School of Biological Sciences, 2.205 Stopford Building, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, UK
Author for correspondence (e-mail: Iain.Hagan{at}man.ac.uk)
Accepted September 10, 2001
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SUMMARY |
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Key words: Mitosis, Tubulin, MTOC, Cytokinesis, Schizosaccharomyces pombe
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INTRODUCTION |
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The microtubule nucleating capacity of the centrosome increases dramatically upon commitment to mitosis (Kuriyama and Borisy, 1981; Snyder and McIntosh, 1975). Visualisation of -tubulinGFP fusions in living cells suggest that this increase arises from increased recruitment of
tubulin complexes to mitotic centrosomes (Khodjakov and Rieder, 1999b). Mitotic recruitment of
tubulin is likely to occur through the modification of existing MTOC proteins or the association of mitosis-specific proteins with this organelle. In Drosophila, recruitment of Asp protein appears to enhance the nucleation capacity of stripped centrosomes in vitro, suggesting that Asp may influence
TURC (
tubulin ring complex) complexes at the centrosome (Avides and Glover, 1999). In the budding yeast Saccharomyces cerevisiae, the Spc110p and Spc72p components of the spindle pole body (SPB) recruit
tubulin complexes to the SPB (Knop and Schiebel, 1997; Knop and Schiebel, 1998). Functional equivalents have not been identified in higher eukaryotes, although compelling data supporting the existence of a molecule related to Spc110 (Tassin et al., 1997). It has recently been reported that Spc110 shares a conserved PACT domain with mammalian pericentrin and mammalian AKAP450 (Flory et al., 2000; Gillingham and Munro, 2000). As this domain targets these two proteins to the centrosome, and pericentrin has been reported to associate with
tubulin (Dictenberg et al., 1998), AKAP450/pericentrin is the prime candidate for an anchor for
tubulin complexes.
Although the proteins required to nucleate microtubules are being identified, the mechanisms by which changes in the microtubule-nucleating capacity of the MTOC are regulated remain unclear. It is also unclear how these changes are coordinated with cell cycle progression to generate specific microtubule arrays. The extensive knowledge of cell cycle controls and dramatic changes in microtubule organisation that accompany mitotic commitment and exit in Schizosaccharomyces pombe make it an ideal model system for studying MTOC regulation and biogenesis.
There are three distinct MTOCs in fission yeast; two are present during mitotic growth whereas the other is specific for the sexual phase of the life cycle (Hagan, 1998; Hagan and Petersen, 2000; Petersen et al., 1998). The principle MTOC, the spindle pole body (SPB), is functionally equivalent to the centrosomes of higher eukaryotes. The fission yeast SPB undergoes cell-cycle-dependent changes in both nucleation competence and localisation, relative to the nuclear envelope (Ding et al., 1997; Masuda et al., 1992). At the end of mitosis, cytoplasmic microtubules are nucleated from the SPBs and a region at the cell equator to generate a post anaphase array (PAA) (Hagan and Yanagida, 1997; Hagan, 1998; Hagan and Hyams, 1988; Horio et al., 1991; Vardy and Toda, 2000).
The equatorial region is also the site where an F-actin ring forms upon commitment to mitosis (Marks and Hyams, 1985). This ring persists until late anaphase when a signal from the septation-inducing network (SIN) promotes its constriction (Le Goff et al., 1999). Spg1, a small GTP-binding protein of the Ras superfamily, lies at the top of the SIN (Schmidt et al., 1997). Spg1 activity is regulated by a two part GAP protein complex composed of Cdc16 and Byr4 (Furge et al., 1998). The protein kinases Cdc7, Sid1 and Sid2 and the products of mob1+, cdc11+, cdc14+, and sid4+ act in this network (Balasubramanian et al., 1998; Chang and Gould, 2000; Fankhauser and Simanis, 1993; Fankhauser and Simanis, 1994; Guertin et al., 2000; Salimova et al., 2000; Schmidt et al., 1997; Sparks et al., 1999). None of these proteins is required for F-actin ring assembly, but they are required to drive the use of this ring for cytokinesis and septation. Other aspects of cell cycle progression proceed normally if the SIN does not function (Balasubramanian et al., 1998; Le Goff et al., 1999; Nurse et al., 1976).
We have used indirect immunofluorescence microscopy and an array of yeast mutants with specific defects in cell cycle progression to show that the equatorial MTOC (EMTOC) is an incomplete ring structure whose integrity requires F-actin but not microtubules. We describe a role for the SIN and the anaphase-promoting complex (APC/C) in the regulation of EMTOC formation and describe data that suggest that these regulatory steps maybe controlled by the polo-like kinase Plo1.
