Involvements of a novel protein, DIA2, in cAMP signaling and spore differentiation during Dictyostelium development

r 2007, Copyright the Authors Differentiation (2008) 76:310–322 DOI: 10.1111/j.1432-0436.2007.00217.x Journal compilation r 2007, International Society of Differentiation

O RI GIN AL AR TI CLE

Kaori Hirata . Aiko Amagai . Soo-Cheon Chae . Shigenori Hirose . Yasuo Maeda

Involvements of a novel protein, DIA2, in cAMP signaling and spore differentiation during Dictyostelium development

Received April 27, 2007; accepted in revised form July 7, 2007

Abstract The novel gene dia2 (differentiation-associated gene 2) was originally isolated as a gene expressed specifically in response to initial differentiation of Dictyostelium discoideum Ax-2 cells. Using dia2AS cells in which the dia2 expression was inactivated by the antisense RNA method, DIA2 protein was found to be required for cAMP signaling during cell aggregation. During late development, the DIA2 protein changed its location from the endoplasmic reticulum (ER) to prespore-specific vacuoles (PSVs) that are specifically present in prespore cells of the slug. In differentiating prestalk cells, however, DIA2 was found to be nearly lost from the cells. Importantly, exocytosis of PSVs from prespore cells and the subsequent spore differentiation were almost completely impaired in dia2AS cells. In addition, spore induction by externally applied 8bromo cAMP was significantly suppressed in dia2AS cells. Taken together, these results strongly suggested that DIA2 might be closely involved in cAMP signaling . ) Kaori Hirata
Aiko Amagai
Yasuo Maeda (* Department of Developmental Biology and Neurosciences Graduate School of Life Sciences Tohoku University Aoba, Sendai 980-8578, Japan Tel: 181 22 795 3789 Fax: 181 22 795 3789 E-mail: [email protected] Soo-Cheon Chae Department of Pathology School of Medicine Wonkwang University Iksan-shi, Chonbuk 570-749 South Korea Shigenori Hirose Departments of Molecular and Human Genetics Biochemistry and Molecular Biology Baylor College of Medicine Houston, TX 77030, USA

and spore differentiation as well as in the initiation of differentiation during Dictyostelium development. Key words DIA2
cAMP
spore differentiation
exocytosis
PSV
mitochondria
Dictyostelium

Introduction In general, growth and differentiation are mutually exclusive, but they are cooperatively regulated during the course of development. Thus, the process of a cell’s transition from growth to differentiation is of general importance not only for the development of organisms but also for the initiation of malignant transformation. Dictyostelium discoideum (strain Ax-2) cells grow and multiply by mitosis as long as nutrients are supplied. Upon exhaustion of nutrients, however, starving cells differentiate to acquire aggregation-competence and they form multicellular structures by means of chemotaxis to cAMP. The cell aggregate (mound) undergoes a series of well-organized movements and differentiation to form a migrating slug. At the slug stage, a clear pattern along the anterior–posterior axis is established; prestalk cells, which differentiate into stalk cells during culmination, are located in the anterior one-fourth, while prespore cells destined to differentiate eventually into spores occupy the posterior three-fourths of the slug. The slug culminates in a fruiting body consisting of a mass of spores and a supporting cellular stalk. The growth and differentiation phases are temporally separated from each other and easily controlled by nutritional conditions. A specific checkpoint (a GDT point, formerly referred to as a PS point) of growth/ differentiation transition (GDT) has been specified in the cell cycle of Ax-2 cells (Maeda et al., 1989). This is the point at which cells are able to exit the cellcycle phase and enter differentiation when placed

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under conditions of nutritional deprivation (reviewed in Maeda, 2005). We have already identified several genes (dia1, dia2, dia3, and carA) that are specifically expressed in response to the initial differentiation from the GDT point (Abe and Maeda, 1994; Chae et al., 1998; Inazu et al., 1999; Hirose et al., 2000). The cAMP receptor 1 (carA) is specifically expressed in cells starved just before the GDT point (Abe and Maeda, 1994), and its product (CAR1) is also essential for differentiation (Sun et al., 1990; Sun and Devreotes, 1991). cAMP acting on CAR1 activates a number of rapid intracellular responses, as exemplified by activation of adenylyl cyclase A (ACA) via heterotrimeric G-proteins, and this activation is achieved by pulsatile cAMP stimuli (Devreotes, 1994; Laub and Loomis, 1998). CAR1dependent events include receptor phosphorylation and an influx of extracellular Ca21 in a G-proteinindependent manner (Milne et al., 1995). Another novel gene, dia2, encodes a lysine- and leucine-rich protein (DIA2) with a predicted molecular mass of 16.9 kDa. The biphasic expression pattern of dia2 during the whole course of development is quite similar to that of carA, and antisense-mediated gene inactivation of dia2 considerably inhibits the earliest step of morphogenesis. This is believed to occur through the reduced expression of carA (Chae et al., 1998). However, the biological significance of dia2 expression in the post-aggregative stages was not determined. In this paper, we report the localization of DIA2 protein and its dynamic changes during development. Based on the results obtained, we also discuss the possible functions of DIA2 in cAMP signaling and cell differentiation, with special emphasis on late development associated with prespore and spore differentiation.

