Lysosomal enzyme inactivation associated with defects in post-translational modification during development in Dictyostelium discoideum

The developmental accumulation of lysosomal alpha-mannosidase-1 activity in Dictyostelium discoideum is controlled at the level of de novo enzyme precursor biosynthesis. Aggregation-deficient mutants are defective with regard to the accumulation of alpha-mannosidase-1 activity beyond 8-16 h of development. We used enzyme-specific monoclonal antibodies to show that the activity defect in aggregation-deficient strains is not due to a lack of alpha-mannosidase-1-precursor synthesis or processing, or to preferential degradation of the mature enzyme protein. Instead, the defect is a result of enzyme inactivation: cells of aggregation-deficient strains contain significant amounts of inactive alpha-mannosidase-1 protein late in development. The alpha-mannosidase-1 inactivation phenotype is associated with a more general defect in lysosomal enzyme modification. A change in the post-translational modification system occurs during normal slime-mold development, as shown by differences in enzyme isoelectric point, antigenicity, and thermolability. We found that this change in modification does not occur in mutant strains blocked early in development. We propose a model in which pleiotropic mutations in early aggregation-essential genes can indirectly affect the accumulation of alpha-mannosidase-1 activity by preventing the expression of a developmentally controlled change in the post-translational modification system, a change which is required for the stability of several lysosomal enzymes late in development.     

Passage of molecules across the intercellular bridge between post-mitotic daughter cells

Living mitotic HeLa cells were microinjected with sodium fluorescein, fluorescein isothiocyanate (FITC)-labelled IgG, and Lucifer Yellow-CH to determine if the intracellular bridge and midbody forms a barrier to the migration of these molecules. All three fluorescent molecules were found to pass across the intercellular bridge. The post-mitotic cells maintained a molecular patency across the intercellular bridge that persisted for considerable lengths of time, extending into the next cell cycle.     

Cell division cycle in mammalian cells : VIII. Mapping of G1 into six segments using temperature-sensitive cell cycle mutants

The G1 blocks in three temperature-sensitive (ts) Syrian hamster cell-cycle mutants have been mapped in relation to other G1 landmarks. Two mutants reported here, ts-559 and ts-694, show defective progression only in G1. When shifted from the permissive temperature of 33 degrees C to the non-permissive temperature of 39 degrees C, G1 cells of these two mutants show no further cell cycle progression, while cells in S, G2 and mitosis progress through the cell cycle but become blocked after entering G1. The two mutants complement each other, and also complement the previously reported mutant ts-550C with blocks in both G1 and G2 of the cell cycle. The locations of the G1 blocks in both ts-559 and ts-694 are before the hydroxyurea arrest point. The G1 ts point in ts-694 is prior to the isoleucine deprivation and serum starvation points, while the G1 block in ts-559 is after the serum starvation point but before the isoleucine block. Other G1 block points which have been reported are in mutants of different species and isolated in different laboratories, causing difficulties for relative positioning of the blocks in G1. The mutants for mapping in this study have been isolated from the same cell line. The G1 ts arrest points of ts-559 and ts-694, and that found in ts-550C, together with nutritional deprivations and metabolic inhibitors, provide seven reference points which divide G1 into six segments, each of which is bracketed by two adjacent points: mitosis, ts-694 block, serum starvation arrest point, ts-559 block, isoleucine deprivation arrest point, ts-550C block, hydroxyurea or excess-thymidine arrest segment.     

Stage-specific cohesion of cell ghosts and plasma-membrane fragments from Dictyostelium discoideum

During fruiting-body construction by Dictyostelium discoideum, the formation and subsequent maintenance of the multicellular assembly involve two stage-specific cohesive systems that are acquired sequentially and are distinguishable on serological and genetic grounds. We demonstrated that both systems, termed aggregation related (AR) and postaggregation related (PAR), can function in vitro. Ghosts prepared from cells of the wild-type and of a cohesion-defective mutant that were harvested during growth and at aggregation and postaggregative stages of fruiting-body construction exhibited the same cohesive properties as the cells from which they were derived. Membrane fragments prepared from the ghosts by mechanical disruption retained these cohesive properties.     

