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Subcellular Localization (subcellular + localization)
Kinds of Subcellular Localization Selected AbstractsThe KCl cotransporter, KCC2, is highly expressed in the vicinity of excitatory synapses in the rat hippocampusEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2001A. I. Gulyás Abstract Immunocytochemical visualization of the neuron-specific K+/Cl, cotransporter, KCC2, at the cellular and subcellular level revealed an area- and layer-specific diffuse labelling, and a discrete staining outlining the somata and dendrites of some interneurons in all areas of the rat hippocampus. KCC2 was highly expressed in parvalbumin-containing interneurons, as well as in subsets of calbindin, calretinin and metabotropic glutamate receptor 1a-immunoreactive interneurons. During the first 2 postnatal weeks, an increase of KCC2 staining was observed in the molecular layer of the dentate gyrus, correlating temporally with the arrival of entorhinal cortical inputs. Subcellular localization demonstrated KCC2 in the plasma membranes. Immunoreactivity in principal cells was responsible for the diffuse staining found in the neuropil. In these cells, KCC2 was detected primarily in dendritic spine heads, at the origin of spines and, at a much lower level on the somata and dendritic shafts. KCC2 expression was considerably higher in the somata and dendrites of interneurons, most notably of parvalbumin-containing cells, as well as in the thorny excrescences of CA3 pyramidal cells and in the spines of spiny hilar and stratum lucidum interneurons. The data indicate that KCC2 is highly expressed in the vicinity of excitatory inputs in the hippocampus, perhaps in close association with extrasynaptic GABAA receptors. A high level of excitation is known to lead to a simultaneous net influx of Na+ and Cl,, as evidenced by dendritic swelling. KCC2 located in the same microenvironment may provide a Cl, extrusion mechanism to deal with both ion and water homeostasis in addition to its role in setting the driving force of Cl, currents involved in fast postsynaptic inhibition. [source] Subcellular localization of proteins labeled with GFP in Xanthomonas citri ssp. citri: targeting the division septumFEMS MICROBIOLOGY LETTERS, Issue 1 2010Paula M.M. Martins Abstract Xanthomonas citri ssp. citri (Xac) is the causal agent of citrus canker, an economically important disease that affects citrus worldwide. To initiate the characterization of essential biological processes of Xac, we constructed integrative plasmids for the ectopic expression of green fluorescent protein (GFP)-labeled proteins within this bacterium. Here, we show that the disruption of the ,-amylase gene (amy), the site of plasmid integration into the bacterial chromosome, does not alter its pathogenesis while abolishing completely the ability of Xac to degrade starch. Furthermore, our GFP expression system was used to characterize ORF XAC3408, a hypothetical protein encoded by Xac that shares significant homology to the FtsZ-stabilizing factor ZapA from Bacillus subtilis (ZapABsu). GFP-XAC3408 expressed in Xac exhibited a septal localization pattern typical of GFP-ZapABsu, which indicates that XAC3408 is the Xac orthologue of the cell division protein ZapABsu. The results demonstrate the potential of GFP labeling for protein functional characterizations in Xac, and, in addition, the Xac mutant strain labeled at the septum constitutes a biological model for the exploration of antibacterial compounds able to inhibit cell division in this plant pathogen. [source] Subcellular localization and phosphorylation of antizyme 2JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2009Noriyuki Murai Abstract Antizymes (AZs) are polyamine-induced proteins that negatively regulate cellular polyamine synthesis and uptake. Three antizyme isoforms are conserved among mammals. AZ1 and AZ2 have a broad tissue distribution, while AZ3 is testis specific. Both AZ1 and AZ2 inhibit ornithine decarboxylase (ODC) activity by binding to ODC monomer and target it to the 26S proteasome at least in vivo. Both also inhibit extra-cellular polyamine uptake. Despite their being indistinguishable by these criteria, we show here using enhanced green fluorescent protein (EGFP)-AZ2 fusion protein that in mammalian cells, the subcellular location of AZ2 is mainly in the nucleus, and is different from that of AZ1. The C-terminal part of AZ2 is necessary for the nuclear distribution. Within a few hours, a shift in the distribution of EGFP-AZ2 fusion protein from cytoplasm to the nucleus or from nucleus to cytoplasm is observable in NIH3T3 cells. In addition, we found that in cells a majority of AZ2, but not AZ1, is phosphorylated at Ser-186, likely by protein kinase CK2. There may be a specific function of AZ2 in the nucleus. J. Cell. Biochem. 108: 1012,1021, 2009. © 2009 Wiley-Liss, Inc. [source] AKIN,1 is Involved in the Regulation of Nitrogen Metabolism and Sugar Signaling in ArabidopsisJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 5 2009Xiao-Fang Li Abstract Sucrose non-fermenting-1-related protein kinase 1 (SnRK1) has been located at the heart of the control of metabolism and development in plants. The active SnRK1 form is usually a heterotrimeric complex. Subcellular localization and specific target of the SnRK1 kinase are regulated by specific beta subunits. In Arabidopsis, there are at least seven genes encoding beta subunits, of which the regulatory functions are not yet clear. Here, we tried to study the function of one beta subunit, AKIN,1. It showed that AKIN,1 expression was dramatically induced by ammonia nitrate but not potassium nitrate, and the investigation of AKIN,1 transgenic Arabidopsis and T-DNA insertion lines showed that AKIN,1 negatively regulated the activity of nitrate ruductase and was positively involved in sugar repression in early seedling development. Meanwhile AKIN,1 expression was reduced