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MATERIALS AND METHODS |
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Indirect immunofluorescence microscopy
Standard anti--tubulin immunofluorescence procedures were used to stain microtubules (Hagan and Hyams, 1988). For detection of
-tubulin, Dmf1/Mid1 and Pk glutaraldehyde was omitted from the fixation step. Anti-Sad1 AP9.2, TAT1 (anti-
tubulin), anti-Mid1/Dmf1 and MAb 336 anti-Pk antibodies were used as described previously (Craven et al., 1998; Hagan and Yanagida, 1995; Sohrmann et al., 1996; Woods et al., 1989). Anti-
-tubulin antibodies were concentrated 10-fold with macrosep filtration columns (Flowgen, UK) and diluted 1/3 or 1/2. Actin was visualised in cells fixed in 80°C methanol using the N350 monoclonal antibody (Lin, 1981) or in formaldehyde-fixed cells with rhodamine-conjugated phalloidin (Sigma) (Marks and Hyams, 1985). DAPI (4,6-diamidine-2-phenyl-indole) and calcofluor staining were as previously described (Moreno et al., 1991; Toda et al., 1981).
Microscopy
Images were obtained using a Hamamtsu SIT camera and NIH image software or a Quantix Photometrics CCD camera with Metamorph software (Universal Imaging). Spindle and cell lengths were determined with Metamorph. A Deltavision system (Applied Precision) was used in conjunction with Softworks software to produce 3D reconstructions of deconvolved serial optical slices through EMTOC-containing cells.
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RESULTS |
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Immunofluorescence microscopy of wild-type cells with a mixture of IH1 and IH2 antibodies revealed that tubulin concentrated at the sites of microtubule nucleation, the SPBs, throughout the cell cycle and at the cell equator from mid-anaphase B until cell separation (Fig. 1). Identical patterns were seen when cells were fixed with formaldehyde or immersed in cold solvent and stained using either antibody alone or both mixed together (data not shown). Similarly, localisation of epitope-tagged
tubulin gave the same distribution as the polyclonal antibodies. The absence of equatorial
tubulin staining during cytokinesis in cut mutants (see below) provided further confirmation that the antibodies specifically recognised
tubulin rather than a component of the F-actin ring. We conclude that IH1 and IH2 recognise S. pombe
tubulin and stain the SPB throughout the cell cycle and the EMTOC at the cell equator towards the end of mitosis.
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The EMTOC and the medial F-actin ring
The localisation of F-actin and tubulin and the frequency of division-related events were monitored in a culture that had been synchronised with respect to cell cycle progression showed that F-actin rings formed much earlier than the EMTOC (Fig. 3D).
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We next asked whether disruption of the equatorial F-actin ring would affect EMTOC structure. The design of this experiment was complicated because F-actin appears to be vital for growth in S. pombe, and cells have to attain a critical cell volume before they will commit to mitosis (Marks and Hyams, 1985; Mitchison and Nurse, 1985). Thus, F-actin disruption by addition of Latrunculin-A (LAT-A) to interphase cells would make it impossible to judge the effect of F-actin depolymerisation on EMTOC formation by inhibiting growth and thus commitment to mitosis when the EMTOC forms. Cells were therefore temporarily arrested at the G2/M boundary without blocking cell growth by incubating cdc25.22 cells at 36°C for 255 minutes (Hagan and Hyams, 1988; Nurse et al., 1976). Upon return to the permissive temperature of 25°C these cells exceeded the critical size threshold for division and so immediately entered mitosis. 90 minutes after returning the culture to 25°C, 40% of the cells had progressed to the point in anaphase B when the EMTOC forms (Fig. 3E, arrowhead). The addition of LAT-A to these cells led to the disappearance of both F-actin rings and EMTOCs from a sample fixed 15 minutes later (Fig. 3E, smaller arrow) (Table 2). As both were still present in control cultures treated with DMSO alone, these manipulations show that F-actin is essential for the structural integrity of the EMTOC. The excellent synchrony in this culture also showed that the frequency of cells with Dmf1/Mid1 rings (Fig. 3F, open circles) declined as the number of cells with an EMTOC increased (Fig. 3F, diamonds).
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We adopted two measures to ensure that microtubules were completely absent from the cells at the restrictive temperature and an additional control to confirm that this apparent absence was not due to processing artifacts. An asynchronous nda3.1828 culture was incubated in an ice/salt bath for 10 minutes to completely depolymerise the microtubules. During this incubation the anti-microtubule drug thiabendazole was added to the culture to a final concentration of 300 µg ml1 and the culture was then incubated at 36°C. 90 minutes later cells were prepared for immunofluorescence microscopy. Before fixation the cultures were split into two, and untreated wild-type cultures were mixed with one of the aliquots seconds before all samples were processed to stain microtubules, tubulin and DNA. Although microtubules were absent in the drug-treated mutant culture, they were present in the dividing, wild-type cells of the mixed samples. We conclude that the inability to see microtubules in the drug-treated cultures reflected a genuine absence rather than a processing artifact. EMTOCs formed as these cells underwent their inappropriate septation event and decondensed their chromosomes to regenerate the diffuse chromatin staining seen in interphase cells (Fig. 4) (Table 3). Thus the EMTOC is a bona fide MTOC rather than a motor-generated coalescence of microtubule ends.
