Localization in the nucleolus and coiled bodies of protein subunits of the ribonucleoprotein ribonuclease P.
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The precise location of the tRNA processing ribonucleoprotein ribonuclease P (RNase P) and the mechanism of its intranuclear distribution have not been completely delineated. We show that three protein subunits of human RNase P (Rpp), Rpp14, Rpp29 and Rpp38, are found in the nucleolus and that each can localize a reporter protein to nucleoli of cells in tissue culture. In contrast to Rpp38, which is uniformly distributed in nucleoli, Rpp14 and Rpp29 are confined to the dense fibrillar component. Rpp29 and Rpp38 possess functional, yet distinct domains required for subnucleolar localization. The subunit Rpp14 lacks such a domain and appears to be dependent on a piggyback process to reach the nucleolus. Biochemical analysis suggests that catalytically active RNase P exists in the nucleolus. We also provide evidence that Rpp29 and Rpp38 reside in coiled bodies, organelles that are implicated in the biogenesis of several other small nuclear ribonucleoproteins required for processing of precursor mRNA. Because some protein subunits of RNase P are shared by the ribosomal RNA processing ribonucleoprotein RNase MRP, these two evolutionary related holoenzymes may share common intranuclear localization and assembly pathways to coordinate the processing of tRNA and rRNA precursors. (+info)
Myotactin, a novel hypodermal protein involved in muscle-cell adhesion in Caenorhabditis elegans.
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In C. elegans, assembly of hypodermal hemidesmosome-like structures called fibrous organelles is temporally and spatially coordinated with the assembly of the muscle contractile apparatus, suggesting that signals are exchanged between these cell types to position fibrous organelles correctly. Myotactin, a protein recognized by monoclonal antibody MH46, is a candidate for such a signaling molecule. The antigen, although expressed by hypodermis, first reflects the pattern of muscle elements and only later reflects the pattern of fibrous organelles. Confocal microscopy shows that in adult worms myotactin and fibrous organelles show coincident localization. Further, cell ablation studies show the bodywall muscle cells are necessary for normal myotactin distribution. To investigate myotactin's role in muscle-hypodermal signaling, we characterized the myotactin locus molecularly and genetically. Myotactin is a novel transmembrane protein of approximately 500 kd. The extracellular domain contains at least 32 fibronectin type III repeats and the cytoplasmic domain contains unique sequence. In mutants lacking myotactin, muscle cells detach when embryonic muscle contraction begins. Later in development, fibrous organelles become delocalized and are not restricted to regions of the hypodermis previously contacted by muscle. These results suggest myotactin helps maintain the association between the muscle contractile apparatus and hypodermal fibrous organelles. (+info)
Ethanol and acetaldehyde elevate intracellular [Ca2+] and stimulate microneme discharge in Toxoplasma gondii.
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One of the first steps in host-cell invasion by the protozoan parasite Toxoplasma gondii occurs when the parasite attaches by its apical end to the target host cell. The contents of apical secretory organelles called micronemes have recently been implicated in parasite apical attachment to host cells. Micronemes are regulated secretory vesicles that discharge in response to elevated parasite intracellular Ca(2+) levels ([Ca2+]i). In the present study we found that ethanol and related compounds produced a dose-dependent stimulation of microneme secretion. In addition, using fluorescence spectroscopy on tachyzoites loaded with the Ca(2+)-sensitive fluorescent dye fura-2, we demonstrated that ethanol stimulated microneme secretion by elevating parasite [Ca2+](i). Furthermore, sequential addition experiments with ethanol and other Ca(2+)-mobilizing drugs showed that ethanol probably elevated parasite [Ca2+](i) by mobilizing Ca(2+) from a thapsigargin-insensitive compartment of neutral pH. Earlier studies have shown that ethanol also elevates [Ca2+](i) in mammalian cells. Thus, because it is genetically tractable, T. gondii might be a convenient model organism for studying the Ca(2+)-elevating effects of alcohol in higher eukaryotes. (+info)
Electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of the lipid molecular species composition of yeast subcellular membranes reveals acyl chain-based sorting/remodeling of distinct molecular species en route to the plasma membrane.
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Nano-electrospray ionization tandem mass spectrometry (nano-ESI-MS/MS) was employed to determine qualitative differences in the lipid molecular species composition of a comprehensive set of organellar membranes, isolated from a single culture of Saccharomyces cerevisiae cells. Remarkable differences in the acyl chain composition of biosynthetically related phospholipid classes were observed. Acyl chain saturation was lowest in phosphatidylcholine (15.4%) and phosphatidylethanolamine (PE; 16.2%), followed by phosphatidylserine (PS; 29.4%), and highest in phosphatidylinositol (53.1%). The lipid molecular species profiles of the various membranes were generally similar, with a deviation from a calculated average profile of approximately +/- 20%. Nevertheless, clear distinctions between the molecular species profiles of different membranes were observed, suggesting that lipid sorting mechanisms are operating at the level of individual molecular species to maintain the specific lipid composition of a given membrane. Most notably, the plasma membrane is enriched in saturated species of PS and PE. The nature of the sorting mechanism that determines the lipid composition of the plasma membrane was investigated further. The accumulation of monounsaturated species of PS at the expense of diunsaturated species in the plasma membrane of wild-type cells was reversed in elo3Delta mutant cells, which synthesize C24 fatty acid-substituted sphingolipids instead of the normal C26 fatty acid-substituted species. This observation suggests that acyl chain-based sorting and/or remodeling mechanisms are operating to maintain the specific lipid molecular species composition of the yeast plasma membrane. (+info)
ECL cell morphology.