upon sugar treatment (including mannitol) and did not affect the activity of sucrose phosphate synthase. The results indicate that AKIN,1 is involved in the regulation of nitrogen metabolism and sugar signaling. [source] DLGdifferentially localizes Shaker K+ -channels in the central nervous system and retina of DrosophilaJOURNAL OF NEUROCHEMISTRY, Issue 6 2002C. Ruiz-Cañada Abstract Subcellular localization of ion channels is crucial for the transmission of electrical signals in the nervous system. Here we show that Discs-Large (DLG), a member of the MAGUK (membrane-associated guanylate kinases) family in Drosophila, co-localizes with Shaker potassium channels (Sh Kch) in most synaptic areas of the adult brain and in the outer membrane of photoreceptors. However, DLG is absent from axonal tracts in which Sh channels are concentrated. Truncation of the C-terminal of Sh (including the PDZ binding site) disturbs its pattern of distribution in both CNS and retina, while truncation of the guanylate kinase/C-terminal domain of DLG induces ectopic localization of these channels to neuronal somata in the CNS, but does not alter the distribution of channels in photoreceptors. Immunocytochemical, membrane fractionation and detergent solubilization analysis indicate that the C-terminal of Sh Kch is required for proper trafficking to its final destination. Thus, several major conclusions emerge from this study. First, DLG plays a major role in the localization of Shchannels in the CNS and retina. Second, localization of DLG in photoreceptors but not in the CNS seems to depend on its interaction with Sh. Third, the guanylate kinase/C-terminal domain of DLG is involved in the trafficking of Shaker channels but not of DLG in the CNS. Fourth, different mechanisms for the localization of Sh Kch operate in different cell types. [source] Subcellular localization of neural-specific NPDC-1 proteinJOURNAL OF NEUROSCIENCE RESEARCH, Issue 6 2005C. Evrard Abstract NPDC-1 is a gene specifically expressed in neural cells when they stop to divide and begin to differentiate. Immunocytochemical study analysis of differentiated PC12 cells transfected with NPDC-tag vectors showed that NPDC-1 is transported in vesicles from the Golgi apparatus to the cell membrane and is then likely internalized into endosomes. The protein colocalized, at least partially, with synaptic vesicle proteins: synaptophysin, synaptobrevin 2, and Rab3 GEP (Rab3 GTP/GDP exchange protein). Moreover, subcellular fractionation of rat brain showed that crude synaptic membrane and crude synaptic vesicle fractions were enriched in NPDC-1. Although NPDC-1 bound Rab3 GEP in vitro, it seems unlikely to be involved in Ca2+ -dependent exocytosis and, thus, in synaptic vesicle trafficking. © 2005 Wiley-Liss, Inc. [source] Subcellular localization of beta-catenin in malignant cell lines and squamous cell carcinomas of the oral cavityJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 7 2002A. Gasparoni Abstract Background:, Beta-catenin, an E-cadherin-associated protein involved in cell,cell adhesion and signaling, has been hypothesized to translocate to the nucleus and activate transcription in several human cancers, including oral squamous cell carcinomas (OSCC). Methods:, In the present study, we analyzed the subcellular localization of beta-catenin in cultures of human oral normal and malignant (cell lines SCC15 and SCC25) keratinocytes and in 24 frozen samples of oral squamous cell carcinomas by a double-staining technique for nucleic acids and beta-catenin. Growth potential, as assessed by cell count at different time periods, was established for normal, SCC15 and SCC25 cell lines; oral squamous cell carcinomas were classified according to the histopathological and malignancy indexes. Results:, Beta-catenin localized at the plasma membrane in the normal and SCC15 cells, not in the SCC25 cells, where it localized mostly in the perinuclear and nuclear areas. In the growth assays, SCC25 cell lines proliferated faster than in normal and SCC15 cells over a period of 6 days (cell numbers were significantly different, P < 0.0001). Carcinoma sections showed a combination of membranous, cytoplasmic and, in few invading epithelial islands of two tumors, nuclear localization of beta-catenin. Conclusions:, In oral squamous cell carcinomas, nuclear beta-catenin staining was observed only within invading islands of two carcinomas deep in the underlying connective tissue. On the basis of this study, we conclude that intranuclear beta-catenin does not appear to be a common finding in oral squamous cell carcinomas and that a clear association between intranuclear beta-catenin and histopathological and malignancy indexes in vivo could not be established. [source] MicroCommentary: Subcellular localization of Escherichia coli osmosensory transporter ProP: focus on cardiolipin membrane domainsMOLECULAR MICROBIOLOGY, Issue 6 2007Eugenia Mileykovskaya Summary The role for specific lipids in the spatial distribution of the membrane proteins and formation of the lipid-protein membrane domains is an emerging theme in the studies of the supramolecular organization of the bacterial cell. A combination of the lipid and protein visualization techniques with manipulation of the cell lipid composition provides a useful tool for these studies. This MicroCommentary reviews the first experimental example demonstrating an involvement of the phospholipid cardiolipin in recruitment of a membrane protein (specifically H+ -osmoprotectant symporter ProP) to the Escherichia coli cell poles. The properties of cardiolipin domains employed in creating a specific environment for structural organization and function of membrane protein complexes are also discussed. [source] Cellular location and temperature-dependent assembly of IncHI1 plasmid R27-encoded TrhC-associated conjugative transfer protein complexesMOLECULAR MICROBIOLOGY, Issue 3 2001Matthew W. Gilmour Conjugal transfer