 3PX, UK. Search for more papers by this author Angela Persico Angela Persico Search for more papers by this author Claudia Cericola Claudia Cericola Search for more papers by this author Gabriele Turacchio Gabriele Turacchio Search for more papers by this author Matteo Bonazzi Matteo Bonazzi Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro, Chieti, Italy Search for more papers by this author Alberto Luini Alberto Luini Search for more papers by this author Daniela Corda Corresponding Author Daniela Corda Search for more papers by this author Author Information Antonino Colanzi1, Cristina Hidalgo Carcedo‡, Angela Persico, Claudia Cericola, Gabriele Turacchio, Matteo Bonazzi1, Alberto Luini and Daniela Corda 1Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro, Chieti, Italy ‡These authors contributed equally to this work *Corresponding author. Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Via Nazionale, 8/A, Santa Maria Imbaro, Chieti 66030, Italy. Tel.: +39 0872 570353; Fax: +39 0872 570412; E-mail: [email protected] of Cell Biology and Oncology, Consorzio Mario Negri Sud, Via Nazionale, 8/A, Santa Maria Imbaro, Chieti 66030, Italy. Tel.: +39 0872 570353; Fax: +39 0872 570412; E-mail: [email protected] The EMBO Journal (2007)26:2465-2476https://doi.org/10.1038/sj.emboj.7601686 Present address: Tumour Cell Biology Laboratory, Cancer Research UK London Research Institute, London WC2A 3PX, UK. Present address: Unité des Interactions Bactéries-Cellules, Institut Pasteur, 75015 Paris, France PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The Golgi ribbon is a complex structure of many stacks interconnected by tubules that undergo fragmentation during mitosis through a multistage process that allows correct Golgi inheritance. The fissioning protein CtBP1-S/BARS (BARS) is essential for this, and is itself required for mitotic entry: a block in Golgi fragmentation results in cell-cycle arrest in G2, defining the 'Golgi mitotic checkpoint'. Here, we clarify the precise stage of Golgi fragmentation required for mitotic entry and the role of BARS in this process. Thus, during G2, the Golgi ribbon is converted into isolated stacks by fission of interstack connecting tubules. This requires BARS and is sufficient for G2/M transition. Cells without a Golgi ribbon are independent of BARS for Golgi fragmentation and mitotic entrance. Remarkably, fibroblasts from BARS-knockout embryos have their Golgi complex divided into isolated stacks at all cell-cycle stages, bypassing the need for BARS for Golgi fragmentation. This identifies the precise stage of Golgi fragmentation and the role of BARS in the Golgi mitotic checkpoint, setting the stage for molecular analysis of this process. Introduction The Golgi complex is the essential station for protein processing and sorting at the center of the secretory pathway. In mammalian cells, this organelle is structured in the form of numerous stacks (usually 70–100; Trucco et al, 2004) that are laterally connected by tubular bridges known as the 'non-compact' zones. This generates a continuous membranous system, the 'Golgi ribbon', that is located in a perinuclear position (Rambourg et al, 1987; Shorter and Warren, 2002). One intriguing aspect of the physiology of the Golgi complex is its mechanism of mitotic inheritance, which involves progressive and reversible disassembly of the ribbon into dispersed elements, allowing the correct partitioning of the Golgi membranes between the daughter cells (Shorter and Warren, 2002; Colanzi et al, 2003). This mitotic fragmentation takes place in two main sequential steps: the pericentriolar Golgi membranes are first converted to scattered tubulo-reticular elements and then these are further fragmented and dispersed throughout the cytoplasm, appearing as the Golgi 'haze' (Colanzi et al, 2003; Axelsson and Warren, 2004; Altan-Bonnet et al, 2006). The molecular mechanisms involved here are partially understood, and through the development of in vitro Golgi fragmentation assays, some of the relevant components have been identified (Acharya et al, 1998; Colanzi et al, 2000, 2003; Sutterlin et al, 2001, 2002; Shorter and Warren, 2002). Among these, a key player is the protein CtBP1-S/BARS (BARS) (Hidalgo Carcedo et al, 2004), which acts by inducing fission of Golgi tubules during both normal trafficking and mitosis (Weigert et al, 1999). A remarkable recent development in this area is that mitotic Golgi fragmentation is required not only for Golgi partitioning but also for entry into mitosis. This was established through a variety of approaches, which showed that inhibition of Golgi fragmentation in living cells results in arrest of the