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Importance of a Myosin II-Containing Progenitor for Actomyosin Ring Assembly in Fission Yeast

2002; Elsevier BV; Volume: 12; Issue: 9 Linguagem: Inglês

10.1016/s0960-9822(02)00790-x

1879-0445

Kelvin C. Wong, Ventris M. D’souza, Naweed I. Naqvi, Fumio Motegi, Issei Mabuchi, Mohan K. Balasubramanian,

Genetics, Aging, and Longevity in Model Organisms

An actomyosin-based contractile ring provides the forces necessary for cell cleavage in several organisms [1Satterwhite L.L. Pollard T.D. Cytokinesis.Curr. Opin. Cell Biol. 1992; 4: 43-52Crossref PubMed Scopus (240) Google Scholar, 2Robinson D.N. Spudich J.A. Towards a molecular understanding of cytokinesis.Trends Cell Biol. 2000; 10: 228-237Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 3Gould K.L. Simanis V. The control of septum formation in fission yeast.Genes Dev. 1997; 11: 2939-2951Crossref PubMed Scopus (157) Google Scholar]. Myosin II is an essential component of the actomyosin ring and has also been detected as a "spot" in interphase Schizosaccharomyces pombe cells [4Bezanilla M. Wilson J.M. Pollard T.D. Fission yeast myosin-II isoforms assemble into contractile rings at distinct times during mitosis.Curr. Biol. 2000; 10: 397-400Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 5Kitayama C. Sugimoto A. Yamamoto M. Type II myosin heavy chain encoded by the myo2 gene composes the contractile ring during cytokinesis in Schizosaccharomyces pombe.J. Cell Biol. 1997; 137: 1309-1319Crossref PubMed Scopus (176) Google Scholar]. It is currently unknown if this myosin II-containing spot is important for cytokinesis. In this study, we characterize this myosin II-containing spot using a combination of genetic and cell biological analyses. Whereas myosin II at the actomyosin ring undergoes rapid turnover, myosin II at the spot does not. Maintenance of the myosin II-containing spot is independent of F-actin function. Interestingly, maintenance of this myosin II spot in interphase requires the function of Rng3p, a UCS domain-containing protein, the Caenorhabditis elegans homolog of which has recently been shown to be a cochaperone for myosin II assembly [6Barral J.M. Hutagalung A.H. Brinker A. Hartl F.U. Epstein H.F. Role of the myosin assembly protein UNC-45 as a molecular chaperone for myosin.Science. 2002; 295: 669-671Crossref PubMed Scopus (191) Google Scholar]. Disassembly of the spot in interphase prevents actomyosin ring formation in the subsequent mitosis, implying that the spot might represent a progenitor that is important for assembly of the actomyosin ring. Given that mitosis represents a short period of the fission yeast cell cycle, organization of this progenitor structure in interphase might ensure proper assembly of the actomyosin ring and successful cell division. An actomyosin-based contractile ring provides the forces necessary for cell cleavage in several organisms [1Satterwhite L.L. Pollard T.D. Cytokinesis.Curr. Opin. Cell Biol. 1992; 4: 43-52Crossref PubMed Scopus (240) Google Scholar, 2Robinson D.N. Spudich J.A. Towards a molecular understanding of cytokinesis.Trends Cell Biol. 2000; 10: 228-237Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 3Gould K.L. Simanis V. The control of septum formation in fission yeast.Genes Dev. 1997; 11: 2939-2951Crossref PubMed Scopus (157) Google Scholar]. Myosin II is an essential component of the actomyosin ring and has also been detected as a "spot" in interphase Schizosaccharomyces pombe cells [4Bezanilla M. Wilson J.M. Pollard T.D. Fission yeast myosin-II isoforms assemble into contractile rings at distinct times during mitosis.Curr. Biol. 2000; 10: 397-400Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 5Kitayama C. Sugimoto A. Yamamoto M. Type II myosin heavy chain encoded by the myo2 gene composes the contractile ring during cytokinesis in Schizosaccharomyces pombe.J. Cell Biol. 1997; 137: 1309-1319Crossref PubMed Scopus (176) Google Scholar]. It is currently unknown if this myosin II-containing spot is important for cytokinesis. In this study, we characterize this myosin II-containing spot using a combination of genetic and cell biological analyses. Whereas myosin II at the actomyosin ring undergoes rapid turnover, myosin II at the spot does not. Maintenance of the myosin II-containing spot is independent of F-actin function. Interestingly, maintenance of this myosin II spot in interphase requires the function of Rng3p, a UCS domain-containing protein, the Caenorhabditis elegans homolog of which has recently been shown to be a cochaperone for myosin II assembly [6Barral J.M. Hutagalung A.H. Brinker A. Hartl F.U. Epstein H.F. Role of the myosin assembly protein UNC-45 as a molecular chaperone for myosin.Science. 2002; 295: 669-671Crossref PubMed Scopus (191) Google Scholar]. Disassembly of the spot in interphase prevents actomyosin ring formation in the subsequent mitosis, implying that the spot might represent a progenitor that is important for assembly of the actomyosin ring. Given that mitosis represents a short period of the fission yeast cell cycle, organization of this progenitor structure in interphase might ensure proper assembly of the actomyosin ring and successful cell division. In fission yeast, there are two members of the type II myosin heavy chain family, Myo2p [5Kitayama C. Sugimoto A. Yamamoto M. Type II myosin heavy chain encoded by the myo2 gene composes the contractile ring during cytokinesis in Schizosaccharomyces pombe.J. Cell Biol. 1997; 137: 1309-1319Crossref PubMed Scopus (176) Google Scholar, 7Balasubramanian M.K. McCollum D. Chang L. Wong K.C. Naqvi N.I. He X. Sazer S. Gould K.L. Isolation and characterization of new fission yeast cytokinesis mutants.Genetics. 1998; 149: 1265-1275Crossref PubMed Google Scholar, 8May K.M. Watts F.Z. Jones N. Hyams J.S. Type II myosin involved in cytokinesis in the fission yeast, Schizosaccharomyces pombe.Cell Motil. Cytoskeleton. 1997; 38: 385-396Crossref PubMed Scopus (97) Google Scholar] and Myo3p/Myp2p [9Bezanilla M. Forsburg S.L. Pollard T.D. Identification of a second myosin-II in Schizosaccharomyces pombe: Myp2p is conditionally required for cytokinesis.Mol. Biol. Cell. 1997; 8: 2693-2705Crossref PubMed Scopus (143) Google Scholar, 10Motegi F. Nakano K. Kitayama C. Yamamoto M. Mabuchi I. Identification of Myo3, a second type-II myosin heavy chain in the fission yeast Schizosaccharomyces pombe.FEBS Lett. 1997; 420: 161-166Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar]. Previous studies have shown that Rlc1p, a component of the actomyosin ring, is a regulatory light chain associated with both Myo2p [11Naqvi N.I. Wong K.C. Tang X. Balasubramanian M.K. Type II myosin regulatory light chain relieves auto-inhibition of myosin-heavy-chain function.Nat. Cell Biol. 2000; 2: 855-858Crossref PubMed Scopus (74) Google Scholar] and Myp2p [12Le Goff X. Motegi F. Salimova E. Mabuchi I. Simanis V. The S. pombe rlc1 gene encodes a putative myosin regulatory light chain that binds the type II myosins myo3p and myo2p.J. Cell Sci. 2000; 113: 4157-4163PubMed Google Scholar]. Here, we have used strains expressing functional Rlc1p-GFP and Myo2p-GFP fusion proteins under their native control sequences to monitor myosin II dynamics through the cell cycle. Time-lapse microscopy analyses in a single focal plane on the constriction of the Rlc1p-GFP ring frequently led to the detection of two spot-like structures (Figure 1A, marked with an arrowhead; out of over 50 cells that were analyzed). Subsequently, some of these spot-like structures appeared to move in a directed manner to the medial region of the daughter cells (Figure 1A). This Rlc1p-GFP spot was shown to be distinct from the spindle pole body by staining cells expressing Rlc1p-GFP with antibodies against a well-characterized SPB protein, Sad1p [13Hagan I. Yanagida M. The product of the spindle formation gene sad1+ associates with the fission yeast spindle pole body and is essential for viability.J. Cell Biol. 1995; 129: 1033-1047Crossref PubMed Scopus (328) Google Scholar](Figure 1B). Additional experiments carried out using the SPB proteins Sid2p [14Sparks C.A. Morphew M. McCollum D. Sid2p, a spindle pole body kinase that regulates the onset of cytokinesis.J. Cell Biol. 1999; 146: 777-790Crossref PubMed Scopus (149) Google Scholar] and Cam1p [15Moser M.J. Flory M.R. Davis T.N. Calmodulin localizes to the spindle pole body of Schizosaccharomyces pombe and performs an essential function in chromosome segregation.J. Cell Sci. 1997; 110: 1805-1812Crossref PubMed Google Scholar] also confirmed this finding (data not shown). Finally, the myosin II-containing spot was distal to the nucleus and localized at the cell tip in approximately 20% of interphase cells (data not shown), again consistent with the conclusion that the myosin II-containing spot is a novel structure independent of the SPB. Given that Rlc1p and Myo2p are characterized in this study, we established that these proteins were components of the same spot structure by double labeling experiments (Figure 1C). Consistent with a previous study, the myosin II spot appeared to move primarily along microtubules [4Bezanilla M. Wilson J.M. Pollard T.D. Fission yeast myosin-II isoforms assemble into contractile rings at distinct times during mitosis.Curr. Biol. 2000; 10: 397-400Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar], and motility was significantly affected upon treatment of cells with the microtubule inhibitor thiabendazole (data not shown). We then investigated if the integrity and motility of the myosin II-containing spot were dependent on F-actin. Latrunculin A (Lat A), a drug that prevents actin polymerization, was added at 100 mM to rlc1gfp cdc25-22 cultures that were heat arrested at 36°C for 4 hr. The myosin II-containing spot was detected in essentially 100% of both DMSO- and Lat A-treated cells (Figure 1D), establishing that F-actin was not required to maintain the integrity of the myosin II-containing spot. Additional experiments (data not shown) established that the motility of the myosin II-containing spot was not compromised in Lat A-treated cells. To investigate if the myosin II-containing spot was present in all cells prior to cytokinesis, the percentage of cells in G1/S or G2/M that had the myosin II-containing spot was determined. Exponentially growing cultures of rlc1gfp cdc10-V50 [16Simanis V. Nurse P. Characterization of the fission yeast cdc10+ protein that is required for commitment to the cell cycle.J. Cell Sci. 1989; 92: 51-56PubMed Google Scholar] and rlc1gfp cdc25-22 [17Russell P. Nurse P. cdc25+ functions as an inducer in the mitotic control of fission yeast.Cell. 1986; 45: 145-153Abstract Full Text PDF PubMed Scopus (695) Google Scholar] were arrested at G1/S and G2/M boundaries, respectively. Essentially, 100% of the cells (at least 200 counted) arrested at G1/S or G2/M exhibited Rlc1p-spot staining, suggesting that the myosin II-containing spot is detected prior to entry into mitosis in all cells (Figure 1E). Myosin II-containing spots were also observed when germinated spores were arrested at the G2/M boundary (Figure 1F, marked with an arrowhead). Based on these data, we conclude that a myosin II spot is detected prior to entry into mitosis in all cells. While the spot appears to originate from a constricted actomyosin ring in many cells, other de novo assembly mechanisms might also participate in its assembly at the medial division site. Cdc12p is also known to localize to a spot-like structure in interphase [18Chang F. Movement of a cytokinesis factor cdc12p to the site of cell division.Curr. Biol. 1999; 9: 849-852Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar]; however, the Cdc12p spot appears to be different from the myosin II spot (F. Chang, personal communication). To address if this myosin II spot