Maurice Sussman

Metabolism of major cell components during slime mold morphogenesis.

Abstract Morphogenetically synchronized Dictyostelium discoideum amoeba were sampled at intervals during development to determine the fates of major cell constituents. Dry weight, total protein and fractions thereof, RNA, free and bound hexoses were followed. None of these except the last appeared to reflect the specific morphogenetic events. Two polysaccharide fractions were encountered which did reflect these events and their syntheses were repressed or disturbed in morphogenetically deficient mutants.

Synchrony of enzyme accumulation in a population of differentiating slime mold cells

Abstract 1. 1. Studies of the cellular slime molds have previously revealed the accumulation of a number of enzymes at different stages during the development of multicellular fruiting structures. 2. 2. A study of the spatial distribution of one of the enenzymes (UDPGlc pyrophosphorylase) during the period of its accumulation revealed a uniform rise in specific activity throughout the developing fruiting body. This suggests that all cells accumulate the enzyme simultaneously rather than as a wave of accumulation starting at a particular region. 3. 3. The distribution of the enzyme UDPGal: polysaccharide transferase served as a control for the techniques employed. The mucopolysaccharide product is known to be restricted to spores, and the enzyme was found to be accumulated only in regions containing incipient spore cells.

Polysaccharides involved in slime-mold development II. Water-soluble acid mucopolysaccharide(s)

Abstract An acid mucopolysaccharide has been isolated from Dictyostelium discoideum and related species. It is trichloroacetic acid and water soluble, ethanol precipitable, and non-dialyzable. In purified preparations approx. 90% of the dry weight is accounted for as galactose, galactosamine and galacturonic acid. The polysaccharide is antigenically reactive. Negligible levels are detected until the terminal stages of fruiting body construction at which time it accumulates to the extent of 1–2% of the dry weight. It fails to appear in morphogenetically deficient mutants and its synthesis seems to be keyed to the overall morphogenetic rate. A novel method for the measurement of a specific polysaccharide antigen in crude cell sonicates is described.

A mutation affecting both rate and pattern of morphogenesis in Dictyostelium discoideum

Abstract At high population densities, mutant Fr-17 of D. discoideum forms only irregular flattened aggregates rather than the mature fruiting structures of the wild type. Yet many of the end products of the normal morphogenesis are present in the mutant. These include: spores, ensheathed clusters of stalk cells, a variety of polysaccharides, antigens known to be associated with the spores and stalks of the wild type, and pigment. In addition the temporal succession of these morphogenetic events is greatly accelerated.

The effect of cyclic AMP on morphogenesis and enzyme accumulation in Dictyostelium discoideum

Abstract When 3′,5′-cyclic AMP is applied externally to migrating slugs and to cell aggregates during particular stages of morphogenesis, it disturbs the normal polarity and dominance relationships among the cells in a specific manner. It also interferes with the developmental programs of at least two enzymes that are needed for fruit construction.

Regulation of enzyme synthesis by slime mold cell assemblies embarked upon alternative developmental programs

Abstract Depending on specified environmental conditions, the developing multi-cellular aggregate of Dictyostelium discoideum can either immediately begin to construct a fruiting body in situ or can transform into a migrating slug and move to a more suitable location. At any time thereafter the latter can be induced to stop migrating and construct a fruiting body. The patterns of accumulation and disappearance of two enzymes have been studied in cell assemblies engaged in either of the developmental programs described above. The enzymes are: uridine disphosphate glucose pyrophosphorylase (EC 2.7.7.9) and uridine diphosphate galactose epimerase (EC 5.1.3.2). In cell assemblies engaged in fruit construction both enzymes accumulate rapidly, the pyrophosphorylase several hours in advance of the epimerase. They reach peaks of specific activity at a given morphogenetic stage, and then disappear, the former partially, the latter completely. In cell assemblies engaged in slug migration the pyrophosphorylase accumulates very slowly to the same activity peak as in assemblies that had fruited directly but does not disappear. The epimerase does not accumulate at all. When migrating slugs are induced to stop moving and start fruiting while this slow round of pyrophosphorylase accumulation is under way, they complete it but in the rapid fashion characteristic of cells which had fruited directly without ever becoming slugs and this is followed by the characteristic sudden disappearance of part of the activity. However, if the slugs are allowed to migrate long enough to have already completed the slow round of pyrophosphorylase accumulation and then are induced to stop moving and start fruiting, they initiate a second rapid round of pyrophosphorylase synthesis while accomplishing the usual first round of epimerase accumulation and disappearance. The fundamental control of these events appears to be at the level of genetic transcription.