of IncHI plasmid DNA between Gram-negative bacteria is temperature sensitive, as mating is optimal between 22°C and 30°C but is inhibited at 37°C. R27, isolated from Salmonella enterica serovar Typhi, is an IncHI1 plasmid of 180 kbp that has been sequenced completely. The gene encoding green fluorescent protein (GFP) was inserted into R27 in frame with trhC. TrhC is a mating pair formation (Mpf) protein that is essential for plasmid transfer and H-pilus production. Fluorescence microscopy allowed visualization of the TrhC,GFP fusion protein, and Escherichia coli cells were examined for the subcellular localization and temperature-dependent production of TrhC,GFP. At 27°C, TrhC,GFP was found at the periphery of cells as discrete foci, indicating an association of TrhC within protein complexes in the bacterial cell membrane, whereas at 37°C, little fluorescence was detected. These foci probably represent the intracellular position of protein complexes involved in conjugative transfer, as the formation of foci was dependent upon the presence of other Mpf proteins. During temperature shift experiments from 37°C to 27°C, a long lag period was required for generation of GFP foci. Conversely, during short shifts from 27°C to 37°C, the GFP foci remained stable. These results suggest that the expression of transfer genes in the Tra2 region of R27 is temperature dependent. Subcellular localization of TrhC was verified by cellular fractionation. Expression patterns of TrhC,GFP were confirmed with immunoblot analysis and reverse transcriptase,polymerase chain reaction (RT,PCR). These results allow us to propose mechanisms to explain the temperature-sensitive transfer of R27. [source] Free IAA in stigmas and styles during pollen germination and pollen tube growth of Nicotiana tabacumPHYSIOLOGIA PLANTARUM, Issue 1 2008Dan Chen Although many studies have emphasized the importance of auxin in plant growth and development, the thorough understanding of its effect on pollen,pistil interactions is largely unknown. In this study, we investigated the role of free IAA in pollen,pistil interactions during pollen germination and tube growth in Nicotiana tabacum L. through using histo and subcellular immunolocalization with auxin monoclonal antibodies, quantification by HPLC and ELISA together with GUS staining in DR5::GUS -transformed plants. The results showed that free IAA in unpollinated styles was higher in the apical part and basal part than in the middle part, and it was more abundant in the transmitting tissue (TT). At the stage of pollen germination, IAA reached its highest content in the stigma and was mainly distributed in TT. After the pollen tubes entered the styles, the signal increased in the part where pollen tubes would enter and then rapidly declined in the part where pollen tubes had penetrated. Subcellular localization confirmed the presence of IAA in TT cells of stigmas and styles. Accordingly, a schematic diagram summarizes the changing pattern of free IAA level during flowering, pollination and pollen tube growth. Furthermore, we presented evidence that low concentration of exogenous IAA could, to a certain extent, facilitate in vitro pollen tube growth. These results suggest that IAA may be directly or indirectly involved in the pollen,pistil interactions. Additionally, some improvements of the IAA immunolocalization technique were made. [source] Inactivation of the CTD phosphatase-like gene OsCPL1 enhances the development of the abscission layer and seed shattering in riceTHE PLANT JOURNAL, Issue 1 2010Hyeonso Ji Summary Although susceptibility to seed shattering causes severe yield loss during cereal crop harvest, it is an adaptive trait for seed dispersal in wild plants. We previously identified a recessive shattering locus, sh-h, from the rice shattering mutant line Hsh that carries an enhanced abscission layer. Here, we further mapped sh-h to a 34-kb region on chromosome 7 by analyzing 240 F2 plants and five F3 lines from the cross between Hsh and Blue&Gundil. Hsh had a point mutation at the 3, splice site of the seventh intron within LOC_Os07g10690, causing a 15-bp deletion of its mRNA as a result of altered splicing. Two transferred DNA (T-DNA) insertion mutants and one point mutant exhibited the enhanced shattering phenotype, confirming that LOC_Os07g10690 is indeed the sh-h gene. RNA interference (RNAi) transgenic lines with suppressed expression of this gene exhibited greater shattering. This gene, which encodes a protein containing a conserved carboxy-terminal domain (CTD) phosphatase domain, was named Oryza sativa CTD phosphatase-like 1 (OsCPL1). Subcellular localization and biochemical analysis revealed that the OsCPL1 protein is a nuclear phosphatase, a common characteristic of metazoan CTD phosphatases involved in cell differentiation. These results demonstrate that OsCPL1 represses differentiation of the abscission layer during panicle development. [source] Molecular cloning and characterization of OsCDase, a ceramidase enzyme from riceTHE PLANT JOURNAL, Issue 6 2008Mickael O. Pata Summary Sphingolipids are a structurally diverse group of molecules based on long-chain sphingoid bases that are found in animal, fungal and plant cells. In contrast to the situation in animals and yeast, much less is known about the spectrum of sphingolipid species in plants and the roles they play in mediating cellular processes. Here, we report the cloning and characterization of a plant ceramidase from rice (Oryza sativa spp. Japonica cv. Nipponbare). Sequence analysis suggests that the rice ceramidase (OsCDase) is similar to mammalian neutral ceramidases. We demonstrate that OsCDase is a bona fide ceramidase by heterologous expression in the yeast double knockout mutant ,ypc1,ydc1 that lacks the yeast ceramidases YPC1p and YDC1p. Biochemical characterization of OsCDase showed that it exhibited classical Michaelis,Menten kinetics, with optimum activity between pH 5.7 and 