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Plo1 was induced in cdc25.22 cells at 25°C. 14 hours later small G2 cells were isolated and incubated at the restrictive temperature of 36°C. This regimen induced actin ring formation and contraction and the formation of a primary septum (Ohkura et al., 1995) (Fig. 7D). It also induced EMTOC formation, however, there was a significant difference between septation and EMTOC formation. Septa continued to accumulate throughout the induction and reached over 80% by six hours (Fig. 7D, filled squares). In contrast, EMTOCs appeared only transiently, peaking around 13% at 150 minutes, before declining to 0 at 250 minutes (Fig. 7D, filled circles). EMTOCs were never seen in cells that had already septated. This indicated that the EMTOC appeared transiently and that it only appeared on the first actin ring that was induced by Plo1 overproduction. Because plo1+ was being over-expressed from a multi-copy plasmid, the variation in plasmid number between cells meant that Plo1 protein accumulated to the critical level that will induce septation at different rates in different cells. This natural variation in absolute levels throughout the population meant that the profile of the EMTOC formation plot was broad. We conclude that EMTOCs formed during the first, but not subsequent rounds, of septation events were induced by Plo1 overproduction.
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DISCUSSION |
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The dispersal of the EMTOC when the F-actin ring was depolymerised suggested that the EMTOC forms by recruitment of tubulin complexes to the F-actin ring. Consistent with earlier reports that the PAA of microtubules is displaced in mid1.366 mutants (Chang et al., 1996), we found that the EMTOC colocalised with the misplaced F-actin ring in a dmf1.6 strain. Thus the EMTOC appears to form through the association of
tubulin complexes with elements of the F-actin ring.
The absence of SPB markers such as Spg1, Cut12, and Sad1 (Bridge et al., 1998; Hagan and Yanagida, 1995; Sohrmann et al., 1998) from the EMTOC underlines the inherent differences between the EMTOC and the SPB. The EMTOC is reminiscent of acentrosomal MTOCs that form during nuclear division in other systems. The mid-body in animal cells and the preprophase band and phragmoplasts of higher plants all form at the site of cytokinesis and lack many of the classic spindle-pole components. It is not clear whether the midbody is a true MTOC that nucleates microtubules de novo during telophase or whether it is assembled from spindle microtubules shed by the centrosomes. Anti-parallel sliding of these microtubules would bring the tubulin caps at their minus ends together to generate a focus of
tubulin in the middle of the cell (Khodjakov and Rieder, 1999a; Mastronarde et al., 1993). However, the inhibition of midbody formation by addition of anti-
-tubulin antibodies to anaphase cells (Julian et al., 1993) argues that microtubule release is not the sole mechanism of midbody formation. Several facts also argue against EMTOC arising as a result of sliding forces within the central spindle. The first is that the EMTOC can form in cells that lack any microtubules. By definition, these cells do not have a central spindle that could slide in an anti-parallel fashion to create an organelle in the central overlap region. Secondly, even if a structure did form as a result of events in the central spindle, this spindle is contained within a sheath of nuclear envelope, and so there is a physical barrier that would stop it from influencing events at the plasma membrane (Tanaka and Kanbe, 1986). Finally all of the microtubules are attached to the spindle pole at this stage of division (Ding et al., 1993). Thus, there are no free microtubules to slide over each other to create this structure. Rather than being influenced by the central spindle, EMTOC behaviour strongly reflects the behaviour of the actin ring, so it would seem that it is forming by recruitment of
-tubulin-containing complexes to the F-actin ring rather than from any influence of the central spindle. Therefore when the central spindle has wandered half way along the cell, the EMTOC still forms at the middle of the cell where the actin ring is cleaving the cell in two, rather than in the spindle overlap zone (Hagan and Hyams, 1988).
Regulating EMTOC formation
tubulin staining of strains with conditional mutations in components of the SIN indicated that this network is required for EMTOC formation towards the end of anaphase B. The conversion of Spg1, the SPB-bound G protein that lies at the top of this network, into its GTP-bound form at the beginning of mitosis results in a physical association with Cdc7 kinase that recruits Cdc7 to the SPB (Sohrmann et al., 1998). A number of changes in markers of SIN activity then occur during the later stages of anaphase B. For example, Cdc7 staining becomes monopolar, Sid1 is recruited to one SPB and the kinase Sid2 associates with the F-actin ring (Guertin et al., 2000; Sohrmann et al., 1996; Sparks et al., 1999). Similarly, epitope-tagged versions of the Spg1 GAP protein, Cdc16, show enhanced affinity for the SPB that lacks Cdc7 (Cerutti and Simanis, 1999).