Materials and methods Cell culture and developmental conditions Vegetative cells of D. discoideum Ax-2 were grown axenically in PS medium (1% Special peptone [Oxoid: Lot No. 333 56412], 0.7% Yeast extract [Oxoid, Basingstoke, Hampshire, UK], 1.5% D-glucose, 0.11% KH2PO4, 0.05% Na2HPO4
12H2O, 40 ng/ml vitamin B12, 80 ng/ml folic acid). Transformed cells were grown axenically by shaking culture in PS-medium containing appropriate concentrations of G418 (40 mg/ml for dia2AS cells, 50 mg/ml for DIA2– GFP cells). To allow cells to differentiate, cells were harvested during the exponential growth phase, washed in Bonner’s salt solution (BSS; 10 mmol/l NaCl, 10 mmol/l KCl, 2.7 mmol/l CaCl2) (Bonner, 1947) as starvation medium, and developed on 1.5% nonnutrient agar at 5  105 cells/cm2 or plastic dishes at 8  105 cells/cm2. This was followed by incubation at 221C. For synchronized development, starved cells on agar plates were incubated at 221C for 2 hr and kept overnight at 41C. Subsequently, they were again transferred to 221C to restart development. To starve cells in suspension cultures, washed cells were shaken at 150 rpm in BSS at a density of 1.0  107 cells/ml at 221C. For cAMP-pulse treatment,

50 nmol/l (final concentration) of cAMP was applied to the starved cell suspensions at intervals of 6 min for 3 or 6 hr. Preparation of the anti-DIA2 antibody Chemically synthesized oligopeptide (77-DQQGSFSRGEKKKK; from amino acids 77–99 of DIA2) with an additional cysteine residue at the C-terminus was conjugated with keyhole limpet hemocyanin (KLH) as a carrier protein (Research Genetics, Huntsville, AL). The KLH-conjugated oligopeptide was injected 4  1 ml subcutaneously (s.c.) into the foot pads of rabbits with Freund’s complete adjuvant. The total amount of the antigen was 5 mg per animal. Five weeks later, a total amount of 1 mg of KLH-conjugated oligopeptide per animal with adjuvant was injected s.c. Samples of blood (about 50 ml) were collected 10 days after the final injection, and aliquoted serum containing the polyclonal anti-DIA2 antibody was stored at  801C. Dissociation of multicellular structures for immunostaining Cell masses at appropriate developmental stages were chemically dissociated in pronase-BAL solution, according to the method of Takeuchi and Yabuno (1970). The dissociated cells were washed three times in BSS, pre-fixed in ice-cold 50% methanol, and fixed in absolute methanol for 10 min on an ice bath. The fixed cells were dried on cleaned coverslips. They were dipped in phosphate-buffered saline (PBS) (10 mmol/l phosphate buffer, pH 7.0, 0.9% NaCl) for 10 min and then stained with various antibodies, as follows: Double immunostaining of DIA2 and prespore-specific vacuoles (PSVs) Chemically dissociated cells were fixed and dipped in PBS on coverslips, as described above. The anti-DIA2 antibody or preimmune rabbit serum, both of which were diluted 1:100 in PBS containing 1% bovine serum albumin (BSA), was placed as a droplet on the coverslip and incubated for 1 hr in a moist chamber at room temperature. The samples were washed in three changes of PBS (10 min for each). Subsequently, rhodamine-conjugated anti-rabbit IgG (Chemicon, Temecula, CA) diluted 1:100 in PBS containing 1% BSA was placed on the coverslip and incubated for 1 hr at room temperature. After washings in PBS, the samples were stained with fluroscene isothiocyanate (FITC)-conjugated anti-Dictyostelium. mucorides spore IgG for 45 min in a moisture chamber at room temperature. After five washes in PBS (5 min for each), the samples were mounted in PBS containing 20% glycerol and observed under a fluorescence microscope. The rhodamine- and FITC-stains in the same optical field were visualized using G- and B2-excitation, respectively. In another experiment, samples were stained with the anti-PSV monoclonal antibody raised against a 36 kDa protein (C-10 antigen) in PSVs, followed by staining with TRITC-conjugated antimouse IgG (Sigma, St. Louis, MO) (Matsuyama and Maeda, 1998). Immunostaining using the anti-calnexin antibody Vegetatively growing DIA2-GFP cells were harvested, washed once with BSS, and resuspended to 1.0  106 cells/ml. Aliquots (30 ml) of the cell suspension were dropped on coverslips and incubated for 20 min. The cells adhering to the coverslips were sandwiched with a thin agarose sheet, basically according to the agarose overlay method (Yumura et al., 1984). Subsequently, the cells were fixed and stained with the anti-calnexin antibody, as reported previously (Muller-Taubenberger et al., 2001). The samples were washed by three changes of PBS containing 100 mmol/l glycine and stained with TRITC-conjugated anti-mouse IgG (Sigma) to detect the anti-calnexin antibody.

312 Western blot analysis Cells were lysed in sodium dodecyl sulfate (SDS)-sample buffer (2% SDS, 62,5 mM Tris-HCl, pH 6.8, 10% glycerol, 42 mM dithiothreitol, 0.005% bromophenol blue) by boiling for 5 min and chilling on ice. The samples were separated by 12% or 15% SDSpolyacrylamide gel electrophoresis, and transferred onto Immunoblot PVDF membranes (Bio-Rad, Hercules, CA). The membranes were blocked overnight with TBS (20 mmol/l Tris-HCl [pH 8.0], 150 mmol/l NaCl) containing 5% skim milk at room temperature. Subsequently, the membranes were probed by the primary antibody diluted 2,000 times with TBS containing 3% skim milk for 1 hr at room temperature. After washing two times in TBS-T (0.05% Tween 20 in TBS) (10 min for each), the membranes were stained for 1 hr with either a horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody or an HRP-conjugated anti-mouse secondary antibody (Amersham Biosciences, Piscataway, NJ) diluted 10,000 times with TBS. Enhanced chemiluminescence (ECL kit, Amersham Biosciences) was used for detection of the HRP.