Early release of the density-dependent inhibition of phosphate uptake and ATP synthesis after src gene expression in chick embryo fibroblasts

Our results showed that the expression of the src gene in chick embryo fibroblasts (CEF) released the density-dependent inhibition (DDI) of phosphate metabolism (phosphate uptake and phosphorylation of small organic compounds). With increasing cell density, phosphate metabolism decreased by 58% in normal CEF and, in contrast, increased by 20% in Rous sarcoma virus (RSV)-transformed CEF. The same change in the DDI was observed in CEF infected by NY68 (a ts mutant for transformation of RSV) and maintained at the permissive temperature (37 degrees C) instead of the restrictive temperature (41.5 degrees C) for the expression of transformation. An interesting feature was that the release of the DDI of phosphate metabolism was an early event in the process of transformation, since it was almost concomitant with the stimulation of the pp60 src kinase activity following the shift from 41.5 to 37 degrees C of NY68 CEF. The phosphorylation of small organic compounds (Po) was more strongly increased by the change in temperature than was 32Pi accumulation. Furthermore, the percentage increases of Po and adenosine triphosphate (ATP) labelling with 32P were similar, suggesting that the expression of src gene enhanced ATP synthesis. In glucose-free medium, the stimulation of Po-labelling was still observed but was decreased. Therefore the activation of glycolytic activity is not an absolute requirement, but is necessary for the maximum effect of transformation on the release of DDI of phosphate metabolism. Oligomycin added in complete medium did not prevent the increase in Po-labelling. From these results, we assumed that ATP turnover was stimulated as a consequence of enhanced ATP degradation. We verified that the stimulation of Po phosphorylation was not a consequence of increased ATP utilization for RNA or protein synthesis. The stimulation of Po labelling was specifically abolished by quercetin. This drug inhibited the transformed cells more strongly than the non-transformed cells.     

Cellular proliferation and hypusine synthesis

Hypusine (N(-)-(4-amino-2-hydroxybutyl) lysine), a spermidine-dependent post-translational protein modification, is synthesized by various mammalian cells in culture. Experiments described in this paper demonstrated a relationship between rates of cellular growth and the synthesis of hypusine. Cells that divide at fast rates have a high rate of hypusine synthesis. In kinetic experiments, a positive relationship is evident between the rates of protein, DNA and hypusine synthesis. Cells seeded at high density, growing non-exponentially, synthesized less hypusine than logarithmically growing cells seeded at low density. Slowing the growth rate of cells by modification of the external milieu also results in a decreased rate of hypusine synthesis. These results provide additional evidence of the association of hypusine with cell proliferation in cultured cell lines and suggest a possible role for this unusual post-translational modification in the complex macromolecular events leading to cellular growth.     

Selective inhibition of calcium-stimulated cation-induced pinocytosis by starvation and inhibitors of protein synthesis in Amoeba proteus

The capacity of Amoeba proteus to form pinocytotic channels after pretreatment with either puromycin, cycloheximide, emetine or a long period of starvation was studied. The effect on pinocytosis of the three inhibitors of protein synthesis was similar. They preferentially affected pinocytosis induced by Na+ with little effect on K+-induced pinocytosis. In Ca2+-deficient media, Na+-induced pinocytosis was inhibited, while the addition of Ca2+ restored channel formation. The degree of inhibition of Na+-induced pinocytosis was influenced by the concentration of Ca2+ in the inducing solution. Selective Ca2+-reversible inhibition of Na+-induced pinocytosis also occurred after starvation or treatment with a proteolytic enzyme, subtilisin. The membrane potential in starved or emetine-treated cells in culture medium was normal and their depolarising response to inducers was not diminished in solutions containing Na+. The resting input resistance of these cells was higher than in normal amoebae, but no significant difference in electrical parameters was observed after pinocytosis was induced. It is suggested that starvation, inhibition of protein synthesis, and enzyme digestion deplete the membrane of structures which are necessary for normal Ca2+ functions during induction of pinocytosis by Na+-like inducers.     

Cytokeratin patterns of human oral epithelia: Differences in cytokeratin synthesis in gingival epithelium and the adjacent alveolar mucosa

Human oral mucosa includes various epithelia that are commonly classified as lining, masticatory, and specialized epithelia. Although adjacent tissues, the gingiva and alveolar mucosa represent two different types of epithelia: the gingiva is cornified and exhibits high rate ridges, whereas the mucosa does not normally cornify and exhibits a relatively smooth-contoured borderline between the epithelium and the underlying connective tissue. We examined the cytokeratin patterns of both epithelia using one- and two-dimensional gel electrophoresis. The gingiva expresses a great complexity of cytokeratins, including significant amounts of components nos. 1, 2, 5, 6, 10, 11, 13, 14, 16, and 17, as well as traces of cytokeratins nos. 4 and 15, i.e., a pattern similar to those of vaginal mucosa and epidermis containing proliferative keratinocytes. In contrast, the alveolar mucosa contains only two major cytokeratins, i.e., nos. 4 and 13, together with two minor amounts of cytokeratins nos. 5, 6, 14, and 17, thus resembling the patterns of certain other stratified, noncornified epithelia, such as the esophagus. Immunofluorescence microscopy using monoclonal antibodies to cytokeratins nos. 4 and 13 revealed the presence of these proteins in the suprabasal layers of alveolar mucosa, whereas in the gingiva, only certain small, suprabasal clusters of cells appeared to contain these cytokeratins. The cytoskeletal differences between gingival and alveolar mucosa are discussed in relation to the differences in their morphology and function, and with respect to pathological processes characteristic of these epithelia.     