cell cycle at the G2 stage (Sutterlin et al, 2002; Hidalgo Carcedo et al, 2004). These approaches included treatments affecting the function of the Golgi matrix protein GRASP-65 (Sutterlin et al, 2002; Preisinger et al, 2005; Yoshimura et al, 2005) as well as the microinjection of an anti-BARS blocking antibody and BARS dominant-negative mutants, and BARS depletion using antisense oligonucleotides (Hidalgo Carcedo et al, 2004). These anti-BARS treatments blocked fragmentation of the Golgi complex and progression through the cell cycle, whereas readdition of recombinant BARS restored these processes (Hidalgo Carcedo et al, 2004). Based on these lines of evidence, the existence of a novel checkpoint that appears to sense the integrity of the Golgi complex, the 'Golgi checkpoint', has been postulated (Sutterlin et al, 2002; Hidalgo Carcedo et al, 2004; Preisinger et al, 2005). The physiological relevance of this checkpoint is witnessed by its link with profound and prolonged inhibition of mitotic entrance (Sutterlin et al, 2002; Hidalgo Carcedo et al, 2004; Yoshimura et al, 2005). Unfortunately, basic aspects of the relationship between Golgi fragmentation and mitotic ingression remain unclear, including the exact cell-cycle phase during which this Golgi checkpoint is triggered (see below) and the precise event in Golgi fragmentation that controls the checkpoint. This information is fundamental for identification of the mechanisms behind the Golgi checkpoint. In particular, a confusing feature here is that it is generally believed that Golgi fragmentation initiates during prophase (Shorter and Warren, 2002). This, however, would be incompatible with the demonstration that inhibition of Golgi fragmentation blocks cell-cycle progression in G2 (Sutterlin et al, 2002; Hidalgo Carcedo et al, 2004), an earlier cell-cycle phase. One possible explanation relates to the ability of mammalian cells to retreat from the earliest mitotic stage (prophase) in the presence of a 'stress' (Mikhailov and Rieder, 2002); therefore, in principle, a cell could sense Golgi fragmentation in early mitosis, and if something is amiss, it would rapidly exit prophase, returning to the premitotic G2 phase. Alternatively, the alterations in the Golgi complex that are necessary for entry into mitosis could occur already during G2; in this case, however, they should be so subtle structurally as to have so far escaped detection. Because BARS controls both Golgi fragmentation and the Golgi checkpoint, a potentially useful approach to address the above questions is likely to be the identification of the precise site of action of BARS in mitotic Golgi partitioning. A relevant hint as to the role of BARS derives from our previous work, which showed that it operates in the fission of Golgi tubules involved in a variety of processes (Weigert et al, 1999; Mironov et al, 2004). This suggests that the role of BARS in Golgi partitioning might be in the severing of the tubular non-compact zones of the Golgi ribbon. Here, using BARS-related molecular tools and a variety of morphological and functional approaches, we clarify these issues and identify an early stage of the Golgi partitioning process that has not been fully characterized so far, namely, the severing of the Golgi ribbon into separate stacks or small groups of stacks. We find that this step occurs in G2 and is necessary to pass the G2/M Golgi checkpoint. Presumably, this is the minimal level of Golgi fragmentation needed to divide the Golgi between daughter cells. BARS is specifically required for severing of the non-compact zones in G2, resulting in ribbon breakdown. The further disassembly of the Golgi complex, which leads to complete fragmentation of this organelle, is BARS independent and takes place only after the cells have entered mitosis. These results set the stage for defining the molecular processes involved in the Golgi checkpoint. Results The Golgi ribbon is severed into isolated stacks during G2 As indicated above, inhibition of fragmentation of the Golgi complex leads to cell-cycle arrest in the G2 phase (Sutterlin et al, 2002; Hidalgo Carcedo et al, 2004), suggesting that Golgi partitioning initiates during G2 rather than, as generally believed, in prophase (Shorter and Warren, 2002). If this is the case, this partitioning most likely involves minor modifications of the general structure of the organelle that have gone unnoticed until now. To explore this possibility, we first used confocal microscopy to investigate the general morphology of Golgi membranes in HeLa cells during G2, taking advantage of the combined staining patterns of anti-phosphorylated-H1 and anti-H3 antibodies to