Advantec, Tokyo, Japan). The filtered media were immediately boiled at 1001C for 10 min to inactivate phosphodiesterase (PDE), followed by evaporation using a spin vacuum to condense the cAMP. The amount of cAMP contained in the samples was measured using the cAMP enzyme immunoassay system (Biotrack, Amersham Biosciences), according to the manufacturer’s instructions.

Induction of spore differentiation by 8-Br-cAMP Spore induction by a membrane-permeable cAMP analogue, 8-BrcAMP, was performed according to the method of Maeda (1988). To allow prespore cells of Ax-2 slugs or dia2AS mounds to differentiate into spores, the cell masses were carefully transferred onto 2% agar containing 0, 5, 10, 15, or 20 mmol/l 8-Br-cAMP and covered with coverslips, followed by incubation in a moist chamber at 221C. After 6 hr of incubation, the samples were fixed in methanol at  401C, stained with the FITC-conjugated anti-PSV antibody, and observed under a fluorescence microscope to monitor the FITC stains.

Isolation of transformants An antisense dia2 transformation vector (pAct15-DIA2AS) was used for preparation of dia2AS cells, as reported previously by Chae et al. (1998). To obtain DIA2-GFP cells, a dia2-gfp expression vector (pDNeo67–dia2–gfp) was prepared as follows: the dia2 cDNA (SSK542) donated by the Dictyostelium cDNA project in Japan (http://www.csm.biol.tsukuba.ac.jp/cDNAproject.html) was inserted into pBluescript II. To prepare the dia2 cDNA without stop codons, the dia2 cDNA with a PstI sequence at the 5 0 end was amplified by polymerase chain reaction using DIA2 PstI-r primer (50 -AAACTGCAGGTTTGGAATAACTTTGATATAATTTTCCA GAA-30 ) and T3 Forward primer (5 0 -ATTAACCCTCACTAA AG-3 0 ). A full length of gfp was digested with HindIII and XbaI, and was ligated in pBluescript II. After digestion of the gfp sequence with SmaI and PstI and of dia2–cDNA–PstI with HincII and PstI, they were ligated to obtain a dia2 cDNA–gfp sequence. The dia2–gfp sequence was then digested by KpnI and XbaI, blunt-ended and ligated with pDNeo67, an expression vector with G418 resistance for Dictyostelium, which had been digested with BamHI and XhoI, blunt-ended, and treated with alkaline phosphatase. DNAs extracted from candidate clones were cut by the restriction enzymes to confirm the direction of the plasmid insertion. To induce the DIA2 protein with GFP at the Cterminal to be expressed, the pDNeo67–dia2–gfp vector was introduced into Ax-2 (clone 8A) cells by electroporation, as described previously (Howard et al., 1988). Transformed cells were first selected in growth medium (PS-medium) containing 10 mg/ml of G418. After 1 week of incubation at 221C, the transformed cells were re-cloned and selected in PS medium containing 50 mg/ml of G418. Preparation of total RNAs and Northern hybridization Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Samples containing 20 mg of RNA were processed as reported previously (Hirose et al., 2000) for Northern hybridization. Determination of cAMP concentrations Total cellular cAMP was extracted from Ax-2 cells at specific developmental stages, using 5% trichloro acetic acid, as described previously (Amagai, 1987). The concentrations of extracted proteins were determined by Bradford’s method using a protein assay CBB solution (Nakarai Tesque, Kyoto, Japan) (Bradford, 1976). To measure the amount of extracellular cAMP released from cells, only media from culture dishes were carefully collected by a syringe and filtered through a cellulose acetate filter (0.2 mm of pore size,

Results The structure of DIA2 protein and its expression pattern during development dia2 was originally isolated as a novel differentiationassociated gene specifically expressed upon differentiation of starved Ax-2 cells from the GDT point. The expression pattern of dia2 mRNA has already been reported by Chae et al. (1998). The dia2 gene encodes a lysine- and leucine-rich protein with a predicted molecular mass of 16.9 kDa (Fig. 1A). Based on a PSORTII search (Horton and Nakai, 1997), DIA2 has an endoplasmic reticulum (ER) targeting signal sequence at the N-terminus that satisfies the  3,  1 rule. Thus, this targeting signal is predicted to be cleaved by a peptidase when DIA2 is translocated to ER (Fig. 1A). The anti-DIA2 antibody used was shown by Western blot analysis to be highly specific (Fig. 1B). As shown in Figure 1C, the expression of DIA2 is developmentally regulated: the amount of DIA2 in vegetatively growing cells was very limited, but increased upon starvation. The maximal expression was attained at t6 (6 hr after starvation) when cAMP signaling for cell aggregation was most prominent. Subsequently, although the level of DIA2 began to decrease gradually up to t18, a small amount of DIA2 was retained during late development (t24–t30) (Fig. 1C). We have demonstrated previously that dia2AS cells expressing the antisense-RNA of dia2 mRNA exhibit delayed aggregation and stop their development at the mound stage (Chae et al., 1998). As expected, the DIA2 expression was found to be markedly delayed and reduced in dia2AS cells throughout development (Fig. 1C). The dia2 gene was originally isolated as one specifically expressed at the initiation of cell differentiation from the GDT point (Chae et al., 1998). Recently, however, several GDT-point-specific genes such as dia1

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and dia3 were found to be weakly expressed during vegetative growth, particularly at high cell densities (Morita et al., 2004). This was also the case for the dia2 expression, as shown in Figure 1D. A part of the differentiation program has been shown to begin before starvation (prestarvation response, PSR). Prestarvation genes such as dscA (discoidin I) and carA are induced as the density of the growing cells increases (Clarke et al., 1988). Interestingly, most of the GDTspecific genes thus far found are prestarvation genes (Maeda, 2005), suggesting that the biological significance of PSR is to allow readily the cells to exit from the GDT point toward the differentiation phase in response to increased cell density. Intracellular localization of DIA2 in preaggregative cells

