Phosphorylation of spore coat proteins during development of Dictyostelium discoideum.
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ABSTRACT:
SUBMITTER: Delaney SJ
PROVIDER: S-EPMC1153146 | biostudies-other | 1983 Jun
REPOSITORIES: biostudies-other
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Project description:The pattern of synthesis of the spore coat proteins during development of Dictyostelium discoideum has been determined by using immunoprecipitation with spore protein antibody. SP170, SP103, 'SP94', SP82, SP76 and SP55 are all first synthesized just prior to the 'Mexican hat' stage of development (16-18h), but the synthesis of SP72 is delayed. This protein is apparently synthesized as a precursor, P66, which is modified during spore maturation to yield SP72. The nature of the modification is unknown. At their peak period of synthesis during early culmination (18-20h), the spore coat proteins account for 5-9% of total protein synthesis. Shortly after synthesis, these proteins are inserted into the spore coat, where all except SP103 become disulphide-cross-linked during the period 24-30h. SP3 does not accumulate until disulphide-cross-linking of the major spore coat proteins occurs and is itself disulphide-cross-linked into the spore coat. Several additional proteins that are accumulated during development have also been identified, namely P31, P25, P21 and P18. P25 first appears at 18-20h and then continues to be made throughout development. P31 synthesis begins at 12-14h and then largely ceases after approx. 20 h of development. The genes for both P21 and P18 are first expressed early in development, starting at 9-12h. P21 synthesis ceases at approx. 14h, but P18 continues to be synthesized throughout the rest of development. The marked differences in the time period of accumulation of these proteins compared with the co-ordinated syntheses of SP170, SP103, 'SP94', SP82, SP76 and SP55 provide a useful system for analysis of the mechanism of temporal gene expression during development.
| S-EPMC1152547 | biostudies-other
Project description:Like the cyst walls of other protists, the spore coat of Dictyostelium discoideum is formed de novo to protect the enclosed dormant cell from stress. Spore coat assembly is initiated by exocytosis of protein and polysaccharide precursors at the cell surface, followed by the infusion of nascent cellulose fibrils, resulting in an asymmetrical trilaminar sandwich with cellulose filling the middle layer. A molecular complex consisting of cellulose and two proteins, SP85 and SP65, is associated with the inner and middle layers and is required for proper organization of distinct proteins in the outer layer. Here we show that, unlike SP85 and other protein precursors, which are stored in prespore vesicles, SP65 is, like cellulose, synthesized just in time. By tagging the SP65 locus with green fluorescent protein, we find that SP65 is delivered to the cell surface via largely distinct vesicles, suggesting that separate delivery of components of the cellulose-SP85-SP65 complex regulates its formation at the cell surface. In support of previous in vivo studies, recombinant SP65 and SP85 are shown to interact directly. In addition, truncation of SP65 causes a defect of the outer layer permeability barrier as seen previously for SP85 mutants. These observations suggest that assembly of the cellulose-SP85-SP65 triad at the cell surface is biosynthetically regulated both temporally and spatially and that the complex contributes an essential function to outer layer architecture and function.
| S-EPMC1951108 | biostudies-literature
Project description:We cloned and sequenced the genes for two of the major proteins found in spore coats of Dictyostelium discoideum. The predicted translation product of each of these genes starts with a hydrophobic signal sequence that is subsequently cleaved. Expression of these spore coat genes is coordinate in prespore cells.
