Etd1p is a novel protein that links the SIN cascade with cytokinesis
2005; Springer Nature; Volume: 24; Issue: 13 Linguagem: Inglês
10.1038/sj.emboj.7600705
ISSN1460-2075
AutoresRafael R. Daga, Agustín Lahoz, Manuel J. Muñoz, Sergio Moreno, Juan Jiménez,
Tópico(s)RNA modifications and cancer
ResumoArticle2 June 2005free access Etd1p is a novel protein that links the SIN cascade with cytokinesis Rafael R Daga Rafael R Daga Centro Andaluz de Biología del Desarrollo, CSIC/Universidad Pablo de Olavide, Carretera de Utrera Km1, Sevilla, Spain Centro de Investigación del Cáncer, CSIC/Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca, SpainPresent address: Department of Microbiology, Columbia University, New York, NY 10032, USA Search for more papers by this author Aurelia Lahoz Aurelia Lahoz Centro Andaluz de Biología del Desarrollo, CSIC/Universidad Pablo de Olavide, Carretera de Utrera Km1, Sevilla, Spain Search for more papers by this author Manuel J Muñoz Manuel J Muñoz Centro Andaluz de Biología del Desarrollo, CSIC/Universidad Pablo de Olavide, Carretera de Utrera Km1, Sevilla, Spain Search for more papers by this author Sergio Moreno Sergio Moreno Centro de Investigación del Cáncer, CSIC/Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca, Spain Search for more papers by this author Juan Jimenez Corresponding Author Juan Jimenez Centro Andaluz de Biología del Desarrollo, CSIC/Universidad Pablo de Olavide, Carretera de Utrera Km1, Sevilla, Spain Search for more papers by this author Rafael R Daga Rafael R Daga Centro Andaluz de Biología del Desarrollo, CSIC/Universidad Pablo de Olavide, Carretera de Utrera Km1, Sevilla, Spain Centro de Investigación del Cáncer, CSIC/Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca, SpainPresent address: Department of Microbiology, Columbia University, New York, NY 10032, USA Search for more papers by this author Aurelia Lahoz Aurelia Lahoz Centro Andaluz de Biología del Desarrollo, CSIC/Universidad Pablo de Olavide, Carretera de Utrera Km1, Sevilla, Spain Search for more papers by this author Manuel J Muñoz Manuel J Muñoz Centro Andaluz de Biología del Desarrollo, CSIC/Universidad Pablo de Olavide, Carretera de Utrera Km1, Sevilla, Spain Search for more papers by this author Sergio Moreno Sergio Moreno Centro de Investigación del Cáncer, CSIC/Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca, Spain Search for more papers by this author Juan Jimenez Corresponding Author Juan Jimenez Centro Andaluz de Biología del Desarrollo, CSIC/Universidad Pablo de Olavide, Carretera de Utrera Km1, Sevilla, Spain Search for more papers by this author Author Information Rafael R Daga1,2, Aurelia Lahoz1, Manuel J Muñoz1, Sergio Moreno2 and Juan Jimenez 1 1Centro Andaluz de Biología del Desarrollo, CSIC/Universidad Pablo de Olavide, Carretera de Utrera Km1, Sevilla, Spain 2Centro de Investigación del Cáncer, CSIC/Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca, Spain *Corresponding author. Centro Andaluz de Biología del Desarrollo, CSIC/Universidad Pablo de Olavide, Carretera de Utrera Km1, 41013 Sevilla, Spain. Tel./Fax: +34 954 349 376; E-mail: [email protected] The EMBO Journal (2005)24:2436-2446https://doi.org/10.1038/sj.emboj.7600705 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In animal cells, cytokinesis occurs by constriction of an actomyosin ring. In fission yeast cells, ring constriction is triggered by the septum initiation network (SIN), an SPB-associated GTPase-regulated kinase cascade that coordinates exit from mitosis with cytokinesis. We have identified a novel protein, Etd1p, required to trigger actomyosin ring constriction in fission yeasts. This protein is localised at the cell tips during interphase. In mitosis, it relocates to the medial cortex region and, coincident with cytokinesis, it assembles into the actomyosin ring by association to Cdc15p. Relocation of Etd1p from the plasma membrane to the medial ring is triggered by SIN signalling and, reciprocally, relocation of the Sid2p–Mob1p kinase complex from the SPB to the division site, a late step in the execution of the SIN, requires Etd1p. These results suggest that Etd1p coordinates the mitotic activation of SIN with the initiation of actomyosin ring constriction. Etd1p