Fig. 1 Structure of DIA2 protein and its expression pattern during development. (A) cDNA sequence of dia2 and its deduced amino acid sequence. The underlined sequence at the N-terminus indicates the signal peptide for endoplasmic reticulum (ER) localization. Open triangles indicate amino acids that fulfill the  1,  3, rule typically conserved in cleavable signal peptides. The DIA2 protein is deduced to be cleaved between amino acid positions 22 and 23. In the dia2 gene, a single intron exists and its position is represented as a filled triangle. The dotted line shows the oligo-peptide used for the preparation of the anti-DIA2 antibody. (B) Immunoblot analysis using the anti-DIA2 antibody. Total proteins were extracted from 1  107 cells of Ax-2 cells after 8 hr of starvation and applied to a lane of 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), followed by Western blotting. It is evident that this antibody detects the DIA2 protein (arrowhead) monospecifically. (C) Expression pattern of DIA2 in dia2AS cells and parental Ax-2 cells. Ax-2 cells and dia2AS cells were starved separately and incubated on 1.5% non-nutrient agar at 5.0  105 cells/ cm2 at 221C. At the indicated times of incubation, cells were collected and lysed with SDS sample buffer. Total proteins were sizefractioned by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and analyzed by Western blotting using the anti-DIA2 antibody. In Ax-2 cells, DIA2 was maximally expressed at 6 hr (t6) of starvation, followed by a gradual decrease. Even after 24 hr of incubation, however, cells at multicellular stages retained a detectable amount of DIA2. In dia2AS cells, it is clear that the amount of DIA2 is significantly reduced throughout development. At t6, little or no DIA2 protein was detected in dia2AS cells (data not shown). The amount of actin contained in each sample is shown as a loading control. (D) DIA2 expression in a cell-density-dependent manner during the vegetative growth phase. Vegetatively growing Ax-2 cells were harvested at the indicated cell density and lysed with SDS sample buffer. Then, the total proteins were sizefractioned by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by Western blot using the anti-DIA2 antibody. It is evident that the amount of DIA2 increases with increased cell densities, indicating that DIA2 expression is under the control of the prestarvation.

As DIA2 has an ER targeting signal sequence at the N-terminus (Fig. 1A), it was supposed to locate predominantly in ER. This was confirmed by Western blottings of cell fractionates and by colocalization of DIA2-GFP with calnexin. As shown in Figure 2A, it is evident that DIA2 is detected in the microsome fraction of starved Ax-2 cells, but not in the nuclear, mitochondrial, or cytosolic fractions. In DIA2-GFP cells, a high level of GFP signals co-localized with calnexin, an ER-localized protein (Muller-Taubenberger et al., 2001) (Fig. 2B). DIA2 was found by Western blottings to be overexpressed in DIA2-GFP cells, as compared with that in parental Ax-2 cells (data not shown). For comparison, we prepared dia2OE cells overexpressing the dia2 gene under control of the actin 6 (A6) promoter, and observed their development. The results showed that both of dia2OE cells and DIA2-GFP cells exhibited normal growth in PS medium and regular morphogenesis after starvation to form fruiting bodies. An antisense-mediated gene inactivation of dia2 results in impaired morphogenesis As reported previously (Chae et al., 1998), dia2AS cells showed abnormal development after starvation, characterized by delayed aggregation, formation of large aggregation streams, and developmental arrest at the mound stage (Fig. 3A). dia2AS cells and parental Ax-2 cells were mixed at various ratios and co-cultured on agar in order to examine whether the phenotypic defects of dia2AS cells are cell-autonomous. It was found that the timing of cell aggregation and the territory size of aggregates were considerably recovered by the presence of parental Ax-2 cells, but that the developmental arrest at the mound stage was scarcely rescued even in the presence of 30% Ax-2 cells (Fig. 3A). In the presence of 30% Ax-2 cells, however, gourd-like cell masses that seemed to be in the transition process from mounds to

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Fig. 2 Intracellular localization of DIA2 protein. (A) Ax-2 cells were harvested during the exponential growth phase and starved in Bonner’s salt solution (BSS). After 6 hr of shaking culture, the cell suspension was centrifuged to isolate cells. Then, the cell pellet was homogenized and fractionated into the indicated fractions by differential centrifugations. Each fraction was analyzed by Western blot using the anti-DIA2 antibody. It is clear that DIA2 is specifically localized in the microsome fraction. The culture medi-

um (supernatant) obtained by the first centrifugation was concentrated and analyzed as an ‘‘extracellular’’ fraction. (B) Vegetative DIA2-GFP cells were washed once with BSS and stained with the antibody raised against calnexin that is known to localize to the endoplasmic reticulum (ER). Although some regions of cells were stained with only the anti-calnexin antibody, the majority of DIA2– GFP fusion signals co-localized with calnexin, as shown in the merged figure (C). Bar, 10 mm.

standing slugs (Fig. 3A-i, arrow; Fig. 3B-c) were sometimes formed. When Ax-2 cells (RFP cells) expressing red fluorescent protein (RFP, DsRed Express) were mixed with dia2AS cells at the ratio of 7:3 and allowed to develop on agar, it was found that the RFP cells were uniformly distributed in aggregation streams (Fig. 3Bb), but that they eventually sorted out into the anterior region of the gourd-like cell mass (Fig. 3B-d). This seems to indicate that Ax-2 cells cannot completely rescue the developmental defects of dia2AS cells, particularly after the mound stage.

bated, during at least the first 16 hr of starvation (Fig. 5B). Table 1 shows much lower percentages of extracellular cAMP to the total cAMP in dia2AS cells than in parental Ax-2 cells. In connection with cAMP signaling, we compared the expression patterns of aca and carA during early develoment of dia2AS cells and parental Ax-2 cells by Northern blot analysis. As a result, both of the aca and carA expressions in dia2AS cells were found to be somewhat delayed and lower compared with those in Ax-2 cells (Fig. 5C).