Antibodies to the most tightly bound proteins in eukaryotic DNA : Formation of immuno-complexes with ‘nuclear matrix’ components

Chromosomal DNA is associated with polypeptides covalently bound to internal DNA ends. Since these polypeptides can only be released from chromosomal DNA by enzymes or other agents hydrolysing phosphodiester bonds they were termed 'the most tightly bound' (MTB) polypeptides in DNA. Antibodies developed against the MTB polypeptides are shown to form immunocomplexes with major 'nuclear matrix' polypeptides as well as with polypeptides which are still associated with 'nuclear matrix' DNA isolated by means of SDS/proteinase K and phenol. Immuno-complex formation is revealed by immunoblotting and by indirect immunofluorescence. Thus, since MTB polypeptides, major 'nuclear matrix' polypeptides and 'nuclear matrix' DNA-associated polypeptides share common antigenic sites they can be considered to be identical or at least closely related. This suggests that a fraction of distinct 'nuclear matrix' polypeptides is either transiently or permanently linked to DNA by covalent bonds. Consistently, isolated eukaryotic 'bulk' DNA is inevitably associated with residual 'nuclear matrix' polypeptides.     

Isolation of a G0-specific ts mutant from a Fischer rat cell line, 3Y1

A ts mutant clone, tsJT60, was isolated from Fisher rat cell line, 3Y1. During the exponential growth at both 34 and 39.5 degrees C, tsJT60 did not appear as ts mutant cells. However, once entered resting state (G0) under serum deprivation at the confluent state, they could re-enter S phase at 34 degrees C but could not at 39.5 degrees C following the stimulation of cells either by the addition of fetal bovine serum or by trypsinization and replating. These and other results suggested that tsJT60 is a G0-specific ts mutant, i.e., the cells have ts defect(s) in the function which is required for the stimulation from the resting state to S phase but not for the progression of the cell cycle in an exponential growth phase.     

Developmental changes in messenger RNAs and protein synthesis in Dictyostelium discoideum

The pattern of proteins synthesized at different stages of differentiation of the slime mold Dictyostelium discoideum was studied by two-dimensional polyacrylamide gel electrophoresis. Of the approximately 400 proteins detected during growth and/or development, synthesis of most continued throughout differentiation. Approximately 100 proteins show changes in their relative rates of synthesis. During the transition from growth to interphase, the major change observed is reduction in the relative rate of synthesis of about 8 proteins. Few further changes are noticeable until the stage of late cell aggregation, when production of about 40 new proteins begins and synthesis of about 10 is reduced considerably. Thereafter, there are few changes in the pattern of protein synthesis. Major changes in the relative rates of synthesis of a number of proteins are found during culmination, but few culmination-specific proteins are observed. In an attempt to understand the molecular basis for these changes, mRNA was isolated from different stages of differentiation and translated in an improved wheat germ cell-free system; the products were resolved on two-dimensional gels. The ratio of total translatable mRNA to total cellular RNA is constant throughout growth and differentiation. Messenger RNAs for many, but not all, developmentally regulated proteins can be identified by translation in cell-free systems. Actin is the major protein synthesized by vegetative cells and by early differentiating cells. The threefold increase in the relative rate of synthesis of actin during the first 2 hr of differentiation and the decrease which occurs thereafter can be accounted for by parallel changes in the amount of translatable actin mRNA. Most of the changes in the pattern of protein synthesis which occur during the late aggregation and culmination stages can also be accounted for by parallel increases or decreases in the amounts of translatable mRNAs encoding these proteins. It is concluded that mRNAs do not appear in a translatable form before synthesis of the homologous protein begins, and that regulation of protein synthesis during development is primarily at the levels of production or destruction of mRNA.     

