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Mol. Cells, Vol. 14, No. 2, pp. 312-317 M olecules and Cells Communication KSMCB 2002 A Glucose-inducible Gene in Schizosaccharomyces pombe, rrg1+, Is Involved in Negative Regulation of G2/M Progression Min Ji Kim, Eun Jung Park1, and Sang Dai Park* School of Biological Sciences, Seoul National University, Seoul 151-742, Korea; 1 Department of Pharmacology, School of Medicine, Ajou University, Suwon 442-749, Korea. (Received June 10, 2002; Accepted July 20, 2002) A glucose-inducible gene in S. pombe is rrg1+. Its mRNA level is rapidly decreased and increased by glucose-depletion and readdition, respectively. The previous study revealed that the rrg1+ expression was regulated by glucose-dependent mRNA stability control. To understand the significance of the glucosedependent expression of rrg1+, the cellular function of rrg1+ was explored. Deletion of the rrg1+ gene from the haploid chromosome of S. pombe cells did not lead to cell lethality but brought about cell size reduction, which was accompanied by fast cell proliferation. In accordance with this result, the overexpression of the Rrg1 protein under the control of the nmt1 promoter produced elongated cells of G2 delay, and consequently resulted in the slowing-down of cell proliferation. In addition, the rrg1+ mRNA level showed cell-cycle dependent changes, peaking at G2/M. These results demonstrate that Rrg1 might be involved in the negative regulation of cell proliferation and G2/M progression for cell size control. Keywords: Cell Proliferation; Cell Size Control; G2/M Progression; Glucose; rrg1+; Schizosaccharomyces pombe. Introduction Glucose-dependent gene expression is one of the most fundamental cellular responses for optimal cell growth, and it occurs on a genome-wide scale. In Saccharomyces cerevisiae, a recent analysis revealed that transcript levels of numerous genes are differentially regulated in response Demo * To whom correspondence should be addressed. Tel: 82-2-880-6689; Fax: 82-2-887-6279 E-mail: [email protected] to varying glucose levels (DeRisi et al., 1997). During the shift from fermentation to respiration upon glucose exhaustion, the cells are subjected to widespread changes in the gene expressions that are involved in fundamental cellular processes, such as protein synthesis and carbohydrate storage as well as carbon metabolism. Among these, the expression of the genes that encodes the low-affinity glucose transporter, glycolytic enzymes, ribosomal proteins, tRNA synthetase, and translation elongation and initiation factors is induced by glucose. A large number of other genes that are involved in the utilization of alternative carbon sources, gluconeogenesis, respiration, and peroxisomal functions are repressed by glucose (DeRisi et al., 1997). Although the genes, whose expression is glucose-inducible, are more abundant than the glucoserepressible genes, more attention has been given to the latter in yeast studies (DeRisi et al., 1997). As glucoseinducible expressions in S. cerevisiae, the transcriptional controls of hexose transporters (HXT) and ribosomal proteins (RP) by glucose have been extensively studied (Ozcan and Johnston, 1999). However, identities of the glucose-regulated genes and molecular mechanisms that underlie the glucose-dependent expressions in the fission yeast S. pombe remain mostly elusive. In nearly all cells, there are two fundamental processes during the cell cycle: one is growth and the other is the splitting event of cell division. These processes must interact in balanced growth, otherwise cell size at division would drift and ultimately lead to cell death. The homeostatic mechanism that maintains cell size is the ‘size control’, which ensures that the processes that lead to division only start when the cell has reached a critical size. Evidence for size control comes from a wide variety of cells. These include Escherichia coli (Donachie, 1968), mammalian fibroblasts (Killander and Zetterberg, 1965; Zetterberg and Killander, 1965), and budding yeast (Murray and Hunt, 1993). However, the most extensive studies on size control have been made with the fission Min Ji Kim et al. yeast S. pombe. In this organism, the individual cell size can be easily determined