Relation of the dia2 expression to cAMP signaling

Preferential translocation of DIA2 into PSVs during prespore differentiation

When cAMP pulses were applied to starved dia2AS cells every 6 min for 6 hr to mimic the pulses realized during aggregation, the delayed aggregation of dia2AS cells was mostly cancelled, but the cAMP pulses could not recover the progress of morphogenesis beyond the mound stage (Fig. 4A). The expression of dia2 mRNA in Ax-2 cells was also accelerated by the cAMP pulses (Iranfer et al., 2003) (Fig. 4B). Surprisingly, however, the dia2 expression in carA-null cells was found to be earlier and higher than in parental Ax-3 cells (Fig. 4C), suggesting that the dia2 expression might be negatively regulated by the cAMP receptor 1 (CAR1). cAMP production in dia2AS cells seemed to be considerably delayed compared with parental Ax-2 cells in which the total cellular cAMP reached the first peak at t8 and then the second peak at t12 (Fig. 5A). Importantly, little or no cAMP was detected in the culture medium, in which dia2AS cells had been incu-

Substantial differences in fine structure have been demonstrated between the prestalk and prespore cells of the slug. Among them, the storage vacuole PSV is believed to be the only organelle that exists in only one of the two cell types (Maeda and Takeuchi, 1969). The PSV is also functionally essential: it fuses with the plasma membrane and the lining membrane is exocytosed from prespore cells to form the outermost layer of the spore cell wall during culmination (Hohl and Hamamoto, 1969; Maeda, 1971). Somewhat surprisingly, the DIA2GFP fusion protein was found to be specifically localized in the posterior prespore region of slugs derived from DIA2-GFP cells (Fig. 6A). Moreover, double staining of dissociated slug cells with the anti-DIA2 antibody and anti-PSV polyclonal antibody (FITCconjugated anti-D. mucoroides spore IgG) showed that DIA2 was strictly localized to PSVs (Fig. 6B). As

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Fig. 3 (A) Partial recovery of developmental defects observed in dia2AS cells in the presence of parental Ax-2 cells. Exponentially growing dia2AS and Ax-2 cells were harvested, washed in Bonner’s salt solution (BSS), and mixed at the indicated ratios. This was followed by the indicated times of incubation on 1.5% non-nutrient agar at 221C. As reported previously (Chae et al., 1998), dia2AS cells alone exhibited considerably delayed aggregation and formed large aggregates (a–c). Each aggregate subdivided into smaller mounds and stopped its development at this stage. dia2AS cells failed to form migrating slugs or fruiting bodies (c). The territory size of aggregating dia2AS cells became normal when mixed with Ax-2 cells, depending on the increased ratios of Ax-2 cells (d–i).

However, the mixed populations failed to form normal-shaped slugs even in the presence of 30% Ax-2 cells. The chimeras occasionally formed gourd-like cell masses (i, arrow). Bar, 1 mm. (B) RFP cells (Ax-2 cells stained with RFP) and dia2AS cells were harvested during the exponential growth phase, washed in BSS, and mixed with Ax-2 in a ratio of 7:3, followed by incubation on 1.5% non-nutrient agar at 221C. Aggregation streams (a, b) and a gourdlike cell mass (c, d) were photographed under a phase-contrast (a, c) or fluorescent microscope (b, d). In the aggregation streams, RFP cells were apparently scattered (b), but RFP cells were eventually sorted out to the anterior region of the gourd-like cell mass (d). Bars, 200 mm. RFP, red fluorescent protein.

expected, the DIA2 protein was exocytosed from prespore cells during sporulation (Fig. 6C). Incidentally, DIA2 was never detected in prestalk cells (Fig. 6B) and stalk cells, indicating that this protein was selectively lost, coupling with prestalk differentiation. To examine a temporal relationship between the appearance of DIA2 and PSV formation, we collected cell masses from the early mound to slug stages, dissociated them chemically, and double-stained as described above (Fig. 7). The results thus obtained showed that the DIA2 staining was recognized in differentiating prespore cells at the early mound stage, but that the

granular staining of PSVs was scarcely detected. This suggests that the translocation of DIA2 into PSVs might occur slightly before mature PSV formation (Fig. 7B, arrows).

DIA2 expression and prespore/prestalk differentiation In slugs derived from Ax-2 cells, about 75% of the total cells are known to be prespore cells. To examine the relation of DIA2 expression to cell-type proportioning, we immunostained chemically dissociated cells from

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Fig. 4 (A) Recovery of an aggregative defect in dia2AS cells by cAMP pulses. Exponentially growing dia2AS cells were harvested, washed, and shaken at 150 rpm in Bonner’s salt solution (BSS) at 1.0  107 cells/ml with or without pulses of 50 nmol/l cAMP every 6 min for 6 hr at 221C. This was followed by incubation on 1.5% non-nutrient agar for the indicated hours at 221C. The cells treated with cAMP pulses started to aggregate at 3 hr, while control cells treated with DDW (distilled deionized water) pulses remained as non-aggregated single cells even after 5 hr of incubation. Both treatments resulted in mounds after 25 hr, but not slugs or fruiting bodies. Bar, 1 mm. (B) Enhanced dia2 expression in Ax-2 cells by cAMP pulses. Ax-2 cells were harvested during exponential growth, washed, and shaken in BSS with or without cAMP pulses, as noted above. At the indicated times of pulsing, total RNAs were extracted, size-fractioned, and analyzed by Northern blot using the dia2 cDNA probe. The cells pulsed with cAMP exhibited earlier and enhanced dia2 expression, compared with control cells. (C) Augmented expression of dia2 in the absence of cAMP receptor 1 (CAR1). Exponentially growing carAnull cells and parental Ax-3 cells were separately harvested, washed, and shaken in BSS at 1.0  107 cells/ml at 221C. At the indicated times of starvation, total RNAs were extracted and analyzed as described above. Unexpectedly, the expression of dia2 was higher in carA-null cells than in parental Ax3 cells.