Biosynthesis of EGF receptor, transferrin receptor and colligin by cultured human keratinocytes and the effect of retinoic acid

The biosynthesis of EGF and transferrin receptor by human keratinocytes in culture has been followed using specific monoclonal antibodies. In addition, keratinocytes are shown to synthesise a Mr 47 000 protein that binds to gelatin-Sepharose. Peptide mapping confirms the identity of this protein with colligin, a newly described cell surface-associated glycoprotein that also binds to native collagens (Kurkinen et al., J biol chem 259 (1984) 5915) [9]. Vitamin A and its analogues have profound effects on the differentiation, morphology and motility of human keratinocytes in culture. We show here that retinoic acid (RA) has no effect on the growth rate of the cells or the synthesis of EGF receptor and colligin, but stimulates the synthesis of transferrin receptor.     

Flow cytometry isolation and improved visualization of sorted mouse chromosomes. Purification of chromosomes X and ISO-1 from cell lines with Robertsonian translocations

While analysis and sorting of human chromosomes by flow cytometry has been widely used, isolation of a pure mouse chromosome remains very difficult, since most murine chromosomes are quite similar in size. To overcome this problem, we have analysed mouse cell lines having either Robertsonian translocations or isochromosomes. The resulting metacentric chromosomes are very different in size and in morphology from normal mouse acrocentric chromosomes. These characteristics have been analysed by computer-monitored flow cytometry, facilitated by improvements in the chromosome extraction procedure. Signals characteristic of the iso-lq chromosome in cell line PCC4 azaR1, and of the normal X chromosome in the mouse strain 22CD have thus been obtained. These chromosomes have been sorted and can be easily recognized by fluorescence microscopy when collected onto serum-albumin-coated microscope slides. The technical modifications made, coupled with the existence of a great diversity of metacentric chromosomes resulting from Robertsonian translocations, should allow the purification of a number of different mouse chromosomes.     

The cellular oncogenes c-myc, c-myb and c-erb are transcribed in defined types of avian hematopoietic cells

The possible role of normal chicken cellular sequences c-erb, c-myb and c-myc, together referred to as c-onc genes and related to the oncogenes of defective avian acute leukemia retroviruses (DLVs), was investigated by determining the accumulation of c-onc RNA in different avian cells an cell lines. Levels of c-myc and in some instances c-myb RNA are elevated in immature hematopoietic cells or cell lines from various lineages but more mature hematopoietic cells, as well as non-hematopoietic cells, contain only low levels. In contrast, the level of c-erb RNA is generally low, but high in a small number of normal bone marrow cells. The results indicate that the cellular homologues of the viral oncogenes are differentially expressed during hematopoiesis. They also indicate that the hypothesis that DLV target cells express their homologous c-onc genes might hold for c-erb, but is not valid in its simple form for c-myc and c-myb.     

Control in previous and present generations of preparation for entry into S phase and the relationship to resting state in 3Y1 rat fibroblastic cells

In both the presence and absence of serum, 3Y1 rat fibroblastic cells synchronized at early S phase by aphidicolin entered M phase 6 h after removal of aphidicolin. However, in the second generation their entry into S phase in the presence of serum was delayed due to the deprivation of serum in the first generation. A similar delaying effect in the second generation was observed when the resting cells were stimulated by serum and then deprived of serum during a period of 8 h preceding mitosis. In both cases, the interval between mitosis and entry into S phase in the second generation was almost equal to that required for the resting cells to enter S phase when stimulated by serum. A similar delaying effect was also observed when the cells, synchronized at early S phase, were kept in suspension culture in the presence of serum for a period in the first generation. Results of a similar type of experiments using various combinations of growth factors showed that, when the G1 period in the second generation was shortened by exposure to growth factors in the first generation, and when the resting cells were stimulated to enter S phase, the same combination of growth factors was required. These and previous results suggest that the preparation for entry into S phase is controlled in both previous and present generations of 3Y1 cells.     

Expression of the neural cell adhesion molecules L1 and N-CAM and their common carbohydrate epitope L2/HNK-1 during development and after transection of the mouse sciatic nerve

The expression of the neural cell adhesion molecules L1 and N-CAM and of their shared carbohydrate epitope L2/HNK-1 was studied during the development and after the transection of mouse sciatic nerves. During development, L1 and N-CAM were detectable on most, if not all, Schwann cells at embryonic day 17, the earliest stage tested. With increasing age, the immunoreactivity was reduced being confined to non-myelinating Schwann cells by post-natal day 10, at which stage the staining pattern resembled that seen in adult sciatic nerves. Double-immunolabelling experiments revealed a complete overlap between L1 and N-CAM antibodies. The L2/HNK-1 epitope was not detectable in developing sciatic nerves until the end of the 2nd post-natal week, when it appeared to be associated with the outer profiles of thick myelin sheets, as also seen in adult sciatic nerves. Three days after the transection of adult sciatic nerves, L1 antigen and N-CAM was detectable in more Schwann cells in the distal nerve end than in untreated control nerves. The peak level of the reappearance of L1 antigen and N-CAM in Schwann cells occurred between 2 and 4 weeks after transection. The reduction of L1-antigen expression to its normal adult level took more than a year, thus recapitulating normal development, but on a more protracted time scale. Similarly, the L2/HNK-1 epitope remained undetectable until the transected nerve had returned to its normal state of myelination, i.e. approximately 1 year after transection.     