because they are cylindrical and growth occurs by length extension (Murray and Hunt, 1993). The size control in S. pombe works by altering the time to mitosis (and division), rather than the rate of growth. (Fantes, 1977). For a population of cells to be able to maintain an average cell size, the cells that are born with a smaller-than-average length must spend more time growing before initiating mitosis and dividing than do the cells that are born with an average cell length. The reverse is true for cells that are born with a larger-thanaverage cell length. The sizing mechanism that links growth to division, or the G2/M cell size checkpoint, has been studied extensively through the analysis of mutant alleles of wee1 (Fantes, 1981; Fantes and Nurse, 1978; Russell and Nurse, 1987; Thuriaux et al., 1978). The G2/M cell size checkpoint is missing in wee1 mutants, so cells initiate mitosis at a length that is much shorter than the wild-type. Currently, the timing of mitosis is determined by antagonistic activities of the Wee1 kinase and the Cdc25 phosphatase, both acting on Tyr15 of Cdc2 (Murray and Hunt, 1993). For optimal cell growth and survival, the S. pombe cells must be able to respond to changes in nutrition by changing the rate of growth and division (Fantes, 1977). Nutritional changes trigger the G2/M cell size control mechanism, shifting the size that is required for mitotic progression so that the cells that grow in the rich medium divide with a longer cell size, but the cells that grow in the poor medium divide with a smaller cell size. Although yeast cells grow well on a variety of carbon sources, they grow fastest on glucose. Therefore, fission yeast cells that grow logarithmically in a glucose medium grow at least 3- to 4-fold faster and produce longer daughter cells than they do after the diauxic shift to ethanol. Also, when faced with severe shortages of nitrogen, the S. pombe cells are able to adapt by altering the G2/M cell size control so that cells arrest in G1 with a much reduced cell size. Without a sexual partner, the arrested cells enter a long-term state of dormancy, also referred to as G0 or quiescence, which allows long-term cell survival and resistance to environmental stresses (Nasim et al., 1989). Rapid response to glucose (rrg1+) has been identified as a glucose-inducible gene in S. pombe, which showed a rapid decrease and increase in the mRNA level by glucose-deprivation and readdition (Kim et al., 2002). The rapid response of the rrg1+ expression is under the posttranscriptional regulation of mRNA stability that is mediated by a downstream region of the poly (A) site. In this study, the cellular function of rrg1+ was explored to find some biological significance of the rapid glucosedependent regulation of its expression. Rrg1 is involved in the negative regulation of cell proliferation and G2/M progression, which provides a reasonable explanation for its glucose-dependent expression. 313 Materials and Methods Strains, culture media, and transformation The S. pombe wild-type haploid strain JY741 (h− ade6-M210 ura4-D18 leu132) that was obtained from Dr. M. Yamamoto (University of Tokyo, Japan) was used for the construction of the deletion mutant. The overproducing cells of the rrg1+. S. pombe temperature-sensitive mutant Q356 (h+ leu1-32 cdc25-22) was a generous gift from Dr. Paul G. Young (Queen’s University, Canada); it was utilized for cell-cycle synchronization by the temperature block-release method (Alfa et al., 1993). The culture media was an Edinburgh minimal medium (EMM) or a YE medium that was supplemented with appropriate amino acids. Transformation of S. pombe cells was routinely performed with 0.1 M LiAc (pH 4.9), as described previously (Alfa et al., 1993). Northern blot analysis For the Northern blot analysis, total RNA was isolated after extraction with phenol/chloroform/SDS, as previously described (Jang et al., 1995; Jin et al., 1996). About 15 µg of total RNA was separated in 1.5% agarose gel that contained 0.67 M formaldehyde, transferred onto a nylon membrane, and hybridized with radiolabeled probes. After stringent washes, the blot was exposed to X-ray film or a phosphorimager (BAS1500; Fuji, Japan). Western blot analysis The cells were grown to 5 × 106 cells/ml and