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Cellular Context (cellular + context)
Selected AbstractsPim-1 kinase phosphorylates and stabilizes RUNX3 and alters its subcellular localizationJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2008Hye-Ryun Kim Abstract The loci of the Pim and Runx gene families have been identified as targets for viral insertions in CD2-myc mice. Synergistic cooperation between Pim and RUNX was also found in the CD2-Runx2 transgenic mouse lymphoma model. RUNX genes have come to prominence recently because of their roles as essential regulators of cell fate in development. Paradoxically, they appear to function either as tumor-suppressor genes or dominant oncogenes according to the cellular context. However, the molecular mechanism of the ambiguous roles played by this family of transcription factors in cancer has remained largely uninvestigated. Here we demonstrate that Pim-1 phosphorylates four Ser/Thr residues within the Runt domain and stabilizes RUNX3 protein. In addition, Pim-1 markedly altered the cellular localization of RUNX3 from the nucleus to the cytoplasm. Our results demonstrate that the subcellular localization of RUNX3 is altered by phosphorylation. We propose that RUNX family members may behave as oncogenes if mislocalized to a cellular micro-compartment. J. Cell. Biochem. 105: 1048,1058, 2008. © 2008 Wiley-Liss, Inc. [source] Kinase suppressor of RAS (KSR) amplifies the differentiation signal provided by low concentrations 1,25-dihydroxyvitamin D3JOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2004Xuening Wang The activity of kinase suppressor of ras (KSR), a kinase or a molecular scaffold upstream from Raf-1, is involved in the MEK/ERK MAP kinase cascade which can signal cell growth, survival, or differentiation, depending on the cellular context. We provide evidence here that KSR is upregulated in HL60 cells undergoing differentiation induced by low (0.3,3 nM) concentrations of 1,25-dihydroxyvitamin D3 (1,25D3), and an antisense oligo (AS), but not a sense oligo, to KSR inhibits this differentiation. The inhibition of differentiation by AS,KSR oligo was less apparent when the concentration of 1,25D3 was increased, suggesting that at the higher concentrations of 1,25D3 KSR is not essential for the signaling of the differentiated phenotype. The reduced differentiation of HL60 cells exposed to AS,KSR was paralleled by reduced phosphorylation of Raf-1 Ser 259, and of p90RSK, used here as read-out for MAPK cascade activity. Conversely, ectopic expression of Flag-tagged wild type KSR potentiated the differentiation-inducing effects of low concentrations of 1,25D3. Additional data suggest that the kinase activity of KSR is required for these effects, as transfection of a kinase inactive KSR construct did not significantly increase the 1,25D3 -induced differentiation. Enzyme assays performed with KSR immunoprecipitated from 1,25D3 -treated cells showed kinase activity when recombinant Raf-1 was used as the substrate, but not when the 1,25D3 -treated cells were pretreated with AS,KSR oligos. Taken together, these data suggest that KSR participates in signaling of monocytic differentiation by augmenting the strength of the signal transmitted through Raf-1 to downstream targets. J. Cell. Physiol. 198: 333,342, 2004© 2003 Wiley-Liss, Inc. [source] Electron Tomography in Plant Cell BiologyJOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 8 2007Thomas J. Haas Abstract This review focuses on the contribution of electron tomography-based techniques to our understanding of cellular processes in plant cells. Electron microscopy techniques have evolved to provide better three-dimensional resolution and improved preservation of the subcellular components. In particular, the combination of cryofixation/freeze substitution and electron tomography have allowed plant cell biologists to image organelles and macromolecular complexes in their native cellular context with unprecedented three-dimensional resolution (4,7 nm). Until now, electron tomography has been applied in plant cell biology for the study of cytokinesis, Golgi structure and trafficking, formation of plant endosome/prevacuolar compartments, and organization of photosynthetic membranes. We discuss in this review the new insights that these tomographic studies have brought to the plant biology field. [source] Nerve Growth Factor-Induced Differentiation Does Not Alter the Biochemical Properties of a Mutant Prion Protein Expressed in PC12 CellsJOURNAL OF NEUROCHEMISTRY, Issue 1 2000Roberto Chiesa Abstract : Insertional and point mutations in the gene encoding the prion protein (PrP) are responsible for familial prion diseases. We have previously generated lines of Chinese hamster ovary cells that express PrP molecules carrying pathogenic mutations, and found that the mutant proteins display several biochemical properties reminiscent of PrPSc, the infectious isoform of PrP. To analyze the properties and effects of mutant PrP molecules expressed in cells with a neuronal phenotype, we have constructed stably transfected lines of PC12 cells that synthesize a PrP molecule carrying a nine-octapeptide insertion. We report here that this mutant PrP acquires scrapie-like properties, including detergent insolubility, protease resistance, and resistance to phospholipase cleavage of its glycolipid anchor. A detergent-insoluble and phospholipase-resistant