Macrocyst Development in Dictyostelium Discoideum. I

J. Cell Set. 55, 341-352 (1982) Printed in Great Britain © Company of Biologists Limited 1982 MACROCYST DEVELOPMENT IN DICTYOSTELIUM DISCOIDEUM. I. INDUCTION OF SYNCHRONOUS DEVELOPMENT BY GIANT CELLS AND BIOCHEMICAL ANALYSIS Y. SAGA AND K. YANAGISAWA Institute of Biological Sciences, University of Tsukuba, Ibaraki 305, Japan SUMMARY In Dictyostelium discoideum, cytological and physiological studies on macrocyst formation revealed that this process consists of at least two steps: the production of giant cells, which are believed to be formed from the fusion of cells of two opposite mating types, and the subsequent induction of macrocyst development by the giant cells. The conditions that had been considered formerly to be required for macrocyst formation, such as darkness and the presence of two cells of complementary mating types in heterothallic strains, were actually required only for the production of the giant cells. Once giant cells are produced, the surrounding cells can aggregate and form macrocysts even in the light. Furthermore, it was demonstrated that giant cells can switch the developmental mode of the surrounding cells to macrocyst formation. That is, if a critical number of the isolated giant cells are introduced into a cell population of a single strain of NC4, which normally would produce only fruiting-bodies, macrocysts are formed instead. When in the presence of giant cells, the development of macrocysts may be initiated by starvation. Therefore, if all cells are made to starve simultaneously development begins and proceeds synchronously. Using this technique of synchronous development, the developmental kinetics of enzyme activities were assayed during macrocyst and fruiting-body formation. Considerable differences in the patterns of those enzyme activities were demonstrated between the two developmental modes of D. discoideum. INTRODUCTION The cellular slime mould Dictyostelium discoideum has two alternative modes of development. One culminates in fruiting-body formation; the other culminates in macrocyst formation and is considered to be a sexual cycle (Clark, Francis & Eisen- berg, 1973; Erdos, Raper & Vogen, 1973; Maclnnes & Francis, 1974). In fruiting-body formation development is initiated by starvation. Accordingly, development can be synchronized easily by subjecting all the cells to starvation simultaneously (White & Sussman, 1961). In contrast, a method of synchronous development has not yet been developed in the case of macrocyst formation. This has made biochemical studies on macrocyst formation difficult. The production of macrocysts in Dictyostelium requires particular conditions, such as the presence of cells of two complementary mating types in heterothallic strains, darkness, humidity, and the appropriate temperature (Blaskovics & Raper, 1957; Clark et al. 1973; Erdos et al. 1973; Nickerson & Raper, 1973; Maclnnes & Francis, 1974; Erdos, Raper & Vogen, 1976). These complicated requirements have made it 342 Y. Saga and K. Yanagisawa difficult to detect precisely a factor(s) that is responsible for the initiation of macrocyst development. Erdos et al. (1976) found that darkness was required only for a certain period in the early stages of macrocyst development. When they shifted a culture with cells of two mating types, initially incubated in the dark, to the light they discovered that macrocyst formation could still take place. Recently, apart from this, O'Day (1979) found that in macrocyst formation cells slightly larger than the surrounding amoebae, called giant cells, appeared in the culture prior to cell aggregation, and that those cells served as centres for aggregation in macrocyst development. We isolated giant cells from the dark-grown mixed cultures of cells of two opposite mating types, and studied their characteristics. We found out why darkness was required only at the beginning of macrocyst development. In this paper we present the results of experiments that demonstrate the function of giant cells in macrocyst development, a method of initiating synchronous development of macrocysts, and the results of a comparative biochemical analysis of macrocyst and fruiting-body formation. MATERIALS AND METHODS Organisms and culture conditions Two strains of D. discoideum, NC4 and HMi, were used. HMi was derived from Vi2, which is a strain of the opposite mating-type to NC4. HMi was kindly provided by Dr R. R. Kay. The strains were maintained on Klebsiella aerogenes on nutrient agar (Sussman, 1966). Formation and isolation of giant cells Giant cells were obtained by the following method. Growth-phase cells of NC4 and HMi, cultured separately on nutrient agar plates, were harvested and suspended together at a ratio of 1:1 in Bonner's salt solution (Bonner, 1947) at a total concentration of 5 x 10' cells/ml. The bacteria were then added to the cell suspension at a final concentration of 1 x io10 cells/ml. The mixture was agitated on a reciprocal