harvested. Total proteins were extracted in a breakage buffer [100 mM Tris-HCl (pH8.0), 20% Glycerol, 1 mM DTT, 5 mM PMSF] (Alfa et al., 1993). About 20 µg of total protein was loaded into each lane on an 8% SDS-polyacrylamide gel and subsequently wet-transferred to a Immobilon-P membrane (Millipore, USA). The blot was probed with a 1:1,000 dilution of a polyclonal anti-Rrg1 antibody. This primary antibody was detected by HRP-conjugated anti-rabbit secondary antibodies (Jackson ImmunoResearch, USA) and Enhanced ChemiLuminescence (ECL, Sigma). Fluorescence microscopy The nucleus was visualized by staining with 2.0 µg/ml of 4′,6′-diamidino-2-phenylindole (DAPI, Sigma) in a mounting medium (Alfa et al., 1993). Septa were visualized by 0.2 mg/ml of Calcofluor (fluorescent brightener, Sigma). Yeast cells were fixed with 3% (w/v) paraformaldehyde, as described previously (Alfa et al., 1993). An indirect immunofluorescence microscopy was performed using monoclonal an anti-tubulin TAT1 antibody, and TRITC-conjugated donkey anti-mouse IgG antibody (Jackson ImmunoResearch, USA). Fluorescence was observed with Zeiss Axiophot and Axioskop 2 with a 100 W light source, Hamamatsu CCD camera and an Openlab2 image-capturing software (Improvision). Results and Discussion G2/M progression and cell proliferation are accelerated in rrg1 deletion mutant Rrg1 is a novel protein, 314 Role of Rrg1 in G2/M Progression A B Fig. 1. Deletion of the rrg1+ gene and its effects on cell proliferation and cell size. A. Strategy for the construction of rrg1 deletion mutant strain. B. Growth curves of the rrg1 deletion mutant. Inoculating the wild-type (JY741) and the rrg1 deletion mutant cells at the same value of OD595, the cells were cultivated continuously at 30°C in a rich medium (YES). At each time point, the portions of the cell cultures were collected and each OD595 value was measured. C. The JY741 (Up) and rrg1 deletion mutant (Bottom) cells were cultivated in a rich medium (YES) and the each exponentially-growing cell was collected for formaldehyde fixation, followed by calcofluor staining and microscopic observation. Scale bar, 10 µm. which has no significant sequence homology to other known proteins. In an attempt to elucidate its cellular function, the rrg1 deletion mutant was generated. The coding region of rrg1+ was substituted with a 1.8 kb HindIII fragment of the ura4+ gene and the recombinant HindIII DNA fragment that was used to transform the JY741 cells (Fig. 1A). The displacement of rrg1+ from the chromosome was confirmed by a Southern blot analysis after selecting the Ura+ cells in the uracil-deficient medium (data not shown). The resulting haploid deletion mutant of rrg1+, MJK1 (h− ade6-M210 ura4-D18 leu1-32 rrg1:: ura4+), was viable, which suggests that the Rrg1 protein is not essential for cell viability. Since the rrg1+ expression is regulated by glucose, a major energy source for cell growth, the effect of the rrg1 deletion on cellular proliferation was examined. The rrg1 null mutant proliferated much faster than the isogenic wild-type cells (Fig. 1B). The estimated doubling time of the mutant was 110 min in a rich (YE) medium, while that of the wild-type cells was 150 min. These results indicate that Rrg1 might negatively control cell proliferation. In addition, the rrg1 deletion mutant divided with into a smaller cell size, compared with the wild-type cells. The mutant produced daughter cells with an average length of 6 µm; whereas, the wild-type cells averaged 7.5 µm (Fig. Fig. 2. Effect of Rrg1-overexpression on cell proliferation. Wild type-cells (JY741) that carried the plasmid that contained the rrg1+ gene under the control of the nmt1 promoter (Rrg1-REP1) were grown in a minimal medium in the presence of 5 µM thiamine. At the time point of 0, the cells were shifted to minimal media with or without thiamine to repress or induce the Rrg1 expression, respectively, and incubated at 30°C. A. The growth curves of the cells that were cultured in the presence ({) or the absence () of thiamine. B. Western blot analyses to confirm the cellular amount of the Rrg1 protein at each time point in the inducing (−T) or in the repressing (+T) conditions. 