Ethionine and Methionine Metabolism by the Chrysomonad Flagellate Ochromonas danica

SYNOPSIS. Growth of Ochromonas danica is competitively inhibited by ethionine. Inhibition can be reversed by methionine. Inhibition indexes of the effect of ethionine on growth and methionine incorporation into proteins are 1 and 4, respectively. Inside the cell, methionine is partially de-methylated and metabolized to form cysteine. Ethionine is partially de-ethylated, and the homocysteine moiety is either re-methylated to form methionine or further metabolized to form cysteine. Ethionine is also incorporated into proteins of O. danica. The kind of metabolic interference, expressed by inhibition of growth, and correlated with incorporation of ethionine, is yet unknown.     

Hypertonic conditions trigger transient plasmolysis,growth arrest and blockage of transporter endocytosis in Aspergillus nidulans and Saccharomyces cerevisiae

Abstract

By using Aspergillus nidulans strains expressing functional GFP-tagged transporters under hypertonic conditions, we noticed the rapid appearance of cortical, relatively static, fluorescent patches (0.5–2.3 μm). These patches do not correspond to transporter microdomains as they co-localize with other plasma membrane-associated molecules, such as the pleckstrin homology (PH) domain and the SsoA t-Snare, or the lipophilic markers FM4-64 and filipin. In addition, they do not show characteristics of lipid rafts, MCCs or other membrane microdomains. Deconvoluted microscopic images showed that fluorescent patches correspond to plasma membrane invaginations. Transporters remain fully active during this phenomenon of localized plasmolysis. Plasmolysis was however associated with reduced growth rate and a dramatic blockage in transporter and FM4-64 endocytosis. These phenomena are transient and rapidly reversible upon wash-out of hypertonic media. Based on the observation that block in endocytosis by hypertonic treatment altered dramatically the cellular localization of tropomyosin (GFP-TpmA), although it did not affect the cortical appearance of upstream (SlaB-GFP) or downstream (AbpA-mRFP) endocytic components, we conclude that hypertonicity modifies actin dynamics and thus acts indirectly on endocytosis. This was further supported by the effect of latrunculin B, an actin depolymerization agent, on endocytosis. We show that the phenomena observed in A. nidulans also occur in Saccharomyces cerevisiae, suggesting that they constitute basic homeostatic responses of ascomycetes to hypertonic shock. Finally, our work shows that hypertonic treatments can be used as physiological tools to study the endocytic down-regulation of transporters in A. nidulans, as non-conditional genetic blocks affecting endocytic internalization are lethal or severely debilitating.     

Identification of the Mitochondrial Genome in the Chrysophyte Alga Ochromonas danica

ABSTRACT. Analysis of total DNA isolated from the Chrysophyte alga Ochromonas danica revealed, in addition to nuclear DNA, two genomes present as numerous copies per cell. The larger genome (?120 kilobase pairs or kbp) is the plastid DNA, which is identified by its hybridization to plasmids containing sequences for the photosynthesis genes rbcL, psbA, and psbC. The smaller genome (40 kbp) is the mitochondrial genome as identified by its hybridization with plasmids containing gene sequences of plant cytochrome oxidase subunits I and II. Both the 120- and 40-kbp genomes contain genes for the small and large subunits of rDNA. The mitochondrial genome is linear with terminal inverted repeats of about 1.6 kbp. Two other morphologically similar species were examined, Ochromonas minuta and Poteriochromonas malhamensis. All three species have linear mitochondrial DNA of 40 kbp. Comparisons of endonuclease restriction-fragment patterns of the mitochondrial and chloroplast DNAs as well as those of their nuclear rDNA repeats failed to reveal any fragment shared by any two of the species. Likewise, no common fragment size was detected by hybridization with plasmids containing heterologous DNA or with total mitochondrial DNA of O. danica; these observations support the taxonomic assignment of these three organisms to different species. The Ochromonas mitochondrial genomes are the first identified in the chlorophyll a/c group of algae. Combining these results with electron microscopic observations of putative mitochondrial genomes reported for other chromophytes and published molecular studies of other algal groups suggests that all classes of eukaryote algae may have mitochondrial genomes < 100 kbp in size, more like other protistans than land plants.     