peaks during cytokinesis and is degraded by the ubiquitin-dependent 26S-proteasome pathway at the end of septation, providing a mechanism to couple inactivation of SIN to completion of cytokinesis. Introduction Cytokinesis in Schizosaccharomyces pombe is similar to that observed in animal cells and is achieved through the use of an actomyosin-based contractile ring (Balasubramanian et al, 2004; Glotzer, 2005). Actomyosin ring assembly is a spatio-temporally regulated process that starts with the determination of the division plane by Mid1p, a protein that shuttles from the nucleus to cell cortex to form a broad band overlying the position of the nucleus before mitosis (Sohrmann et al, 1996; Paoletti and Chang, 2000). Upon entry into mitosis, myosin (Myo2) and its regulatory light chains (Rlc1p, Cdc4p) are recruited to this broad band (Motegi et al, 2004). This is followed by the arrival of Cdc12p, which nucleates actin polymerization, and Cdc15p, which is also required for actin ring assembly (Carnahan and Gould, 2003; Kovar et al, 2003; Wu et al, 2003). This primary broad band, composed of actin and myosin together with many other ring components, is compacted prior to anaphase B into a thinner mature ring (Wu et al, 2003). After ring assembly, actin patches become enriched along side the actomyosin ring. F-actin patch localisation is cell cycle dependent. During interphase, actin patches accumulate at the growing ends of the cells, while at the onset of mitosis, patches assemble at the medial region, where they might be required for septum synthesis (Marks et al, 1986; Pelham and Chang, 2001). By the end of anaphase B, once the two sets of chromosomes have been segregated, the septum initiation network (SIN) triggers ring contraction and septum deposition behind the contracting ring. A number of genes are required for SIN function, including spg1/sid3, cdc7, cdc11, sid4, cdc14, sid1, sid2 and cps1 (Simanis, 2003). The absence of any of these proteins allows normal actomyosin ring assembly but cells fail to initiate ring constriction. All these components function in a conserved GTPase-regulated protein kinase cascade located at the spindle pole body (SPB)—the yeast equivalent of the centrosome of higher eukaryotes (Ding et al, 1997; McCollum and Gould, 2001). The nucleotide binding state of Spg1p, a small GTPase of the Ras superfamily, is of key importance in SIN activity. In interphase cells, Spg1p is in the GDP-bound inactive form. As the mitotic spindle forms during metaphase, Spg1p becomes GTP-bound and active at both SPBs until anaphase B, when it reconverts to the inactivated (GDP-bound) form at one of the two SPBs (Schmidt et al, 1997; Cerutti and Simanis, 1999). The protein kinase Cdc7p is asymmetrically recruited to the SPB that maintains the activated form of Spg1p (Sohrmann et al, 1998) and Sid1p–Cdc14p binds to this activated SPB. The recruitment of Sid1p–Cdc14p to the SPB depends on inactivation of the Cdc2p/Cdc13p (Cdk1/cyclinB) protein kinase complex at the end of anaphase (Guertin et al, 2000). The SIN signal is then transduced to the division site by the Sid2p–Mob1p protein kinase complex. This complex relocalises from the SPB to the cell division site and triggers medial ring constriction and septation (Salimova et al, 2000; Hou et al, 2004). Cell wall material is deposited behind the constricting ring by the septum synthesis machinery, which includes Cps1p/Drc1p/Bgs1p, the catalytic subunit of β 1-3 glucan synthase (Liu et al, 1999). Regulation of Spg1p is critical for the control of the SIN. Cells that overexpress spg1 undergo multiple rounds of septation but never complete cell separation (Schmidt et al, 1997). The GTPase-activating proteins (GAPs) Byr4p and Cdc16p negatively regulate Spg1p by promoting nucleotide hydrolysis, which converts active GTP-bound Spg1p to the inactive GDP-bound form (Furge et al, 1998). GTPases are also activated by guanine nucleotide exchange factors (GEFs) (Schmidt and Hall, 2002), although GEFs for Spg1p have not yet been identified. In budding yeast, the equivalent pathway to the SIN is known as the MEN (mitotic-exit network) (Bardin and Amon, 2001). In this yeast, the Tem1 GTPase (Spg1p in S. pombe) is negatively regulated by the GAP complex