Regulation of functionally related enzymes during alternative developmental programs

Abstract Cell aggregates of Dictyostelium discoideum can either construct fruiting bodies directly at the site of aggregation or transform into migrating slugs and move away. The latter can be induced at any time by appropriate environmental signals to stop migrating and construct fruits. In this and a previous publication ( P. C. Newell and M. Sussman , J. Mol. Biol. , 49 (1970)627), the patterns of accumulation and disapperance of three enzymes have been examined during these alternative developmental programs. The enzymes are: UDP-glucose pyrophosphorylase (EC 2.7.7.9), UDP-galactose 4-epimerase (EC 5.1.3.2), and UDP-galactose : polysaccharide galactosyl-transferase. The results indicate that: 1. 1. The performances of all three enzymes differ drastically but consistently in these two developmental modes in respect to times at which the activities begin to accumulate, the periods of accumulation, and the periods of disappearance. There is no evidence that the three are coordinately controlled with respect to any of the above. 2. 2. When slugs are induced to stop migrating and to start fruiting they accomplish a complete second round of pyrophosphorylase and of transferase accumulation and a complete first round or epimerase accumulation (having synthesized none as slugs). 3. 3. The activities may accumulate rapidly, slowly, or not at all but where the developmental program does call for a round of accumulation, a peak level on specific activity characteristic of each enzyme is always attained. 4. 4. The basic control of these events appears to be at the level of genetic transcription.

Accumulation of uridine diphosphoglucose pyrophosphorylase in Dictyostelium discoideum via preferential synthesis

Abstract At the end of the exponential growth phase, the enzyme UDP-glueose pyrophosphorylase is present in the vegetative cells of Dictyostelium discoideum NC4 (haploid) at a low level (about 0.05% of total protein). During the initial stages of fruiting body construction, while the cells are entering into multicellular aggregates, the enzyme level remains constant, but increases dramatically thereafter reaching a peak (about 0.5% of total protein) at the end of fruiting body construction, and then partially decreasing. Previous studies have shown that both the accumulation and disappearance are keyed to the flow of morphogenetic events. In this study, cells were labeled with amino acids for different periods throughout the sequence. The enzyme was quantitatively immune-precipitated from crude cell extracts, the precipitate was washed and redissolved, and the enzyme protein separated by acrylamide gel electrophoresis in order to estimate the differential incorporation ratio, i.e. disints min in enzyme protein per 10 8 cells disints min in total protein per 10 8 cells × 100 for each labeling period. During the initial stages, when the enzyme level remained relatively constant, this ratio was about 0.03 to 0.04%. As the enzyme began to accumulate it rose progressively, attaining levels of 0.6 to 0.8% toward the end of fruiting body construction before declining. The data are not consistent with the theory of Gustafson and Wright (1973) that differential turnover controls the level of this enzyme during the development of D. discoideum. They are consistent with the conclusion that directed changes in the differential rate of synthesis of UDP-glucose pyrophosphorylase is the controlling element. The estimates of enzyme content are based on a value for the specific enzyme activity of 100,000 units/mg enzyme, which had been determined previously using samples of the enzyme purified to apparent physical homogeneity. This figure has been confirmed in the present study by quantitative immuneprecipitation of the enzyme from crude extracts of homogeneously labeled cells. The method can be generally used to determine if a specific biological activity estimate obtained with a purified protein is consistent with its activity when measured before or during purification.

Polysaccharides involved in slime-mold development I. Water-soluble glucose polymer(s)

Abstract A water-soluble glucose polysaccharide fraction, synthesized and degraded during cellular slime-mold morphogenesis has been isolated and characterized. The kinetics of its appearance and disappearance are correlated with the overall rate of morphogenesis. Mutant strains incapable of accomplishing all or parts of the normal morphogenetic sequence exhibit altered patterns of synthesis and degradation.

The regulatory program for UDPgalactose polysaccharide transferase activity during slime mold cytodifferentiation: Requirement for specific synthesis of ribonucleic acid

Abstract During specific stages of cytodifferentiation in Dictyostelium discoideum , the enzyme UDPgalactose polysaccharide transferase appears, accumulates, is preferentially released into the extracellular space and then disappears. The data presented here demonstrate that both the accumulation and disappearance are inhibited by exposure of the cells to actinomycin D. The actinomycin-sensitive period for enzyme accumulation is of relatively short duration (about one-third of the total time needed for morphogenesis), begins several hours after the start of morphogenesis, and 4–5 h in advance of the appearance of the enzyme itself. During this period, a simple linear relation exists between the time at which actinomycin was added and the amount of enzyme activity which ultimately accumulated. Comparison between the wild type and a temporally deranged mutant showed that both the time of initiation and the duration of the actinomycin-sensitive period (as well as of the accumulation and disappearance of the enzyme) are controlled by the overall regulatory program.