MEK1 inactivates Myt1 to regulate Golgi membrane fragmentation and mitotic entry in mammalian cells
2012; Springer Nature; Volume: 32; Issue: 1 Linguagem: Inglês
10.1038/emboj.2012.329
ISSN1460-2075
AutoresJulien Villeneuve, Margherita Scarpa, M. Bellido, Vivek Malhotra,
Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle14 December 2012free access Source Data MEK1 inactivates Myt1 to regulate Golgi membrane fragmentation and mitotic entry in mammalian cells Julien Villeneuve Julien Villeneuve Cell and Developmental Biology Programme, Centre for Genomic Regulation (CRG), Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain Search for more papers by this author Margherita Scarpa Margherita Scarpa Cell and Developmental Biology Programme, Centre for Genomic Regulation (CRG), Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain Search for more papers by this author Maria Ortega-Bellido Maria Ortega-Bellido Cell and Developmental Biology Programme, Centre for Genomic Regulation (CRG), Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain Search for more papers by this author Vivek Malhotra Corresponding Author Vivek Malhotra Cell and Developmental Biology Programme, Centre for Genomic Regulation (CRG), Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain Search for more papers by this author Julien Villeneuve Julien Villeneuve Cell and Developmental Biology Programme, Centre for Genomic Regulation (CRG), Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain Search for more papers by this author Margherita Scarpa Margherita Scarpa Cell and Developmental Biology Programme, Centre for Genomic Regulation (CRG), Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain Search for more papers by this author Maria Ortega-Bellido Maria Ortega-Bellido Cell and Developmental Biology Programme, Centre for Genomic Regulation (CRG), Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain Search for more papers by this author Vivek Malhotra Corresponding Author Vivek Malhotra Cell and Developmental Biology Programme, Centre for Genomic Regulation (CRG), Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain Search for more papers by this author Author Information Julien Villeneuve1,2, Margherita Scarpa1,2, Maria Ortega-Bellido1,2 and Vivek Malhotra 1,2,3 1Cell and Developmental Biology Programme, Centre for Genomic Regulation (CRG), Barcelona, Spain 2Universitat Pompeu Fabra (UPF), Barcelona, Spain 3Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain *Corresponding author. CRG—Centre de Regulació Genòmica, PRBB Building, Dr Aiguader, 88, Barcelona 08003, Spain. Tel.:+34 93 316 0235; Fax:+34 93 3969 983; E-mail: [email protected] The EMBO Journal (2013)32:72-85https://doi.org/10.1038/emboj.2012.329 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The pericentriolar stacks of Golgi cisternae are separated from each other in G2 and fragmented extensively during mitosis. MEK1 is required for Golgi fragmentation in G2 and for the entry of cells into mitosis. We now report that Myt1 mediates MEK1's effects on the Golgi complex. Knockdown of Myt1 by siRNA increased the efficiency of Golgi complex fragmentation by mitotic cytosol in permeabilized and intact HeLa cells. Myt1 knockdown eliminated the requirement of MEK1 in Golgi fragmentation and alleviated the delay in mitotic entry due to MEK1 inhibition. The phosphorylation of Myt1 by MEK1 requires another kinase but is independent of RSK, Plk, and CDK1. Altogether our findings reveal that Myt1 is inactivated by MEK1 mediated phosphorylation to fragment the Golgi complex in G2 and for the entry of cells into mitosis. It is known that Myt1 inactivation is required for CDK1 activation. Myt1 therefore is an important link by which MEK1 dependent fragmentation of the Golgi complex in G2 is connected to the CDK1 mediated breakdown of Golgi into tubules and vesicles in mitosis. Introduction The pericentriolar stacks of Golgi cisternae are separated from each other in G2 (Shima et al, 1998). The Golgi stacks continue to fragment until metaphase and appear to be composed of small tubules and vesicles (Jesch and Linstedt, 1998; Jokitalo et al, 2001; Axelsson and Warren, 2004; Pecot and Malhotra, 