This thesis is about the role of the surf ace cell membrane of Dictyostelium discoideum during the aggregation of these cells. The physical basis of this engagement lies in the presence of the relevant antigen receptor(s) (antibodies) on the plasma membrane of the cell. The presence of receptors on the cell surface has been visualized in numerous experiments using radioactively labeled or fluorescently labeled antigens.
The changes are apparently due to changes in the internal concentrations of various cyclic nucleotides caused by lectin binding. The nature of the intercellular contacts formed during aggregation is not well understood. The experiments described in Chapter I show this. the only requirement for obtaining this state appears to be a series of biochemical events triggered by the absence of an available food source.
Perhaps the most thoroughly researched aspect of slime mold development is the study of the so-called "developmental".
INHIBITION OF THE DEVELOPMENT OF DICTYOSTELIUM DISCOIDEUN BY
The cellular slime mold Dictyostelium discoideum provides a unique, well-adapted system for the study of cellular interactions in morphogenesis, and the role of the cell membrane in these interactions. The membranes appear to work by preventing the cells from becoming competent to aggregate, rather than by blocking the actual formation of the intercellular bonds. As a result of these experiments, and to avoid the problem of bacterial contamination of the test plates, membranes from the aggregation phase were routinely autoclaved before being mixed with cells for plating.
These results imply that once cells have progressed normally through the first 8-9 hours of development, they become impervious to the action of the membranes. Specifically, if such cells are harvested and plated eight hours after the depletion of the bacterial food source, they can begin to aggregate. Membranes in concentrations up to 1.5 mg/plate are unable to prevent subsequent aggregation and development of the cells, although aggregation is delayed by 1-12 hours depending on the membrane concentration.
Most of the theories proposed to explain the data visualized the cell-cell. The development of the cellular slime form involves a well-defined transition from a unicellular to a multicellular mode of existence. The specific gravity of the membranes isolated on sucrose gradients in these experiments is characteristic of m€:ll.brane material (Steck and Wallach, 1970).
Finally, chemical and enzymatic treatments demonstrate the biological activity of the membranes used in. The ability of this membrane preparation to inhibit aggregation in developing cells is not the result of a simple physical blockade of the cell surface factors. Difficulties in separating cells from membranes after plating prevent a direct evaluation of the developmental capacity of the treated cells.
Removal of the food source appears to be the only necessary environmental condition for the acquisition of aggregation competence. However, experiments described in the next article show that the nebrane-cell interaction causes changes in the expression of minor aspects of the cell's developmental program.
EFFECTS OF DIFFERENTIATED MEMBRANES ON THE DEVELOPMENTAL
In the preceding article (Tuchman et al., 1973) we described the preparation and properties of a partially purified plasma membrane fraction isolated from developing cells of the cellular slime mold Dictyostelium discoidetun. However, such membranes were found to be unable to inhibit the reaggregation of mechanically disturbed cells that had previously been allowed to proceed normally through the first 12 h of development. Gerisch, 1968) and not to block the physical events involved in the formation of the actual cell-cell contacts.
It has been shown in many different biological systems that biochemical interactions on the outside of the cell surface can affect, and in some cases directly, events inside the cells (Anderson and Huebner, 1968; Lilien, 1969; Sutherland et al. al., 1965; Kohn and Fuchs, 1971). In this article, we describe experiments designed to shed light on the effect of membrane treatment on some of the biochemical events that make up what is known about the slime mold developmental program. Partially purified membranes isolated from aggregated cells of this species have been shown to affect the induction of some of these enzymes in ways that may provide some clues to the mechanism of the cell-membrane interaction and the nature of the slime mold developmental program.
Cells were plated with and without membranes and allowed to develop for various times, after which they were harvested from the filter by washing with 3 ml PDF (per liter: 0.111 g CaC12, 4.6 g NaH. Sar::ples harvested after 22 h , were sonicated for two 30 second periods to allow for the increased strength of the spore coating.If any activity was found, it remained constant throughout the plating period and was subtracted from the final activity of the cells plus membranes.
