The movement of coiled bodies visualized in living plant cells by the green fluorescent protein.
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Coiled bodies are nuclear organelles that contain components of at least three RNA-processing pathways: pre-mRNA splicing, histone mRNA 3'- maturation, and pre-rRNA processing. Their function remains unknown. However, it has been speculated that coiled bodies may be sites of splicing factor assembly and/or recycling, play a role in histone mRNA 3'-processing, or act as nuclear transport or sorting structures. To study the dynamics of coiled bodies in living cells, we have stably expressed a U2B"-green fluorescent protein fusion in tobacco BY-2 cells and in Arabidopsis plants. Time-lapse confocal microscopy has shown that coiled bodies are mobile organelles in plant cells. We have observed movements of coiled bodies in the nucleolus, in the nucleoplasm, and from the periphery of the nucleus into the nucleolus, which suggests a transport function for coiled bodies. Furthermore, we have observed coalescence of coiled bodies, which suggests a mechanism for the decrease in coiled body number during the cell cycle. Deletion analysis of the U2B" gene construct has shown that the first RNP-80 motif is sufficient for localization to the coiled body. (+info)
Substitution rates of organelle and nuclear genes in sharks: implicating metabolic rate (again).
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Rates of nucleotide substitution for nuclear genes are thought to be governed primarily by the number of germ line replication events (the so-called "generation time" hypothesis). In contrast, rates of mitochondrial DNA evolution appear to be set primarily by DNA damage pathways of mutation mediated by mutagenic by-products of oxidative phosphorylation (the so-called "metabolic-rate" hypothesis). Comparison of synonymous substitution rates estimated for the mitochondrial cytochrome b gene and nuclear-encoded dlx, hsp70, and RAG-1 genes in mammals and sharks shows that rates of molecular evolution for sharks are approximately an order of magnitude slower than those for mammals for both nuclear and mitochondrial genes. In addition, there is significant positive covariation of substitution rate for mitochondrial and nuclear genes within sharks. These results, interpreted in light of the pervasiveness of DNA damage by mutagenic by-products of oxygen metabolism to both nuclear and mitochondrial genes and coupled with increasing evidence for cross-genome activity of DNA repair enzymes, suggest that molecular clocks for mitochondrial and nuclear genes may be set primarily by common mutational mechanisms. (+info)
Effects of heat shock on the functional morphology of cell organelles observed by video-enhanced microscopy.
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In living astrocytes and MDCK cells we observed morphological phenomena during and after heat shock (HS) utilizing our new perfusable microchamber system, which monitors pH, pO(2), pCO(2), and temperature. By means of electronic light microscopy and confocal laser scanning microscopy, mitochondria were demonstrated to swell and to reduce their motility. The specific fluorescent probe MitoTracker Green revealed that the mitochondrial morphology changed from a rodlike into an annular shape with a central vacuole-findings which were corroborated by transmission electron microscopy. After HS (shift from 37 degrees C to 45 degrees C for 15 min) the mitochondrial membrane potential (DeltaPsi(m)) was depressed in most but not all mitochondria as monitored with the fluorescent probe JC-1. The dual emission images of JC-1 illustrated a heterogeneous red staining of distinct areas of single mitochondria. The shape changes as well as the drop of the membrane potential of the mitochondria indicated severe cellular stress and a direct intervention on the mitochondrial permeability transition. (+info)
Establishment of the dorsal-ventral axis in Xenopus embryos coincides with the dorsal enrichment of dishevelled that is dependent on cortical rotation.
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Examination of the subcellular localization of Dishevelled (Dsh) in fertilized Xenopus eggs revealed that Dsh is associated with vesicle-like organelles that are enriched on the prospective dorsal side of the embryo after cortical rotation. Dorsal enrichment of Dsh is blocked by UV irradiation of the vegetal pole, a treatment that inhibits development of dorsal cell fates, linking accumulation of Dsh and specification of dorsal cell fates. Investigation of the dynamics of Dsh localization using Dsh tagged with green fluorescent protein (Dsh-GFP) demonstrated that Dsh-GFP associates with small vesicle-like organelles that are directionally transported along the parallel array of microtubules towards the prospective dorsal side of the embryo during cortical rotation. Perturbing the assembly of the microtubule array with D(2)O, a treatment that promotes the random assembly of the array and the dorsalization of embryos, randomizes translocation of Dsh-GFP. Conversely, UV irradiation of the vegetal pole abolishes movement of Dsh-GFP. Finally, we demonstrate that overexpression of Dsh can stabilize beta-catenin in Xenopus. These data suggest that the directional translocation of Dsh along microtubules during cortical rotation and its subsequent enrichment on the prospective dorsal side of the embryo play a role in locally activating a maternal Wnt pathway responsible for establishing dorsal cell fates in Xenopus. (+info)
The nonmuscle myosin regulatory light chain gene mlc-4 is required for cytokinesis, anterior-posterior polarity, and body morphology during Caenorhabditis elegans embryogenesis.