is incorporated into the actomyosin ring during mitosis, a rlc1gfp cdc25-22 strain was synchronized at the G2/M boundary and was released to 24°C to allow mitosis to proceed (Figure 1G). At the G2/M boundary, Rlc1p was seen as a spot in the vicinity of the medial region of the cell. As mitosis proceeded, the spot became part of the cortical network of patches surrounding the nucleus (Figure 1G, marked with arrowhead; 20 cells were studied by time-lapse microscopy). This cortical network then focused into a tight actomyosin ring, which constricted at the end of cytokinesis. Thus, the components of the myosin II-containing spot are ultimately incorporated into the actomyosin ring (Figure 1G). Similar results were obtained with a strain expressing a functional Myo2p-GFP fusion (data not shown). These time-lapse studies indicated that the myosin II spot was ultimately incorporated into the actomyosin ring at mitosis. Myosin II is present in a spot that appears to be a progenitor of the actomyosin ring, a structure with a high turnover rate [10Motegi F. Nakano K. Kitayama C. Yamamoto M. Mabuchi I. Identification of Myo3, a second type-II myosin heavy chain in the fission yeast Schizosaccharomyces pombe.FEBS Lett. 1997; 420: 161-166Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 11Naqvi N.I. Wong K.C. Tang X. Balasubramanian M.K. Type II myosin regulatory light chain relieves auto-inhibition of myosin-heavy-chain function.Nat. Cell Biol. 2000; 2: 855-858Crossref PubMed Scopus (74) Google Scholar]. Therefore, we utilized fluorescence-recovery after photobleaching (FRAP) experiments [19Jacobson K. Derzko Z. Wu E.S. Hou Y. Poste G. Measurement of the lateral mobility of cell surface components in single, living cells by fluorescence recovery after photobleaching.J. Supramol. Struct. 1976; 5: 417-428Crossref PubMed Google Scholar] to examine the dynamics of myosin II in the spot and at the actomyosin ring. The rlc1gfp cdc25-22 and myo2gfp cdc25-22 strains were synchronized at G2/M, and a region of interest confined to the vicinity of the myosin II-containing spot was subjected to photobleaching. In 50 independent experiments, the myosin II-containing spot fluorescence was abrogated immediately after photobleaching and did not recover even after 10 min (Figure 2A; data not shown for Myo2p-GFP). As an internal control, the synchronized cultures of rlc1gfp cdc25-22 and myo2gfp cdc25-22 were released to 24°C for 30 min to allow mitosis to occur, following which, Rlc1p (Figure 2B) and Myo2p (data not shown) at the actomyosin ring were photobleached and their recovery monitored. These cells recovered fluorescence at the actomyosin ring, subsequent to photobleaching, and proceeded through mitosis and cytokinesis in all cases (Figure 2B). These observations substantiate the fact that the photobleaching was not lethal to the cells. These data indicate that the components of the myosin II-containing spot do not turn over appreciably (Figure 2A). However, the situation is different at the actomyosin ring, where there is evidence of significant myosin II turnover, as indicated by FRAP experiments (Figure 2B). Thus, the myosin II in the interphase spot might represent a form distinct from the myosin II at the actomyosin ring. Since the myosin II-containing spot is seen in interphase, cells were blocked in interphase, and the role of actomyosin ring assembly factors (cdc3, cdc4, cdc8, cdc12, cdc15, rng2, rng3, and rng5) and the components of the SIN signaling pathway (cdc7, spg1, and sid2) in maintenance of the myosin II-containing spot were analyzed. To determine this, 11 temperature-sensitive mutants (rng2-D5 [20Eng K. Naqvi N.I. Wong K.C. Balasubramanian M.K. Rng2p, a protein required for cytokinesis in fission yeast, is a component of the actomyosin ring and the spindle pole body.Curr. Biol. 1998; 8: 611-621Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar], rng3-65 [21Wong K.C. Naqvi N.I. Iino Y. Yamamoto