6.0. OsCDase activity was enhanced in the presence of Ca2+, Mg2+, Mn2+ and Zn2+, but inhibited in the presence of Fe2+. OsCDase appears to use ceramide instead of phytoceramide as a substrate. Subcellular localization showed that OsCDase is localized to the endoplasmic reticulum and Golgi, suggesting that these organelles are sites of ceramide metabolism in plants. [source] All-trans retinoic acid affects subcellular localization of a novel BmNIF3l protein: functional deduce and tissue distribution of NIF3l gene from silkworm (Bombyx mori),ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 4 2010Jianqing Chen Abstract A novel cDNA sequence encoding a predicted protein of 271 amino acids containing a conserved NIF3 domain was found from a pupal cDNA library of silkworm. The corresponding gene was named BmNIF3l (Bombyx mori NGG1p interacting factor 3-like). It was found by bioinformatics that BmNIF3l gene consisted of five exons and four introns and BmNIF3l had a high degree of homology to other NIF3-like proteins, especially in the N-terminal and C-terminal regions. A His-tagged BmNIF3l fusion protein with a molecular weight of approximately 33.6,kDa was expressed and purified to homogeneity. We have used the purified fusion protein to produce polyclonal antibodies against BmNIF3l for histochemical analysis. Subcellular localization revealed that BmNIF3l is a cytoplasmic protein that responds to all-trans retinoic acid (ATRA). Western blotting and real-time reverse transcription polymerase chain reaction showed that the expression level of BmNIF3l is higher in tissues undergoing differentiation. Taken together, the results suggest that BmNIF3l functions in transcription. © 2010 Wiley Periodicals, Inc. [source] Expression of Na+/HCO3, co-transporter proteins (NBCs) in rat and human skeletal muscleACTA PHYSIOLOGICA, Issue 1 2004J. M. Kristensen Abstract Aim:, Sodium/bicarbonate co-transport (NBC) has been suggested to have a role in muscle pH regulation. We investigated the presence of NBC proteins in rat and human muscle samples and the fibre type distribution of the identified NBCs. Methods and results:, Western blotting of muscle homogenates and sarcolemmal membranes (sarcolemmal giant vesicles) were used to screen for the presence of NBCs. Immunohistochemistry was used for the subcellular localization. The functional test revealed that approximately half of the pH recovery in sarcolemmal vesicles produced from rat muscle is mediated by bicarbonate-dependent transport. This indicates that the NBCs are preserved in the vesicles. The western blotting experiments demonstrated the existence of at least two NBC proteins in skeletal muscle. One NBC protein (approximately 150 kDa) seems to be related to the kidney/pancreas/heart isoform NBC1, whereas the other protein (approximately 200 kDa) is related to the NBC4 isoform. The two NBC proteins represent the electrogenic isoforms named NBCe1 and NBCe2. Membrane fractionation and immunofluorescence techniques confirmed that the two NBCs are located in the sarcolemmal membrane as well as in some internal membranes, probably the T-tubules. The two NBCs localized in muscle have distinct fibre type distributions. Conclusions:, Skeletal muscle possesses two variants of the sodium/bicarbonate co-transporter (NBC) isoforms, which have been called NBCe1 and NBCe2. [source] Connexins, cell motility, and the cytoskeletonCYTOSKELETON, Issue 11 2009Stephan Olk Abstract Connexins (Cx) comprise a family of transmembrane proteins, which form intercellular channels between plasma membranes of two adjoining cells, commonly known as gap junctions. Recent reports revealed that Cx proteins interact with diverse cellular components to form a multiprotein complex, which has been termed "Nexus". Potential interaction partners include proteins such as cytoskeletal proteins, scaffolding proteins, protein kinases and phosphatases. These interactions allow correct subcellular localization of Cxs and functional regulation of gap junction-mediated intercellular communication. Evidence is accruing that Cxs might have channel-independent functions, which potentially include regulation of cell migration, cell polarization and growth control. In the current review, we summarize recent knowledge on Cx interactions with cytoskeletal proteins and highlight some aspects of their role in cellular motility. Cell Motil. Cytoskeleton 66: 1000,1016, 2009. © 2009 Wiley-Liss, Inc. [source] In vivo phosphorylation of regulatory light chain of myosin II in sea urchin eggs and its role in controlling myosin localization and function during cytokinesisCYTOSKELETON, Issue 2 2008Ryota Uehara Abstract Phosphorylation of myosin regulatory light chain (RLC) at Ser19 (mono-phosphorylation) promotes filament assembly and enhances actin-activated ATPase activity of non-muscle myosin, while phosphorylation at both Ser19 and Thr18 (di-phosphorylation) further enhances the ATPase activity. However, it has not well been addressed which type of phosphorylation is important in regulating myosin during cytokinesis. Here, we investigated subcellular localization in sea urchin eggs of mono-phosphorylated and di-phosphorylated RLC by both quantitative biochemical and spatiotemporal cytological approaches. Mono-phosphorylated RLC was dominant in the equatorial cortex throughout the whole process of cytokinesis. Inhibition of myosin light chain kinase (MLCK) decreased mono-phosphorylated RLC both in the cortex and in the cleavage furrow, and blocked both formation and contraction of the contractile ring. Two different types of ROCK inhibitor gave inconsistent results: H1152 blocked both RLC mono-phosphorylation in the cleavage furrow and contraction of the contractile ring, while Y27632 affected neither the mono-phosphorylation nor cell division. These results suggest that there may be other targets of H1152 than ROCK, which is involved in the RLC phosphorylation in the cleavage furrow. Furthermore, it was revealed that localization of myosin heavy chain in the cleavage furrow, but not in the cortex, was perturbed by inhibition of RLC mono-phosphorylation. These results suggested that RLC mono-phosphorylation by more than two RLC kinases play a main role in regulation and localization of myosin in the dividing sea urchin eggs. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source] Xenopus axin-related protein: A link between its centrosomal localization and function in the Wnt/,-catenin pathwayDEVELOPMENTAL DYNAMICS, Issue 1 2010Evguenia M. Alexandrova Abstract The Wnt/,-catenin signaling pathway regulates cell proliferation and cell fate determination in multiple systems. However, the subcellular localization of Wnt pathway components and the significance of this localization for the pathway regulation have not been extensively analyzed. Here we report that Xenopus Axin-related protein (XARP), a component of the ,-catenin destruction complex, is localized to the centrosome. This localization of XARP requires the presence of the DIX domain and an adjacent region. Since other components of the Wnt pathway have also been shown to associate with the centrosome, we tested a hypothesis that the ,-catenin destruction complex operates at the centrosome. However, XARP mutants with poor centrosomal localization revealed an enhanced rather than decreased ability to antagonize the Wnt/,-catenin pathway. Our data are consistent with the idea that the inactivation of XARP at the centrosome is an important regulatory point in Wnt signaling. Developmental Dynamics 239:261,270, 2010. © 2009 Wiley-Liss, Inc. [source] The role of twist during palate development,DEVELOPMENTAL DYNAMICS, Issue 10 2008Wenli Yu Abstract In palatogenesis, the MEE (Medial Edge Epithelium) cells disappear when palates fuse. We hypothesize that the MEE cells undergo EMT (Epithelial-Mesenchymal Transition) to achieve mesenchyme confluence. Twist has an important role in EMT for tumor metastasis. The purpose of this study was to analyze Twist function during palatal fusion. Twist protein was expressed in palatal shelves and MEE both in vivo and in vitro just prior to fusion. Twist mRNA increased in chicken palates 3 and 6 hr after TGF,3 treatment. Palatal fusion was decreased when cultured palatal shelves were treated with 200 nM Twist siRNA and the subcellular localization of ,-catenin was altered. Twist mRNA decreased in palatal shelves treated with TGF,3 neutralizing antibody or LY294002, a specific phosphatidylinositol-3 kinase (PI-3K) inhibitor. In summary, Twist is downstream of TGF,3 and PI-3K pathways during palatal fusion. However, decreasing Twist with siRNA did not completely block palate fusion, indicating that the function of Twist may be duplicated by other transcription factors. Developmental Dynamics 237:2716,2725, 2008. © 2008 Wiley-Liss, Inc. [source] Asymmetric localization of numb in the chick somite and the influence of myogenic signalsDEVELOPMENTAL DYNAMICS, Issue 3 2006Tamara Holowacz Abstract Whereas Notch signaling is known to play an essential role in the formation of somites, its role during later stages of somite maturation is less well understood. Here, we examine the signals and transcription factors that control the expression of the Notch antagonist, Numb, during somite maturation in the chick embryo. Numb mRNA is present in the epithelial somite and is increased in expression in the forming myotome. Numb protein displays a very specific subcellular localization and dynamic expression during somite maturation. Numb protein is asymmetrically localized in a cortical crescent on the basal side of dividing cells in the dorsomedial lip of the dermomyotome and is subsequently uniformly distributed throughout differentiated myotomal cells. Treatment of somites with either the combination of Wnt-3a and Shh, or ectodermal signals plus noggin, both of which induce somitic myogenesis, did not significantly affect Numb transcript levels but did lead to a dramatic increase in the levels of Numb protein, which was uniformly distributed throughout the cytoplasm of the resultant myotubes. Forced expression of MyoD in somites similarly induced high levels of Numb protein throughout the cytoplasm, without affecting Numb mRNA levels. We also found that signals that promote somitic myogenesis or forced MyoD expression induced expression of the Notch ligand, Serrate-2. Our findings suggest that Notch signals are specifically repressed in the myotome and that asymmetric expression of Numb in dividing cells of the dorsomedial lip of the dermomyotome may modulate whether these cells continue to divide or differentiate into myotomal cells. Developmental Dynamics 235:633,645, 2006. © 2006 Wiley-Liss, Inc. [source] Identification of BOIP, a novel cDNA highly expressed during spermatogenesis that encodes a protein interacting with the orange domain of the hairy-related transcription factor HRT1/Hey1 in Xenopus and mouseDEVELOPMENTAL DYNAMICS, Issue 4 2003Reginald Van Wayenbergh Abstract Hairy-related transcription factor (HRT/Hey) genes encode a novel subfamily of basic helix-loop-helix (bHLH) transcription factors related to the Drosophila hairy and Enhancer-of-split (E(spl)) and the mammalian HES proteins that function as downstream mediators of Notch signaling. Using the yeast two-hybrid approach, a previously uncharacterized protein was identified in Xenopus that interacts with XHRT1 (originally referred to as bc8), one member of the HRT/Hey subclass. This protein is evolutionarily conserved in chordates. It binds to sequences adjacent to the bHLH domain of XHRT1 known as the Orange domain and has been named bc8 Orange interacting protein (BOIP). BOIP shows a rather uniform subcellular localization and is recruited to the nucleus upon binding to XHRT1. In Xenopus, XBOIP mRNA is detected by RNase protection analysis throughout embryogenesis. In the adult, the strongest expression is detected in testis. In