Randall L. Dimond

Visualization of antigenic proteins on Western blots

A new technique for the detection of antibodies bound to proteins blotted onto nitrocellulose paper was developed. The method is rapid, sensitive, and does not require radioactive probes. Proteins transferred to nitrocellulose paper are first reacted with primary antibody followed by reaction with an alkaline phosphatase conjugated second antibody. The phosphatase activity is then visualized using an agar gel impregnated with the histochemical phosphatase stain 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (J. P. Horwitz, J. Chua, M. Noel, J. T. Donatti, and J. Freisler (1966) J. Med. Chem. 9, 447; Sigma Chemical Co., Technical bulletin No. 710-EP (1978]. Antigen-antibody complexes give rise to sharp, permanent blue stained bands both on the nitrocellulose paper and in the agar overlay gel. This procedure allows detection of bands containing less than 20 ng of protein.

A sequence of dependent stages in the development of Dictyostelium discoideum

Abstract Eleven marker enzymes which accumulate during discrete stages of development in Dictyostelium discoideum were followed in two independent temperature-sensitive mutant strains. Strain TS2 has a temperature-sensitive period during aggregation and remains as a smooth lawn at the nonpermissive temperature (27°C). It develops normally at 22°C. Strain DTS6 has a temperature-sensitive lesion in the post-aggregation stage and fails to form slugs at 27°C. Early enzymes accumulate in these strains at the nonpermissive temperature but late stage-specific enzymes fail to accumulate at 27°C. The pattern of accumulation of specific enzymes in these and other morphological mutants defines a linear dependent pathway of at least eight steps which determines temporal differentiation in this organism. Development in Dictyostelium is also dependent on environmental cues which determine the onset of differentiation and the preparation for culmination.

Regulation and secretion of early developmentally controlled enzymes during axenic growth in Dictyostelium discoideum

The four earliest developmentally controlled enzymes in the cellular slime mold, Dictyostelium discoideum, accumulate during axenic growth in rich media. We have shown that at low cell titers the specific activities of N-acetylglucosaminidase, alpha-mannosidase, leucine aminopeptidase, and alanine transaminase are each at very low or vegetative levels comparable to amoebae which have been grown on bacteria as the food source. During the exponential phase of growth all four enzymes accumulate dramatically reaching cellular specific activities at least as high as during development. The magnitude of this accumulation is influenced by alterations in the growth medium. We suggest that these results, combined with those of prior investigations, indicate that a restricted segment of early development is initiated during axenic growth. This means that growth and early development are not mutually exclusive events in this organism. The secretion of lysosomal enzymes is also affected by the composition of the growth media. In all media, including growth in bacterial suspensions, lysosomal enzymes are secreted in significant quantities. There is a correspondence in the effects of media composition on the secretion of these enzymes and on the regulation of developmentally controlled enzymes during axenic growth. The secretion of lysosomal enzymes that are not developmentally regulated is affected in these media, suggesting that the regulation and secretion of these enzymes are under separate control. It is clear that studies of the regulation of lysosomal enzymes in this organism must take into account the secretion of the enzymes as well as their cellular specific activities to properly reflect levels of gene expression.

Isolation and characterization of mutations affecting UDPG pyrophosphorylase activity in Dictyostelium discoideum

A procedure for screening large numbers of clones for an enzyme activity was used to isolate mutations which affect UDPG pyrophosphorylase activity (EC 2.7.7.9) in the cellular slime mold Dictyostelium discoideum. Five strains were recovered which have little or no UDPG pyrophosphorylase activity. Ten other strains were found which have significant activity in vivo which is rapidly inactivated upon cell lysis. These strains have permitted us to evaluate the role of UDPG pyrophosphorylase during growth and development. The enzyme affects the growth rate of the cells but is not essential for growth. However, during development the lack of enzyme activity leads to cell death and lysis. Strains which lack UDPG pyrophosphorylase accomplish early developmental events but are unable to culminate. However, certain biochemical and cytological differentiations associated with late stages were observed.