composed of Bfa1 and Bud2 (Cdc16p–Byr4p in fission yeast), while the putative GEF Lte1 activates it (Bardin and Amon, 2001). A number of regulatory mechanisms are known to coordinate SIN activity with mitosis (Krapp et al, 2004). In addition to signalling cytokinesis, the SIN regulates the localisation of Clp1p/Flp1p, a conserved Cdc14-like phosphatase. Clp1p/Flp1p is released from the nucleolus during mitosis by SIN signalling, and the non-nucleolar Clp1p/Flp1p antagonises Cdc2p/Cdc13p (CDK) activity, which is inhibitory to SIN. This feedback loop connecting SIN with CDK activity ensures that SIN is active only when CDK activity drops (Cueille et al, 2001; Trautmann et al, 2001). Dma1p, a spindle checkpoint protein, prevents cytokinesis during spindle checkpoint arrest by inhibiting SIN activity, thereby providing another mechanism by which mitotic exit is coordinated with SIN signalling (Guertin et al, 2002b). As in the case of mitotic events, the SIN should be coordinated with cytokinetic events, but regulatory mechanisms connecting SIN with initiation, progression or exit from cytokinesis are poorly understood. By using ethanol as a temperature-independent conditional method, we identified Etd1p, a novel protein essential for cytokinesis (Jimenez and Oballe, 1994). Characterisation of this protein revealed a novel mechanism connecting SIN activity with cytokinesis. Results Etd1p is a new protein required for cytokinesis Many genes required for cytokinesis in yeasts have been identified in screenings for temperature-sensitive mutants, but not all genes yield thermo-sensitive mutant alleles. To identify new genes, we used ethanol as a conditional system that operates independently of temperature. Two large groups of mutants were isolated as ethanol-sensitive mutants (ets) and ethanol-dependent mutants (etd) (Jimenez and Oballe, 1994). One of the mutants belonging to the latter group, etd1-1, produced elongated and multinucleate cells without a septum under restrictive conditions (absence of ethanol), a characteristic phenotype of S. pombe cells defective in cytokinesis (Figure 1A and B). Figure 1.etd1 is an essential gene required for cytokinesis. (A) etd1-1 is a conditional mutant that requires ethanol for growth. Wild-type and etd1-1 cells were grown at 25°C in YE medium containing 6% ethanol (permissive condition) and then replica-plated to medium without ethanol (restrictive condition). (B) etd1-1 cells grown under permissive (+ethanol) and restrictive conditions (−ethanol) for 4, 8 and 12 h were fixed and stained with DAPI to visualise DNA and calcofluor to visualise cell wall material. (C) One copy of the etd1 open reading frame (ORF) was replaced by ura4 (scheme) to construct a deletion allele (etd1Δ) in a diploid strain. Tetrad dissection of asci from this etd1Δ/etd1+ diploid strain is shown (left panel). etd1Δ spores were germinated in medium lacking uracil (middle panel) and the etd1Δ spores fixed and stained with DAPI (right panel). (D) Strong expression of etd1 also impairs cytokinesis. etd1-1 mutant cells expressing etd1 (cDNA) under the nmt1 thiamine-repressible promoter were grown in minimal medium containing thiamine. To determine the phenotype associated with overexpression of etd1, these cells were washed three times with thiamine-free medium and finally resuspended in medium with or without thiamine for 18 h at 25°C. Cells were fixed and stained with DAPI and calcofluor. Bars: 3 μm. Download figure Download PowerPoint The etd1 gene encodes an uncharacterised 391-amino-acid polypeptide (Jimenez and Oballe, 1994). Etd1p sequence is not conserved in other eukaryotes and conventional sequence analysis failed to provide any information about its putative function. To determine whether etd1 was essential, one copy of the etd1 gene was replaced with the ura4 gene in a diploid S. pombe strain. Spores deleted for etd1 (etd1Δ) germinated and accumulated multiple nuclei without septation, an identical phenotype to that of etd1-1 mutant cells under restrictive conditions (Figure 1C). Thus, etd1 encodes an essential new protein required for cytokinesis in fission yeast. To further analyse the function of etd1, we examined the effect of increased expression of the etd1 gene. A cDNA encoding