2004). In late anaphase/telophase, the fragmented Golgi membranes fuse and assemble into stacks of Golgi cisternae by a process that also involves membrane export from the ER, and finally the stacks are localized to the pericentriolar region of the cells after cytokinesis (Lucocq et al, 1987; Lucocq and Warren, 1987; Colanzi et al, 2007; Persico et al, 2009). This process thus ensures the partitioning of Golgi membranes into daughter cells during cell division (Shorter and Warren, 2002). Surprisingly, inhibiting fragmentation of the Golgi complex in G2 prevents or delays entry of cells into mitosis (Sutterlin et al, 2002; Preisinger et al, 2005; Colanzi et al, 2007; Feinstein and Linstedt, 2007). What is the role of the pericentriolar stacks of Golgi cisternae and why does inhibiting stack separation and dispersal arrest cells in G2? Incubation of isolated Golgi membranes or permeabilized cells, with mitotic cytosol and an ATP-regenerating system causes extensive Golgi membrane fragmentation, and these procedures have revealed the involvement of three different kinases: the mitogen activated kinase kinase (MEK1), polo like kinase (Plk), and cyclin dependent kinase CDK1 (Misteli and Warren, 1994; Acharya et al, 1998; Lowe et al, 1998; Colanzi et al, 2000, 2003; Sutterlin et al, 2001). MEK1 and polo like kinase (Plk) are required for the dispersal of the pericentriolar Golgi apparatus into smaller stacks and fragments, which then breakdown into small tubules and vesicles by a CDK1 dependent process (Lowe et al, 1998; Kano et al, 2000; Sutterlin et al, 2001; Shorter and Warren, 2002). A protein called Golgi matrix protein 130 (GM130) localized on the cis side of the Golgi membranes is phosphorylated by CDK1 at Ser25 (Lowe et al, 1998). However, new recent evidences suggest that phosphorylation of Ser25 in GM130 is not required for Golgi membrane fragmentation and mitotic progression (Sundaramoorthy et al, 2010). Plk binds to the CDK1 phosphorylated Golgi membrane associated protein GRASP65; however the net effect of this reaction on the Golgi complex during mitosis is poorly understood (Lin et al, 2000). MEK1 phosphorylates the Golgi membrane protein GRASP55 (Feinstein and Linstedt, 2008). Importantly, depletion of GRASP55 alleviates the requirement of MEK1 in the process of Golgi membrane fragmentation. The data suggests that GRASP55 is required for connecting Golgi stacks laterally; phosphorylation by MEK1/Extracellular signal-regulated kinase (ERK) inactivates the function of GRASP55, which leads to the separation of Golgi stacks from each other in G2 and thus the entry of cell into mitosis (Feinstein and Linstedt, 2008). However, the function of MEK1 in the Golgi membrane fragmentation is also reported to be independent of its well-characterized targets ERK1/ERK2 (Acharya et al, 1998). For example, deletion of the N-terminal residues of MEK1 required for binding ERK1/ERK2 does not affect its ability to fragment Golgi membranes (Colanzi et al, 2000). There are also reports of a specific spliced variant of ERK, ERK1c, as the target of MEK1 dependent Golgi membrane fragmentation (Shaul and Seger, 2006). It is likely that MEK1 has additional targets on the Golgi membranes or that MEK1 does not directly phosphorylate GRASP55 to catalyse Golgi stacks separation. As mentioned above, the overall breakdown of Golgi complex into tubules and vesicles is mediated sequentially by MEK1 and CDK1, respectively. How are these events connected? We have tested the hypothesis that these sequential events are connected by the ER-Golgi complex associated kinase called Myt1, which is expressed only in the metazoans (Liu et al, 1997). Our assumption is based on the following facts. Myt1 phosphorylates CDK1 on Thr14 and Tyr15 (Mueller et al, 1995) and the Myt1 phosphorylated CDK1 is inactive (Booher et al, 1997). Inactivation of Myt1 is therefore necessary for the activation of CDK1 and entry into mitosis. MEK1 is known to phosphorylate Myt1 via p90RSK (90 kDa ribosomal S6 kinase) and this event is required for the entry of Xenopus oocytes into meiosis (Palmer et al, 1998). We now report that MEK1 inactivates Myt1 in mammalian somatic cells. We show that this reaction