1C). Cell growth is tightly coupled with cell-cycle progression in order to maintain constant cell size; this sizing mechanism is linked to mitotic control in S. pombe (Breeding et al., 1998; Murray and Hunt, 1993). Therefore, the reduced cell size of the rrg1 mutant implies that the mitotic control was altered and G2/M transition was initiated faster in the absence of Rrg1, which indicates Rrg1 involvement in G2/M cell size control. G2/M cell size control, which coordinates cell growth and division, is one of the most fundamental processes during cell proliferation (Fantes, 1977). Cells regulate their division by this size control in accordance with nutritional conditions; cells accelerate in a favored environment and slow down in scarcity. Rrg1 appears to be involved in a part of the slowing-down of the cell cycle at G2/M for this control. However, it is not likely that Rrg1 plays a major role in the cell cycle regulation, because the cell size changes that originate from deletion or overproduction (see Fig. 3 and next paragraph) of Rrg1 were not comparable to those shown in mutants of key regulators for G2/M progression, Min Ji Kim et al. 315 Table 1. Distribution of cells on each phase of cell cycle when Rrg1 is overproduced. (%) Rrg1-REP1 (+T) Rrg1-REP1 (-T) Interphase Anaphase Cytokinesis 85.4 96.4 11.7 1.8 2.9 1.8 All of 171 and 608 cells were counted for repressed (+T) and induced (-T) conditions, respectively. cell growth. In contrast, the rrg1 deletion mutant showed a reduced cell-doubling time, as well as a decrease in cell size, which indicates that Rrg1 may also affect the cell growth rate. These results, therefore, suggest that Rrg1 might play a role in the negative regulation of cell proliferation and G2/M progression. Fig. 3. Morphologies of G2 delayed cells with overproduced Rrg1. The JY741 cells that carried the Rrg1-overproducing plasmid under the control of the nmt1 promoter were cultured with (A) or without (B−H) thiamine to repress or induce the Rrg1 expression, respectively. The cells were fixed with formaldehyde and stained with 4′,6′-diamidino-2-phenylindole (DAPI) for microscopic observation. (A, B) Under light and fluorescent microscopic observation. Scale bar, 10 µm. (E) Indirect immunofluorescence assay with anti-tubulin antibody. (F−H) Calcofluor and DAPI staining. such as wee1 or cdc25 (Murray and Hunt, 1993). Therefore, Rrg1 seems to be an accessory component of the checkpoint system for “fine-tuning” of cell cycle progression. However, the cellular function of Rrg1 does not seem to be restricted to the negative regulation of G2/M progression, compared with the case of Wee1. Although the temperature-sensitive mutant of wee1 are smaller when grown at a restrictive temperature (35°C) than they are when grown at a permissive temperature (25°C), the duration of the cell cycle at both 35 and 25°C is identical to that of a wild-type strain that is grown under the same conditions (Murray and Hunt, 1993). When the wee1ts cells were shifted from 25 to 35°C, the threshold cell size that is required to pass the mitotic entry checkpoint is greatly reduced. Therefore, the length of the cell cycle is dramatically reduced since the cells no longer have to double in size in order to divide. However, subsequent cycles at 35°C are of normal duration since the cells (smaller at birth) must spend the same amount of time to again double in size before division under the constant Overexpression of Rrg1 leads to G2 delay To confirm the negative role of Rrg1 in G2/M progression, the effect of the Rrg1 overexpression was investigated (Fig. 2). The overproduced Rrg1, under the control of the nmt1 thiamine-repressible promoter (Maundrell, 1993), was detected in a Western blot analysis after thiamine was washed out of the medium (Fig. 2B). As shown in Fig. 2A, the rate of cell proliferation was immediately reduced after the amount of Rrg1 was increased. Interestingly, among the Rrg1-overproduced cells, the cells in the interphase (mononucleated cells) accumulated, while the percentages of the binuclear and septated cells were greatly reduced, which represent the processes of anaphase and cytokinesis in mitosis, respectively (Table 1). This suggests that the slow growth of the Rrg1-overproduced cells might result from the inhibition of the mitotic entry. In addition, the length of the