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Using immunohistochemistry at the conventional light, confocal and electron microscopic levels, we have demonstrated that rat stomach ECL cells store histamine and pancreastatin in granules and secretory vesicles, while histidine decarboxylase occurs in the cytosol. Furthermore the ECL cells display immunoreactivity for vesicular monoamine transporter type 2 (VMAT-2), synaptophysin, synaptotagmin III, vesicle-associated membrane protein-2, cysteine string protein, synaptosomal-associated protein of 25 kDa, syntaxin and Munc-18. Using electron microscopy in combination with stereological methods, we have evidence to suggest the existence of both an exocytotic and a crinophagic pathway in the ECL cells. The process of exocytosis in the ECL cells seems to involve a class of proteins that promote or participate in the fusion between the granule/vesicle membrane and the plasma membrane. The granules take up histamine by VMAT-2 from the cytosol during transport from the Golgi zone to the more peripheral parts of the cells. As a result, they turn into secretory vesicles. As a consequence of stimulation (e.g., by gastrin), the secretory vesicles fuse with the cell membrane to release their contents by exocytosis. The crinophagic pathway was studied in hypergastrinemic rats. In the ECL cells of such animals, the secretory vesicles were found to fuse not only with the cell membrane but also with each other to form vacuoles. Subsequent lysosomal degradation of the vacuoles and their contents resulted in the development of lipofuscin bodies. (+info)
The AtCAO gene, encoding chlorophyll a oxygenase, is required for chlorophyll b synthesis in Arabidopsis thaliana.
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Chlorophyll b is synthesized from chlorophyll a and is found in the light-harvesting complexes of prochlorophytes, green algae, and both nonvascular and vascular plants. We have used conserved motifs from the chlorophyll a oxygenase (CAO) gene from Chlamydomonas reinhardtii to isolate a homologue from Arabidopsis thaliana. This gene, AtCAO, is mutated in both leaky and null chlorina1 alleles, and DNA sequence changes cosegregate with the mutant phenotype. AtCAO mRNA levels are higher in three different mutants that have reduced levels of chlorophyll b, suggesting that plants that do not have sufficient chlorophyll b up-regulate AtCAO gene expression. Additionally, AtCAO mRNA levels decrease in plants that are grown under dim-light conditions. We have also found that the six major Lhcb proteins do not accumulate in the null ch1-3 allele. (+info)
Kinesin and dynein mutants provide novel insights into the roles of vesicle traffic during cell morphogenesis in Neurospora.
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BACKGROUND: Kinesin and cytoplasmic dynein are force-generating molecules that move in opposite directions along microtubules. They have been implicated in the directed transport of a wide variety of cellular organelles, but it is unclear whether they have overlapping or largely independent functions. RESULTS: We analyzed organelle transport in kinesin and dynein single mutants, and in a kinesin and dynein double mutant of Neurospora crassa. Remarkably, the simultaneous mutation of kinesin and dynein was not lethal and resulted in an additive phenotype that combined the features of the single mutants. The mutation of kinesin and dynein had opposite effects on the apical and retrograde transport, respectively, of vesicular organelles. In the kinesin mutant, apical movement of submicroscopic, secretory vesicles to the Spitzenkorper - an organelle in the hyphal apex - was defective, whereas the predominantly retrograde movement of microscopic organelles was only slightly reduced. In contrast, the dynein mutant still had a prominent Spitzenkorper, demonstrating that apical transport was intact, but retrograde transport was essentially inhibited completely. A major defect in vacuole formation and dynamics was also evident. In agreement with the observations on apical transport, protein secretion into the medium was markedly inhibited in the kinesin mutant but not in the dynein mutant. CONCLUSIONS: Transport of secretory vesicles is necessary but not sufficient for normal apical extension. A component of retrograde transport, presumably precursors of the vacuole system, is also essential. Our findings provide new information on the role microtubule motors play in cell morphogenesis and suggest that kinesin and cytoplasmic dynein have largely independent functions within separate pathways. (+info)
Thapsigargin has similar effect on p53 protein response to benzo[a]pyrene-DNA adducts as TPA in mouse skin.
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The level of p53 tumor suppressor protein increases in response to DNA damage caused by benzo[a]pyrene (B[a]P). The most used tumor promoter in the two step mouse skin carcinogenesis model, 12-O-tetradecanoylphorbol-13-acetate (TPA) decreases this response in mouse skin. In this study the effect of another promoter, thapsigargin was tested on B[a]P-induced p53 response using immunohistochemistry, western blotting and immunoelectron microscopy. We also studied the localization of p53 protein after treatments with BP and TPA or thapsigargin. Thapsigargin had a TPA-like effect on the acute induction of p53 protein related to benzo[a]pyrene-7, 8-diol-9,10-epoxide-DNA adducts in the skin of C57BL/6 mouse. After B[a]P treatment, there was slightly more putatively wild-type p53 protein in nuclei than in cytoplasm of the cells. Neither TPA nor thapsigargin affected the localization of p53 protein. Since both compounds increase the level of intracellular calcium, the inhibition of the p53 response may depend on the level of intracellular calcium. Inhibition of the putatively genome-protecting increase in p53 protein may be one of the critical effects of tumor promoters. (+info)