One of the first signs of cytokinesis, and so presumably of SIN activation, is the disassociation of Dmf1/Mid1 from the F-actin ring (Bähler et al., 1998; Sohrmann et al., 1998). Data from highly synchronised mitoses show a remarkable correlation between loss of Dmf1/Mid1 from the ring and EMTOC formation. Taken with our inability to detect equatorial tubulin and Dmf1/Mid1 in the same cells (data not shown), these data suggest that there is a change in ring structure or regulation that results in Mid1 loss and EMTOC formation. We consider the simple model that Dmf1/Mid1 mobilisation exposes a docking site for a
tubulin complex unlikely because we did not see premature EMTOC formation in dmf1.6 cells at the restrictive temperature (data not shown). Nor did we see EMTOC formation in dmf1.6 sints double mutants (data not shown). A more attractive possibility is that Dmf1/Mid1 dispersal and EMTOC formation are mediated by the same effector that is an integral part or target of the SIN. However, activation of the SIN alone was insufficient to drive EMTOC formation, even though it did induce repeated rounds of the changes in F-actin ring behaviour associated with septation. This indicated that additional regulatory pathways must control EMTOC formation. The targeting of one or more proteins for destruction by ubiquitination by the APC/C is one of these. Strains harbouring conditional mutations in the APC/C components Cut9 and Cut4 or the proteosome-targeting protein Cut8 undergo a normal cytokinesis and yet do not form an EMTOC. APC/C activity is known to target Cdc13/cyclinB and the securin Cut2 for destruction (Funabiki et al., 1996). EMTOC formation in cut2 mutants and in strains expressing non-degradable Cut2 indicates that targeting of Cut2 for destruction by APC/C is not required for EMTOC formation (data not shown). APC/C must therefore target Cdc13/cyclin B or a presently unidentified molecule for destruction to promote EMTOC formation. Loss of MPF activity can be a key factor in facilitating cytokinesis (He et al., 1997); however, APC/C mutants eventually undergo cytokinesis. Because an EMTOC does not form during these leak-through cytokinesis events, it would appear that the specific APC/C mutant alleles selected in the cut mutant screen (Hirano et al., 1986) have sufficient residual activity to eventually drive MPF inactivation below a cytokinesis threshold without enabling sufficient proteolytic activity to permit the proteolysis event that is required for EMTOC formation.
The link between APC/C and SIN activity in EMTOC regulation identified here bears a striking resemblance to the crosstalk between the APC/C and the mitotic exit network (MEN) in budding yeast (Shirayama et al., 1999). The MEN is equivalent to the SIN. Highly related molecules function at similar points in each network (Balasubramanian et al., 2000). The similarity between the two pathways is highlighted by the ability of S. pombe Cdc7 to complement a temperature-sensitive mutation in the homologous S. cerevisiae MEN component, Cdc15p (Fankhauser and Simanis, 1994). The sole essential role for S. cerevisiae APCcdc20 is to direct the destruction of Pds1p and Clb5p. Pds1p destruction releases Cdc14p from the nucleolus (Shirayama et al., 1999). Cdc14p is a phosphatase that apparently forces cells out of mitosis by dephosphorylating MPF substrates (Visintin et al., 1998). Cdc14p release is also regulated by the activity of the MEN (Shou et al., 1999; Visintin et al., 1999). Thus both the APC/C and MEN impinge upon Cdc14. The requirement for the function of all components of the SIN to enable EMTOC formation suggests that an analogous final SIN effector could control EMTOC formation.
Plo1 and EMTOC formation
The polo-like kinase Plo1 seems to form a bridge between the two pathways regulating EMTOC formation. Like SIN activation, overproduction of Plo1 in interphase cells induces F-actin ring formation, F-actin ring constriction and primary septum synthesis. However, unlike activation of the SIN, overproduction of Plo1 also drives EMTOC formation on the first actin ring that it induces. As overproduction of Plo1 activates the SIN (Tanaka et al., 2001), and yet SIN activation alone can not drive EMTOC formation, Plo1 overproduction must stimulate EMTOC formation by doing more than simply activating the SIN. Polo-like kinases appear to regulate APC/C activity in a number of different systems (Glover et al., 1998; Nigg, 1998). Most significantly the budding yeast polo-like kinase Cdc5p appears to promote mitotic exit through the activation of APCCdh1/Hct1 and the concomitant destruction of Clb2 (Charles et al., 1998; Shirayama et al., 1999). The relationship between Plo1 and the S. pombe APC/C awaits investigation, but the data presented here are consistent with Plo1 functioning as an APC/C regulator. Plo1 may therefore trigger EMTOC formation by activating both the APC/C and the SIN (Fig. 8A, red). An alternative model is suggested by studies of centrosome function in Drosophila. Recent data show that polo kinase regulates the centrosomal recruitment of Asp that is associated with the enhanced microtubule-nucleation capacity of salt stripped centrosomes in cell extracts (Avides and Glover, 1999; do Carmo Avides et al., 2001). Plo1 could therefore play a more direct role in controlling EMTOC formation in fission yeast. As such, it could either operate independently of the SIN and APC/C (Fig. 8B, yellow) or it could act as a downstream effector that is dependent upon the activity of both pathways (Fig. 8B, blue).