Complete large subunit ribosomal RNA sequences from the heterokont algae ochromonas danica, nannochloropsis salina, and tribonema aequale, and phylogenetic analysis

The large subunit ribosomal RNA sequences from the heterokont algae Ochromonas danica, Nannochloropsis salina, and Tribonema aequale were determined. These sequences were combined with small subunit ribosomal RNA sequences in order to carry out a phylogenetic analysis based on neighbor-joining, maximum parsimony, and maximum likelihood methods. Our results indicate that heterokont fungi and heterokont algae each are monophyletic, and confirm that they together form a monophyletic group called ``stramenopiles.' Within the heterokont algae, the eustigmatophyte Nannochloropsis salina either clusters with the chrysophyte Ochromonas danica or forms a sister group to a cluster comprising the phaeophyte Scytosiphon lomentaria and the xanthophyte Tribonema aequale. The alveolates were identified as the closest relatives of the stramenopiles, but the exact order of divergence between the eukaryotic crown taxa could not be established with confidence. Received: 22 November 1996 / Accepted: 14 February 1997     

Internalization of surface HLA-DR molecules by human epidermal Langerhans cells: analysis by flow cytometry and confocal microscopy

Langerhans cells (LC) play a pivotal role in antigen processing and presentation to T cells during delayed-type hypersensitivity reaction in the skin. Antigen presentation involves the interaction between the class II molecules of MHC (HLA-DR) expressed by LC and T receptor of CD4⁺ T lymphocytes. It is now recognized that class II molecules are internalized into LC and can be associated with processed immunogenic peptides. This process involves receptor-mediated endocytosis. The aim of this study was to investigate the time-course of endocytosis of HLA-DR by freshly isolated human LC. Epidermal cells, obtained from normal skin samples, were labeled by indirect immunofluoresence using anti-HLA-DR monoclonal antibodies (MAb). The cell suspension was incubated at 37°C for different periods (15, 30, 45, 60 and 90 min) and then analyzed by flow cytometry and confocal microscopy. Flow cytometry analysis showed decreased HLA-DR molecule expression by LC after incubation at 37°C. Confocal microscopic analysis showed different strain patterns depending on the incubation time: (1)T=0, continuous peripheral staining; (2)T=15 min, patchy peripheral staining; (3)T=30 min, patches or intracellular vesicular staining; (4)T=45 min, intracellular vesicular staining; (5)T=60 min, diffuse intracellular staining; (6)T=90 min, aggregated staining. In our study model, flow cytometry provides quantitative information for the HLA-DR endocytosis, whereas confocal microscopy provides qualitative results concerning the intracellular distribution of internalized HLA-DR molecules. The use of the two complementary techniques allows us to characterize the spontaneous endocytosis of HLA-DR molecule by freshly isolated LC. Thisin vitro study model might be useful for testing the sensitizing potential of different chemical substances.Abbreviations Ab antibodies - APC antigen-presenting cells - BG Birbeck granules - DNCB 1-chloro-2,4-dinitrobenzene - DNFB 2,4-dinitrofluorobenzene - DTAF dichlorotriazinylfluorescein - FSC forward light scatter - LC Langerhans cells - LSCM laser scanning confocal microscopy - MHC major histocompatibility complex - MAb monoclonal antibodies - PFA paraformaldehyde - SSC side light scatter     

Calcineurin regulates coelomocyte endocytosis via DYN-1 and CUP-4 in Caenorhabditis elegans

C. elegans coelomocytes are macrophage-like scavenger cells that provide an excellent in vivo system for the study of clathrin-mediated endocytosis. Using this in vivo system, several genes involved in coelomocyte endocytosis have been identified previously. However, the detailed mechanism of endocytic pathway is still unknown. Here, we report a new function of calcineurin, an evolutionarily conserved Ca²⁺/calmodulin-dependent Ser/Thr protein phosphatase, in coelomocyte endocytosis. We found that calcineurin mutants show defective coelomocyte endocytosis. Genetic analysis suggests that calcineurin and a GTPase, dynamin (DYN-1), may function upstream of an orphan receptor, CUP-4, to regulate endocytosis. Therefore, we propose a model in which calcineurin may regulate coelomocyte endocytosis via DYN-1 and CUP-4 in C. elegans.     