the mouse, high levels of BOIP mRNA are also found in adult testis. No expression is detected in the embryo and in any of the other adult organs tested. In situ hybridization revealed that BOIP transcripts were detected almost exclusively in round spermatids and that this expression overlaps with that of Hey1 (HRT1), which is expressed throughout spermatogenesis. In view of the importance of the Orange domain for HRT/Hey function, the newly identified BOIP proteins may serve as regulators specifically of HRT1/Hey1 activity. Developmental Dynamics 228:716,725, 2003. © 2003 Wiley-Liss, Inc. [source] Protein kinase C and extracellular signal regulated kinase are involved in cardiac hypertrophy of rats with progressive renal injuryEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 2 2004H. Takahashi Abstract Increased cardiovascular mortality is an unresolved problem in patients with chronic renal failure. Cardiac hypertrophy is observed in the majority of patients with chronic renal failure undergoing haemodialysis. However, the mechanisms, including signal transduction pathways, responsible for cardiac hypertrophy in renal failure remain unknown. We examined the subcellular localization of protein kinase C (PKC) isoforms and phosphorylation activities of 3 mitogen-activated protein (MAP) kinase families in hypertrophied hearts of progressive renal injury rat model by subtotal nephrectomy (SNx). We also examined the effects of a novel angiotensin II type-1 receptor antagonist, CS-866, on the PKC translocation, MAP kinase activity and cardiac hypertrophy in SNx rats. The left ventricle/body weight ratios were significantly larger in SNx rats than in sham rats at 1, 2, and 4 weeks after surgery. The translocation of PKC, and , isoforms to membranous fraction was observed in SNx rat hearts at 1, 2, and 4 weeks after surgery. Activation of extracellular signal regulated kinase (ERK) 1/2, but not p38 MAP kinase and c-Jun N-terminal kinase (JNK), was observed at 1 and 2 weeks after surgery. Angiotensin II receptor blockade with CS-866 (1 mg kg,1 day,1) prevented cardiac hypertrophy, PKC translocation and ERK1/2 activation in SNx rats without significant changes in blood pressure. These data suggest that PKC and ERK1/2 are activated by an angiotensin II receptor-mediated pathway and might play an important role in the progression of cardiac hypertrophy in renal failure. [source] Constitutive expression of the FK506 binding protein 51 (FKBP51) in bone marrow cells and megakaryocytes derived from idiopathic myelofibrosis and non-neoplastic haematopoiesisEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 4 2004Oliver Bock Abstract: Objectives:, Overexpression of FK506 binding protein 51 (FKBP51) in megakaryocytic progenitor cells generated from purified CD34+ cells in patients with idiopathic myelofibrosis (IMF) has been demonstrated. It has been suggested that FKBP51 is involved in the dysregulation of the apoptotic programme with consecutive prolongation of cell survival. The knowledge of FKBP51 and its expression in bone marrow cells and mature megakaryocytes in non-neoplastic haematopoiesis and IMF is sparse. Methods:, To evaluate a potential overexpression of FKBP51 in patients with IMF (n = 37) compared with non-neoplastic haematopoiesis (n = 31), total bone marrow cells as well as single megakaryocytes, isolated by laser microdissection, were quantitatively analysed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). By applying immunohistochemistry, FKBP51 gene expression was correlated with staining pattern and cellular localisation of the corresponding FKBP51 protein. Results:, We demonstrated that FKBP51 is constitutively expressed in non-neoplastic haematopoiesis. FKBP51 gene expression by total bone marrow cells as well as megakaryocytes was not significantly different in IMF. FKBP51 protein expression could be localised to myeloid progenitor cells as well as megakaryocytes. In particular, megakaryocytes were stained almost exclusively nuclear for FKBP51. No differences in expression patterns between both IMF and control cases could be demonstrated. Conclusions:, For the first time, FKBP51 expression, in particular gene expression and subcellular localization was described in bone marrow cells of non-neoplastic and neoplastic haematopoiesis grown in vivo. We conclude that FKBP51 could be temporarily overexpressed in megakaryocytic progenitors rather than contribute to the accumulation of mature megakaryocytes in IMF. [source] SUMOylation attenuates c-Maf-dependent IL-4 expressionEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 4 2010Bo-Shiou Lin Abstract The function of transcription factors can be critically regulated by SUMOylation. c-Maf, the cellular counterpart of v-maf oncogene, is a potent transactivator of the IL-4 gene in Th2 cells. We found in a yeast two-hybrid screen that c-Maf can interact with Ubc9 and PIAS1, two key enzymes of the SUMOylation pathway. In this study, we report that c-Maf co-localized with these two SUMO (small ubiquitin-like modifier) ligases in the nucleus and that c-Maf can be SUMOylated in vitro and also in primary Th2 cells. We also demonstrated that lysine-33 is the dominant, if not the only, SUMO acceptor site of c-Maf. SUMOylation of c-Maf attenuated its transcriptional activity. Reciprocally, a SUMOylation resistant c-Maf was more potent than WT-c-Maf in driving IL-4 production in c-Maf-deficient Th2 cells. Furthermore, we showed that ablation of the SUMO site did not alter the subcellular localization or the stability of c-Maf protein but instead enhanced its recruitment to the Il4 -promoter. We conclude that SUMOylation at lysine-33 is a functionally critical post-translational modification event of c-Maf in Th cells. [source] Overexpression of GAP-43 modifies the distribution of the receptors for myelin-associated growth-inhibitory proteins in injured Purkinje axonsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2009Simona