etd1 was cloned in the pREP3X vector under the control of the thiamine-repressible nmt1 promoter (Maundrell, 1993). The pREP3X-etd1 plasmid was transformed into etd1-1 mutant cells and in wild-type cells. Under repressed conditions (+thiamine), the nmt1:etd1 construction provided sufficient Etd1p expression to rescue the lethal phenotype of the etd1-1 mutant (see Figure 1D). Under derepressed conditions (−thiamine), Etd1p overproduction generated elongated and multinucleate cells in both etd1-1 mutant and wild-type backgrounds (Figure 1D and data not shown). Thus, the phenotypic defect caused by an excess of Etd1p was identical to that produced by a deficiency of this protein, suggesting that Etd1p functions in a stoichiometric protein complex. To better understand the role of Etd1p in cytokinesis, we examined the localisation of the protein in S. pombe cells. A strain was constructed that expressed an Etd1p-GFP fusion from the thiamine-repressible nmt41x promoter. A single copy of the construction was integrated at the leu1 locus. Under expression conditions, the resulting Etd1p-GFP fusion protein was functional and able to complement the lethality of the etd1-1 mutant and the etd1 null allele. In interphase cells, Etd1p-GFP was located at the cell cortex and was more concentrated at the cell tips (Figure 2A, cell 1). In early anaphase, Etd1p-GFP became concentrated in the medial region of the cell cortex as a broad band (Figure 2A, cell 2) and finally as a ring late in anaphase before septation (Figure 2A, cell 3). At the time of septum formation, Etd1p-GFP spread into the cell (Figure 2A, cell 4) and once the primary septum is formed, it appears as a double layer at the cell equator (Figure 2A, cell 5). Finally, during cell separation, Etd1p-GFP signal disappeared from the middle of the cell (Figure 2A, cell 6). An identical localisation was observed when the Etd1p-GFP fusion was expressed under its own promoter on a multicopy plasmid or from its normal chromosomal locus, although fluorescence was almost undetectable in this latter case (data not shown). In spheroplasts lacking the cell wall, Etd1p remained associated with the cell periphery, suggesting that this protein is associated with the cell cortex or anchored to the cell membrane (Figure 2B). Figure 2.Etd1p localises to the division site at the end of mitosis. (A) Low-level expression of Etd1p-GFP (driven by the nmt41x promoter) was used to analyse the in vivo localisation of Etd1p. Cell 1 is in interphase, cell 2 is in early anaphase, cell 3 in late anaphase, cell 4 is undergoing septation, cell 5 formed the primary septum and cell 6 completed cytokinesis. GFP images (upper panels) and phase contrast (lower panels) are shown. Asterisks indicate localisations of Etd1p in cells 1 and 2. (B) Cell wall digested with novozyme and cell wall-free spheroplasts observed by fluorescence microscopy. Etd1p-GFP was associated with the plasma membrane. (C, D) cdc25-22 cells expressing Etd1p-GFP were synchronised by a cdc25-22 block–release protocol after 4 h of incubation at 36°C. Time-lapse images of living cells were collected every 5 min for 2 h after release at 25°C, using a confocal microscope. Two stacks of images were captured, one with a step size of 1 μm between focal planes (C) and the other of 0.3 μm serial sections to reconstruct three-dimensional images of the cell (D). (E) Time-lapse images in Etd1p-GFP Cdc7p-GFP living cells were used to determine precisely the transition of Etd1p from the cell tip to the cell centre during interphase (cell 1), entry into mitosis (cell 2)—as determined by Cdc7p association to the SPB—and initiation of anaphase (cell 3)—according to SPB segregation. (F) Localisation (asterisks) of Etd1p-GFP or Cdc15p-GFP—actomyosin ring marker—in nda3-arrested cells in metaphase (nda3-KM311 mutant background, incubated for 3 h at the restrictive temperature of 20°C). Cells incubated at the permissive temperature are shown as a control (32°C). In arrested cells, Cdc15p was always found in the medial ring while Etd1p was mainly in the cell tips (about 82%, cell 1) and a small fraction was at the middle of the cell (about 18%, cell 2). (G) Analysis of the relocation of Etd1p-GFP from a broad band (cell 1) to a compact