Ax-2 slugs and dia2AS mounds with the anti-PSV antibody. There was no significant difference in the proportion of prespore cells between the two (data not shown). Moreover, we could not detect any difference in the number of PSVs contained in prespore cells or the staining intensity by the PSV antibody between the two samples. As shown in Figure 8A, however, the amount of the C-10 antigen contained on PSVs seemed to be slightly less in dia2AS cells than in Ax-2 cells. In this experiment, all of the cells plated were collected without filtration, and therefore non-aggregated single cells were contained in the samples used. A certain number of dia2AS cells remained as non-aggregated single cells even after a prolonged time of starvation, as compared with Ax-2 cells that mostly participated in cell aggregate formation and differentiation. Therefore, it is likely that

the observed less amount of the C-10 antigen in the dia2AS preparation might be due to an increase in the number of undifferentiated single cells in their population. In fact, no difference was detected in the amount of the C-10 antigen between Ax-2 slugs and dia2AS mounds, when only cell masses were collected by removal of undifferentiated single cells and small loose aggregates through a nylon filter (pore size, 50 mm). Ax-2 cells normally form fruiting bodies by 30 hr post starvation, but dia2AS cells largely arrest their development at the mound stage and fail to form fruiting bodies. After a prolonged time (460 hr) of incubation, the dia2AS mounds began to collapse into smaller cell masses or single cells. This seemed to be correlated to a marked decrease of the C-10 antigen in dia2AS cells at the time-point of t60 (Fig. 8A).

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(8-Br-cAMP), we placed Ax-2 slugs or dia2AS mounds on agar plates containing various concentrations (0–20 mmol/l) of 8-Br-cAMP and covered them with coverslips. This was followed by incubation for 6 hr in a moisture chamber at 221C. As a result, we found that PSVs in Ax-2 prespore cells are extensively exocytosed to form spores in the presence of 5 mmol/l 8-Br-cAMP, while spore differentiation characterized by the exocytosis of PSVs is considerably suppressed in dia2AS prespore cells (Fig. 8B). Essentially the same results were obtained using 10, 15, or 20 mmol/l 8-Br-cAMP.

Discussion

Fig. 5 Impaired cAMP release from dia2AS cells. (A) Exponentially growing Ax-2 cells and dia2AS cells were harvested separately, washed in Bonner’s salt solution, and plated in 24-well plates (1 ml of cell suspension per well) at 8.0  105 cells/cm2 at 221C. At the indicated times of stationary cultures, culture media (supernatants) were carefully collected, and the remaining cells were extracted with trichloric acid to measure intracellular cAMP. Preparation of samples and measurements of cAMP were performed as described in ‘‘Materials and methods.’’ (A) Changes in the amount of intracellular cAMP contained in Ax-2 cells (filled square) and dia2AS cells (open square). Increase of intracellular cAMP in dia2AS cells was delayed and seemed to be slightly reduced, compared with that in parental Ax-2 cells. Error bars indicate the standard deviations (SD) in three independent experiments. (B) Changes in the amount of extracellular cAMP released from Ax-2 cells (filled triangle) and dia2AS cells (open triangle). A significant amount of cAMP was released from Ax-2 cells during the first 6 hr of starvation, but extracllular cAMP was scarcely noticed in the samples of dia2AS cells during the course of the experiment. Error bars indicate the standard deviations (SD) in three independent experiments. (C) Expressions of aca and carA during the development of dia2AS cells and parental Ax-2 cells, analyzed by Northern blot. It is evident that both aca and carA expression in dia2AS cells are somewhat delayed with a lower concentration as compared with Ax-2 cells

Involvement of DIA2 in exocytosis of PSVs The DIA2:GFP fusion protein was found to be exocytosed during spore differentiation to form the spore cell wall (data not shown). Thereupon, to determine whether the PSV secretion from prespore cells to form spores is affected by application of 8-bromo-cAMP

The dia2 gene was initially identified as a novel gene that is specifically expressed during the transition from growth to differentiation at the GDT point of the Dictyostelium cell cycle (Chae et al., 1998). Antisensemediated gene inactivation considerably inhibited the progress of differentiation, presumably through the impaired cAMP signaling. The impaired differentiation in the dia2AS cells was partially recovered by externally added cAMP pulses. In this study, the dia2 gene was found to be a prestarvation gene as well as a differentiation-associated gene, as is the case for dia1 and dia3. The PSR has been regarded as a preparative event that allows cells to sense the population density and begin preparing for differentiation. We have attempted to destroy the dia2 gene by homologous recombination, but failed to obtain dia2-null cells in spite of many trials. This seems to indicate that the DIA2 protein is essential for cell growth as well as for differentiation, thus resulting in failure to prepare the knockout strains. Consequently, we isolated the knockdown transformants (dia2-RNAi cells) of DIA2 by the RNAi method. In dia2-RNAi cells, the expression level of DIA2 protein was significantly decreased, resulting in a growth rate slower than that of DIA2-RNAi cells (data not shown). This again indicates that DIA2 is necessary for cell growth, as is the case for the Hsp90 family including mitochondrial Hsp90L (TRAP-1). At first glance, the amount of cAMP released from starved dia2AS cells seemed to be very limited, compared with that from aggregation-competent Ax-2 cells. However, the amount of extracellular AMP is known to be totally determined by extracellular cAMP PDE and PDE inhibitor (PDI) as well as by cAMP synthesis, all of which are developmentally regulated (Franke and Kessin, 1981; Hall et al., 1993). In the present work, we have not measured temporal changes of PDE and PDI activities during the course of development. Therefore, we must remark that although our results are suggestive of a defect of cAMP secretion in dia2AS cells, they do not directly demonstrate this. Although our knowledge