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ACKNOWLEDGMENTS |
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REFERENCES |
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Avides, M. D. and Glover, D. M. (1999). Abnormal spindle protein, asp, and the integrity of mitotic centrosomal microtubule organizing centers. Science 283, 1733-1735.
Bähler, J., Steever, A. B., Wheatley, S., Wang, Y. L., Pringle, J. R., Gould, K. L. and McCollum, D. (1998). Role of polo kinase and Mid1p in determining the site of cell division in fission yeast. J. Cell Biol. 143, 1603-1616.
Balasubramanian, M. K., McCollum, D., Chang, L., Wong, K. C. Y., Naqvi, N. I., He, X. W., Sazer, S. and Gould, K. L. (1998). Isolation and characterization of new fission yeast cytokinesis mutants. Genetics 149, 1265-1275.
Balasubramanian, M. K., McCollum, D. and Surana, U. (2000). Tying the knot: linking cytokinesis to the nuclear cycle. J. Cell Sci. 113, 1503-1513.
Bridge, A. J., Morphew, M., Bartlett, R. and Hagan, I. M. (1998). The fission yeast SPB component Cut12 links bipolar spindle formation to mitotic control. Genes Dev. 12, 927-942.
Cerutti, L. and Simanis, V. (1999). Asymmetry of the spindle pole bodies and spg1p GAP segregation during mitosis in fission yeast. J. Cell Sci. 112, 2313-2321.
Chang, F., Woollard, A. and Nurse, P. (1996). Isolation and characterization of fission yeast mutants defective in the assembly and placement of the contractile actin ring. J. Cell Sci. 109, 131-142.
Chang, L. and Gould, K. L. (2000). Sid4p is required to localize components of the septation initiation pathway to the spindle pole body in fission yeast. Proc. Natl. Acad. Sci. USA 97, 5249-5254.
Charles, J. F., Jaspersen, S. L., Tinker-Kulberg, R. L., Hwang, L., Szidon, A. and Morgan, D. O. (1998). The Polo-related kinase Cdc5 activates and is destroyed by the mitotic cyclin destruction machinery in S. cerevisiae. Curr. Biol. 8, 497-507.[Medline]
Craven, R. A., Griffiths, D. J. F., Sheldrick, K. S., Randall, R. E., Hagan, I. M. and Carr, A. M. (1998). Vectors for the expression of tagged proteins in Schizosaccharomyces pombe. Gene 221, 59-68.[Medline]
Dictenberg, J. B., Zimmerman, W., Sparks, C. A., Young, A., Vidair, C., Zheng, Y. X., Carrington, W., Fay, F. S. and Doxsey, S. J. (1998). Pericentrin and -tubulin form a protein complex and are organized into a novel lattice at the centrosome. J. Cell Biol. 141, 163-174.
Ding, R., McDonald, K. L. and McIntosh, J. R. (1993). 3-Dimensional reconstruction and analysis of mitotic spindles from the yeast, Schizosaccharomyces pombe. J. Cell Biol. 120, 141-151.[Abstract]
Ding, R., West, R. R., Morphew, M. and McIntosh, J. R. (1997). The spindle pole body of Schizosaccharomyces pombe enters and leaves the nuclear envelope as the cell cycle proceeds. Mol. Biol. Cell 8, 1461-1479.[Abstract]
do Carmo Avides, M., Tavares, A. and Glover, D. M. (2001). Polo kinase and Asp are needed to promote the mitotic organizing activity of centrosomes. Nat. Cell Biol. 3, 421-424.[Medline]
Fankhauser, C. and Simanis, V. (1993). The Schizosaccharomyces pombe cdc14 gene is required for septum formation and can also inhibit nuclear division. Mol. Biol. Cell 4, 531-539.[Abstract]
Fankhauser, C. and Simanis, V. (1994). The Cdc7 protein-kinase is a dosage-dependent regulator of septum formation in fission yeast. EMBO J. 13, 3011-3019.[Abstract]
Flory, M., Moser, M. J., Monnat, R. J. and Davis, T. N. (2000). Identification of a human centrosomal calmodulin-binding protein that shares homology with pericentrin. Proc. Natl. Acad. Sci. USA 97, 5919-5923.
