David A. Knecht

Cell motility and chemotaxis in Dictyostelium amebae lacking myosin heavy chain

Dictyostelium amebae have been engineered by homologous recombination of a truncated copy of the myosin heavy chain gene (heavy meromyosin (HMM) cells) and by transformation with a vector encoding an antisense RNA to myosin heavy chain mRNA (mhcA cells) so that they lack native myosin heavy chain protein. In the former case, cells synthesize only the heavy meromyosin portion of the protein and in the latter case they synthesize negligible amounts of the protein. Surprisingly, it was demonstrated that both cell lines are viable and motile. In order to compare the motility of these cells with normal cells, the newly developed computer-assisted Dynamic Morphology System (DMS) was employed. The results demonstrate that the average HMM or mhcA ameba moves at a rate of translocation less than half that of normal cells. It is rounder and less polar than a normal cell, and exhibits a rate of cytoplasmic expansion and contraction roughly half that of normal cells. In a spatial gradient of cAMP, the average ameba of HMM or mhcA exhibits a chemotactic index of +0.10 or less, compared to the chemotactic index of +0.50 exhibited by normal cells. Finally, the initial area, rate of expansion, and final area of pseudopods are roughly half that of normal cells. The five fastest HMM amebae (out of 35 analyzed in detail) moved at an average rate of translocation equal to that of normal amebae, and exhibited an average chemotactic index of +0.34. In addition, the average rate of cytoplasmic flow in fast HMM cells was equal to that of the average normal ameba. However, fast HMM amebae still exhibited the same defects in pseudopod formation that were exhibited by the entire HMM cell population. These results suggest that myosin heavy chain is involved in the fine tuning and efficiency of pseudopod formation, but is not essential for the basic behavior of pseudopod expansion.

Developmental regulation of Dictyostelium discoideum actin gene fusions carried on low-copy and high-copy transformation vectors.

The Dictyostelium discoideum genome contains an estimated 17 to 20 actin genes. We report the identification of a new member of this multigene family, actin 15, and its complete nucleotide sequence and transcription initiation sites. We constructed transformation vectors carrying either the actin 15 promoter fused to the neomycin phosphotransferase gene from transposon Tn903 or the actin 6 promoter fused to the neomycin phosphotransferase gene from Tn5. Cells transformed with the actin 15 vector carried less than five copies of vector DNA, while cells transformed with the actin 6 vector carried more than 200 copies. In both cases, the vector appeared to be integrated into the chromosome as a tandem array. Gene fusion RNAs transcribed from the actin 15 and actin 6 vectors were regulated like endogenous actin genes during D. discoideum development. DNA sequences required for temporal and cell type-specific regulation of these genes were contained within 2.8 kilobases of 5 noncoding DNA for actin 15 and 0.7 kilobases of 5 noncoding DNA for actin 6.

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.

Surface glycoprotein, gp24, involved in early adhesion of Dictyostelium discoideum

A membrane glycoprotein of 24,000 Da (gp24) was purified from developed cells of Dictyostelium discoideum and shown to neutralize a crude antiserum (R695) that blocks EDTA-sensitive cell-cell adhesion during the early developmental stages of this organism. Purified gp24 was used to raise rabbit polyclonal antibodies and mouse monoclonal antibodies. Rabbit antiserum R851 was shown to be highly specific to gp24 by both Western analysis and immunoprecipitation. IgG of R851 is able to block adhesion of dissociated cells swirled in suspension. Adhesion of wild-type cells is blocked by R851 antibodies during the first 8 hr of development but not thereafter when other adhesion mechanisms come into play. The glycoprotein gp80 plays an essential role in the second adhesion system that appears during the aggregation stage of D. discoideum. By adding both anti-gp24 and anti-gp80 antibodies, adhesion of aggregation stage cells could be blocked. Late in development a third adhesion mechanism appears that is not blocked by either antibodies to gp24 or gp80 or both antibodies together. Western analysis and immunoprecipitation with monoclonal antibody mLJ11, specific for gp24, indicated that gp24 is absent in cells growing exponentially on bacteria but is rapidly synthesized and accumulated following the initiation of development. Synthesis of gp24 is maximal during the first 4 hr of development and then continues at a reduced rate throughout the remainder of development. The coordinate appearance of gp24 and EDTA-sensitive cell-cell adhesion as well as the ability of this glycoprotein to neutralize the adhesion blocking activity of R695 and R851 antibodies indicates that it plays a role in early cell-cell adhesion.