M. Balasubramanian M.K. Fission yeast Rng3p: an UCS-domain protein that mediates myosin II assembly during cytokinesis.J. Cell Sci. 2000; 113: 2421-2432Crossref PubMed Google Scholar], cdc3-124 [22Balasubramanian M.K. Hirani B.R. Burke J.D. Gould K.L. The Schizosaccharomyces pombe cdc3+ gene encodes a profilin essential for cytokinesis.J. Cell Biol. 1994; 125: 1289-1301Crossref PubMed Scopus (203) Google Scholar], cdc4-8 [23McCollum D. Balasubramanian M.K. Pelcher L.E. Hemmingsen S.M. Gould K.L. Schizosaccharomyces pombe cdc4+ gene encodes a novel EF-hand protein essential for cytokinesis.J. Cell Biol. 1995; 130: 651-660Crossref PubMed Scopus (139) Google Scholar], cdc8-110 [24Balasubramanian M.K. Helfman D.M. Hemmingsen S.M. A new tropomyosin essential for cytokinesis in the fission yeast S. pombe.Nature. 1992; 360: 84-87Crossref PubMed Scopus (196) Google Scholar], myo2-E1 [7Balasubramanian M.K. McCollum D. Chang L. Wong K.C. Naqvi N.I. He X. Sazer S. Gould K.L. Isolation and characterization of new fission yeast cytokinesis mutants.Genetics. 1998; 149: 1265-1275Crossref PubMed Google Scholar], cdc12-112 [25Chang F. Drubin D. Nurse P. cdc12p, a protein required for cytokinesis in fission yeast, is a component of the cell division ring and interacts with profilin.J. Cell Biol. 1997; 137: 169-182Crossref PubMed Scopus (330) Google Scholar], cdc15-140 [26Fankhauser C. Reymond A. Cerutti L. Utzig S. Hofmann K. Simanis V. The S. pombe cdc15 gene is a key element in the reorganization of F-actin at mitosis.Cell. 1995; 82: 435-444Abstract Full Text PDF PubMed Scopus (221) Google Scholar], cdc7-24 [27Fankhauser C. Simanis V. The cdc7 protein kinase is a dosage dependent regulator of septum formation in fission yeast.EMBO J. 1994; 13: 3011-3019Crossref PubMed Scopus (137) Google Scholar], spg1-106 [7Balasubramanian M.K. McCollum D. Chang L. Wong K.C. Naqvi N.I. He X. Sazer S. Gould K.L. Isolation and characterization of new fission yeast cytokinesis mutants.Genetics. 1998; 149: 1265-1275Crossref PubMed Google Scholar], and sid2-250 [7Balasubramanian M.K. McCollum D. Chang L. Wong K.C. Naqvi N.I. He X. Sazer S. Gould K.L. Isolation and characterization of new fission yeast cytokinesis mutants.Genetics. 1998; 149: 1265-1275Crossref PubMed Google Scholar]) expressing Rlc1p-GFP were constructed. To achieve synchrony in S phase prior to heat inactivation, all 12 strains (Rlc1p-GFP in wild-type or with 11 independent mutations) were treated with 12 mM hydroxyurea (HU) at 24°C for 6–8 hr. All strains were then shifted to 36°C in the presence of HU for 4 hr to inactivate the temperature-sensitive proteins. The heat-arrested wild-type strain (Figure 3A) and mutants cdc3-124, cdc8-110, cdc12-112, cdc15-140, myo2-E1, cdc7-24, sid2-250, and spg1-106 (data not shown) displayed a prominent myosin II-containing spot. In all cases, spots were observed in 55%–70% of the HU-treated cells (500 cells counted for each strain) that were incubated at 36°C for 4 hr. Myosin II-containing spots that were significantly weaker in intensity than those observed in wild-type cells were detected in 60%–70% of HU- and heat-arrested cdc4-8 and rng2-D5 cells (data not shown). The myosin II spots in cdc4-8 and rng2-D5 were detectable only using 100 W lamps or confocal microscopy, but not using conventional 50 W fluorescence lamps. Interestingly, the myosin II-containing spot was completely abolished and was not detected in HU-treated and heat-arrested rng3-65 cells (Figure 3A; 0/500 cells). Thus, our data established that the function of the UCS domain containing myosin II assembly factor, Rng3p, is essential for maintenance of the myosin II-containing spot that is incorporated into the actomyosin ring [21Wong K.C. Naqvi N.I. Iino Y. Yamamoto M. Balasubramanian M.K. Fission yeast Rng3p: an UCS-domain protein that mediates myosin II assembly during cytokinesis.J. Cell Sci. 2000; 113: 2421-2432Crossref PubMed Google Scholar]. Given that myosin