Funabiki, H., Yamano, H., Kumada, K., Nagao, K., Hunt, T. and Yanagida, M. (1996). Cut2 proteolysis required for sister-chromatid separation in fission yeast. Nature 381, 438-441.[Medline]
Furge, K. A., Wong, K., Armstrong, J., Balasubramanian, M. and Albright, C. F. (1998). Byr4 and Cdc16 form a two component GTPase activating protein for the Spg1 GTPase that controls septation in fission yeast. Curr. Biol. 8, 947-954.[Medline]
Gillingham, A. K. and Munro, S. (2000). The PACT domain, a conserved centrosomal targeting motif in the coiled coil proteins AKAP450 and pericentrin. EMBO reports 1, 524-529.
Glover, D. M., Hagan, I. M. and Tavares, A. (1998). Polo kinases: a team that plays throughout mitosis. Genes Dev. 12, 3777-3787.
Guertin, D. A., Chang, L., Irshad, F., Gould, K. L. and McCollum, D. (2000). The role of Sid1p kinase and Cdc14p in regulating the onset of cytokinesis in fission yeast. EMBO J. 19, 1803-1815.
Hagan, I. and Yanagida, M. (1995). The product of the spindle formation gene sad1+ associates with the fission yeast spindle pole body and is essential for viability. J. Cell Biol. 129, 1033-1047.[Abstract]
Hagan, I. and Yanagida, M. (1997). Evidence for cell cycle-specific, spindle pole body-mediated, nuclear positioning in the fission yeast Schizosaccharomyces pombe. J. Cell Sci. 110, 1851-1866.
Hagan, I. M. (1998). Fission yeast microtubules. J. Cell Sci. 111, 1603-1612.
Hagan, I. M., Gull, K. and Glover, D. M. (1998). Poles apart? Spindle pole bodies and centrosomes differ in ultrastructure yet their function and regulation is conserved. In Mechanisms of cell division: frontiers in molecular biology (ed. D. M. Glover and S. Endow). Oxford University Press, Oxford.
Hagan, I. M. and Hyams, J. S. (1988). The use of cell-division cycle mutants to investigate the control of microtubule distribution in the fission yeast Schizosaccharomyces pombe. J. Cell Sci. 89, 343-357.[Abstract]
Hagan, I. M. and Petersen, J. (2000). The microtubule organizing centers of Schizosaccharomyces pombe. In the centrosome in cell replication and reproduction, Vol. 49 (ed. R. E. Palazzo and G. P. Schatten) 133-154. Academic Press, San Diego.
Harlow, E. and Lane, D. (1988). Antibodies: a laboratory manual. Cold Spring Harbor Laboratory, New York.
He, X. W., Patterson, T. E. and Sazer, S. (1997). The Schizosaccharomyces pombe spindle checkpoint protein Mad2p blocks anaphase and genetically interacts with the anaphase-promoting complex. Proc. Natl. Acad. Sci. USA 94, 7965-7970.
Hirano, T., Funahashi, S., Uemura, T. and Yanagida, M. (1986). Isolation and characterization of Schizosaccharomyces pombe cut mutants that block nuclear division but not cytokinesis. EMBO J. 5, 2973-2979.
Horio, T., Uzawa, S., Jung, M. K., Oakley, B. R., Tanaka, K. and Yanagida, M. (1991). The fission yeast -tubulin is essential for mitosis and is localized at microtubule organizing centers. J. Cell Sci. 99, 693-700.[Abstract]
Hyman, A. and Karsenti, E. (1998). The role of nucleation in patterning microtubule networks. J. Cell Sci. 111, 2077-2083.
Julian, M., Tollon, Y., Lajoiemazenc, I., Moisand, A., Mazarguil, H., Puget, A. and Wright, M. (1993). -tubulin participates in the formation of the midbody during cytokinesis in mammalian cells. J. Cell Sci. 105, 145-156.
Khodjakov, A. and Rieder, C. L. (1999a). Centrosome behavior in vertabrate somatic cells as defined by -tubulin/GFP. Mol. Biol. Cell. 10, 725.
Khodjakov, A. and Rieder, C. L. (1999b). The sudden recruitment of g-tubulin to the centrosome at the onset of mitosis and its dynamic exchange throughout the cell cycle, do not require microtubules. J. Cell Biol. 146, 585-596.
Knop, M. and Schiebel, E. (1997). Spc98p and Spc97p of the yeast -tubulin complex mediate binding to the spindle pole body via their interaction with Spc110p. EMBO J. 16, 6985-6995.