identify late-G2 cells (Hendzel et al, 1997; Hidalgo Carcedo et al, 2004). When labelled with antibodies against giantin, the Golgi ribbon in S phase appeared as a large continuous perinuclear formation, either compact or elongated, with alternating heavily and lightly stained areas that possibly represent stacks and non-compact zones (Figure 1A). A characteristic continuous ribbon was present in most of the cells found in S phase (Figure 1B). During late G2 (1–2% of the cells), the Golgi complex showed breaks that interrupted the continuity of the Golgi membranes (with a considerable increase in discrete Golgi objects) in more than 90% of the G2 cells (Figure 1A and B), indicating that some morphological modification was taking place. Then, in late prophase/prometaphase, the pericentriolar Golgi stacks broke down into smaller fragments, and finally, between prometaphase and early anaphase, the Golgi membranes underwent further fragmentation and were diffusely dispersed throughout the cytosol (mitotic Golgi haze) (Figure 1A), in agreement with previous observations (Shorter and Warren, 2002). Similar results were obtained when staining the Golgi with an anti-GM130 antibody (not shown). Figure 1.Morphology of Golgi membranes in HeLa cells through the cell cycle. (A) HeLa cells were grown on coverslips and fixed and labelled with anti-phosphohistone-H1 and -H3 polyclonal antibodies (pH1/pH3) as markers of different cell-cycle phases (Hidalgo Carcedo et al, 2004), and with a giantin antibody to label the Golgi complex. Images were acquired using a confocal microscope set at maximal resolution. For quantitative analysis of Golgi phenotypes, fixed imaging conditions were applied to all of the images. Scale bar, 5 μm. (B) Percent distribution of Golgi phenotypes as described in Materials and methods. Data shown are representative of a total of 70–120 cells for each experimental condition across three independent experiments. Download figure Download PowerPoint To analyze the morphology of Golgi membranes during G2 at the ultrastructural level, HeLa cells were induced to accumulate in G2 with an 18-h incubation with bisbenzimide, a topoisomerase-I inhibitor that activates the DNA damage checkpoint and promotes the accumulation of cells in G2 (Supplementary Figure S1) (Dubey and Raman, 1983). A survey of the Golgi by electron microscopy showed that the ribbon in non-treated (interphase) cells consisted of stacks of variable sizes, which were often aligned along their main axis and appeared connected by tubular non-compact zones, often visible even in random thin sections (Figure 2A). G2-blocked cells did not show major differences either in the number of cisternae that composed the single stacks or in the average diameter of the stacks; however, when compared with non-treated cells, they showed stacks that were isolated (i.e. not interconnected by tubules) in most cases and were not longitudinally aligned (Figure 2A). To define more precisely the connectivity of the Golgi ribbon in G2, we used serial sections to examine the ribbon in three-dimensional space. This analysis showed that while nearly all of the control stacks were interconnected, the stacks of G2 cells were either isolated or connected in small groups of two to four stacks. The ribbon connectivity was also quantified in thin sections by assessing the fraction of the stacks that were isolated or connected in small groups. Although this method does not reveal connections outside the plane of the section, it is quite suitable to compare G2 with control conditions. Again, the differences indicated a large loss of ribbon connectivity during G2 (Figure 2B). Thus, these data are consistent with confocal microscopy observations and indicate that during G2, the alterations in the global structure of the Golgi complex consist of the disappearance of the interstack tubular connections. Figure 2.Ultrastructure of Golgi membranes during G2 and interphase. (A) Non-treated HeLa cells and HeLa cells induced to accumulate in G2 with bisbenzimide were processed for electron microscopy. Representative images of thin sections of non-treated (interphase) and G2-blocked (G2) cells are shown. Note that Golgi membranes are organized as stacks (GS) connected by non-compact zones (arrows) in non-synchronized HeLa cells, and as isolated stacks (GS) during G2. Scale bar, 0.5 μm. (B) Morphometric analysis of the ribbon extension in thin sections from non-treated (interphase) and G2-blocked HeLa cells (G2), as indicated. Stack connectivity represents the relative percent distribution of Golgi stacks found isolated, in groups of 2–3 or of at least four connected stacks. The data are representative