is required for the process by which Stacks of Golgi cisternae are separated from each other -independent of CDK1- in G2 and promote entry of cells into mitosis. Surprisingly, the MEK1 dependent inactivation of Myt1 is RSK and CDK1 independent. The description of our findings on the involvement of Myt1 in Golgi membrane reorganization and mitotic entry follows. Results Myt1 knockdown promotes early entry into mitosis HeLa cells were arrested in S-phase or mitosis (pre-metaphase) by incubation for 18 h with thymidine or nocodazole, respectively, and the cell lysates western blotted with an anti-Myt1 antibody. Compared with S-phase cells, Myt1 in mitotic cells migrated as a higher mol.wt polypeptide (Figure 1A). The total cell lysate of cells arrested in mitosis was incubated with λ protein phosphatase and probed by western blotting with an anti-Myt1 antibody. Phosphatase treatment shifted Myt1 to the size observed in non-mitotic cells (Figure 1B). This confirms the mitosis specific phosphorylation of Myt1 reported by Nishida and colleagues (Nakajima et al, 2008). To investigate the kinetics of Myt1 phosphorylation during mitotic progression, HeLa cells were arrested in S-phase with a double thymidine block, thymidine was then removed and the cells placed in normal thymidine free medium. At the indicated times, cells were lysed and the lysates western blotted with an anti-Myt1 antibody. Myt1 phosphorylation peaked at 10 h post release from the double thymidine block, and contrary to a previous report (Nakajima et al, 2008), Myt1 was dephosphorylated and not degraded after M-phase (Figure 1C). Figure 1.Myt1 knockdown promotes early entry into mitosis. (A) HeLa cells were incubated for 18 h with DMSO (asynchronized cells), thymidine (S-phase cells) or nocodazole (mitotic cells), and Myt1 expression was analysed by western blotting the total cell lysate. Western blotting with an anti-β-actin antibody was used as a loading control. (B) Lysates from cells synchronized in mitosis were treated with or without λ protein phosphatase and analysed by western blotting with an anti-Myt1 and an anti-β-actin antibody, respectively. (C) HeLa cells were arrested in S-phase with a double thymidine block, the cells were washed to remove thymidine and at the indicated times the total cell lysates were western blotted with an anti-Myt1 antibody. Western blotting with an anti-β-actin antibody was used as a loading control and western blotting with an anti-cyclin-B1 antibody was used to monitor the progression of cells into mitosis. (D) HeLa cells were transfected with control or Myt1 specific siRNA oligos and after 48 and 72 h, respectively, the levels of Myt1 were analysed by western blotting the total cell lysates. Western blotting with an anti-β-actin antibody was used as a loading control. (E) Quantitation of Myt1 protein upon siRNA transfection. (F) The percentage of cells in mitosis (mitotic index) was determined by staining DNA with DAPI and an anti-phospho-histone H3 antibody at the indicated times after thymidine release for control and Myt1 siRNA transfected cells. 400 cells were counted for each time point (mean±s.d., n=3). (G) The percentage of cells in late G2 in the total number of cells in late G2 and all stages of mitosis was determined at the indicated times after thymidine release for control and Myt1 siRNA transfected cells. For each time point, 400 cells were counted (mean±s.d., n=3).Source data for this figure is available on the online supplementary information page. Source Data for Figure 1 [embj2012329-sup-0001-SourceData-S1.pdf] Download figure Download PowerPoint Is Myt1 required for mitotic entry and progression under our experimental conditions? HeLa cells were transfected with control and Myt1 specific siRNA oligos, and the cell lysates western blotted with an anti-Myt1 antibody. Transfection with Myt1 specific siRNA oligo reduced Myt1 level by 80% compared to control cells (Figures 1D and E). The same procedure of Myt1 knockdown was repeated in cells arrested in S-phase with a double thymidine block, the cells were then washed to remove thymidine and incubated in normal medium. At the indicated times, the cells were stained with