Fluorescence in the Migrating Pseudoplasmodium of the Cellular Slime Mold

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CELL STRUCTURE AND FUNCTION 19: 159-163 (1994) 1994 by Japan Society for Cell Biology Fluorescence in the Migrating Pseudoplasmodium of the Cellular Slime Mold Dictyostelium mucoroides Saburo

1 CELL STRUCTURE AND FUNCTION 19: (1994) 1994 by Japan Society for Cell Biology Fluorescence in the Migrating Pseudoplasmodium of the Cellular Slime Mold Dictyostelium mucoroides Saburo Uchiyama1*, Shin-ichi Nagai1, Keizo Maruyama2 department of Biology, Dokkyo University School of Medicine, Mibu, Tochigi , laboratory of Biology, Faculty of Integrated Human Studies, Kyoto University, Kyoto 606, Japan Key words: cellular slime mold/dictyostelium mucoroides/fluorescence microscopy/cell differentiation/localization ABSTRACT. Fluorescence was observed under light microscope in living cells cell mass of Dictyostelium mucoroides. The fluorescence was localized in the vacuoles of live vegetative cells. While the cell mass of D. discoideum does not have a stalk during migration period, the cell mass of D. mucoroides has a stalk that forms at the beginning of the migration period. Wewere able to observe a preferential loss of the fluorescent vacuoles from the cells of the stalk from the stalk-forming cells at the tip region of the slug. Although the fluorescence was also present in the mature spore mass of Z>. mucoroides, the fluorescence was not observed in the spores, but rather in the spaces between the spores within the spore mass. The fluorescent vacuoles in the cells of vegetative stage of migrating slug stage may be related to the interspore fluorescence in the spore mass. Possible roles of the fluorescent substance(s) in amoebae, slugs spore masses were discussed. Amoebaeof the cellular slime mold Dictyostelium mucoroides proliferate by binary fission, using bacteria as a food source. After deprivation of bacteria, the homogeneouspopulation of slime mold cells aggregates to form a slug-shaped mass of cells (pseudoplasmodium) on a solid substratum. The cell mass ofd. mucoroides has a stalk that forms at the beginning of the migration period, during which time the cell mass crawls over the surface of the substratum. Eventually the mass of cells generates a fruiting body, which consists of a mass of spores a supporting cellular stalk. The cells grown in liquid nutrient mediumremain stable for an extended period of time prior to cell lysis (18). This period is referred to as the stationary phase. There have been several reports that stationary phase cells are significantly different from either growing or developing cells (14, 16, 23). It is knownthat the cellular slime molds contain fluorescent substance(s). A major intracellular fluorescent product of D. discoideum cells, dictyopterin, 6-(D-threo-l,2-dihydroxypropyl)-pterin, has been isolated from vegetative cells (12). It has been reported that D. discoideum cells secrete lumazine (the demination product of pterin), an extracellular fluorescent product that has been linked with their ability to aggregate (19, 20). We previously observed fluorescence in the live cells of D. discoideum during their growth morphogenesis (21). In present study, we examined the localization of To whomall correspondences should be addressed. 159 fluorescence in the cells cell mass of D. mucoroides by fluorescence microscopy. While the cell mass of D. discoideum forms a stalk after the migration period, the cell mass ofd. mucoroides has a stalk from the start of the migration period. MATERIALS AND METHODS The cellular slime mold, Dictyostelium mucoroides, was used in all experiments. The cells were grown in a liquid medium that contained the following per liter: 10 g bacto-peptone (Difco, Detroit, U.S.A.), 10g glucose, 0.96g Na2HPO4-12H2O, 1.45 g KH2PO4 (22). Escherichia coli strain B/r was the food source for the myxoamoebae. Cultures in 30 ml of the nutrient broth were shaken on a reciprocating shaker (100 strokes per min) at 22 C. The cells were harvested in the middle of the logarithmic phase of growth (2-5 x 106 cells /ml), in the full growth phase (2-3 x 107 cells/ml) in the stationary phase, that is, the full grown cells were further incubated for 24 h in the same medium. The cells were washed free of bacteria with Bonner's salt solution (2), then observed by fluorescence microscopy. To obtain cell masses cells were grown on solid nutrient mediumwith E. coli as the food source. Cells were harvested with a glass rod. The solid nutrient mediumcontained the same constituents as described above for the liquid mediumexcept for the addition of 20 g of agar (Difco, Detroit, U.S.A.) per liter (2). Cells (2 x 108) were washed free of bacteria with Bonner's salt solution allowed to develop on a square cellophane membrane(about 1 square cm) placed on 2%plain