Knop, M. and Schiebel, E. (1998). Receptors determine the cellular localization of a -tubulin complex and thereby the site of microtubule formation. EMBO J. 17, 3952-3967.
Kuriyama, R. and Borisy, G. G. (1981). Microtubule-nucleating activity of centrosomes in Chinese-hamster ovary cells is independent of the centriole cycle but coupled to the mitotic-cycle. J. Cell Biol. 91, 822-826.
Le Goff, X., Utsig, S. and Simanis, V. (1999). Controlling septation in fission yeast: finding the middle and timing it right. Curr. Genet. 35, 571-584.[Medline]
Lin, J. J.-C. (1981). Monoclonal antibodies against myofibrillar components of rat skeletal muscle decorate the intermediate filaments of cultured cells. Proc. Natl. Acad. Sci. USA 78, 2335-2339.[Abstract]
Marks, J. and Hyams, J. S. (1985). Localization of F-actin through the cell-division cycle of Schizosaccharomyces pombe. Eur. J. Cell. Biol. 39, 27-32.
Mastronarde, D. N., McDonald, K. L., Ding, R. and McIntosh, J. R. (1993). Interpolar Spindle Microtubules in Ptk Cells. J. Cell Biol. 123, 1475-1489.[Abstract]
Masuda, H., Sevik, M. and Cande, W. Z. (1992). In vitro microtubule nucleating activity of spindle pole bodies in fission yeast Schizosaccharomyces pombe cell cycle-dependent activation in Xenopus cell-free-extracts. J. Cell Biol. 117, 1055-1066.[Abstract]
Maundrell, K. (1990). Nmt1 of fission yeast is a highly transcribed gene completely repressed by Thiamine. J. Biol. Chem. 265, 10857-10864.
Mitchison, J. M. and Nurse, P. (1985). Growth in cell length in the fission yeast Schizosaccharomyces pombe. J. Cell Sci. 75, 357-376.[Abstract]
Moreno, S., Klar, A. and Nurse, P. (1991). Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Meth. Enzymol. 194, 795-823.[Medline]
Mulvihill, D. P., Petersen, J., Ohkura, H., Glover, D. M. and Hagan, I. M. (1999). Plo1 kinase recruitment to the spindle pole body and its role in cell division in Schizosaccharomyces pombe. Mol. Biol. Cell. 10, 2771-2785.
Murone, M. and Simanis, V. (1996). The fission yeast dma1 gene is a component of the spindle assembly checkpoint, required to prevent septum formation and premature exit from mitosis if spindle function is compromised. EMBO J. 15, 6605-6616.[Abstract]
Nigg, E. A. (1998). Polo-like kinases: positive regulators of cell division from start to finish. Curr. Opin. Cell Biol. 10, 776-783.[Medline]
Nurse, P., Thuriaux, P. and Nasmyth, K. (1976). Genetic control of the cell division cycle in the fission yeast Schizosaccharomyces pombe. Mol. Gen. Genet. 146, 167-178.[Medline]
Ohkura, H., Hagan, I. M. and Glover, D. M. (1995). The conserved Schizosaccharomyces pombe kinase Plo1, required to form a bipolar spindle, the actin ring, and septum, can drive septum formation in G1 and G2 cells. Genes Dev. 9, 1059-1073.[Abstract]
Petersen, J., Heitz, M. J. and Hagan, I. M. (1998). Conjugation in S. pombe: Identification of a microtubule organising centre, a requirement for microtubules and a role for Mad2. Curr. Biol. 8, 963-966.[Medline]
Pichova, A., Kohlwein, S. D. and Yamamoto, M. (1995). New arrays of cytoplasmic microtubules in the fission yeast Schizosaccharomyces pombe. Protoplasma 188, 252-257.
Radcliffe, P., Hirata, D., Childs, D., Vardy, L. and Toda, T. (1998). Identification of novel temperature-sensitive lethal alleles in essential -tubulin and nonessential
2-tubulin genes as fission yeast polarity mutants. Mol. Biol. Cell. 9, 1757-1771.
Salimova, E., Sohrmann, M., Fournier, N. and Simanis, V. (2000). The S. pombe orthologue of the S. cerevisiae mob1 gene is essential and functions in signalling the onset of septum formation. J. Cell Sci. 113, 1695-1704.