form of the mutant protein is also released spontaneously into conditioned medium. These scrapie-like biochemical properties are quantitatively similar to those seen in Chinese hamster ovary cells and are not affected by differentiation of the PC12 cells into sympathetic neurons by nerve growth factor. Moreover, there is no detectable effect of mutant PrP expression on the morphology or viability of the cells in either the differentiated or undifferentiated state. These results indicate that conversion of mutant PrP into a PrPSc -like form does not depend critically on the cellular context, and they suggest that mutant PrP expressed in cultured cells, even those having the phenotype of differentiated neurons, is not neurotoxic. [source] Mechanisms of neurodegenerative diseases: Insights from live cell imagingJOURNAL OF NEUROSCIENCE RESEARCH, Issue 3 2008Carina Weissmann Abstract Pathologic alterations in protein dynamics such as changes in protein degradation, accumulation of misfolded proteins, and deficits in cellular transport mechanisms are a common feature of most if not all neurodegenerative diseases. Live cell imaging studies promise to contribute to a better understanding of the molecular mechanisms underlying these diseases by visualizing the turnover, accumulation, and transport of proteins in a living cellular context in real time. In this review, we discuss recent work in which different live cell imaging approaches are applied in cellular models of amyotrophic lateral sclerosis, polyQ diseases, and tauopathies as paradigmatic examples of diseases with different types of alterations in protein dynamics. It becomes evident that live cell imaging studies provide new insights into different aspects of protein dynamics, such as the understanding that aggregates are not as static as concluded from previous studies but exhibit a remarkable molecular exchange and that the dynamicity state of the neuronal cytoskeleton might have a critical role in neuronal degeneration. It can be anticipated that live cell imaging studies will lead to a more dynamic view of protein turnover and aggregation, which may aid in identifying drugs that specifically interfere with disease-related changes. © 2007 Wiley-Liss, Inc. [source] Notch signaling promotes astrogliogenesis via direct CSL-mediated glial gene activationJOURNAL OF NEUROSCIENCE RESEARCH, Issue 6 2002Weihong Ge Abstract In the developing central nervous system (CNS), Notch signaling preserves progenitor pools and inhibits neurogenesis and oligodendroglial differentiation. It has recently been postulated that Notch instructively drives astrocyte differentiation. Whether the role of Notch signaling in promoting astroglial differentiation is permissive or instructive has been debated. We report here that the astrogliogenic role of Notch is in part mediated by direct binding of the Notch intracellular domain to the CSL DNA binding protein, forming a transcriptional activation complex onto the astrocyte marker gene, glial fibrillary acidic protein (GFAP). In addition, we found that, in CSL,/, neural stem cell cultures, astrocyte differentiation was delayed but continued at a normal rate once initiated, suggesting that CSL is involved in regulating the onset of astrogliogenesis. Importantly, although the classical CSL-dependent Notch signaling pathway is intact and able to activate the Notch canonical target promoter during the neurogenic phase, it is unable to activate the GFAP promoter during neurogenesis. Therefore, the effect of Notch signaling on target genes is influenced by cellular context in regulation of neurogenesis and gliogenesis. © 2002 Wiley-Liss, Inc. [source] Laminar variation in neuronal viability and trophic dependence in neocortical slicesJOURNAL OF NEUROSCIENCE RESEARCH, Issue 5 2001Mary M. Niblock Abstract Organotypic slices are used frequently in studies of central nervous system development and function because they provide excellent experimental access with significant preservation of cellular context and relationships. Within a slice, however, a variety of factors may cause individual classes of neurons to respond differently to the culture environment. Differences in deafferentation, cellular maturation, trophic dependence and ongoing naturally occurring cell death may produce changes in the neuronal population that are transparent to the experimenter but that could affect experimental results significantly. In this study, we examined the distribution and prevalence of cell death among neurons in each cortical layer in organotypic slices. In addition, we assessed the ability of several neurotrophic factors to ameliorate neuronal death in each cortical layer. Within the first 24 hr in culture, there was striking laminar variation in the extent of neuronal death in culture, which could not be accounted for by the pattern of programmed cell death in vivo. In addition, neurons in the six layers of the neocortex differed in the degree to which they could be rescued by neurotrophic factors. These data suggest that differential neuronal death and rescue are important considerations in studies utilizing organotypic slices and may represent particularly confounding variables in studies of effects of trophic factors in such preparations. J. Neurosci. Res. 65:455,462, 2001. © 2001 Wiley-Liss, Inc. [source] Multiple large filament bundles observed in Caulobacter crescentus by electron