Major changes in gene expression occur during at least four stages of development of Dictyostelium discoideum

The spectrum of proteins synthesized at different stages of development of the cellular slime mold Dictyostelium discoideum was analyzed by two-dimensional (2D) gel electrophoresis. Of the approximately 400 proteins detected by this method 189 show changes in their relative rate of synthesis. Most of these changes occur during four distinct stages of development: commencement of development immediately following removal of nutrients (early interphase), early aggregation, late aggregation, and culmination. During commencement the synthesis of 19 proteins begins, the relative rate of synthesis of 21 other proteins increases, and 16 proteins show a rapid decrease in their synthetic rate. During early aggregation the largest change occurs in the spectrum of proteins being synthesized. Specifically, the synthesis of 29 new proteins begins and an increase occurs in the relative synthetic rate of 43 others. During late aggregation, when tight cell-cell contacts form, a reduction takes place in the synthetic rate of most of these induced proteins in addition to the synthesis of 12 new proteins. At least two of these induced proteins are synthesized exclusively in prespore and eventually spore cells. Finally, during culmination, 23 new proteins begin to be synthesized and the synthetic rate of 12 other proteins increases. Five of the 23 newly synthesized proteins appear to be stalk-cell specific. In general, synthesis of spore-cell specific proteins begins just following the formation of tight aggregates while stalk-cell specific proteins are induced during culmination. The relative amounts of mRNAs coding for most of the early developmentally regulated proteins have been estimated by their translation in rabbit reticulocyte lysates and subsequent analysis of protein products by 2D gel electrophoresis. For most of those proteins whose rate of synthesis increases in vivo following starvation there is a parallel increase in the cellular level of the functional mRNAs encoding them. This suggests that the genes coding for these mRNAs may be under transcriptional control. In contrast, the mRNAs coding for most of the proteins whose synthetic rate decreases early in development are under translational control and persist in the cell in an inactive state.

The use of tolerization in the production of monoclonal antibodies against minor antigenic determinants

An initial attempt to prepare monoclonal antibodies specific for the Dictyostelium discoideum lysosomal enzyme beta-glucosidase was unsuccessful. All of the antibodies resulting from this fusion recognized an extremely immunogenic epitope that is present on all of the lysosomal enzymes of Dictyostelium. In two succeeding fusions, changes in the immunization schedule intended to increase the immune response to enzyme-specific epitopes were not entirely successful. Although nine hybridomas producing antibodies specific for beta-glucosidase resulted from these two fusions, most (70%) of the cell lines isolated secrete antibodies that recognize the shared, immunodominant epitope. Moreover, the nine beta-glucosidase-specific antibodies proved to be of limited utility since none recognize the native enzyme. Therefore, we attempted to tolerize a BALB/c mouse to the common epitope by injecting the lysosomal enzyme, N-acetylglucosaminidase, within 40 h after birth. As an adult, this animal was immunized with beta-glucosidase. Fusion of the spleen cells from this mouse with myeloma cells resulted in the isolation of nine hybridoma lines that produce antibodies specific for beta-glucosidase. No antibodies reactive with the common epitope were detected. These results suggest that tolerization may provide a means whereby an undesired class of antibody-producing cell lines can be selectively eliminated from the products of a fusion.

Secretory mutants in the cellular slime mold Dictyostelium discoideum.

Our initial studies have shown that the cellular slime mold Dictyostelium discoideum is a particularly suitable organism for the study of lysosomal enzyme secretion. During appropriate stages in the life cycle, secretion is prominent for a number of lysosomal enzymes. The methods described here have been developed to investigate various aspects of the secretion process. Moreover, our evidence that regulation of the secretory system is influenced by environmental changes and by cell differentiation indicates that this organism may be useful for studying the functional regulation of this organellar system. To a large degree these types of studies have been limited in the past due to the lack of an appropriate experimental system. The ability to isolate secretory mutants affecting the secretion of lysosomal enzymes adds another dimension to investigations using D. discoideum. In our initial attempts we have been successful in isolating a variety of different types of mutants that alter the secretion of one or more lysosomal enzymes. While the results are in agreement with the results of our physiological investigations, they also indicate much more heterogeneity in the lysosomal system than we had previously suspected. The indications that many of our secretory mutants may also affect modification of the enzymes is also intriguing. This observation may also help to explain the fact that many of these strains are defective in normal development. Together with the immunological methods available in this organism for studying posttranslational modification, the mutants may be valuable in deciphering the relationship between modification of lysosomal enzymes and their proper localization and secretion from the cell. Thus, Dictyostelium discoideum may become as useful for the study of some questions of cell biology as it has been for development.