Sambrook, J., Fritch, E. F. and Maniatis, T. (1989). Molecular Cloning a Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
Samejima, I. and Yanagida, M. (1994). Identification of cut8+ and cek1+, a novel protein-kinase gene, which complement a fission yeast mutation that blocks anaphase. Mol. Cell. Biol. 14, 6361-6371.[Abstract]
Schmidt, S., Sohrmann, M., Hofmann, K., Woolard, A. and Simanis, V. (1997). The Spg1 GTPase is an essential dosage-dependent inducer of septum formation in Schizosaccharomyces pombe. Genes Dev. 11, 1519-1534.[Abstract]
Shirayama, M., Toth, A., Galova, M. and Nasmyth, K. (1999). APCCdc20 promotes exit from mitosis by destroying the anaphase inhibitor Pds1 and cyclin Clb5. Nature 402, 203-207.[Medline]
Shou, W. Y., Seol, J. H., Shevchenko, A., Baskerville, C., Moazed, D., Chen, Z. W. S., Jang, J., Charbonneau, H. and Deshaies, R. J. (1999). Exit from mitosis is triggered by Tem1-dependent release of the protein phosphatase Cdc14 from nucleolar RENT complex. Cell 97, 233-244.[Medline]
Snyder, J. A. and McIntosh, J. R. (1975). Initiation and growth of microtubules from mitotic centres in lysed mammalian cells. J. Cell Biol. 67, 744-760.[Abstract]
Sohrmann, M., Fankhauser, C., Brodbeck, C. and Simanis, V. (1996). The Dmf1/Mid1 gene is essential for correct positioning of the division septum in fission yeast. Genes Dev. 10, 2707-2719.[Abstract]
Sohrmann, M., Schmidt, S., Hagan, I. and Simanis, V. (1998). Asymmetric segregation on spindle poles of the Schizosaccharomyces pombe septum-inducing protein kinase Cdc7p. Genes Dev. 12, 84-94.
Sparks, C. A., Morphew, M. and McCollum, D. (1999). Sid2p, a spindle pole body kinase that regulates the onset of cytokinesis. J. Cell Biol. 146, 777-790.
Stearns, T., Evans, L. and Kirschner, M. (1991). -tubulin is a highly conserved component of the centrosome. Cell 65, 825-836.[Medline]
Tanaka, K. and Kanbe, T. (1986). Mitosis in fission yeast Schizosaccharomyces pombe as revealed by freeze substition electron microscopy. J. Cell Sci. 80, 253-268[Abstract]
Tanaka, K., Petersen, J., MacIver, F., Mulvihill, D. P., Glover, D. M. and Hagan, I. M. (2001). The role of Plo1 kinase in mitotic commitment and septation in Schizosaccharomyces pombe. EMBO J. 20, 1259-1270.
Tassin, A. M., Celati, C., Paintrand, M. and Bornens, M. (1997). Identification of an Spc110p-related protein in vertebrates. J. Cell Sci. 110, 2533-2545.
Tatebe, H. and Yanagida, M. (2000). Cut8, essential for anaphase, controls localization of 26S proteosome, facilitating destruction of cyclin and Cut2. Curr. Biol. 10, 1329-1338.[Medline]
Toda, T., Yamamoto, M. and Yanagida, M. (1981). Sequential alterations in the nuclear chromatin region during mitosis of the fission yeast Schizosaccharomyces pombe video fluorescence microscopy of synchronously growing wild-type and cold-sensitive cdc mutants by using a DNA-binding fluorescent-probe. J. Cell Sci. 52, 271-287.[Abstract]
Vardy, L. and Toda, T. (2000). The fission yeast -tubulin complex is required in G1 phase and is a component of the spindle assembly checkpoint. EMBO J. 19, 6098-6111.
Visintin, R., Craig, K., Hwang, E. S., Prinz, S., Tyers, M. and Amon, A. (1998). The phosphatase Cdc14 triggers mitotic exit by reversal of CDK- dependent phosphorylation. Mol. Cell 2, 709-718.[Medline]
Visintin, R., Hwang, E. S. and Amon, A. (1999). Cfi1 prevents premature exit from mitosis by anchoring Cdc14 phosphatase in the nucleolus. Nature 398, 818-823.[Medline]
Wiese, C. and Zheng, Y. (2000). A new function for the tubulin ring complex as a microtubule minus-end cap. Nat. Cell Biol. 2, 358-364.[Medline]
Woods, A., Sherwin, T., Sasse, R., Macrae, T. H., Baines, A. J. and Gull, K. (1989). Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal-antibodies. J. Cell Sci. 93, 491-500.[Abstract]
Yamada, H., Kumada, K. and Yanagida, M. (1997). Distinct subunit functions and cell cycle regulated phosphorylation of 20S APC/cyclosome required for anaphase in fission yeast. J. Cell Sci. 110, 1793-1804.
Yamashita, Y. M., Nakaseko, Y., Samejima, I., Kumada, K., Yamada, H., Michaelson, D. and Yanagida, M. (1996). 20S cyclosome complex formation and proteolytic activity inhibited by the cAMP/PKA pathway. Nature 384, 276-279.[Medline]
Zachariae, W. and Nasmyth, K. (1999). Whose end is destruction: cell division and the anaphase promoting complex. Genes Dev. 13, 2039-2058.