Developmental consequences of the lack of myosin heavy chain in Dictyostelium discoideum

Two different Dictyostelium discoideum cell lines that lack myosin heavy chain protein (MHC A) have been previously described. One cell line (mhcA) was created by antisense RNA inactivation of the endogenous mRNA and the other (HMM) by insertional mutagenesis of the endogenous myosin gene. The two cell lines show similar developmental defects; they are delayed in aggregation and become arrested at the mound stage. However, when cells that lack myosin heavy chain are mixed with wild-type cells, some of the mutant cells are capable of completing development to form mature spores. The pattern of expression of a number of developmentally regulated genes has been examined in both mutant cell lines. Although morphogenesis becomes aberrant before aggregation is completed, all of the markers that we have examined are expressed normally. These include genes expressed prior to aggregation as well as prespore genes expressed later in development. It appears that the signals necessary for cell-type differentiation are generated in the aborted structures formed by cells lacking MHC A. The mhcA cells have negligible amounts of MHC A protein while the HMM cells express normal amounts of a fragment of the myosin heavy chain protein similar to heavy meromyosin (HMM). The expression of myosin light chain was examined in these two cell lines. HMM cells accumulate normal amounts of the 18,000-D light chain, while the amount of light chain in mhcA cells is dramatically reduced. It is likely that the light chains assemble normally with the HMM fragment in HMM cells, while in cells lacking myosin heavy chain (mhcA) the light chains are unstable.

DNA sequences required for expression of a Dictyostelium actin gene.

A 2.8‐kb fragment of 5′ non‐coding DNA from the Dictyostelium actin 15 gene has previously been shown to contain all of the cis‐acting DNA sequence elements required for normal developmentally‐regulated transcription of actin gene fusion RNAs when reintroduced into the genome by DNA‐mediated transformation. Deletion analysis of this promoter fragment indicates that all of the necessary information is contained within a 270‐bp fragment of actin 15 5′ non‐coding DNA. This fragment contains four short G/C‐rich repeated sequences that are also found in other co‐regulated Dictyostelium actin genes. A 12‐bp consensus sequence, AAAAATGGGG/ATT, is present in the regions essential for expression of two different Dictyostelium actin genes, actin 6 and actin 15, but is absent from an actin gene showing a different temporal pattern of developmental regulation. Deletion analysis and DNase I footprinting implicate this sequence as a functional cis‐acting element required for transcription of the actin 15 gene.

Signal transduction, chemotaxis, and cell aggregation in Dictyostelium discoideum cells without myosin heavy chain☆

Dictyostelium discoideum cells have been generated that lack myosin heavy chain (MHC) due to antisense RNA inactivation of the endogenous mRNA or to insertional mutagenesis of the myosin gene. These cells retain chemotactic movement in gradients of the chemoattractant cAMP. Furthermore, cAMP does induce many biochemical and physiological responses in aggregative cells, including binding of cAMP to surface receptors, modification, and down-regulation of the receptor; activation of adenylate and guanylate cyclase, secretion of cAMP; and the association of actin to the Triton-insoluble cytoskeleton. Cells lacking MHC were found to have a requirement for bivalent cations in the medium for optimal chemotaxis and cell aggregation.

Precursors of alpha and beta globin messenger RNAs.

Abstract Several structural properties of the pulse-labeled globin messenger RNA precursor molecules that sediment faster than steady-state (10 S) globin mRNA (Curtis & Weissmann, 1976; Ross, 1976; Kwan et al., 1977; Bastos & Aviv, 1977) are investigated. The radioactive, globin-specific RNA resolves into two peaks during electrophoresis in polyacrylamide gels prepared in 98% formamide. The molecular weights of the larger and the smaller RNAs are 600,000 and 280,000, respectively, as compared with the average molecule weight of cytoplasmic globin mRNA, 200,000. When RNA from cells labeled for 10 minutes is analyzed by formamide gel electrophoresis, a precursor larger than 600,000 Mr is not detected. Most, if not all, of the molecules in the 600,000 and 280,000 Mr RNA fractions are polyadenylated. The 600,000 Mr RNA contains β globin mRNA sequences, but little, if any, a globin sequences; the 280,000 Mr RNA contains both α and β sequences. At steady-state the 14-day-old mouse fetal liver erythroblast contains approximately 50 molecules of the 600,000 Mr RNA, 1000 molecules of the 280,000 Mr RNAs, and 60,000 molecules of mature globin mRNA. The half-lives of the 600,000 and 280,000 Mr RNAs calculated using steady-state levels are 2 minutes and 17 minutes, respectively. These data demonstrate that β globin gene sequences are transcribed into a molecule (β-precursor) that is threefold larger than mature β globin mRNA. In contrast, the largest transcript of the α globin genes detected in these experiments is only 1.4-fold larger than mature α globin mRNA. The possibility that the 280,000 Mr, β-specific RNA is a processing intermediate generated by cleavage of the 600,000 Mr β-precursor is discussed, along with alternative hypotheses.

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.

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.