of more than 80 Golgi containing sections for each experimental condition in three independent experiments. Download figure Download PowerPoint Next, we further assessed the loss of ribbon integrity by a different quantitative approach based on analysis of the diffusion-mediated process of fluorescence recovery after photobleaching (FRAP) of Golgi-resident enzymes (Lippincott-Schwartz and Patterson, 2003). Golgi enzymes diffuse along the length of the Golgi ribbon, as revealed by their fast FRAP (Cole et al, 1996); thus, if the tubular connections between adjacent stacks are severed, there should be a block in diffusion of the enzymes between stacks, as has been previously reported (Mironov et al, 2004). HeLa cells were transfected with the medial-Golgi-resident enzyme galactosyltransferase fused to GFP (GalT-GFP; Cole et al, 1996) and induced to accumulate in G2 with bisbenzimide (Supplementary Figure S1). Then, a region corresponding to about half of the Golgi membranes was bleached by repeated laser illumination at high intensity and the FRAP of GalT-GFP was examined. Figure 3A and B illustrate the pattern and time course of the FRAP of the bleached area. In non-treated (interphase) cells, recovery of GalT-GFP fluorescence (normalized to the unbleached areas) was rapid, consistent with an intact Golgi ribbon (Figure 3A–C, interphase), and reached a plateau in 5 min. In contrast, in cells in G2, GalT-GFP FRAP reached a much lower plateau than that seen in control cells (Figure 3A–C, G2), suggesting that in G2, the continuity of the Golgi ribbon is interrupted. Figure 3.BARS mediates fission of the Golgi ribbon during G2. (A–C) HeLa cells were transfected with GalT-GFP and left non-treated (interphase/Int) or were induced to accumulate in G2 with bisbenzimide (C, G2 blocked). They were then mock microinjected (G2(−)) or microinjected with BARS-DN SBD (G2+SBD/SBD) and fluorescent dextran as microinjection marker and subjected to FRAP analysis after bleaching about 50% of the Golgi mass. (A) Representative images of GalT-GFP HeLa cells before bleaching (Pre-bleach), at the end of bleaching (Bleach) and 300 s after bleaching (300 s). The bleached areas are delineated by white-bordered rectangles. Scale bar, 5 μm. (B) Time courses of FRAP of interphase (top), G2 (middle) and BARS-DN-SBD-microinjected G2 (bottom) cells illustrated in (A). Fluorescence intensities in bleached areas were monitored every 5 s. Recovery curves of fluorescence intensities are normalized to non-bleached areas and corrected for background versus times. (C) Mean FRAP (±s.d.) 300 s after bleaching. Data are from analysis of 18–20 cells for each condition, and from four independent experiments performed as in (A). Statistical significances were evaluated by Student's t-test: (a) versus (b), P<0.0005; (b) versus (c), P<0.008 and (a) versus (c), P<0.03. (D) HeLa cells were transfected with GalT-GFP and left overnight non-treated (Int) or to accumulate in S phase with aphidicolin (G2 enriched). Eleven to thirteen hours after aphidicolin washout, the G2-enriched cells were microinjected with recombinant GST (8 mg/ml) (GST), recombinant BARS-DN SBD (8 mg/ml) (SBD) or the p50-2 anti-BARS antibody (3–6 mg/ml) (Ab), with fluorescent dextran as microinjection marker. Finally, the cells were subjected to FRAP after bleaching 15–20% of the Golgi mass. Mean FRAP (±s.d.) measured 300 s after bleaching and derived from 32–38 cells for each experimental condition, from five independent experiments. Statistical significances evaluated by Student's-t test: (a) versus (b), P<0.0001; (b) versus (c), P<0.0001; (b) vs (d), P<0.0001 and (a) versus (c), P<0.2. (E) HeLa cells arrested in S phase using aphidicolin (Hidalgo Carcedo et al, 2004). After 1 h, cells were microinjected with recombinant GST (8 mg/ml), generic IgG (3-6 mg/ml), recombinant BARS-DN SBD (8 mg/ml) (SBD) or the p50-2 anti-BARS antibody (3–6 mg/ml) (Ab), with FITC-conjugated dextran as microinjection marker; after 13 h, they were fixed and labelled for cell-cycle phase (Hoechst 33258; not shown), as detailed in (Hidalgo Carcedo et al, 2004). More than 200 cells were microinjected for each condition. The relative mitotic index was calculated after measuring the percentages of microinjected cells in mitosis normalized to non-microinjected cells on the same coverslip. Means (±s.d.) from three independent experiments. Download figure Download PowerPoint To rule out the possibility that the reduced FRAP seen in G2 is due to nonspecific effects of bisbenzimide, we set up an alternative approach to assess FRAP in cells in G2. HeLa cells were transfected with GalT-GFP and induced to accumulate at the