Fine Structure of the Cell Surface and Golgi Apparatus of Ochromonas*

SYNOPSIS. Ochromonas danica has an unusually flexible cell surface capable of producing projections of varying sizes and shapes: large projections, 340–360 nm long, and small projections, 50–110 nm long. These projections have been demonstrated by transmission and scanning electron microscopy; some of them may break off into the medium and be the source of extracellular membranes and vesicles reported in the cell-free O. danica growth medium. Ruthenium red stained the acid mucopolysaccharide layer just outside the cell surface as well as small blebs at the cell surface. The Golgi complex of O. danica, Ochromonas malhamensis, Ochromonas sociabilis and Ochromonas sp. produced small coated vesicles which may move toward and fuse with the plasma membrane. The role of the several vesicles is unknown but possible functions are discussed.     

Endocytosis and its inhibitors in basidiomycetous fungus <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

Endocytosis is a complex process of absorption from the environment (and subsequent distribution within the cell) of soluble substances, macromolecules, microparticles, etc. by means of vesicles developed by cytoplasmic membrane. Endocytosis in animal and human cells is actively and successfully studied. Thus, classification of this process in the animals (based only on the peculiarities of primary vesicle formation) includes up to ten different endocytosis pathways. Modern knowledge about endocytosis in mycelial fungi is not so extensive; therefore, its study in this group of organisms is a topical and promising direction in fundamental and applied mycology. In the present work, we studied the effect of six different inhibitors (acting both on the assembly of actin/tubulin cytoskeleton and on the formation of different types of endocytosis) on the dynamics of endocytosis in phytopathogenic heterobasidial fungus Rhizoctonia solani. The estimation of the effect of inhibitors was conducted by means of microscopic analysis of the absorption of the fluorescent marker of endocytosis AM4-64 by mycelial cells. As a result of the conducted study, four types of the inhibitor effect on the R. solani endocytosis were detected: from the complete absence of the effect to severe suppression of different stages of fungal endocytosis. It was found that four of six inhibitors used for the suppression of endocytosis in the animals and human have a suppressive effect on endocytosis of R. solani. This indicates the conservative nature of some endocytosis mechanisms in the studied fungus and probably in mycelial fungi in general. Different hypotheses concerning principles of the effect of studied inhibitors on endocytosis activity of fungi were suggested.     

A Wiskott–Aldrich syndrome protein is involved in endocytosis in Aspergillus nidulans

Endocytosis is vital for hyphal tip growth in filamentous fungi and is involved in the tip localization of various membrane proteins. To investigate the function of a Wiskott–Aldrich syndrome protein (WASP) in endocytosis of filamentous fungi, we identified a WASP ortholog-encoding gene, wspA, in Aspergillus nidulans and characterized it. The wspA product, WspA, localized to the tips of germ tubes during germination and actin rings in the subapical regions of mature hyphae. wspA is essential for the growth and functioned in the polarity establishment and maintenance during germination of conidia. We also investigated its function in endocytosis and revealed that endocytosis of SynA, a synaptobrevin ortholog that is known to be endocytosed at the subapical regions of hyphal tips in A. nidulans, did not occur when wspA expression was repressed. These results suggest that WspA plays roles in endocytosis at hyphal tips and polarity establishment during germination.     

Isolation of the Flagellar Swelling and Identification of Retinal in the Phototactic Flagellate, Ochromonas danica (Chrysophyceae)

Ochromonas danica, a freshwater, planktonic chrysophyte, is capable of sensing the light conditions of its environment. This biflagellate alga has a swelling near the base of the short flagellum and a chloroplastidic stigma in close association with it. A procedure is described for the isolation of this three dimensional flagellar swelling, the presumed photoreceptor. In contrast to an earlier method developed for the isolation of the paraflagellar swelling from Euglena gracilis, the protocol reported here for Ochromonas results in higher yields that should facilitate future biochemical investigations and could open avenues of investigation for the isolation and purification of the presumptive receptor protein. To verify the hypothesis that a rhodopsin-like protein might be present in this alga, we applied a standard extraction procedure successfully used in the identification of retinal. We here report the purification and identification of all-trans retinal in Ochromonas cells by column chromatography, HPLC and GC-MS. Since retinal is the chromophore of rhodopsin-like proteins, this finding may suggest that in these unicellular algae, too, a rhodopsin-like protein could be the photoreceptor pigment.     