| S-EPMC363674 | biostudies-other
Project description:Vegetative and developed amoebae of Dictyostelium discoideum gain traction and move rapidly on a wide range of substrata without forming focal adhesions. We used two independent assays to quantify cell-substrate adhesion in mutants and in wild-type cells as a function of development. Using a microfluidic device that generates a range of hydrodynamic shear stress, we found that substratum adhesion decreases at least 10 fold during the first 6 hr of development of wild type cells. This result was confirmed using a single-cell assay in which cells were attached to the cantilever of an atomic force probe and allowed to adhere to untreated glass surfaces before being retracted. Both of these assays showed that the decrease in substratum adhesion was dependent on the cAMP receptor CAR1 which triggers development. Vegetative cells missing talin as the result of a mutation in talA exhibited slightly reduced adhesive properties compared to vegetative wild-type cells. In sharp contrast to wild-type cells, however, these talA mutant cells did not show further reduction of adhesion during development such that after 5 hr of development they were significantly more adhesive than developed wild type cells. In addition, both assays showed that substrate adhesion was reduced in 0 hr cells when the actin cytoskeleton was disrupted by latrunculin. Consistent with previous observations, substrate adhesion was also reduced in 0 hr cells lacking the membrane proteins SadA or SibA as the result of mutations in sadA or sibA. However, there was no difference in the adhesion properties between wild type AX3 cells and these mutant cells after 6 hr of development, suggesting that neither SibA nor SadA play an essential role in substratum adhesion during aggregation. Our results provide a quantitative framework for further studies of cell substratum adhesion in Dictyostelium.
| S-EPMC4172474 | biostudies-literature
Project description:The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis; however, the mechanism by which CotH affects germination is unclear. Here, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology.
| S-EPMC4922149 | biostudies-literature
Project description:In Dictyostelium, sporulation occurs synchronously as prespore cells approach the apex of the aerial stalk during culmination. Each prespore cell becomes surrounded by its own coat comprised of a core of crystalline cellulose and a branched heteropolysaccharide sandwiched between heterogeneous cysteine-rich glycoproteins. The function of the heteropolysaccharide, which consists of galactose and N-acetylgalactosamine, is unknown. Two glycosyltransferase-like genes encoding multifunctional proteins, each with predicted features of a heteropolysaccharide synthase, were identified in the Dictyostelium discoideum genome. pgtB and pgtC transcripts were modestly upregulated during early development, and pgtB was further intensely upregulated at the time of heteropolysaccharide accumulation. Disruption of either gene reduced synthase-like activity and blocked heteropolysaccharide formation, based on loss of cytological labeling with a lectin and absence of component sugars after acid hydrolysis. Cell mixing experiments showed that heteropolysaccharide expression is spore cell autonomous, suggesting a physical association with other coat molecules during assembly. Mutant coats expressed reduced levels of crystalline cellulose based on chemical analysis after acid degradation, and cellulose was heterogeneously affected based on flow cytometry and electron microscopy. Mutant coats also contained elevated levels of selected coat proteins but not others and were sensitive to shear. Mutant spores were unusually susceptible to hypertonic collapse and damage by detergent or hypertonic stress. Thus, the heteropolysaccharide is essential for spore integrity, which can be explained by a role in the formation of crystalline cellulose and regulation of the protein content of the coat.
| S-EPMC2620749 | biostudies-literature
Project description:ATP-binding cassette (ABC) transporters can translocate a broad spectrum of molecules across the cell membrane including physiological cargo and toxins. ABC transporters are known for the role they play in resistance towards anticancer agents in chemotherapy of cancer patients. There are 68 ABC transporters annotated in the genome of the social amoeba Dictyostelium discoideum. We have characterized more than half of these ABC transporters through a systematic study of mutations in their genes. We have analyzed morphological and transcriptional phenotypes for these mutants during growth and development and found that most of the mutants exhibited rather subtle phenotypes. A few of the genes may share physiological functions, as reflected in their transcriptional phenotypes. Since most of the abc-transporter mutants showed subtle morphological phenotypes, we utilized these transcriptional phenotypes to identify genes that are important for development by looking for transcripts whose abundance was unperturbed in most of the mutants. We found a set of 668 genes that includes many validated D. discoideum developmental genes. We have also found that abcG6 and abcG18 may have potential roles in intercellular signaling during terminal differentiation of spores and stalks.