Foscarin Abstract Neurons with enhanced intrinsic growth capabilities can elongate their axons into non-permissive territories, but the mechanisms that enable the outgrowing processes to overcome environmental inhibition are largely unknown. To address this issue, we examined adult mouse Purkinje cells that overexpress the axonal growth-associated protein GAP-43. After injury, these neurons exhibit sprouting along the intracortical neuritic course and at the severed stump in the white matter. To determine whether GAP-43-overexpressing Purkinje cells are responsive to extrinsic inhibitory cues, we investigated the content and subcellular localization of major receptors for myelin-associated inhibitory proteins, PlexinB1 and the Nogo receptor (NgR) with the related co-receptors LINGO-1 and p75. Expression of these molecules, estimated by measuring perikaryal immunostaining intensity and Western blot, was not different in wild-type or transgenic mice, and it was not overtly modified after axotomy. Following injury, however, the content of PlexinB1 was significantly reduced in GAP-43-overexpressing neurites. Furthermore, in the same axons the distribution of both PlexinB1 and NgR was altered, being inverse to that of GAP-43. Labelling for the two receptors was conspicuously reduced on the axonal surface and it was almost undetectable in the outgrowing sprouts, which showed strong GAP-43 immunoreactivity. These observations indicate that although GAP-43 overexpression does not modify the expression of receptors for myelin-associated inhibitory factors, it interferes with their subcellular localization and exposure on the neuritic membrane. Therefore, GAP-43 promotes axon growth by multiple synergistic mechanisms that potentiate the intrinsic motility of the elongating processes, while reducing their sensitivity to environmental inhibition. [source] The type 1 cannabinoid receptor is highly expressed in embryonic cortical projection neurons and negatively regulates neurite growth in vitroEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2008Tania Vitalis Abstract In the rodent and human embryonic brains, the cerebral cortex and hippocampus transiently express high levels of type 1 cannabinoid receptors (CB1Rs), at a developmental stage when these areas are composed mainly of glutamatergic neurons. However, the precise cellular and subcellular localization of CB1R expression as well as effects of CB1R modulation in this cell population remain largely unknown. We report that, starting from embryonic day 12.5, CB1Rs are strongly expressed in both reelin-expressing Cajal-Retzius cells and newly differentiated postmitotic glutamatergic neurons of the mouse telencephalon. CB1R protein is localized first to somato-dendritic endosomes and at later developmental stages it localizes mostly to developing axons. In young axons, CB1Rs are localized both to the axolemma and to large, often multivesicular endosomes. Acute maternal injection of agonist CP-55940 results in the relocation of receptors from axons to somato-dendritic endosomes, indicating the functional competence of embryonic CB1Rs. The adult phenotype of CB1R expression is established around postnatal day 5. By using pharmacological and mutational modulation of CB1R activity in isolated cultured rat hippocampal neurons, we also show that basal activation of CB1R acts as a negative regulatory signal for dendritogenesis, dendritic and axonal outgrowth, and branching. Together, the overall negative regulatory role in neurite development suggests that embryonic CB1R signaling may participate in the correct establishment of neuronal connectivity and suggests a possible mechanism for the development of reported glutamatergic dysfunction in the offspring following maternal cannabis consumption. [source] The spatio-temporal and subcellular expression of the candidate Down syndrome gene Mnb/Dyrk1A in the developing mouse brain suggests distinct sequential roles in neuronal developmentEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2008Barbara Hämmerle Abstract It is widely accepted that the neurological alterations in Down syndrome (DS) are principally due to modifications in developmental processes. Accordingly, a large part of the research on DS in recent years has focused on chromosome 21 genes that influence brain development. MNB/DYRK1A is one of the genes on human chromosome 21 that has raised most interest, due to its relationship with the brain functions that are altered in DS. Although a number of interesting experimental mouse models for DS are being developed, we still know little about the expression of Mnb/Dyrk1A during mouse brain development. Here, we report that Mnb/Dyrk1A displays a rather dynamic spatio-temporal expression pattern during mouse central nervous system development. Our data indicate that Mnb/Dyrk1A is specifically expressed in four sequential developmental phases: transient expression in preneurogenic progenitors, cell cycle-regulated expression in neurogenic progenitors, transient expression in recently born neurones, and persistent expression in late differentiating neurones. Our results also suggest that the subcellular localization of MNB/DYRK1A, including its translocation to the nucleus, is finely regulated. Thus, the MNB/DYRK1A protein kinase could be a key element in the molecular machinery that couples sequential events in neuronal development. This rich repertoire of potential functions in the developing central nervous system is suitable to be linked to the neurological alterations in DS through the use of mouse experimental models. [source] Regulation of ,FosB transcriptional activity by Ser27 phosphorylationEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2007Paula G. Ulery Abstract The transcription factor, ,FosB, is an important mediator of the long-term plasticity induced in brain by chronic exposure to drugs of abuse, stress, or several other psychoactive