The management of metabolic energy storage during the life cycle of mayflies: a comparative field investigation of the collector-gatherer <Emphasis Type="Italic">Ephemera danica</Emphasis> and the scraper <Emphasis Type="Italic">Rhithrogena semicolorata</Emphasis>

The concentration and seasonal dynamics of the major energy storage components, triglycerides and glycogen, were measured in two species of mayfly (Rhithrogena semicolorata and Ephemera danica) with contrasting life cycle strategies living in a small mountain stream. E. danica is a burrowing, semivoltine collector-gatherer; R. semicolorata is univoltine and scrapes periphyton from stones. This is the first publication which focuses on the role of metabolic energy sources during the larval life span of two mayfly species until the larvae emerge. Although triglycerides are the major energy reserve in both species (>84% of total energy storage) throughout the whole larval development their seasonal dynamic differed considerably. In R. semicolorata the triglyceride concentration declined during the last weeks prior to emergence in both sexes. The same pattern was found in female larvae of E. danica, but not in male E. danica. It is suggested that females use triglycerides in the last larval stages for egg maturation, which is completed in the last larval instar. In male E. danica the triglyceride concentrations remained high until emergence, presumably due to their high energy demands as adults for their swarming flights. Glycogen concentrations did not show such a difference between species and sexes. Its significance as a storage substrate for energy is rather low; however, concentrations decreased in both species and sexes prior to emergence.     

Endocytosis in mouse blastocysts: Characterization and quantification of the fluid phase component

Fluid phase endocytosis in mouse blastocysts was characterized using the fluid phase marker, ³H-dextran, which did not bind to the membrane. This nonsaturable uptake occurred via an energy-requiring process, with only 20% accountable by diffusion as indicated by analysis at 4°C. Insulin stimulated uptake of ³H-dextran by 30% (P<0.05) over the first hr. The rate of uptake then decreased in both control and insulin-treated blastocysts. However, by 2 hr, insulin-treated blastocysts contained 38% more ³H-dextran (38%; P<0.01) than control blastocysts. Incubation of blastocysts in protein-free medium increased ³H-dextran uptake to a rate equivalent to 12% of the blastocyst volume/min (1,500 ± 240 pliter/hr), compared to 4.5% and 1.5% of the blastocyst volume/min for uptake in the presence of 0.1 g BSA/I and 10 g BSA/I, respectively. Confocal microscopic studies of fluorescently labelled dextran uptake in blastocysts, cultured in the absence of BSA, showed an increase in weak fluorescence labelling in the trophectoderm cells of blastocysts, compared to blastocysts cultured in the presence of BSA. There was no diffusion of fluorescence label into the blastocoel cavity. This is consistent with fluid being endocytosed, possibly by a large number of small pinocytic vesicles. Thus fluid-phase endocytosis in blastocysts is stimulated by insulin, increasing the delivery of nutrient-containing fluid into blastocysts. In the absence of protein, embryos also increase fluid uptake, possibly in an attempt to maintain the rate of supply of protein nutrient to trophectoderm cells. An analysis of the rate of protein delivery in both adsorbed and dissolved phases is presented, which reveals the potential for significant contributions of both phases of endocytosis to blastocyst metabolism in vivo. © 1995 Wiley-Liss, Inc.     

A block of endocytosis of the yeast cell wall integrity sensors Wsc1 and Wsc2 results in reduced fitness in vivo

The response to cell surface stress in yeast is mediated by a set of five plasma membrane sensors. We here address the relation of intracellular localization of the sensors Wsc1, Wsc2, and Mid2 to their turnover and signaling function. Growth competition experiments indicate that Wsc2 plays an important role in addition to Wsc1 and Mid2. The two Wsc sensors appear at the bud neck during cytokinesis and employ different routes of endocytosis, which govern their turnover. Whereas Wsc1 uses a clathrin-dependent NPFDD signal, Wsc2 relies on a specific lysine residue (K495). In end3 and doa4 endocytosis mutants, both sensors accumulate at the plasma membrane, and a hypersensitivity to cell wall-specific drugs and to treatment with zymolyase is observed. A haploid strain in which endocytosis of the two sensors is specifically blocked displays a reduced fitness in growth competition experiments. If the Mid2 sensor is mobilized by the addition of an endocytosis signal, it mimics the dynamic distribution of the Wsc sensors, but is unable to complement the specific growth defects of a wsc1 deletion. These data suggest that sensor distribution is not the major determinant for its specificity.     