2 S. Uchiyama et al. agar in a humid atmosphere at 22 C. After an appropriate incubation period, the square cellophane membrane,on which the slugs fruiting bodies had developed, was transferred onto a glass side, covered with a glass coverslip, slightly compressed by the coverslip for microscopic examination. A slug was squeezed between the glass silde the coverslip for observation of individual cells. For observation of individual spores, a spore masswas smearedonto a glass slide. Fluorescence microscopy was carried out using a Nikon Microphot microscope (Nihonkougaku Co., Tokyo Japan) equipped with filter sets for selective autonomous/fitc (fluorescein isothiocyanate) fluorescence. The excitation wavelength was around 365 nm the emission wavelength was cut off below 410 nm. Photographs were taken on Fuji color film with ASA100. RESULTS Fluorescent vacuoles were observed in the growing amoebae of Dictyostelium mucoroides (Fig. 1). The intensity of the fluorescence of the vacuoles decreased in the full grown cells, compared with that of cells in the middle of the logarithmic growing phase. Further decrease of the intensity of fluorescence of the vacuoles occurred in the stationary phase cells which were incubated for the extended period of time (24 h) in the same medium after full growth. The cell mass ofd. mucoroides has a stalk that forms at the beginning of the migration period, in contrast to the cell mass of D. discoideum. The stalk cells are formed in the tip region of the slug. Figure 2 shows bright fluorescence over the whole slug, although the fluorescence is weak in the tip region of the slug as well as in the stalk which is located in the cylindrical axis of the migrating slug. To observe the fluorescent vacuoles of the cells, a slug was squeezed between a glass slide a coverslip. Figure 3 shows the loss of fluorescent vacuoles from the cells in the tip region those in the stalk. As shown in Fig. 4, fluorescent vacuoles were maintained in almost all cells located in the fluorescent region of a slug. This observation suggests that the fluorescent vacuoles are preferentially lost from the cells involved in the process of stalk cell differentiation. The fluorescence lost from stalk cells was not released into the external environment of the slug at a level that could be detected microscopically. In the mature fruiting body, the spore mass was fluorescent, although the fluorescence was not present in the individual spores, but rather present in the spaces between the spores (Fig. 5). This is consistent with the results from studies of the spore mass ofd. discoideum as described elsewhere (21). DISCUSSION In this study we observed the localization of fluorescence in amoebae,slugs, spore masses ofd. mucoroides. Our observations coincide with those previously made for D. discoideum (21), the exception being the loss of fluorescent vacuoles from the tip region of D. mucoroides. It has been reported that D. discoideum cells secreted fluorescent substance (lumazine), which is responsible for the extracellular fluorescent products linked with their aggregation ability (19, 20). In D. mucoroides cells, we observed the loss of the fluorescent vacuoles in the cells of the tip region of the slug in the stalk cells. In fruiting body, the stalk cells the spores themselves did not show fluorescence, but the Fig. 1. Corresponding light fluorescence micrographs of growing D. mucoroides cells. Light micrograph of cells (A) fluorescence micrograph of cells (B). Bright fluorescent vacuoles are present in the cells. Scale bar indicates 50 /jm. 160

Fluorescence in the Cell Mass of D. mucoroides Fig. 2. Corresponding light fluorescence micrographs of a slug which has been silghtly compressed by a coverslip.