beginning of S phase by synchronization with aphidicolin, a DNA polymerase inhibitor (see Hidalgo Carcedo et al, 2004). After the aphidicolin washout, the number of cells in mitosis increased with time, peaking 13 h after removal of aphidicolin. GalT-GFP FRAP was then evaluated in interphase (non-synchronized) cells and in a G2-enriched cell population represented by the non-mitotic cells seen 11–13 h after aphidicolin washout, which is prevalently composed of cells in various stages of G2 (Supplementary Figure S1). Even under these conditions, the FRAP in G2-enriched cells measured 5 min after bleaching was significantly reduced compared with that of interphase cells (Figure 3D), in spite of the non-complete G2 synchrony. Indeed, a small fraction of the non-mitotic cell population had high FRAP values, most likely because this represented cells that were not in G2, in which, therefore, the ribbon had not yet undergone fragmentation. Altogether, this demonstrates that during G2, the non-compact zones through which the Golgi enzymes diffuse along the ribbon (Cole et al, 1996) become fragmented, resulting in the production of isolated stacks or small groups of Golgi stacks. As this Golgi fragmentation step occurs in G2, it might be the Golgi-related event that is required for mitotic entry. Since BARS is essential for mitotic entry, to address this point, we analyzed its role in this early Golgi fragmentation. BARS is required for fission-dependent cleavage of the non-compact zones in G2 We have previously reported that BARS promotes the fission of tubules originating from the rims of Golgi stacks under a variety of conditions (Weigert et al, 1999). It thus seems likely that the role of BARS in Golgi mitotic partitioning might primarily be the severing of the inter-stack tubular connections. Although our previous investigations into the role of BARS in Golgi fragmentation were obtained in NRK cells (Hidalgo Carcedo et al, 2004), since our experiments on cell-cycle synchronization were performed in the HeLa cell system, we addressed this here by first also assessing the role of BARS on mitotic ingression in HeLa cells. HeLa cells were transfected with GalT-GFP and induced to accumulate at the beginning of S phase by aphidicolin treatment. After aphidicolin washout, the cells were microinjected with either the BARS substrate-binding domain (SBD), which acts as a dominant-negative BARS (BARS-DN SBD), or the p52 affinity-purified anti-BARS blocking antibody to block BARS function. The cells were then fixed 13 h after aphidicolin washout to evaluate the number of cells in mitosis, as previously described (Hidalgo Carcedo et al, 2004). In cells microinjected with BARS-DN SBD or with the anti-BARS blocking antibody, the number of cells undergoing mitosis was greatly reduced compared with control (GST- or IgG-microinjected) cells (Figure 3E), indicating that BARS controls mitotic entry also in HeLa cells. Having verified this, we tested the effect of BARS blockers on G2-specific fission of the non-compact zones using the above-described FRAP assay in HeLa cells. We first tested the effects of inhibition of BARS activity in bisbenzamide-blocked G2 cells. As shown in Figure 3A–C, microinjection of BARS-DN SBD prevented the reduction in FRAP in G2-blocked cells, indicating that the G2 division of the ribbon into isolated stacks was inhibited. We also used FRAP with a G2-enriched cell population as described above. Under these conditions, in cells where BARS was inhibited by microinjection of BARS-DN SBD or the anti-BARS blocking antibody, the FRAP was markedly greater than that of control (GST-microinjected) G2-enriched cells; indeed, it was comparable with that seen in interphase cells (Figure 3D). Microinjection of recombinant BARS or generic IgGs in interphase cells did not have any effect on the FRAP (data not shown). These data indicate that BARS is required for G2-specific severing of the Golgi ribbon into stacks. Since BARS also controls mitotic entrance (Hidalgo Carcedo et al, 2004), this indicates that ribbon fragmentation in G2 is the event that controls progression into mitosis. BARS function is dispensable for G2/M transition if the Golgi complex is in the form of isolated stacks An alternative possibility, however, is that BARS influences mitotic entrance in ways unrelated to Golgi fragmentation. Albeit unlikely, this possibility is not formally ruled out by the data above. To address this point, we reasoned that if BARS-dependent fission of non-compact tubular zones is the essential BARS effect for passing through the Golgi checkpoint, then BARS should be required for entry into mitosis only in cells that have a normal