318 Table 1 Difference in the amount of extracellular cAMP between Ax-2 and dia2AS cells during aggregation

Ax-2 cells dia2AS cells

Times (hr)

Total cAMP (mean  SD) (pmol)

Extracellular cAMP (mean  SD) (pmol)

Percentage of extracellular cAMP (mean  SD) (%)

6 9 11 12

9.3  3.6 6.2  3.1 4.6  1.9 5.2  3.3

0.92  0.76 0.48  0.20 0.06  0.06 0.07  0.10

9.4  6.6 8.8  3.3 1.5  1.6 1.4  1.4

Starved Ax-2 and dia2AS cells were allowed to develop in Bonner’s salt solution (BSS) at 8.0  105 cells/cm2 at 221C. After the indicated times (hr) of starvation, the concentrations of intracellular and extracellular cAMP in each sample were determined, as described in ‘‘Materials and methods’’ and the legend of Fig. 5. The ratio of extracellular cAMP to the total cAMP (intracellular cAMP plus extracellular cAMP) is shown in the right-most column. Data collected from three independent experiments. Significant difference between Ax-2 and dia2AS (po0.05).

of the mechanisms regulating the cAMP secretion during cell aggregation has remained rudimentary, it has now been shown that the ACA is enriched in vesicles located in the rear of individual cells; the asymmetric distribution of ACA provides a compartment from which cAMP is secreted to locally act as a chemoattractant (Kriebel et al., 2003). This seems to indicate

that at least a part of cAMP synthesized in the cells may be secreted by exocytosis of cAMP-containing vesicles through DIA2. Noc2 has been proposed to regulate exocytosis in both endocrine and exocrine cells, and actually the Noc2/Rab27 complex has been shown to be a crucial constituent of the early stage of isopreterenolstimulated amylase secretion (Imai et al., 2006). In

Fig. 6 (A) Preferential translocation of DIA2 protein into presporespecific vacuoles (PSVs) in differentiating prespore cells. Phasecontrast (left) and fluorescent (right) micrographs of a migrating slug derived from DIA2-GFP cells. The outline of the slug in the right image was drawn as a white dotted line, and the anterior prestalk region located in the right dark. Bar, 200 mm. (B) DIA2 specifically localized in PSVs of prespore cells. Slugs formed from Ax-2 cells were dissociated, fixed, and double-stained with the antiDIA2 antibody and fluroscene isothiocyanate (FITC)-conjugated anti-Dictyostelium mucoroides spore IgG. It is evident that DIA2 is

exclusively located in PSVs of prespore cells but not in prestalk cells. The outline of a prestalk cell was drawn as a white dotted line. Bar, 10 mm. (C) Behavior of the DIA2 protein during sporulation. During the early stage of spore differentiation, a strong staining by the anti-DIA2 antibody was observed near the cell cortex of prespore cells in which PSVs were presumably just exocytosed (a, arrow). Coupled with subsequent spore maturation, DIA2 seems to be exclusively located in the spore wall (b, arrowheads). Their signals became weaker, however, possibly because of DIA2 release into the extracellular space (c). Bar, 10 mm.

319

Fig. 7 (A) Temporal and spatial changes of DIA2 and presporespecific vacuoles (PSVs) during multicellular development. Starved Ax-2 cells were plated on 1.5% non-nutrient agar at 5.0  105 cells/ cm2, incubated for 2 hr at 221C, and then kept overnight at 41C to synchronize the progression of development. Subsequently, the plates were re-transferred to 221C and incubated for the indicated times. Cell developmental structures were collected, dissociated, and fixed in absolute methanol. This was followed by double staining of the samples with the anti-DIA2 antibody and fluroscene isothiocyanate (FITC)conjugated anti-Dictyostelium mucoroides spore IgG (anti-PSV antibody), as described above. The upper-most drawing shows the gross morphology of cell developmental structures at the indicated times of incubation after cold treatment. At the early mound stage (a–c), all of the cells were stained weakly by the DIA2 antibody but not by the

anti-PSV antibody. The faint green color seen in (c) does not show positive staining by the anti-PSV antibody and is at the level of selffluorescence. At the middle mound stage (d–g), a marked increase of DIA2 was observed in a limited number of cells along with a small number of cytoplasmic granules (PSVs) in the same cells. At the late mound and slug stages (h–k, l–o), PSVs containing DIA2 increased in number, indicating colocalizaion of DIA2 and PSVs in prespore cells. Bar, 10 mm. (B) Enlarged images of A (d–g). In the cell indicated by an arrow, cytoplasmic granules containing DIA2 are observed (a), but these granules barely stained with the antiPSV antibody. This seems to indicate that DIA2 may accumulate in cytoplasmic granules (pre-PSV) of differentiating prespore cells before prespore-specific vacuole (PSV) formation. DIA2 is almost completely eliminated from prestalk cells. Bar, 10 mm.