II-containing spots that were significantly fainter in appearance were detected in cdc4-8 and rng2-D5 mutants, it is possible that these proteins are also important for maintenance of the myosin II-containing spot. We then released the heat- and HU-arrested rng3-65 mutant to the permissive temperature in the absence of HU to address if the myosin II-containing spot is important for subsequent actomyosin ring assembly. Interestingly, following release to the permissive temperature, neither medial spots nor actomyosin rings containing myosin II were detected in rng3-65 mutant cells (Figure 3B; approximately 500 cells counted). In contrast, actomyosin rings were detected in 90% (approximately 500 cells counted in two independent experiments) of wild-type cells (Figure 3B) and 45%–90% (approximately 500 cells counted in 2 independent experiments) of other mutants with normal-looking myosin II-containing spots (data not shown for cdc3-124, cdc8-110, cdc12-112, cdc15-140, myo2-E1, cdc7-24, sid2-250 and spg1-106) undergoing mitosis that were treated similarly. In addition, myosin II-containing spots were detected in over 50% of these cells with a single nucleus. As in released rng3-65 cells, actomyosin ring assembly defects were noticed in cdc4-8 and rng2-D5 mutants (0% out of approximately 500 cells in each case with actomyosin rings), in which very faint myosin II-containing spots were noticed. As expected, actomyosin ring assembly defects led to the accumulation of cells with up to four nuclei, in a manner similar to that observed in the rng mutants. Interestingly, actomyosin ring assembly defects were noticed even 8 hr after shift-down to the permissive temperature. The finding that actomyosin ring assembly defects are noticed in released rng3-65, cdc4-8, and rng2-D5 mutants suggests that a normal myosin II-containing spot is important for cytokinesis. However, it is still possible that a threshold level of functional Rng3-65p, Rng2-D5p, and Cdc4-8p might not have accumulated (even after 8 hr) in these released cells, leading to cytokinesis defects. The protein levels of Cdc4-8p following downshift is comparable to that in wild-type cells (data not shown), suggesting that insufficient levels of Cdc4-8p are not a contributing factor in failure to assemble the actomyosin ring. The finding that myosin II-containing spots are not reassembled in released rng3-65 cells suggests that spot assembly de novo might be a very inefficient process and might normally be assembled from a preexisting actomyosin ring upon ring constriction. In this study, we have shown that a spot containing myosin II is detected in all cells prior to mitosis at the cell division site. While the spot containing myosin II appears to emanate from a constricting actomyosin ring in several cells, it is unclear if this represents the sole mechanism for assembly of the spots, since myosin II spots are also seen in germinated spores. Thus, the spot of myosin II might be assembled either de novo or from remnants of a constricting/disassembling actomyosin ring. Intriguingly, inactivation of the spot by the use of temperature-sensitive mutants prevents reassembly of this spot, suggesting that assembly of the spot from remnants of the constricting/disassembling actomyosin ring might predominate, while de novo assembly might operate as a back-up mechanism. Consistent with this possibility, we find that components of the spot undergo minimal turnover compared to components of the actomyosin ring that undergo appreciable turnover. We have provided evidence that the UCS domain protein Rng3p is required for the assembly and maintenance of the spot and that the loss of the myosin II-containing spot leads to actomyosin ring assembly defects in the subsequent mitosis. An attractive hypothesis is that the assembly of the actomyosin ring during mitosis, an event that lasts approximately less than a tenth of the S. pombe cell cycle, might require a preexisting template structure, perhaps to expedite the assembly of the multicomponent actomyosin ring. The spot of myosin II that is assembled in interphase and incorporated into the actomyosin ring might represent such a structure. S. pombe strains used in this study and their genotypes are listed in Table 1. Media used for vegetative growth (YES and EMM2) and genetic methods were as described in Moreno et al. [28Moreno S. Klar A. Nurse P. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe.Methods Enzymol. 1991; 194: 795-823Crossref PubMed Scopus (3042) Google Scholar]. Genetic crosses and sporulation were performed on YPD agar plates.Table 1Schizosaccharomyces pombe Strains Used in This StudyStrainGenotypeSourceMBY 175cdc25-22 ura4-D18 h−K. GouldMBY 624rlc1gfp leu1-32 ura4-D18 h−This studyMBY 628rlc1gfp cdc25-22 h+This studyMBY 640rlc1gfp cdc10-V50 h−This studyMBY 658rlc1gfp sid2gfp cdc25-22This studyMBY 659rlc1gfp cam1gfp cdc25-22This studyMBY 660sid2gfp cdc25-22This studyMBY 937myo2gfp cdc25-22This studyMBY1201rlc1gfp rng3-65 ura4-D18 ade6-M210 leu1-32 h+This studyMBY1202rlc1gfp rng2-D5 ura4-D18 ade6-M210 leu1-32 h+This studyMBY1203rlc1gfp cdc15-140 ura4-D18 ade6-M210 leu1-32 h−This studyMBY1204rlc1gfp cdc12-112 ura4-D18 ade6-M210 leu1-32 h−This studyMBY1206rlc1gfp cdc4-8 ura4-D18 ade6-M210 h−This studyMBY1254pREP81gfp-cdc4+leu1-32 ura4-D18 h−This study Open table in a new tab S. pombe cells were fixed for 1 min with 3.7% formaldehyde, and DNA was stained with 4′6,-diamidino-2-phenylindole (DAPI). DAPI was dissolved in antifade (1 mg/ml p–phenylenediamine dissolved in 50% glycerol) and used at 1 μg/ml. All still fluorescence microscopy was done with a Leica DMLB microscope, and appropriate sets of filters and images were captured using an Optronics DEI-750T cooled CCD (charge-coupled device) camera and Leica QWIN software. Image processing was done on Adobe PhotoShop 5.5 and assembled using Canvas 5. All fluorescence recovery after photobleaching (FRAP) experiments were carried out on the Zeiss laser scanning microscope (LSM) 510 using a 458-nm argon laser at 25% transmission, 100% power, and 200 iterations in less than a second. Quantitation of the rate of recovery after photobleaching was made using NIH image 1.62. For imaging of GFP-fusion proteins in live S. pombe cells, cultures were grown in appropriate selective media until mid-exponential phase, and 1 μl cell culture was mounted on a borosilicate glass slide (Matsunami Trading) and covered with a cover slip (Matsunami Trading), and the edges were sealed with VALAP (vaseline/lanolin/paraffin, 1:1:1). For temperature-shift experiments, cultures were grown at the restrictive temperature for the indicated time before release to permissive temperature for a minimum of 10 min before 1 μl cell culture was mounted on a borosilicate glass slide. Time-lapse microscopy was carried out using a LEICA DM IRBE inverted microscope equipped with a LEICA N Plan 100×/1.25 oil objective and an Orca II C4742-98 CCD camera (Hamamatsu). Images were obtained with the Metaview (Universal Imaging), processed, and analyzed using Adobe PhotoShop 5.5, Quicktime 4 (Apple), and NIH image 1.62. Individual frames for time-lapse microscopy were captured in tagged image file format (TIFF) at appropriate intervals. All microscopy observations were done in a single focal plane. Many thanks are due to all members of the IMA-yeast laboratories, and, in particular, to Drs. Jianhua Liu, Snezhana Oliferenko, David Balasundaram, Thirumaran Thanabalu, Suniti Naqvi, Ms. Srividya Rajagopalan, and Mr. Mithilesh Mishra for encouragement, critical reading of the manuscript, discussion, and suggestions. We also thank Dr. Fred Chang for sharing unpublished results. This work was supported by research funds from the National Science and Technology Board, Singapore to M.K.B. and by grant-in-aid for Specially Promoted Research from the Ministry of Education, Science, Sports and Culture of Japan to I.M.