ring (cell 2), in relation to Cdc7p-GFPU as described in panel E. Download figure Download PowerPoint To better characterise the cell cycle dynamics of Etd1p localisation, cdc25-22 mutant cells expressing Etd1p-GFP were synchronised by a temperature block–release protocol. This procedure causes a G2-phase cell cycle block at the restrictive temperature after which entry into mitosis is synchronously induced by shifting the culture to the permissive temperature. In G2 cells, Etd1p-GFP was observed at the cell tips (Figure 2C and D, time 0) and prior to anaphase began to relocate to the cell centre, about 15–20 min after the release (Figure 2C). Between 20 and 40 min after the release, Etd1p-GFP was observed as a broad band at the cell cortex in the middle region of the cell (Figure 2C, time 20–40 min; Figure 2D, time 35 min). At late anaphase, Etd1p compacted into a ring at the site of cytokinesis (Figure 2C and D, time 55 min). After this, Etd1p was observed in the region of septum formation, spreading in a centripetal manner into the cell as the actomyosin ring contracted (Figure 2D, time 75 min). This observation suggests that it is probably associated with the ingressing plasma of the cleavage furrow. Between 75 and 95 min after the release, coinciding with septum formation, Etd1p-GFP was observed along both sides of the septum (Figure 2C and D, time 80–95 min). Finally, after degradation of the primary septum, and simultaneous to cell separation, the Etd1p-GFP signal decreased strikingly (Figure 2C, time 100–115 min). Overall, localisation and dynamics of Etd1p agree with a role of this protein in cytokinesis. Dynamics of SPBs can be used to determine more precisely the cell cycle-dependent localisation of Etd1p. Cdc7p is particularly useful to this end because this protein binds to the SPB at the G2–M transition and, after SPB duplication, it remains associated with only one of the two poles of the spindle during anaphase B (Sohrmann et al, 1998). Using Cdc7p-GFP, we found that Etd1p-GFP initiated its localisation at the cell centre after association of Cdc7p to the SPB and before SPB duplication (metaphase), being more concentrated as the SPB duplicated and initiated separation (anaphase A) (Figure 2E, cells 2 and 3 respectively). In agreement with this observation, only a reduced fraction of cells showed Etd1p in the medial cortex region in metaphase-arrested cells by using the nda3-KM311 mutation (Figure 2F). In these nda3-KM311-arrested cells, the actomyosin ring was already formed, as revealed by the medial ring component Cdc15p-GFP (Figure 2F), indicating that Etd1p arrives to the middle cortex after medial ring assembly. At the time that Cdc7 is seen on only one SPB, in anaphase B, Etd1p compacted to a tight ring (Figure 2G, cell 2). Thus, Etd1p localises to the cell tips during interphase, relocates to the cell centre shortly coincident with the metaphase to anaphase transition, after actomyosin ring assembly, and compacts to a medial ring during anaphase B (see Supplementary data, Movie 1). Etd1p is essential for actomyosin ring constriction To determine whether Etd1p associates to the actomyosin ring, we examined its location in different types of actomyosin ring mutants. Mid1p is a key factor for the central positioning of the cytokinetic ring (Sohrmann et al, 1996). The location and dynamics of Etd1p and Mid1p at the cell centre are very similar (Celton-Morizur et al, 2004); however, in mid1-deleted cells, Etd1p-GFP localised at the randomly positioned actomyosin rings found in these mutant cells (Figure 3A). Therefore, medial ring components rather than Mid1p could be involved in the medial ring localisation of Etd1p. Cdc8p tropomyosin is an essential protein required to form F-actin rings (Balasubramanian et al, 1992; Arai et al, 1998). We therefore analysed the localisation of Etd1p-GFP in cdc8-110 mutant cells and found that, at the restrictive temperature of 36°C, Etd1p never formed a ring (Figure 3B, upper panels). The S. pombe Cdc15p is also required for medial ring formation during cytokinesis (Fankhauser et al, 1995; Carnahan and Gould, 2003). Overexpression of cdc15 is sufficient to drive medial actin recruitment in G2-arrested cells, indicating that Cdc15p plays a key