3 Fluorescence in the Cell Mass of D. mucoroides Fig. 2. Corresponding light fluorescence micrographs of a slug which has been silghtly compressed by a coverslip. Light micrograph of the slug with a stalk (A). Fluorescence micrograph of the same slug (B). Bright fluorescence is observed over the whole slug, except for the tip region the stalk (see also Fig. 4). Scale bar indicates 200 fim. surroundings of the spores did. The fluorescent vacuoles in the cells of vegetative stage of migrating slug stage maybe related to the interspore fluorescence in the spore mass. As to the fluorescence in the stalk region of D. discoideum (21), we might observe false or residual fluorescence, since, as to be described elsewhere, fluorescence spectra using a microspectrophotometersuggested the exisence of non-specific low noisy fluorescence in the stalk region. The above observation coincides with the weak fluorescence in the stalk region of D. mucoroides observed in this paper. In Saccharomyces cerevisiae, the fluorescence in the culture medium has been attributed to NAD(P)H(9). In Escherichia coli, an unknown fluorophore(s) that displayed an emission spectrum very similar to that of NAD(P)Hwas shown to be a biochemically distinct compound.this fluorophore(s) is responsible for the fluorescence of a culture of E. coli, with fluorescence 161 Fig. 3. Corresponding light fluorescence micrographs of the tip region of a slug that has been compressed between a coverslip a glass slide. Light micrograph of the tip region of the slug (A). The stalk cells are observed as cells with a clear cell wall. Fluorescence micrograph of the slug (B). Bright fluorescence is not observed in the cells of the tip region or the stalk region. Scale bar indicates 50 jim. being emitted by both the cells the medium (9). The fluorescence of dictyopterin, a major pteridine isolated from vegetative cells ofd. discoideum (12), is very similar to that of NAD(P)Hin mitochondria (1). The contribution of natural fluorophores to the fluorescence observed is still unknownin D. mucoroides cells. The fluorescent substance(s) is thought to be involved in the photoreception associated with phototaxis. Localized fluorescence has been widely observed in the flagella /or the eyespot of brown golden algae (3, 10, 1 1). By microspectrophotometry, pterin- flavin-like fluorescent substances were observed in the paraflagellar body of Euglena gracilis (17). Furthermore, the pterin- flavin-like fluorescent substances were extracted from isolated flagella of Euglena gracilis (7). Since D. discoideum amoebaealso exhibit phototactic responses (4, 8), the fluorescent vacuoles in the cellular slime mold might be involved in the photoreception as-

S. Uchiyama et al. tip rgion of the slug. In the slug, therefore, it may be hard to correlate phototaxis with the functional role of the fluorescent substance(s).

4 S. Uchiyama et al. tip rgion of the slug. In the slug, therefore, it may be hard to correlate phototaxis with the functional role of the fluorescent substance(s). Ultraviolet irradiation of washed spores of D. discoideumresulted in a prolonged delay of the emergenceof amoebaefrom swollen spores (15). Since the fluorescent substance(s) absorbs harmful ultraviolet light emits the fluorescent light in a harmless form, the fluorescent substance(s) may play a role in protecting the spores from ultraviolet light in the field. Furthermore, spores are capable of continuing to the next generation, while stalk cells are not. This fact is consistent with the obser- vations presented in the present paper, namely, that the fluorescent substance(s) is present in the spaces between the spores surrounds the spores in the spore mass (Fig. 5), but it is not present in either the stalk or the stalk-forming region (Fig. 3). Fig. 4. Fluorescence micrograph of the bright fluorescent region in aalmost slug that has been compressed between a coverslip a glss slide. all cells contain bright fluorescent vacuoles. Scale bar indi- REFEREN CES cates 50 //m. 1. Avi-Dor, Y., Olson, J.M., Doherty, M.D., N.O Fluorescence of pyridine nucleotides dria. sociated with phototaxis. /. Biol. Chem, 237: Kaplan, in mitochon- 2. Bonner, J.T Evidence for the formation of cell aggregates by chemotaxis in the development of the slime mold Dictyostelium discoideum. J. Exp. Zool., 106: Coleman, A.W The auto fluorescent flagellum: a new Schmidt et al. (17) reported the presence of blue fluorescent spots that were distinct from the fluorescent par- aflagellar body of Euglena gracilis, although the functional role of the former fluorescence was unclear. In Dictyostelium, the functional role of the fluorescent substance(s) in amoebae requires further investigation. Although slugs demonstrate phototaxis (13), the phototactic response of the slug is based on a lens effect of the cylindrical body which consists of about 105 cells (5, 6). Furthermore, as demonstrated in this study, fluorescent vacuoles are preferentially lost from the cells at the phylogenetic 4. Fisher, enigma. P.R., directional FisherP.R., 24: Williams, K.L Multi- phototaxis by Dictyostelium discoideum amoebae. FEMS Microbiol 5. J. Phycol., Hader, D.-P., Lett., Smith, 29: E., Williams,K.L Anextracel- lular chemical signal controlling phototactic behavior by D. dis- coideum slugs. Cell, 23: Francis, D.W Some studies 7. on phototaxis stelium. J. Cell. Comp. Physiol, 64: Gall, P., Keiner, P., Dornemann, D., of DictyoSenoer, H., Brodhun, B., Hader, D.-P Pterin- flavin-like fluorescence associated with isolated flagella of Euglena graci8. lis. Photochem. Hader, D.-P., Photobiol., Claviez, 51: M., Merkl, R., Gerisch, G Responses of Dictyostelium discoideum amoebae to local stimulation by light. Cell Biol. Int. Rep., 7: Hottiger, H. Bailey, fluorescence nor intracellular NAD(P)H levels in Escherichia Biotechnol., 36: J.E Neither total culture fluorescence are indicative of coli MG Appl. Microbiol Kawai, H A flavin-like auto fluorescent substance in the posterior flagellum of golden brown algae. J. Phycol. 24: ll. Kawai, H Green flagellar auto fluorescence in brown algal swarmers their phototactic responses. Bot. Mag. Tokyo, 115: 12. Klein, R., Thiery, R., Tatischeff, I Dictyopterin, 6-(D-threo-l,2-dihydroxypropyl)-pterin, a new natural isomer of L-biopterin. Isolation from vegetative cells of Dictyostelium discoideum identification. Eur. J. Biochem., 187: Loomis, W.F Phototaxis. InDictyostelium discoideum, A Developmental System. Academic Press, New York. pp. 63- Fig. 5. Fluorescence micrograph of spores smeared on a glass slide. Note that the spores are not fluorescent, but that the interspore space is. Scale bar indicates fim. 162