Isolation and characterization of intraspecific cybrids : Effect of mitochondrial DNA on their cellular properties

Cybrid clones were obtained by fusing whole cells of rat glioma C6BU-1, resistant to 5-bromodeoxyuridine (BrdU), with cytoplasts of embryonic rat 3Y1CAP cells, resistant to chloramphenicol (CAP), in selective medium with BrdU and CAP. The clones resistant to BrdU and CAP were confirmed to be cybrids by chromosome and mtDNA analyses. More than half the mtDNA of all the cybrid clones was from the 3Y1CAP cells. After cultivation of a cybrid clone Y22 for 3 months in the absence of CAP, subclones were isolated. One subclone Y22-22 contained predominantly mitochondrial DNA (mtDNA) from the 3Y1CAP cells. Using this subclone, the effects of the mitochondrial genome on cellular properties were examined. The growth patterns, expression of glioma-specific beta-adrenergic receptor, and composition of the major proteins of C6BU-1 cells were not affected by transmitted mtDNA from the 3Y1CAP cells. This procedure for isolating cells containing predominantly foreign mtDNA will be useful in studies on the interaction between genomes of the mitochondria and nucleus.     

Identification and analysis of Dictyostelium actin genes,a family of moderately repeated genes

Plasmid M6 has been shown to contain sequences complementary to two related abundant mRNA species which differ in length by 100 nucleotides and code for Dictyostellum actin. M6 complementary RNA was isolated by hybridization to immobilized M6 DNA and translated in vitro. The product is identical to major forms of in vivo labeled actin in both mobility on two-dimensional gels and two-dimensional fingerprints of tryptic peptides. Both plasmid M6 and a second plasmid complementary to the actin mRNA complementary region in M6, pDd actin 2 (McKeown et al., 1978), direct the synthesis in minicells of a number of similar polypeptides that are not seen in minicells containing other recombinant plasmids. Three of these polypeptides are similar in two-dimensional gel mobility to Dictyostelium actin and bind to DNAase I agarose.The repetition frequency of isolated restriction fragments from actin mRNA complementary plasmid M6 has been examined. The data from two different experimental approaches (DNA excess hybridizations using plasmid DNA as probe, and hybridization of plasmid probe to DNA blot filters of restriction enzyme-digested Dictyostelium DNA) indicate that the mRNA complementary region is reiterated 15–20 times. When an actin cDNA probe is used in the same experiments, the results suggest that the entire coding region is reiterated.When the two major actin mRNA species are separated and independently translated, each appears to code for one of the two major actin species. The results suggest that there are at least two different functional genes, and possibly more, for Dictyostelium actin.     

Growth and differentiation of fetal rat small intestinal epithelium in tissue culture: Relationship to fetal age

Explants of small intestinal tissue have been cultured from fetal and young rats (from 13-day fetuses to 3-week-old rats). Growth of morphologically typical epithelial cells was obtained from explants of tissue from 14–20 day fetuses. Optimal growth was obtained using tissue from 17-day fetuses with outgrowth from the explant being observed 1-day after explant. Eighty per cent of explants developed epithelial growth by 11 days in culture. Initially, the epithelial outgrowth showed no morphological evidence of differentiation but after 5–10 days in culture differentiation into goblet or elongated cells with alkaline phosphatase activity occurred. Cells with brush borders and goblet cells were identified using electron microscopy. No differentiation occurred if the explant was removed even though growth continued.It was very difficult to culture tissue from fetuses older than 20 days' gestation, and when small intestine of 18–20-day fetuses was divided into two parts (proximal and distal) and cultured separately, growth of epithelial cells from explants of the proximal segment was less successful than that of the distal segment, indicating that the growth ability of these epithelial cells in vitro was closely related to tissue maturation in vivo. In contrast to the apparent relationship between fetal age and successful growth of intestinal epithelial cells, squamous epithelial cells of the esophagus could be grown from explants of 14-day fetus through newborn and 3-week-old rats.