role in the establishment of the medial actomyosin ring. In cdc15-140 mutant cells under restrictive conditions (Fankhauser et al, 1995), we also failed to detect Etd1p-GFP localised as a ring (Figure 3B, lower panels). In both cdc8-110 or cdc15-140 mutants at the restrictive conditions, Etd1p-GFP remained at the cell tips or as a diffuse central band in mitotic cells but it was not assembled into the medial ring. Therefore, we conclude that Etd1p requires the actin ring for its proper localisation to the division site. Figure 3.Assembly of Etd1p-GFP into the actomyosin ring. (A) Localisation of Etd1p-GFP in mid1-deleted cells (mid1Δ) and wild-type cells (wt). (B) Medial ring cdc8-110 and cdc15-140 thermo-sensitive mutant cells expressing Etd1p-GFP were grown to mid-exponential phase at 25°C; half of the culture was shifted to 36°C for 4 h, and living cells were photographed. (C) Protein extracts prepared from cells expressing Etd1p-GFP, Cdc15p-13Myc or both were immunoprecipitated with anti-Myc antibodies; the immunoprecipitates were run on SDS–PAGE gels and probed with anti-Myc and anti-GFP antibodies. Western blots of total extracts were also probed with anti-Myc and anti-GFP antibodies to check the levels of tagged proteins. Download figure Download PowerPoint Recruitment of Etd1p into the medial ring could take place through an interaction with Cdc15p. This is based on the fact that cells expressing Cdc15p tagged with HA or GFP in combination with HA-tagged Etd1p showed a synthetic lethal cdc phenotype (data not shown). To investigate a possible physical interaction between Etd1p and Cdc15p, strains carrying a plasmid expressing Etd1p-GFP (tagged at the N-terminus), Cdc15p-Myc (tagged at the N-terminus) or both were constructed. Protein extracts were prepared from these strains and the association between these proteins was determined in co-immunoprecipitation experiments. As shown in Figure 3C, anti-Myc immunoprecipitates contained Etd1p-GFP, demonstrating that Etd1p interacts physically with Cdc15p. Similarly, Cdc15p was detected in anti-GFP immune complexes (data not shown). Thus, Etd1p may localise to the actomyosin ring by association with Cdc15p. Cytokinesis in S. pombe cells requires actomyosin ring assembly and F-actin patch rearrangement from the cell tips to the division site. Cdc15p is involved in both processes (Fankhauser et al, 1995). F-actin cables are also involved in the formation of the actomyosin ring (Arai and Mobuchi, 2002). To determine whether Etd1p has a role in any of these events, we analysed the assembly of Cdc15p-GFP, the formation of F-actin cables and dynamics of Crn1p-GFP (coronin), a marker for actin patches (Pelham and Chang, 2001). As shown in Figure 4A–C, neither of these processes was affected in etd1-1 mutant cells, suggesting that Etd1p functions downstream of Cdc15p and F-actin patch recruitment in cytokinesis. However, in etd1-1 mutant cells, the medial ring marked with Cdc15p-GFP seems to fail constriction. Figure 4.Actomyosin ring is assembled in the etd1-1 mutant but fails to contract. (A) Assembly of Cdc15p-GFP—a key component of the actomyosin ring—was imaged in living wild-type (upper panels) or etd1-1 mutant cells under the restrictive condition (lower panels) by time-lapse microscopy at 5 min intervals on a single focal plane with a step size of 0.3 μm. (B) Rhodamine-conjugated phalloidin was used to stain F-actin structures in etd1-1 mutant cells under the restrictive condition. Actin cables and patches are indicated. (C) The S. pombe coronin homologue crn1 tagged with GFP was used as a marker of F-actin patches. Living cells were imaged as described in panel A. (D) The myosin regulatory light-chain Rlc1p tagged with GFP was used as a marker of the actomyosin ring. Living cells were imaged as described in panel A. Download figure Download PowerPoint To better determine a role of Etd1p in actomyosin ring constriction, we used the myosin regulatory light chain (encoded by the rlc1 gene) tagged with GFP as a ring marker (Le Goff et al, 2000). Time-lapse images of rlc1-GFP in wild-type and etd1-1 living cells progressing from G2 to cytokinesis were obtained. In wild-type cells, the actomyosin ring assembled early during mitosis (in metaphase) and