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Using RNA-mediated genetic interference in a phenotypic screen, we identified a conserved nonmuscle myosin II regulatory light chain gene in Caenorhabditis elegans, which we name mlc-4. Maternally supplied mlc-4 function is required for cytokinesis during both meiosis and mitosis and for establishment of anterior-posterior (a-p) asymmetries after fertilization. Reducing the function of mlc-4 or nmy-2, a nonmuscle myosin II gene, also leads to a loss of polarized cytoplasmic flow in the C. elegans zygote, supporting models in which cytoplasmic flow may be required to establish a-p differences. Germline P granule localization at the time of cytoplasmic flow is also lost in these embryos, although P granules do become localized to the posterior pole after the first mitosis. This result suggests that a mechanism other than cytoplasmic flow or mlc-4/nmy-2 activity can generate some a-p asymmetries in the C. elegans zygote. By isolating a deletion allele, we show that removing zygotic mlc-4 function results in an elongation phenotype during embryogenesis. An mlc-4/green fluorescent protein transgene is expressed in lateral rows of hypodermal cells and these cells fail to properly change shape in mlc-4 mutant animals during elongation. (+info)
Characterization of integrin-tetraspanin adhesion complexes: role of tetraspanins in integrin signaling.
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Tetraspanins (or proteins from the transmembrane 4 superfamily, TM4SF) form membrane complexes with integrin receptors and are implicated in integrin-mediated cell migration. Here we characterized cellular localization, structural composition, and signaling properties of alpha3beta1-TM4SF adhesion complexes. Double-immunofluorescence staining showed that various TM4SF proteins, including CD9, CD63, CD81, CD82, and CD151 are colocalized within dot-like structures that are particularly abundant at the cell periphery. Differential extraction in conjunction with chemical cross-linking indicated that the cell surface fraction of alpha3beta1-TM4SF protein complexes may not be directly linked to the cytoskeleton. However, in cells treated with cytochalasin B alpha3beta1-TM4SF protein complexes are relocated into intracellular vesicles suggesting that actin cytoskeleton plays an important role in the distribution of tetraspanins into adhesion structures. Talin and MARCKS are partially codistributed with TM4SF proteins, whereas vinculin is not detected within the tetraspanin-containing adhesion structures. Attachment of serum-starved cells to the immobilized anti-TM4SF mAbs induced dephosphorylation of focal adhesion kinase (FAK). On the other hand, clustering of tetraspanins in cells attached to collagen enhanced tyrosine phosphorylation of FAK. Furthermore, ectopic expression of CD9 in fibrosarcoma cells affected adhesion-induced tyrosine phosphorylation of FAK, that correlated with the reorganization of the cortical actin cytoskeleton. These results show that tetraspanins can modulate integrin signaling, and point to a mechanism by which TM4SF proteins regulate cell motility. (+info)
Inhibition of polyamine synthesis arrests trichomonad growth and induces destruction of hydrogenosomes.
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Trichomonad parasites such as Tritrichomonas foetus produce large amounts of putrescine (1,4-diaminobutane), which is transported out of the cell via an antiport mechanism which results in the uptake of a molecule of spermine. The importance of putrescine to the survival of the parasite and its role in the biology of T. foetus was investigated by use of the putrescine analogue 1, 4-diamino-2-butanone (DAB). Growth of T. foetus in vitro was significantly inhibited by 20 mM DAB, which was reversed by the addition of exogenous 40 mM putrescine. High-performance liquid chromatography analysis of 20 mM DAB-treated T. foetus revealed that putrescine, spermidine, and spermine levels were reduced by 89, 52, and 43%, respectively, compared to those in control cells. The DAB treatment induced several ultrastructural alterations, which were primarily observed in the redox organelles termed hydrogenosomes. These organelles were progressively degraded, giving rise to large vesicles that displayed material immunoreactive with an antibody to beta-succinyl-coenzyme A synthetase, a hydrogenosomal enzyme. A protective role for polyamines as stabilizing agents in the trichomonad hydrogenosomal membrane is proposed. (+info)
FliL is a membrane-associated component of the flagellar basal body of Salmonella.
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FliL is one of the least understood proteins in the flagellar systems of Salmonella and Escherichia coli. There is no apparent mutant phenotype associated with it, even when virtually the entire coding sequence has been eliminated. In this study it has been shown that FliL is a cytoplasmic membrane protein associated with the basal body. Although it has a sequence that conforms to the consensus cleavage site for lipoproteins, FliL does not undergo cleavage or modification under physiological conditions. (+info)