stimuli. We have previously demonstrated that the casein kinase 2 (CK2)-mediated phosphorylation of a highly conserved N-terminal serine (Ser27) plays a critical role in regulating ,FosB's unusual stability, while it does not affect that of the full-length FosB protein. In the present study, we analysed whether CK2 and Ser27 phosphorylation also play a role in regulating ,FosB's transcriptional activity. Our findings indicate that CK2 activation increases ,FosB's transactivation potential, while CK2 inhibition decreases it. Further, we show that preventing Ser27 phosphorylation by mutating the site to Ala results in a significant decrease in ,FosB transactivation, without affecting ,FosB's subcellular localization or DNA-binding affinity. In contrast, Ser27 does not seem to play a role in the transactivation potential of full-length FosB. These findings constitute the first evidence of a role for phosphorylation in ,FosB's transcriptional activity. [source] Central nervous system neurons acquire mast cell products via transgranulationEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2005M. Wilhelm Abstract Resting and actively degranulating mast cells are found on the brain side of the blood,brain barrier. In the periphery, exocytosis of mast cell granules results in the release of soluble mediators and insoluble granule remnants. These mast cell constituents are found in a variety of nearby cell types, acquired by fusion of granule and cellular membranes or by cellular capture of mast cell granule remnants. These phenomena have not been studied in the brain. In the current work, light and electron microscopic studies of the medial habenula of the dove brain revealed that mast cell-derived material can enter neurons in three ways: by direct fusion of the granule and plasma membranes (mast cell and neuron); by capture of insoluble granule remnants and, potentially, via receptor-mediated endocytosis of gonadotropin-releasing hormone, a soluble mediator derived from the mast cell. These processes result in differential subcellular localization of mast cell material in neurons, including free in the neuronal cytoplasm, membrane-bound in granule-like compartments or in association with small vesicles and the trans-Golgi network. Capture of granule remnants is the most frequently observed form of neuronal acquisition of mast cell products and correlates quantitatively with mast cells undergoing piecemeal degranulation. The present study indicates that mast cell-derived products can enter neurons, a process termed transgranulation, indicating a novel form of brain,immune system communication. [source] Activity-dependent subcellular localization of NAC1EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2005Laxman Korutla Abstract The expression of the transcriptional regulator NAC1 is increased in the nucleus accumbens of rats withdrawn from cocaine self-administration, and in vivo studies indicate that the up-regulation is a compensatory mechanism opposing the acute effects of cocaine. Both mammalian two-hybrid assay and punctate localization largely in the nucleus suggest NAC1 is a transcriptional regulator. However, in this report it is shown that in differentiated PC12 and Neuro2A cells, as well as in primary cortical neurons, NAC1 is diffusely expressed not only in the cell nucleus but also in cytoplasm. Blockade of spontaneous electrical activity by tetrodotoxin prevented the diffuse expression of NAC1, and depolarization with high potassium concentrations induced diffuse cellular localization in non-differentiating cells. The use of protein kinase C (PKC) inhibitors and activator, as well as the systematic mutation of potential PKC phosphorylation sites in NAC1, demonstrated that phosphorylation of residue S245 by PKC is a necessary event inducing diffuse NAC1 expression outside of the nucleus. These observations indicate a potential non-transcriptional role for NAC1 in the brain. [source] Distribution and regulation of L-type calcium channels in deep dorsal horn neurons after sciatic nerve injury in ratsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2005E. Dobremez Abstract Deep dorsal horn neurons are involved in the processing of nociceptive information in the spinal cord. Previous studies revealed a role of the intrinsic bioelectrical properties (plateau potentials) of deep dorsal horn neuron in neuronal hyperexcitability, indicating their function in pain sensitization. These properties were considered to rely on L -type calcium currents. Two different isotypes of L -type calcium channel alpha 1 subunit have been cloned (CaV1.2 and CaV1.3). Both are known to be expressed in the spinal cord. However, no data were available on their subcellular localization. Moreover, possible changes in CaV1.2 and CaV1.3 expression had never been investigated in nerve injury models. Our study provides evidence for a differential expression of CaV1.2 and CaV1.3 subunits in the somato-dendritic compartment of deep dorsal horn neurons. CaV1.2 immunoreactivity is restricted to the soma and proximal dendrites whereas CaV1.3 immunoreactivity is found in the whole somato-dendritic compartment, up to distal dendritic segments. Moreover, these specific immunoreactive patterns are also found in electrophysiologically identified deep dorsal horn neurons expressing plateau potentials. After nerve injury, namely total axotomy or partial nerve ligation, CaV1.2 and CaV1.3 expression undergo differential changes, showing up- and down-regulation, respectively, both at the protein and at the mRNA levels. Taken together, our data support the role of L-type calcium channels in the control of intrinsic biolectrical regenerative properties. Furthermore, CaV1.2 and CaV1.3 subunits may have distinct and specific roles in sensory processing in the dorsal horn of the spinal cord, the former being most likely involved in long-term changes after nerve injury. [source] | ||||||||||||||||||||||||||||||||||