mononucleated cells increased up to 14−19 µm in the induced condition (-T), compared with 12 µm in the repressed condition (+T) of the Rrg1 expression (Figs. 3A and 3B). The elongated cell also demonstrated that mitosis was hindered, even after the proper cell size was reached in the Rrg1-overexpression. Moreover, the elongated cells had an altered shape in the nuclear chromatin region; they appeared elongated and loose (Fig. 3C). This phenomenon was not accompanied by a α-tubulin signal (compare segregated binucleus with α-tubulin and elongated mononucleus without α-tubulin in Figs. 3D and 3E). This result suggests that the abnormally elongated nuclear shape in the Rrg1-overexpressed cells was not the outcome of defective chromatin condensation with normal mitotic spindle, which was easily observed in mutants of chromatin condensation. The lack of spindle microtubules indicates that the Rrg1-overexpressed cells were halted at G2 rather than in mitosis, and that the elongated nuclear morphologies could then be 316 Role of Rrg1 in G2/M Progression A B Fig. 4. Periodic changes in the rrg1+ mRNA level during cell cycle. A. Synchronously-dividing cells were generated by the temperature block-release method of cdc25-22 cells. After release to the permissive temperature (25°C), the cells were taken for RNA isolation at each time point and the Northern blot was probed for the indicated genes. B. The mRNA levels of rrg1+ and cdc22+ (indicator for G1/S) were quantified using a Imagemaster ID (Pharmacia Biotech) and plotted with a septation index. concluded as a general phenomenon that is shown in G2arrested cells (Nasim et al., 1989). The G2 delay by the Rrg1 overexpression was leaky, and there were cells that initiated and completed the mitosis in a small portion (Table 1), which eventually led to cell proliferation until the stationary phase (Fig. 2A). However, a considerable portion of the Rrg1-overproduced cells that undergo mitosis showed an abnormal accumulation of septum material (Figs. 3F, 3G, and 3H). The fluorescent signal for the septum material that was stained by calcofluor still remained, even after the cell fission was complete. This implies that there were defects in the relocation of the septum material. These results raised the possibility that the Rrg1 overexpression might affect cytokinesis, as well as G2 delay. Cell-cycle dependent expression of rrg1+ The Rrg1 role in the G2/M progression raised a question for the cellcycle dependent expression of rrg1+. To address this possibility, the transcript level of rrg1+ was measured during the cell cycle. For this purpose, a synchronous cell population was generated by the block-release method of temperature-sensitive cdc25-22 cells (Alfa et al., 1993). Also, the rrg1+ mRNA level was monitored after rescue from the G2 arrest (Fig. 4). The mRNA levels of cdc22+ and H2A1 were used as indicators for the G1/S peak (Lowndes et al., 1992) and peak at the S phase, respectively; whereas, ura4+ had a constant mRNA level during the cell cycle. The Northern blot analysis revealed that the rrg1+ transcript level slowed periodicity during the cell cycle with a summit at the G2/M phase, according to the levels of the indicator mRNAs and septation index (Fig. 4B). This cell-cycle dependent expression of rrg1+ that peaked at G2/M may support the possible role of Rrg1 in G2/M. The cellular function of Rrg1 in cell proliferation and cell cycle regulation provides a plausible explanation for its glucose-dependent expression. Since glucose is a central energy source for cell proliferation, it is quite likely that the genes that function for basic cellular growth (such as rrg1+) are under the control of glucose. In addition, the cellular growth-related genes need to be tightly regulated by environmental cues for optimal cell growth, which clearly explains the rapid response of the rrg1+ expression to glucose and other fermentable sugars (Kim et al., 2002). Considering the currently-available information about the glucose-dependent expression in S. pombe, the knowledge about the molecular regulation of the rrg1+ expression and its cellular role is quite informative in understanding how cells respond to glucose in fission yeast. 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