Dictyostelium, the aggregative defect of dia2AS cells was partially rescued when mixed with parental Ax-2 cells, presumably by active cAMP secretion from aggregation-competent Ax-2 cells. The partial rescue of cell ag-

gregation was also observed when cAMP pulses were externally applied to starved dia2AS cells. The tip of the migrating slug has the ability to function as an aggregation center that secretes cAMP. This signal is relayed

320

Fig. 8 (A) Changes in the amount of C-10 antigen contained in prespore-specific vacuoles (PSVs) during development of Ax-2 cells and dia2AS cells. Starved Ax-2 and dia2AS cells were developed separately on 1.5% non-nutrient agar at 5.0  105 cells/ cm2 at 221C. After the indicated times of incubation, cells or cell developmental structures were collected and lysed with SDS sample buffer. Total proteins were size-fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by Western blot using the anti-PSV monoclonal antibody that specifically recognizes the C-10 antigen of the 36 kDa. In dia2AS cells, the synthesis of C-10 antigen was delayed and its amount was slightly reduced, as compared with that of parental Ax-2 cells. The amount of actin contained in each sample is shown as a loading control. (B) Spore induction by application of 8-Br-cAMP. Ax-2 slugs and dia2AS mounds were transferred separately onto 2% agar containing 0 or 5 mmol/l 8-Br-cAMP and incubated for 6 hr at 221C to monitor spore differentiation from prespore cells, as described in ‘‘Materials and methods.’’ It is evident that most of the PSVs are exocytosed from Ax-2 prespore cells to form spores with the C-10 antigen at the surface, while spore induction by 8-Br-cAMP is greatly suppressed in dia2AS prespore cells. (a–d) Fluorescence photomicrographs; (e–h) Phase-contrast photomicrographs. Bar, 50 mm.

to the posterior cells, allowing the entire slug cells to move in the direction of the tip. Accordingly, a failure of dia2AS cells to develop to the slug stage might be due to impaired cAMP secretion from dia2AS cells. Alternatively, it is also possible that dia2AS cells may have reduced sensitivity to cAMP, as compared with parental Ax-2 cells. Although the mechanisms by which the DIA2 protein is specifically translocated into PSVs during late development and lost preferentially from differentiating prestalk cells are presently unknown, it is interesting to speculate upon its possible role in relation to the genesis of PSVs during prespore differentiation. Before PSV formation, mitochondria in differentiating prespore cells undergo drastic transformation to form unique vacuoles (M vacuoles) derived from the mitochondrial membrane and engulfed cytoplasm. The mitochondrial molecular chaperon HSP90L (DdTRAP1) is located in PSV as well as in the mitochondria (Yamaguchi et al., 2005). More importantly, it has been shown immunocytochemically that a PSVspecific antigen (C-10) is also present in the Golgi cisternae that fused the M vacuoles (Matsuyama and Maeda, 1998). During the intermediate stage of PSV maturation, the C-10 antigen is noticed in a limited part

of the mitochondria adjacent to the PSV-mitochondrion complexes as well as in the lining membrane of PSVs. An ER-localized molecular chaperone (glucoseregulated protein 94; Dd-GRP94) has been shown through a biochemical assay to be contained in purified PSVs as well as in ER (Srinivasan et al., 2001; Alexander et al., 2003), and later confirmed by immunoelectron microscopy using the anti-Dd-GRP94 antibody (Yamaguchi et al., 2005). As DIA2 seems to be involved in membrane fusion, it is quite likely that the DIA2 protein itself may be readily transported into the PSV via fusion of ER-derived Golgi vesicles with the M vacuole, as appeared to be the case for Dd-GRP94 and C-10 antigen. The PSV–mitochondrion complex is twisted at the junction and eventually they separate to become the respective organelles. Thus, it is evident that both the mitochondria and Golgi complexes are cooperatively implicated in PSV formation. Here, it seems critical to contemplate briefly the functional significance of the M vacuole. It can be easily imagined that mitochondria efficiently supply many chemically highenergized compounds such as ATP to the M vacuole, thus providing a favorable compartment for biosynthesis of the macromolecules constituting PSVs. Moreover, the M vacuole might provide a well-made compartment that

321

does not fuse with lysosomes, in contrast with autophagic vacuoles that are well developed in prestalk cells and involved in the digestion of various macromolecules including DIA2 with the help of lysosomes. The PSV is a functionally essential structure and it is exocytosed from prespore cells to form the outermost layer of spore cell wall during culmination. In adipocytoes and myosytes, it has been shown that the glucose transporter GLUT4 and the aminopeptidase IRAP are the major cargo proteins of GLUT4 storage vesicles, and that protein kinase Akt/PKB is required for insulinstimulated exocytosis of GLUT4 to the cell membrane (Gonzalez and McGraw, 2006). As reported previously (Chae et al., 1998), dia2AS cells stopped their development at the mound stage, thus resulting in a failure of spore and stalk differentiation. More importantly, spore induction by externally applied 8-Br-cAMP was considerably suppressed in dia2AS cells. This seems to indicate that DIA2 might be closely involved in the exocytotic secretion of PSVs during sporulation as well as in cAMP signaling during cell aggregation. Alternatively, however, it is also possible that DIA2 is necessary for the expression during prespore differentiation of other components that are needed for the final events in spore maturation in response to 8-Br-cAMP.

Conclusion In summary, a novel small protein, DIA2, was found to be predominantly located in ER during early development, and then selectively translocated into PSVs during late development. Importantly, exocytosis of PSVs that is required for spore wall formation was considerably impaired in dia2AS cells. Considering the data presented here, it is quite possible that a novel protein, DIA2, may be tightly involved in spore differentiation as well as in cAMP signaling, presumably as one of the key factors required for developmental regulation of the membrane traffic such as exocytosis. Acknowledgments We thank Drs. Matthew Cabral and Waleed Nasser for their critical reading of the manuscript and valuable comments. We are also grateful to Dr. Aneette Muller-Taubenberger for kindly providing the anti-calnexin antibody. This work was supported by a Grant-in-Aid (No. 16370030 and 16657020) from JSPS. This work was also funded by the Mitsubishi Foundation.

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