Binding and Uptake of RGD-Containing Ligands to Cellular α v β 3 Integrins

The cyclic peptide, cRGDf[N(me)]V, binds to the α _v β ₃ integrin and can disrupt binding of the integrin to its natural ligands in the extracellular matrix. In this work, the ability of a water-soluble, fluorescently labeled variant of the RGD-containing peptide (cRGDfK-488) to bind to integrins on human umbilical vascular endothelial cells (HUVEC) and subsequently undergo endocytosis was characterized. This information was compared to the binding and uptake properties of an α _v β ₃ integrin-specific monoclonal antibody, LM609X. The specificity of the RGD-containing peptide is assessed by comparison with control peptide that does not bind to the α _v β ₃ integrin, cRADfK-488. Using a high purity construct, it is shown that the RGD ligand exhibits dissociation constants in the micromolar range whereas LM609X exhibits dissociation constants in the nanomolar range. However, the RGD ligand showed greater uptake following incubation at temperatures which permit endocytosis. A 7.4-fold increase in uptake of the RGD peptide was observed following a 1 h incubation with HUVEC at 37°C (an endocytosis permissive temperature), as compared to that at 4°C (an endocytosis prohibitive temperature). In contrast, only a 1.9-fold increase in cell-associated fluorescence was observed for similar incubations with LM609X. Results from fluorescence microscopy supports the notion that the RGD peptide is rapidly endocytosed at 37°C as compared to LM609X. These results are discussed with regard to previous work indicating that RGD ligands enter cells by integrin-independent pathways. These studies provide well-controlled measures of how RGD ligands stimulate endocytosis. This may be of considerable interest for intracellular delivery of ligand-associated drugs in anti-angiogenic applications.     

Competition of Ethionine and Methionine for Aminoacyl-tRNA Formation in an In Vitro System from Ochromonas danica*

Aminoacyl-tRNA synthetase and tRNA were isolated from the chrysomonad Ochromonas danica. The mutual effect of methionine and ethionine, and the effect of other amino acids on methionyl- and ethionyl-tRNA formation, were tested in an in vitro system. The tRNA^Met had a similar accepting capacity for either methionine or ethionine. Ethionine and methionine, but none of the other amino acids tested, competed for the same aminoacyl-tRNA synthetase. The K_m of methionine was 0.88 × 10^–5 M, and that of ethionine 5 × 10^–4 M. Ethionine inhibited methionine binding; K_i 3.4 × 10^–4 M. The respective values in a similar system isolated from E. coli were 2.2 × 10^–5, 1.95 × 10^–3, and 1.95 × 10^–3.     

Embryonic gut differentiation in nematodes: endocytosis of macromolecules and its experimental inhibition

During embryogenesis of Caenorhabditis elegans cytoplasmic components are transferred from nongut cells into the developing gut primordium and an exo/endocytosis mechanism has been hypothesized (Bossinger and Schierenberg 1992). To test endocytotic activity of the gut primordium, we compared the uptake of different fluorochrome-conjugated marker molecules in two nematode species, C. elegans and Cephalobus spec., which differ in the pattern of early cleavage and cell-cell communication. We found no uptake of dextran (as a marker for pinocytosis) but rapid internalization of 30-fold larger transferrin molecules (as a marker for receptor-coupled endocytosis) into the differentiating gut primordium in both nematodes. The two studied species differ with respect to when this process starts. While the uptake of macromolecules in the fast developing C. elegans is first observed at a stage when essentially all cells of the hatching juvenile have been generated, in the slow developing Cephalobus endocytosis begins during the early proliferation phase when only two gut precursor cells are present. We found that the polysulfated hydrocarbon dye trypan blue and the cationic amphiphilic drug chlorpromazine both inhibit endocytosis into the gut primodium.     