a DNA binding dye DAPI to identify mitotic cells, and with an anti-phospho-histone H3 antibody to identify cells in late G2-phase and in mitosis (Colanzi et al, 2007). In control cells, a mitotic index (cells in mitosis/total number of cells) of 25% was observed at 10 h after removal of the cells from S-phase block. Knockdown of Myt1 did not increase the mitotic index, however, the peak of mitotic index was observed at 8 h post removal from the S-phase block (Figure 1F). In other words, Myt1 knockdown changed the kinetics of entry into mitosis, and the peak of mitotic index was observed at 8 h instead of 10 h. To further test the involvement of Myt1 in G2 to M-phase transition, we counted the number of cells in late G2-phase (uncondensed DNA and specific punctate phospho-histone H3 staining, as also shown by Corda and colleagues (Colanzi et al, 2007)) and the total number of cells that were in late G2 and M-phase (all the cells that are phospho-histone H3 positive from late G2 to all the mitotic stages). In control and Myt1 siRNA transfected cells, 6 h after thymidine release, 70% of phospho-histone H3 positive cells were in G2. Interestingly, 8 h after thymidine release, 40% of phospho-histone H3 positive control cells were still in G2 whereas in Myt1 knockdown cells, only 20% of phospho-histone H3 positive cells were in G2 (Figure 1G). These results suggested that Myt1 knockdown increases the kinetics of G2-M transition. The role of Myt1 in the organization of Golgi complex We investigated the role of Myt1 in Golgi complex organization and function in interphase cells. HeLa cells were transfected with control or Myt1 specific siRNA oligos and after 48 h, the cells were fixed and visualized by fluorescence microscopy with an anti-TGN46 (of the Trans Golgi Network) and anti-GM130 (of the early Golgi cisternae) antibody, respectively. Myt1 knockdown did not affect the overall organization of the Golgi complex (Figure 2A). Moreover, fluorescence recovery after photobleaching an area of the Golgi membranes revealed no obvious kinetic difference between control and Myt1 specific siRNA transfected cells. Thus unlike the dissociation of Golgi stacks from each other upon GRASP55 knockdown in HeLa cells (Feinstein and Linstedt, 2008), Myt1 knockdown does not affect the lateral connexions between Golgi stacks (Figure 2B). HeLa cells stably expressing signal sequence and V5 tagged horseradish peroxidase (ss-HRP) were transfected with control, Myt1, or SCFD1 (a Sec family domain containing 1) specific siRNA oligos and the quantity of HRP secreted by the cells was monitored by chemiluminescence as described previously (von Blume et al, 2009). Knockdown of SCFD1, which is involved in vesicular transport between the ER and the Golgi apparatus, significantly inhibited HRP secretion, whereas knockdown of Myt1 was without any obvious effect (Figure 2C). Altogether, these data reveal that Myt1 knockdown does not affect Golgi membrane organization and protein secretion in non-mitotic HeLa cells. Figure 2.Myt1 knockdown does not modify Golgi organization and function in S-phase but promotes fragmentation of the Golgi complex in late G2. (A) HeLa cells grown on coverslips were transfected with control or Myt1 specific siRNA oligos. 48 h after transfection, cells were fixed and processed for immunofluorescence microscopy with DAPI and antibodies to GM130 and TGN46, respectively. Scale bar is 10 μm. (B) HeLa cells expressing ManII-GFP were transfected with control or Myt1 specific siRNA oligo. 48 h after transfection, the central area of the Golgi complex was bleached and the recovery of fluorescence monitored for 400 s. The fluorescence recovery was quantified as the ratio of GFP fluorescence of the bleached and the unbleached Golgi membrane area and normalized. The rate of recovery in control and Myt1 siRNA transfected cells was plotted and shown (mean±s.d., n=3, >10 cells each). (C) The media from control, SCFD1 and Myt1 knockdown HeLa cells stably expressing ss-HRP were used to detect HRP secretion by chemiluminescence. HRP activity in the medium was normalized to the total HRP activity in the cell lysates (mean±s.d., n=3, *P<0.05). (D) Left panel. Control