0 Gradients were separated by bottom pumping and enzyme activity was immediately assessed in aliquots. The level of initial activity (O hours) and the amount of growth depends on the concentration at which the cells are taken from the growth medium and on the composition of the medium. Coated membranes alone were also analyzed and activity was subtracted from total cells and membranes.
ACETYLGLUCOSAMINIDASE
Cells were plated with and without added membranes and harvested at intervals and assayed as described in Methods. Tyrosine transaminase normally undergoes a twofold increase in activity, reaching a maximum after eighteen hours of development. Similarly, there are no changes in UDPG pyrophosphorylase activity in membrane-treated cells.
The very large increase (about fifteenfold) and subsequent decrease observed in untreated cells are absent and activity remains constant at the 0-hour level. The situation in membrane-treated cells in the case of B-glucosidase, shown in Figure 5, is similar to that of tyrosine transaminase and N-acetylglucosaminidase. The normal activity profile represents the composite activities of two isozymes, of which only the second is considered an integral feature of the developmental program (Coston and Loomis, 1969).
Instead, the first isozyme gradually decreases during the first 15 hours of development, after which the measurable activity remains constant. Another developmentally controlled enzyme whose activity consists of two isozymes is shown in Figure 6. Cells were seeded with and without added membranes and harvested and assayed at intervals.
Cells were seeded with and without added membranes and harvested and assayed at intervals as described in Methods.
UDPG PYROPHOSPHORYLASE
PREFERENTIAL SYNTHESIS OF ACTIN DURING EARLY DEVELOPMENT
Due to its relative simplicity, biochemical sophistication and ease of handling in the laboratory, Dictyostelium makes an attractive candidate for an intensive study of the molecular mechanisms that control development in eukaryotes. In this article we describe the patterns of protein synthesis during development and the purification and identification of the main component as slime mold lactin. The labeled cells were harvested in 0.01M Tris HCl pH 6.5, centrifuged, resuspended in 1 ml of the same buffer, and frozen in liquid nitrogen.
0 After centrifugation, the upper 1 ml of the tui'e was removed and tested for the presence of depolymerized actin. This means that a very large number of proteins are produced, which is greater than the power of the dissolving gel. Arrowheads on the left side of the gel indicate bands that are new at that site or markedly increase in intensity at the indicated time.
A curved background line was drawn as shown in the figure and the extent of the main band (band B; RF 0.55) was marked. Due to the molecular weight of the strongly labeled band, 47,000, we speculated that it might be slime mold actin. About 90% of the material:·. on the gel is located in a band running at RF 0. 55. 1. The molecular weight of this band was calculated from the logarithmic plot of the standards as shown in Figure 5 and estimated to be 47,000.
In the absence of detergent, 0.1 percent of the count in the membrane pellet was released after 5 hours of cold stirring. Thirty minutes after detergent addition, 10 percent of the total was released. Aliquots of released material were mixed with purified slime mold actin and processed as described in the legend to Figure 5.
Gels of samples labeled at this later period may not accurately reflect major events that occur in only one of the two cell types present at this stage of development. Since the background is much lower in this region of the gel, these bands may actually be present in lower nolar amounts than those species in the molecular weight region of the gel. The most remarkable feature of Dictyostelium protein synthesis as revealed on these gels is the finding that a single type of protein, band B, increases from 4-6 percent of total protein synthesis in vegetative cells to 22 percent in the first two hours of development. nent.
This protein has been identified as slime mold actin by comparing its properties with purified actin. To do this, slime mold lactin was first purified by a modification of a previously developed procedure for isolation. The purified material was further identified as actin by its characteristic behavior upon addition of KCl to a solution of the protein subunits.
The properties of the purified actin were then shown to be equal to those of the highly labeled material. It therefore appears that the protein synthesized in very large quantities during the first hours of development is slime mold. These workers firmly established the identity of the filaments as actin by a variety of criteria (Pollard and Korn, 1973).
Highly purified plasma membrane preparations show associated actin filaments localized on the inner (cytoplasmic) surface of the membranes, which form characteristic arrowhead structures after interaction with heavy meromyosin. Thus, the bulk of the actin appears to be loosely bound to the membrane, while a small fraction appears to be tightly bound. It is not clear why the slime mold cells should require such a greatly increased oxidation of actin at the beginning of development.