initiated constriction late in anaphase, between 30 and 35 min after assembly (Figure 4D, upper panels). In etd1-1 mutant cells, actomyosin ring assembled as in wild type, but the ring failed to constrict and finally collapsed (Figure 4D, lower panels, and Supplementary data, Movie 2A and B). Thus, Etd1p is not required for actomyosin ring assembly, but is essential for ring contraction. A role of Etd1p in SIN signalling Actomyosin ring contraction requires proper ring assembly and the activation of the SIN. Since etd1-1 mutant cells assembled a normal medial ring, we wondered whether Etd1p might be required for SIN signalling. In fact, Etd1p-defficient cells resembled sin mutants (see Figure 1). The protein kinase complex Sid2p–Mob1p functions at a late stage of the SIN pathway by transmitting the signal from the SPB to the medial ring to initiate cytokinesis (Salimova et al, 2000; Hou et al, 2004). We produced Mob1-GFP and Sid2p-GFP constructions and determined that, as previously described (Salimova et al, 2000; Hou et al, 2004), Mob1p-GFP localised to both SPBs during mitosis and at the division site during septation in wild-type cells (Figure 5A, upper panels). In Etd1p-deficient cells, the SPB localisation of Mob1p-GFP remained unaffected, but notably, Mob1p-GFP did not relocalise to the division site (Figure 5A, lower panels). We were unable to construct a sid2-GFP etd1-1 double mutant due to negative genetic interaction between these two alleles (data not shown). However, in a strain deleted for etd1 (etd1Δ) kept alive by expressing etd1 from the weak nmt81x promoter (etd1Δnmt81x:etd1), we observed that the localisation of Sid2p-GFP to the cleavage site also required Etd1p (see below). We thus conclude that Etd1p is required for the transduction of the Sid2p–Mob1p signal from the SPB to the division site. Figure 5.Effects of Etd1p on SIN signalling. (A) Localisation of Mob1p-GFP to the division site depends on Etd1p. Mob1p-GFP was imaged in live wild-type cells (upper panels) or etd1-1 mutant cells (lower panels) under restrictive conditions (−ethanol) by time-lapse microscopy at 5 min intervals on a single focal plane. (B) Etd1p is required to maintain SIN active. Cdc7p-GFP was imaged by time-lapse microscopy at 5 min intervals on a single focal plane in living wild-type cells (upper panels) and etd1-1 mutant cells (lower panels) under restrictive conditions (−ethanol). Fluorescence intensity was quantified (arbitrary units) and represented for each SPB (uSPB, up; dSPB, down). (C) Hyperactivation of SIN does not bypass the requirement of Etd1p for Sid2p-GFP localisation. Sid2p-GFP was localised in cells with (−thiamine) or without Etd1p (+thiamine) under a normal (25°C) or hyperactive (34°C) SIN cascade. Cells of the etd1Δ nmt81x:etd1 sid2-GFP cdc16-116 strain expressing etd1 were grown to mid-exponential phase at 25°C. Thiamine was added to half of the culture, and after 2 h, half of each subculture was shifted to 34°C. Sid2p-GFP was observed in living cells after 4 h. Bar: 3 μm. Download figure Download PowerPoint Since Etd1p associates to the actomyosin ring and is required for SIN signalling, this new protein might be a ring component required for the recruitment of Sid2–Mob1 complexes to this structure, that is, a downstream element of the SIN cascade localised at the medial ring. If this were the case, activation of upstream elements of SIN should take place normally in etd1-mutant cells, and similarly, ectopic activation of the SIN alone should not bypass the requirement of Etd1p to relocate Sid2p or Mob1p from the SPB to the actomyosin ring. To analyse upstream activation of SIN, we studied the localisation of Cdc7p-GFP in etd1-1 mutant cells by time-lapse microscopy. The Spg1p GTPase localises to the SPBs throughout the cell cycle. In interphase cells, Spg1p is GDP-bound, but upon entry into mitosis, it converts into the GTP-bound form. Spg1p is then active at both SPBs until anaphase B, when it converts back into the inactive GDP-bound form at one of the two SPBs. Cdc7p only binds the active (GTP-bound) form of Spg1p (Sohrmann et al, 1998). Thus, Cdc7p is an excellent marker for monitoring upstream activation of the SIN cascad
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