Golgi ribbon, and would instead be dispensable when the ribbon organization is lost. A previous indication of this comes from studies using the microtubule depolymerizing agent nocodazole, which divides the Golgi ribbon into isolated stacks (Hidalgo Carcedo et al, 2004). Nocodazole, however, might have severe side effects. Thus, to test this prediction, we used LdlG-CHO cells, which lack the Golgi protein GM130 (Vasile et al, 2003). When grown at 34°C, LdlG-CHO cells show an apparently normal Golgi structure as revealed by immunofluorescence microscopy (Figure 4A); however, since GM130 is required for maintenance of the Golgi ribbon (Puthenveedu et al, 2006), LdlG-CHO cells lack the ribbon organization and have Golgi membranes in the form of isolated but fully functional, ministacks that are clustered around the nucleus (Marra et al, 2007). Moreover, if LdlG-CHO cells are transfected with GM130, they reacquire normal Golgi ribbon structure (Marra et al, 2007). Based on these considerations, BARS should not be required for mitotic entry in LdlG-CHO cells, which possess no Golgi ribbon organization; in contrast, in GM130-transfected LdlG-CHO cells, where the Golgi ribbon can reform, BARS should be necessary for entrance into mitosis. This prediction was tested using the BARS blockers: microinjection of the anti-BARS blocking antibody into LdlG-CHO cells had no effect on entry into mitosis, whereas the same treatment in GM130-transfected LdlG-CHO cells blocked them in G2 (Figure 4B). An analogous case in a different cell line is described below. These results therefore establish a causal link between inhibition of severing of the Golgi ribbon in G2 by BARS blockers and inhibition of mitotic entrance. Notably, once the cells had entered mitosis, further fragmentation of the Golgi complex in cells microinjected with BARS blockers was essentially identical to that of control (IgG microinjected) cells, indicating that after entry into mitosis, BARS does not have an essential role in the subsequent stages of Golgi fragmentation (not shown). Figure 4.Inhibition of BARS does not affect progression into mitosis in cells that do not possess an intact Golgi ribbon. (A) CHO, LdlG (CHO cells defective in GM130 expression) and LdlG/GM130 (LdlG cells stably expressing GM130) cells (as indicated) were fixed and treated for immunofluorescence microscopy. The structure of the Golgi complex was monitored using an anti-giantin antibody. Scale bar, 5 μm. (B) NRK (normal rat kidney), LdlG and LdlG/GM130 cells (as indicated) arrested in S phase using aphidicolin (Hidalgo Carcedo et al, 2004). After 1 h, cells were microinjected with generic IgG (3–6 mg/ml; IgG) or the p50-2 BARS antibody (3–6 mg/ml; Ab). After a further 7 h (NRK) or 14 h (LdlG and LdlG/GM130), cells were fixed and labelled for microinjection (goat antibody to rabbit IgG; not shown) and cell-cycle phase (Hoechst 33258; not shown), as detailed in (Hidalgo Carcedo et al, 2004). More than 200 NRK and 600 LdlG and LdlG/GM130 cells were microinjected per sample. The relative mitotic index was calculated after measuring the percentage of microinjected cells in mitosis normalized to non-microinjected cells on the same coverslip. Means (±s.d.) from three independent experiments. Download figure Download PowerPoint Therefore, these data identify the first stage of the partitioning process, the severing of the Golgi ribbon into separate stacks (which is necessary to pass the G2/M checkpoint), as the target of the action of BARS in the mitotic entry process. Cells derived from BARS knockout embryos lose both Golgi ribbon organization and the Golgi mitotic checkpoint Our previous (Hidalgo Carcedo et al, 2004) and present data all converge toward an essential role for BARS in Golgi fragmentation and entry into mitosis. Thus, an apparent discrepancy arises between these data and those based on a BARS knockout (KO) mouse (Hildebrand and Soriano, 2002). These KO data reveal that although this BARS KO is embryonically lethal, a cell line derived from immortalized fibroblasts obtained from 8-day embryos (CtBP90 mouse embryo fibroblasts (MEFs)) shows normal proliferation (Hildebrand and Soriano, 2002). Moreover, as shown in Figure 5, the mitotic cycle and the mode of Golgi complex partitioning during mitosis in CtBP90 MEFs appears normal, at least at the immunofluorescence level. We therefore examined how CtBP90 MEFs compensate for a lack of BARS with respect to these mitotic events. Figure 5.Morphology of Golgi membranes in CtBP90 cells through the cell cycle. CtBP90 cells were grown on coverslips and fixed and labelled with anti-phosph
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