and Myt1 siRNA transfected HeLa cells stably expressing ManII-GFP were arrested in S-phase with a double thymidine block. Cells were washed to remove thymidine, incubated for 8 h in thymidine free medium, fixed and stained with an anti-phospho-histone H3 antibody and visualized by fluorescence microsopy. The images show cells in G2. Scale bar is 10 μm. Right panel. Percentage of cells with fragmented Golgi in S-phase and G2 in control and in Myt1 knockdown cells. For each condition, 200 cells on 2 different coverslips were counted (mean±s.d., n=3, *P<0.05). Download figure Download PowerPoint Preventing fragmentation of the pericentriolar Golgi complex in G2 is known to prevent or delay entry of the cells into mitosis (Sutterlin et al, 2002; Preisinger et al, 2005; Colanzi et al, 2007; Feinstein and Linstedt, 2007). Is Myt1 required for the fragmentaion of Golgi complex in G2? Control and Myt1 siRNA transfected cells stably expressing Mannosidase II (ManII)-GFP were arrested in S-phase with a double thymidine block. The cells were then washed to remove thymidine and incubated in normal medium. After 8 h, the cells were stained with an anti-phospho-histone H3 antibody to identify the cells in G2. We visualized the organization of Golgi membranes in these cells stably expressing ManII-GFP by fluorescence microscopy. Quantitation of this data revealed that the Golgi complex was fragmented in 60% of the control cells in G2. However, 80% of the cells transfected with Myt1 siRNA had fragmented Golgi complex in G2 (Figure 2D). Myt1 knockdown did not affect the Golgi membrane organization in cells arrested in S-phase by thymidine treatment (Figure 2D). Taken together these findings indicate that Myt1 knockdown by siRNA promotes Golgi membrane fragmentation in G2. We then tested whether Myt1 was required for fragmentation of the Golgi complex in permeabilized HeLa cells incubated with mitotic cytosol (Acharya et al, 1998). Briefly, permeabilized HeLa cells stably expressing ManII-GFP were incubated with cytosol prepared from HeLa cells treated with thymidine (S-phase block) or nocodazole (pre-metaphase block), and an ATP-regenerating system at 32°C for 1 h. The cells were visualized by fluorescence microscopy and revealed extensive fragmentation of the Golgi complex in 65% of permeabilized cells incubated with mitotic cytosol (Figure 3A). Figure 3.Fragmentation of the Golgi complex with mitotic cytosol in permeabilized cells. (A) Left panel. HeLa cells stably expressing ManII-GFP were grown on coverslips and incubated with thymidine for 12 h before permeabilization. Permeabilized cells were incubated with an ATP-regenerating system and either KHM buffer (top panel), S-phase cytosol (centre panel), or mitotic cytosol (bottom panel), at 32°C for 1 h. Cells were fixed and visualized by fluorescence microscopy. Scale bar is 10 μm. Right panel. Quantitation of the experimental data. 200 cells on 2 different coverslips were counted (mean±s.d., n=3, *P<0.05) for each experimental condition to obtain the percentage of cells with fragmented Golgi complex. (B) Left panel. Total cell lysate, S-phase, and mitotic cytosol were western blotted with the antibodies shown. Right panel. The level of the same (indicated) proteins was analysed by western blotting the total cell lysate of non-permeabilized cells and cells permeabilized for 1, 2 and 3 min with digitonin and washed with 1M KCl in KHM buffer.Source data for this figure is available on the online supplementary information page. Source Data for Figure 3B [embj2012329-sup-0002-SourceData-S2.pdf] Download figure Download PowerPoint Myt1 is anchored to the cytoplasmic surface of the Golgi membranes and the ER and we tested whether it was contained in the mitotic cytosol preparation used for the fragmentation of the pericentriolar Golgi complex in permeabilized HeLa cells. Permeabilized HeLa cells and the cytosol prepared from S-phase (thymidine treated cells) and mitotic cytosol (nocodazole treated cells) were analysed by western blotting. Myt1 is detected in the permeabilized HeLa cells but not in the S-phase or in the mitotic cytosol preparations (Figure 3B). Therefore, Myt1 is not contained in the S-phase or