Developmental changes in the modification of lysosomal enzymes in Dictyostelium discoideum.

Evidence has been found for a generalized change in the post-translational modification of lysosomal enzymes during development of Dictyostelium discoideum. The physical and antigenic properties of four developmentally regulated lysosomal enzymes, N-acetylglucosaminidase, beta-glucosidase, alpha-mannosidase, and acid phosphatase, have been examined throughout the life cycle. In vegetative cells, a single major isoelectric species is detected for each enzymatic activity on native nonequilibrium isoelectric focusing gels. Between 6 and 10 hr of development, all activities, including the preformed enzyme, become less negatively charged, resulting in a modest but reproducible shift in the isoelectric focusing pattern. This alteration is not detected by native gel electrophoresis at constant pH. As development continues, the specific activity of beta-glucosidase, alpha-mannosidase, and acid phosphatase continues to increase and coincidentally, new, less acidic isozymic bands of activity can be observed on both gel systems. Some of these new isozymes accumulate preferentially in anterior cells, while others accumulate preferentially in posterior cells of migrating slugs. N-Acetylglucosaminidase does not increase in specific activity late in development and no new isozymic species appear. Using a monoclonal antibody that reacts with sulfated N-linked oligosaccharides shared by vegetative lysosomal enzymes in D. discoideum, the antigenicity of the developmental isozymes has been characterized. All of the enzymatic activity present during vegetative growth and early development is immunoprecipitable. However, the less negatively charged isozymes that accumulate after aggregation are not recognized by the antibody. Nonantigenic acid phosphatase and alpha-mannosidase are found in both anterior and posterior cells from migrating pseudoplasmodia. Since each enzyme is coded by a single structural gene, these results suggest that the isozymes present late in development arise from the synthesis of the same polypeptides with altered post-translational modifications. The appearance of anterior and posterior specific isozymes is likely to be the result of cell type specific changes in the glycoprotein modification pathway for newly synthesized proteins.

Chapter 7 Defining the Intracellular Localization Pathways followed by Lysosomal Enzymes in Dictyostelium discoideum

Publisher Summary This chapter describes the biochemical and genetic methods used to define both the intracellular pathways followed by lysosomal enzymes and the molecular nature of the “sorting” signals found on the proteins. Using biochemical methods, the lysosomal enzymes α-man and β-glu in Dictyostelium are synthesized as precursor polypeptides that are cotranslationally translocated into the lumen of the endoplasmic reticulum (ER) and N-glycosylated. The precursors move at different rates from the RER to the Golgi complex, where they are sulfated and sorted into two classes. One class, containing the majority of the precursor polypeptides, is directed to lysosomes where they are rapidly cleaved to mature forms of the enzymes. The other class of precursors is rapidly secreted from cells. The different intracellular transport rates for the α-man and β-glu precursors support the existence of a transport-mediating receptor. The genetic approach involved the isolation and characterization of mutants altered in the secretion, modification, processing, and/or localization of lysosomal enzymes. Through biochemical analysis of these mutants, one can identify and characterize the molecular components involved in the processing and transport of this group of enzymes.

Regulation of lysosomal α-mannosidase-1 synthesis during development in Dictyostelium discoideum☆

The cellular specific activity of lysosomal α-mannosidase-1 increases dramatically during development in Dictyostelium discoideum. α-Mannosidase-1 is composed of two subunits (Mr = 58,000 and 60,000) which are derived from a common precursor polypeptide (Mr = 140,000). Using enzyme-specific monoclonal antibodies we have determined that throughout development (a) the relative rate of precursor biosynthesis closely parallels the rate of accumulation of cellular enzyme activity and (b) the newly synthesized precursor is efficiently processed to mature enzyme (t12 < 10 min). This indicates that the developmental accumulation of α-mannosidase-1 activity is primarily controlled by de novo enzyme synthesis. Furthermore, the change in the relative rate of enzyme precursor synthesis can be accounted for by an increase in the cellular level of functional α-mannosidase-1 mRNA during development.