shaker (120 strokes/min), in flasks 5-6 times the volume of the mixture, in the dark at 22 °C. After 20-22 h cultivation, cells were harvested, washed three times with Bonner's salt solution by centrifugation and resuspended in a 5 mM-EDTA, 17 mM-phosphate buffer (pH 6-5). Giant cells were isolated by straining the suspension through a nylon mesh (10 fun pore size). Fixation and staining procedure Washed cells were fixed with 60 % methanol (4 °C), placed on slides, and air-dried. The slides were then treated with trypsin (o-i % tryps in in 0-85% NaCl solution) for 30-90 s, washed with water, and stained with 10% Giemsa stain in phosphate buffer (pH 6-8). Synchronous development of macrocysts and fruiting-bodies Development of macrocysts was synchronized by the following method. NC4 cells, which were harvested from nutrient agar plates, were washed and suspended, together with the isolated giant cells, at a ratio of 1000:2 in Bonner's salt solution at a concentration of 5 x io7 cells/ml. The mixture was spread on a non-nutrient agar plate (5x10* cells/cm1) and then incubated at 22 °C in either the dark or the light. Synchronous development of fruiting- bodies was achieved by incubating washed NC4 cells on a non-nutrient agar under exactly the same conditions, except that no giant cells were added. Macrocyst development in D. discoideum 343 Enzyme assays Cells were taken at various times during development, washed and stored as pellets at — 20 °C. Frozen pellets were thawed and suspended in the following solutions: (I) cold water for assays of TV-acetylglucosaminidase, a-D-mannosidase, /9-D-glucosidase and alkaline phosphatase; (II) 20 mM-potassium phosphate buffer, 1 mM-MgSO4 (pH 7-4) for assay of cellular phosphodiesterase; (III) o-i M-tricine buffer (pH 7-4) for assay of UDP-glucose pyrophosphorylase. Cell suspensions containing 1 x io7 cells were sonicated and used for the enzyme assays. iV-acetylglucosaminidase, a-D-mannosidase and alkaline phosphatase were measured by the method of Loomis (1969a, b, 1970). /?-D-glucosidase was measured by the method of Coston & Loomis (1969), phosphodiesterase as described by Malchow, NSgele, Schwarz & Gtrisch (1972), and UDP-glucose pyrophosphorylase by the method of Hames (1976). Proteins were determined by the method of Lowry, Rosebrough, Fair & Randall (1951) using bovine serum albumin as a standard. Specific activities of the enzymes were expressed as nanomoles per min per mg protein. All of the enzyme assays were performed at least three times. RESULTS Formation of giant cells and their role in macrocyst development Cells of two strains of complementary mating type, NC4 and HM1, were suspended together with bacteria in Bonner's salt solution. The mixture was then shaken in a flask in the dark. Under these conditions numerous macrocysts were formed after cultivation for a few days. We examined cell growth in the mixed dark-grown culture, and found that the number of cells increased rather rapidly during the early period of cultivation, but then very slowly after 10 h of cultivation (*10) as shown in Fig. 1. In contrast, when cells were cultured in the light, the number of cells increased con- tinuously until the stationary phase was reached (approx. 2 x io7 cells/ml) at t^-t^. No macrocysts were produced in this case. In the dark-grown culture, we found that very large cells began to appear at about tw and the number of these cells increased with time until it reached a maximum of about 13-14 % of the total cells. These large cells were fixed, stained with Giemsa, and observed. We found that they were all multinucleated and some of them had more than 50-60 nuclei, as shown in Fig. 2D. The reduction of the rate of increase in the number of cells observed after 10 h of cultivation in the dark-grown culture (Fig. 1) is conceivably due to the formation of these multinucleated large cells. These large cells seem to be similar to the giant cells described previously by O'Day (1979). According to O'Day, these giant cells, which were binucleated zygotes and slightly larger than the surrounding cells, appeared in the early stage of macrocyst development and served as the centres for cell aggregation, which subsequently form macrocysts. Our large giant cells are multinucleated and differ from O'Day's giant cells in their morphology. It is certain, however, that our giant cells also play an important role in macrocyst formation, as they never appear in either mixed cultures grown in the light (Fig. 2 A, B) or cultures of single strains grown in the dark; that is, under the conditions in which macrocysts fail to be produced, these giant cells are not found. Furthermore, we found that once the giant cells were produced in the dark-grown mixed culture, macrocysts were formed even if the culture was shifted from dark to light conditions or the bacteria were removed. In order to study in more detail the role of giant cells in macrocyst formation, the 344 Y.

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