cryotomographyMOLECULAR MICROBIOLOGY, Issue 1 2006Ariane Briegel Summary While the absence of any cytoskeleton was once recognized as a distinguishing feature of prokaryotes, it is now clear that a number of different bacterial proteins do form filaments in vivo. Despite the critical roles these proteins play in cell shape, genome segregation and cell division, molecular mechanisms have remained obscure in part for lack of electron microscopy-resolution images where these filaments can be seen acting within their cellular context. Here, electron cryotomography was used to image the widely studied model prokaryote Caulobacter crescentus in an intact, near-native state, producing three-dimensional reconstructions of these cells with unprecedented clarity and fidelity. We observed many instances of large filament bundles in various locations throughout the cell and at different stages of the cell cycle. The bundles appear to fall into four major classes based on shape and location, referred to here as ,inner curvature', ,cytoplasmic', ,polar' and ,ring-like'. In an attempt to identify at least some of the filaments, we imaged cells where crescentin and MreB filaments would not be present. The inner curvature and cytoplasmic bundles persisted, which together with their localization patterns, suggest that they are composed of as-yet unidentified cytoskeletal proteins. Thus bacterial filaments are frequently found as bundles, and their variety and abundance is greater than previously suspected. [source] Experimental and computational tools useful for (re)construction of dynamic kinase,substrate networksPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 23 2009Chris Soon Heng Tan Abstract The explosion of site- and context-specific in vivo phosphorylation events presents a potentially rich source of biological knowledge and calls for novel data analysis and modeling paradigms. Perhaps the most immediate challenge is delineating detected phosphorylation sites to their effector kinases. This is important for (re)constructing transient kinase,substrate interaction networks that are essential for mechanistic understanding of cellular behaviors and therapeutic intervention, but has largely eluded high-throughput protein-interaction studies due to their transient nature and strong dependencies on cellular context. Here, we surveyed some of the computational approaches developed to dissect phosphorylation data detected in systematic proteomic experiments and reviewed some experimental and computational approaches used to map phosphorylation sites to their effector kinases in efforts aimed at reconstructing biological signaling networks. [source] Sequence-specific inhibition of RNA polymerase III-dependent transcription using Zorro locked nucleic acid (LNA)THE JOURNAL OF GENE MEDICINE, Issue 1 2008Rongbin Ge Abstract Background RNA polymerase III (pol III)-dependent transcripts are involved in many fundamental activities in a cell, such as splicing and protein synthesis. They also regulate cell growth and influence tumor formation. During recent years vector-based systems for expression of short hairpin (sh) RNA under the control of a pol III promoter have been developed as gene-based medicines. Therefore, there is an increasing interest in means to regulate pol III-dependent transcription. Recently, we have developed a novel anti-gene molecule ,Zorro LNA (Locked Nucleic Acid)', which simultaneously hybridizes to both strands of super-coiled DNA and potently inhibits RNA polymerase II-derived transcription. We have now applied Zorro LNA in an attempt to also control U6 promoter-driven expression of shRNA. Methods In this study, we constructed pshluc and pshluc2BS plasmids, in which U6 promoter-driven small hairpin RNA specific for luciferase gene (shluc) was without or with Zorro LNA binding sites, respectively. After hybridization of Zorro LNA to pshluc2BS, the LNA-bound plasmid was cotransfected with pEGFPluc into mammalian cells and into a mouse model. In cellular experiments, cotransfection of unhybridized pshluc2BS, Zorro LNA and pEGFPluc was also performed. Results The results showed that the Zorro LNA construct efficiently inhibited pol III-dependent transcription as an anti-gene reagent in a cellular context, including in vivo in a mouse model. Conclusions Thus, this new form of gene silencer ,Zorro LNA' could potentially serve as a versatile regulator of pol III-dependent transcription, including various forms of shRNAs. Copyright © 2007 John Wiley & Sons, Ltd. [source] Rho kinase,dependent activation of SOX9 in chondrocytesARTHRITIS & RHEUMATISM, Issue 1 2010Dominik R. Haudenschild Objective The transcription factor SOX9 directly regulates the expression of the major proteoglycans and collagens comprising the cartilage extracellular matrix. The DNA binding activity and cellular localization of SOX9 is controlled through posttranslational modifications, including phosphorylation. The activity of Rho kinase (ROCK) has profound effects on the actin cytoskeleton, and these effects are instrumental in determining the phenotype and differentiation of chondrocytes. However, the mechanisms linking ROCK to altered chondrocyte gene expression remain unknown. The purpose of the present study was to test for a direct interaction between ROCK and SOX9. Methods Human SW1353 chondrosarcoma cells were transfected with constructs coding for RhoA, ROCK, Lim kinase, and SOX9. The