| S-EPMC3743828 | biostudies-literature
Project description:BackgroundMany microbial phenotypes are the product of cooperative interactions among cells, but their putative fitness benefits are often not well understood. In the cellular slime mold Dictyostelium discoideum, unicellular amoebae aggregate when starved and form multicellular fruiting bodies in which stress-resistant spores are held aloft by dead stalk cells. Fruiting bodies are thought to be adaptations for dispersing spores to new feeding sites, but this has not been directly tested. Here we experimentally test whether fruiting bodies increase the rate at which spores are acquired by passing invertebrates.ResultsDrosophila melanogaster accumulate spores on their surfaces more quickly when exposed to intact fruiting bodies than when exposed to fruiting bodies physically disrupted to dislodge spore masses from stalks. Flies also ingest and excrete spores that still express a red fluorescent protein marker.ConclusionsMulticellular fruiting bodies created by D. discoideum increase the likelihood that invertebrates acquire spores that can then be transported to new feeding sites. These results thus support the long-hypothesized dispersal benefits of altruism in a model system for microbial cooperation.
| S-EPMC4038703 | biostudies-literature
Project description:Cytokinins (CKs) are a family of evolutionarily conserved growth regulating hormones. While CKs are well-characterized in plant systems, these N6-substituted adenine derivatives are found in a variety of organisms beyond plants, including bacteria, fungi, mammals, and the social amoeba, Dictyostelium discoideum. Within Dictyostelium, CKs have only been studied in the late developmental stages of the life cycle, where they promote spore encapsulation and dormancy. In this study, we used ultra high-performance liquid chromatography-positive electrospray ionization-high resolution tandem mass spectrometry (UHPLC-(ESI+)-HRMS/MS) to profile CKs during the Dictyostelium life cycle: growth, aggregation, mound, slug, fruiting body, and germination. Comprehensive profiling revealed that Dictyostelium produces 6 CK forms (cis-Zeatin (cZ), discadenine (DA), N6-isopentenyladenine (iP), N6-isopentenyladenine-9-riboside (iPR), N6-isopentenyladenine-9-riboside-5' phosphate (iPRP), and 2-methylthio-N6-isopentenyladenine (2MeSiP)) in varying abundance across the sampled life cycle stages, thus laying the foundation for the CK biosynthesis pathway to be defined in this organism. Interestingly, iP-type CKs were the most dominant CK analytes detected during growth and aggregation. Exogenous treatment of AX3 cells with various CK types revealed that iP was the only CK to promote the proliferation of cells in culture. In support of previous studies, metabolomics data revealed that DA is one of the most significantly upregulated small molecules during Dictyostelium development, and our data indicates that total CK levels are highest during germination. While much remains to be explored in Dictyostelium, this research offers new insight into the nature of CK biosynthesis, secretion, and function during Dictyostelium growth, development, and spore germination.
| S-EPMC6920973 | biostudies-literature
Project description:Dictyostelium discoideum is a social amoeba, which on starvation develops from a single-cell state to a multicellular fruiting body. This developmental process is accompanied by massive changes in gene expression, which also affect non-coding RNAs. Here, we investigate how tRNAs as key regulators of the translation process are affected by this transition. To this end, we used LOTTE-seq to sequence the tRNA pool of D. discoideum at different developmental time points and analyzed both tRNA composition and tRNA modification patterns. We developed a workflow for the specific detection of modifications from reverse transcriptase signatures in chemically untreated RNA-seq data at single-nucleotide resolution. It avoids the comparison of treated and untreated RNA-seq data using reverse transcription arrest patterns at nucleotides in the neighborhood of a putative modification site as internal control. We find that nucleotide modification sites in D. discoideum tRNAs largely conform to the modification patterns observed throughout the eukaroytes. However, there are also previously undescribed modification sites. We observe substantial dynamic changes of both expression levels and modification patterns of certain tRNA types during fruiting body development. Beyond the specific application to D. discoideum our results demonstrate that the developmental variability of tRNA expression and modification can be traced efficiently with LOTTE-seq.
| S-EPMC8163159 | biostudies-literature