the mitotic cytosol used in our experimental procedures. We then transfected HeLa cells stably expressing ManII-GFP with control and Myt1 specific siRNA oligos for 48 h. Cells were permeabilized, washed to remove cytoplasmic proteins, incubated with S-phase or mitotic cytosol, and the organization of the Golgi complex monitored by fluorescence microscopy. Incubation of HeLa cells transfected with control siRNA oligo and mitotic cytosol revealed fragmented Golgi complex in 60% of the cells. The percentage of HeLa cells with fragmented Golgi complex increased to almost 100% in cells transfected with Myt1 specific siRNA oligos (Figure 4A). Thus knockdown of Myt1 potentiated the ability of mitotic cytosol to fragment Golgi complex. However, this effect was mitotic cytosol specific as there was no change in the organization of the Golgi complex in Myt1 knockdown cells incubated with S-phase cytosol (Figure 4A). To further ascertain the effect of Myt1 knockdown on fragmentation of the Golgi complex by mitotic cytosol, we incubated HeLa cells transfected with control or Myt1 specific siRNA oligos with serial dilutions of mitotic cytosol. After 1 h at 32°C, the organization of the Golgi membranes was monitored by fluorescence microscopy. We found that 2-fold diluted mitotic cytosol was ineffective in fragmenting Golgi complex in cells transfected with control siRNA oligo. However, 4-fold diluted mitotic cytosol fragmented Golgi membranes in cells transfected with Myt1 specific siRNA oligos (Figure 4B). This further suggests that depletion of Myt1 increases the potency of mitotic cytosol to fragment Golgi complex. Figure 4.Myt1 inactivation promotes fragmentation of the Golgi complex. (A) Left panel. HeLa cells stably expressing ManII-GFP were transfected with control or Myt1 specific siRNA oligo. After incubation with thymidine for 12 h, cells were permeabilized, salt-washed and incubated with S-phase cytosol (top panel) or mitotic cytosol (bottom panel), and visualized by fluorescence microscopy. Scale bar is 40 μm. Right panel. Percentage of cells with fragmented Golgi upon incubation with S-phase or mitotic cytosol in control and in Myt1 knockdown cells. For each condition, 200 cells on 2 different coverslips were counted (mean±s.d., n=3, *P<0.05). (B) HeLa cells stably expressing ManII-GFP were transfected with control or Myt1 specific siRNA oligo. After incubation with thymidine for 12 h, permeabilized and salt-washed cells were incubated with KHM buffer, S-phase cytosol, or serial dilutions of mitotic cytosol. The organization of Golgi membranes was analysed by fluorescence microscopy. Scale bar is 20 μm. (C) Left panel. HeLa cells stably expressing ManII-GFP were transfected with Myc-Myt1 wild type (WT) plasmid and Myc-Myt1 kinase dead (KD) plasmid. After incubation with thymidine for 12 h, permeabilized and salt-washed cells were incubated with S-phase cytosol (top panel) or with mitotic cytosol (bottom panel) and visualized by fluorescence microscopy. Transfected cells were identified by staining with an anti-Myc antibody. Scale bar is 15 μm. Right panel. Percentage of cells with fragmented Golgi upon incubation with S-phase or mitotic cytosol in untransfected cells, cells expressing Myc-Myt1 WT, or Myc-Myt1 KD. For each condition, 200 cells on 2 different coverslips were counted (mean±s.d., n=3, *P<0.05). Download figure Download PowerPoint To determine whether the kinase activity of Myt1 was required for fragmentation of the Golgi complex, HeLa cells were transfected with a wild type (WT) or a kinase dead (KD) variant of Myc-Tagged Myt1. HeLa cells stably expressing ManII-GFP were permeabilized and incubated in the presence of an ATP-regenerating system at 32°C, for 1 h with S-phase or mitotic cytosol, respectively. The organization of the Golgi membranes was monitored by fluorescence microscopy. Transfection of the WT or the KD form of Myt1 had no effect on the organization of Golgi complex in permeabilized cells incubated with S-phase cytosol. However, incubation of cell expressing Myt1-WT with mitotic cytosol decreased the number of cells with fragmented Golgi complex (from 60–65% to under 40%) whereas the expression of the KD form