The Sla1 adaptor‐clathrin interaction regulates coat formation and progression of endocytosis

Clathrin‐mediated endocytosis is a fundamental transport pathway that depends on numerous protein‐protein interactions. Testing the importance of the adaptor protein‐clathrin interaction for coat formation and progression of endocytosis in vivo has been difficult due to experimental constrains. Here, we addressed this question using the yeast clathrin adaptor Sla1, which is unique in showing a cargo endocytosis defect upon substitution of 3 amino acids in its clathrin‐binding motif (sla1^AAA) that disrupt clathrin binding. Live‐cell imaging showed an impaired Sla1‐clathrin interaction causes reduced clathrin levels but increased Sla1 levels at endocytic sites. Moreover, the rate of Sla1 recruitment was reduced indicating proper dynamics of both clathrin and Sla1 depend on their interaction. sla1^AAA cells showed a delay in progression through the various stages of endocytosis. The Arp2/3‐dependent actin polymerization machinery was present for significantly longer time before actin polymerization ensued, revealing a link between coat formation and activation of actin polymerization. Ultimately, in sla1^AAA cells a larger than normal actin network was formed, dramatically higher levels of various machinery proteins other than clathrin were recruited, and the membrane profile of endocytic invaginations was longer. Thus, the Sla1‐clathrin interaction is important for coat formation, regulation of endocytic progression and membrane bending.      

Altered sterol synthesis and its relationship to fluid-phase endocytosis in a macrophage cell line P388D1

Summary In a previous study glucocorticoids have been shown to depress the rate of fluid-phase endocytosis in a macrophage cell line, P388D₁. This effect was observed when either fluorescein-labeled dextran or horseradish peroxidase (HRP) was used to measure endocytosis. In this report the relationship between cholesterol synthesis and endocytosis was examined in light of the ability of glucocorticoids to inhibit cholesterol biosynthesis. Two known inhibitors of cholesterol biosynthesis, ML-236B and 25-hydroxycholesterol (25-OH), were compared with dexamethasone (dex) for the ability to suppress endocytosis in cells grown in media supplemented with either 10% whole or delipidized neonatal bovine serum (NBS). In 10% whole serum all inhibitors reduced the uptake of HRP after 12 h incubation. Dexamethasone (1 μM) suppressed endocytosis by 30% whereas 25-OH (2.5 μM) and ML-236B (11.6 μM) inhibited by 38 and 52%, respectively. Supplementation of the growth medium with mevalonolactone (3.4 mM) prevented the inhibition of endocytosis by ML-236B. In contrast, mevalonolactone supplementation did not prevent either dex or 25-OH from suppressing endocytosis. The same pattern of results was obtained when cultures were grown in delipidized NBS. After 4 h all inhibitors caused a decrease in amount of [¹⁴C]acetate incorporated into both nonsaponifiable lipids and digitonin precipitable sterols. Although dex inhibited cholesterol biosynthesis, total cellular cholesterol was unaffected by dex treatment after 24 h incubation. It is suggested that in addition to suppressing mevalonate synthesis, 25-OH, and by analogy dex, may act at some metabolic site(s) distal to the formation of mevalonate. This investigation was supported, in part, by a Public Health Service Research grant (CA-08315) from the National Cancer Institute, Bethesda, MD.     

Defective calmodulin‐dependent rapid apical endocytosis in zebrafish sensory hair cell mutants

Vertebrate mechanosensory hair cells contain a narrow “pericuticular” zone which is densely populated with small vesicles between the cuticular plate and cellular junctions near the apical surface. The presence of many cytoplasmic vesicles suggests that the apical surface of hair cells has a high turnover rate. The significance of intense membrane trafficking at the apical surface is not known. Using a marker of endocytosis, the styryl dye FM1‐43, this report shows that rapid apical endocytosis in zebrafish lateral line sensory hair cells is calcium and calmodulin dependent and is partially blocked by the presence of amiloride and dihydrostreptomycin, known inhibitors of mechanotransduction channels. As seen in lateral line hair cells, sensory hair cells within the larval otic capsule also exhibit rapid apical endocytosis. Defects in internalization of the dye in both lateral line and inner ear hair cells were found in five zebrafish auditory/vestibular mutants: sputnik, mariner, orbiter, mercury, and skylab. In addition, lateral line hair cells in these mutants were not sensitive to prolonged exposure to streptomycin, which is toxic to hair cells. The presence of endocytic defects in the majority of zebrafish mechanosensory mutants points to a important role of apical endocytosis in hair cell function. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 424–434, 1999