5 Fluorescence in the Cell Mass of D. mucoroides Malkinson, A.M. Ashworth, J.M Adenosine 3',5 -cyclic monophosphate concentrations phosphodiesterase activities during axenic growth differentiation of cell in the cellular slime mould Dictyostelium discoideum. Biochem. J., 134: Okaichi, K RNAsynthesis during germination of UVirradiated Dictyostelium discoideum spores. /. Radiat. Res., 28: Rossomo, E.F., Steffek, A.J., Mujwid, D.K., Alexer, S Scanning electron microscope observations on cell surface change during aggregation of Dictyostelium discoideum. Exp. Cell Res., 85: Schmidt, W., Gall, P., Senger, H., Furuya, M Microspectrophotometry flavin-like fluorescence in of Euglena gracilis. the paraflagellar body. Pterin- Planta, 182: Soll, D.R., Yarger, J., Mirick, M Stationary phase the cell cycle of Dictyostelium discoideum in liquid nutrient medium. /. Cell Sci., 20: Tatischeff, I. Klein, R Fluorescent products secreted by Dictyostelium discoideum cells which are able to aggregate. FEBS Lett., 138: Tatischeff, I., Klein, R., Tham, G Extracellular lumazine from aggregating Dictyostelium discoideum cells. Influence of ph on its fluorescence. Hoppe-Seyler's Z. Physiol. Chem., 365: Uchiyama, S., Nagai, S., Maruyama, K Localization of fluorescent substances in the cellular slime mold Dictyostelium discoideum cells during growth development. /. Plant Res., 106: Uchiyama, S., Okamoto, K., Takeuchi, I Repression of rrnasynthesis induced by disaggregation in Dictyostelium discoideum. Biochim. Biophys. Ada., 562: Weeks, G Agglutination of growing differentiating cells of Dictyostelium discoideum by concanavalin A. Exp. Cell Res., 76: {Received for publication, February 1 7, 1994 accepted April 12, 1994) 163

Adventures in Multicellularity

Adventures in Multicellularity The social amoeba (a.k.a. slime molds) Dictyostelium discoideum Dictyostelium discoideum the most studied of the social amoebae / cellular slime molds predatory soil amoeba