of Myt1 increased the number of cells with fragmented Golgi complex to 80% (Figure 4C). Taken together, these results strongly suggest that Myt1 inhibits fragmentation of the Golgi complex by mitotic cytosol; its knockdown, or overexpression of a KD form, increases the efficiency of this reaction. MEK1 regulates Golgi membrane fragmentation via Myt1 Plk is known to phosphorylate and inactivate Myt1 (Nakajima et al, 2003; Inoue and Sagata, 2005). Is Myt1 involved in Plk dependent Golgi complex fragmentation by mitotic cytosol? We depleted Plk from mitotic cytosol by immunoadsorption with an anti-Plk antibody. This procedure resulted in greater than 70% depletion of Plk from the mitotic cytosol (Figure 5A and B). Permeabilized HeLa cells were incubated with control or Plk-depleted mitotic cytosol and after 1 h incubation at 32°C, in the presence of an ATP- regenerating system visualized by fluorescence microscopy. Incubation of permeabilized cells with Plk-depleted mitotic cytosol significantly inhibited fragmentation of the Golgi complex (Figure 5C). We then tested the effect of Myt1 knockdown on Plk dependent Golgi complex fragmentation. HeLa cells were transfected with control or Myt1 specific siRNA oligos as described above and the cells were incubated with either control or Plk-depleted mitotic cytosol. Fluorescence microscopy revealed that knockdown of Myt1 did not alleviate the requirement of Plk for the mitotic cytosol dependent Golgi complex fragmentation (Figure 5D). This suggests that Myt 1 is not downstream of the Plk mediated Golgi fragmentation process. Figure 5.Plk does not regulate fragmentation of the Golgi complex via Myt1. (A) Mock and Plk-depleted mitotic cytosol were western blotted with an anti-Plk antibody. Western blotting with an anti-CDK1 antibody was used as a loading control. (B) Quantification of Plk levels in mitotic cytosol upon immunodepletion. (C) Left panel. After incubation with thymidine for 12 h, permeabilized and salt-washed cells were incubated with mock or Plk-depleted mitotic cytosol, and an ATP-regenerating system for 1 h at 32°C. The organization of the Golgi membranes was visualized by fluorescence microscopy. Scale bar is 10 μm. Right panel. Percentage of cells with fragmented Golgi upon incubation with mock or Plk-depleted mitotic cytosol. For each condition, 200 cells were counted on 2 different coverslips (mean±s.d., n=3, *P<0.05). (D) Left panel. HeLa cells stably expressing ManII-GFP were transfected with control or Myt1 specific siRNA oligo, and after incubation with thymidine for 12 h, permeabilized and salt-washed cells were incubated with mock or Plk-depleted mitotic cytosol and an ATP-regenerating system. The organization of the Golgi membranes was visualized by fluorescence microscopy. Scale bar is 10 μm. Right panel. Percentage of cells with fragmented Golgi in the experimental conditions describe above. For each condition, 200 cells were counted on 2 different coverslips (mean±s.d., n=3, *P<0.05). Download figure Download PowerPoint Is Myt1 required for MEK1 dependent Golgi complex fragmentation by mitotic cytosol? Permeabilized HeLa cells were incubated with mitotic cytosol in the presence of DMSO (control), the MEK1 inhibitor PD98058 (PD) or U0126, the RSK inhibitor SL0101 or BI-D1870, and the CDK1 inhibitor olomoucine or RO-3306. The inhibitory activity of these chemicals on their respective kinases was confirmed and shown in Supplementary Figure S1. After 1 h of incubation at 32°C, in the presence of an ATP-regenerating system, the cells were visualized by fluorescence microscopy. PD or U0126 treatment inhibited fragmentation of the Golgi complex in 75% of the cells (Figure 6A and Supplementary Figure S2). This confirms our previous findings that MEK1 is required for mitotic cytosol dependent Golgi complex fragmentation (Acharya et al, 1998; Colanzi et al, 2000, 2003). RSK inhibitor SL0101 or BI-D1870 did not block Golgi complex fragmentation by mitotic cytosol. This suggests that RSK, which is required for MEK1 dependent Myt1 phosphorylation (and its inactivation in Xenopus egg extracts (Palmer et al, 1998)) is not important f
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