interaction between ROCK and SOX9 was tested on purified proteins, and was verified within a cellular context using induced overexpression and activation of the Rho pathway. The effects of SOX9 transcriptional activation were quantified with a luciferase reporter plasmid containing SOX9 binding sites from the COL2A1 enhancer element. Results SOX9 was found to contain a consensus phosphorylation site for ROCK. In vitro, ROCK directly phosphorylated SOX9 at Ser181, and the overexpression of ROCK or the activation of the RhoA pathway in SW1353 chondrosarcoma cells increased SOX9Ser181 phosphorylation. ROCK caused a dose-dependent increase in the transcription of a SOX9-luciferase reporter construct, and increased phosphorylation and nuclear accumulation of SOX9 protein in response to transforming growth factor , treatment and mechanical compression. Conclusion These results demonstrate a new interaction that directly links ROCK to increased cartilage matrix production via activation of SOX9 in response to mechanical and growth factor stimulation. [source] Microbial systems engineering: First successes and the way aheadBIOESSAYS, Issue 4 2010Sven Dietz Abstract The first promising results from "streamlined," minimal genomes tend to support the notion that these are a useful tool in biological systems engineering. However, compared with the speed with which genomic microbial sequencing has provided us with a wealth of data to study biological functions, it is a slow process. So far only a few projects have emerged whose synthetic ambition even remotely matches our analytic capabilities. Here, we survey current technologies converging into a future ability to engineer large-scale biological systems. We argue that the underlying synthetic technology, de novo DNA synthesis, is already rather mature , in particular relative to the scope of our current synthetic ambitions. Furthermore, technologies towards rationalizing the design of the newly synthesized DNA fragment are emerging. These include techniques to implement complex regulatory circuits, suites of parts on a DNA and RNA level to fine tune gene expression, and supporting computational tools. As such DNA fragments will, in most cases, be destined for operating in a cellular context, attention has to be paid to the potential interactions of the host with the functions encoded on the engineered DNA fragment. Here, the need of biological systems engineering to deal with a robust and predictable bacterial host coincides with current scientific efforts to theoretically and experimentally explore minimal bacterial genomes. [source] Role of Network Branching in Eliciting Differential Short-Term Signaling Responses in the Hypersensitive Epidermal Growth Factor Receptor Mutants Implicated in Lung CancerBIOTECHNOLOGY PROGRESS, Issue 3 2008Jeremy Purvis We study the effects of EGFR inhibition in wild-type and mutant cell lines upon tyrosine kinase inhibitor TKI treatment through a systems level deterministic and spatially homogeneous model to help characterize the hypersensitive response of the cancer cell lines harboring constitutively active mutant kinases to inhibitor treatment. By introducing a molecularly resolved branched network systems model (the molecular resolution is introduced for EGFR reactions and interactions in order to distinguish differences in activation between wild-type and mutants), we are able to quantify differences in (1) short-term signaling in downstream ERK and Akt activation, (2) the changes in the cellular inhibition EC50 associated with receptor phosphorylation (i.e., 50% inhibition of receptor phosphorylation in the cellular context), and (3) EC50 for the inhibition of activated downstream markers ERK-(p) and Akt-(p), where (p) denotes phosphorylated, upon treatment with the inhibitors in cell lines carrying both wild-type and mutant forms of the receptor. Using the branched signaling model, we illustrate a possible mechanism for preferential Akt activation in the cell lines harboring the oncogenic mutants of EGFR implicated in non-small-cell lung cancer and the enhanced efficacy of the inhibitor erlotinib especially in ablating the cellular Akt-(p) response. Using a simple phenomenological model to describe the effect of Akt activation on cellular decisions, we discuss how this preferential Akt activation is conducive to cellular oncogene addiction and how its disruption can lead to dramatic apoptotic response and hence remarkable inhibitor efficacies. We also identify key network nodes of our branched signaling model through sensitivity analysis as those rendering the network hypersensitive to enhanced ERK-(p) and Akt-(p); intriguingly, the identified nodes have a strong correlation with species implicated in oncogenic transformations in human cancers as well as in drug resistance mechanisms identified for the inhibitors in non-small-cell lung cancer therapy. [source] Tankyrase is necessary for canonical Wnt signaling during kidney developmentDEVELOPMENTAL DYNAMICS, Issue 7 2010Courtney M. Karner Abstract Recent studies using small molecule antagonists have revealed that the poly(ADP-ribose) polymerases (PARPs) Tankyrase 1 and 2 are critical regulators of canonical Wnt signaling in some cellular contexts. However, the absence of any activity during zebrafish embryogenesis suggested that the tankyrases may not be general/core components of the Wnt pathway. Here, we show that Tnks1 and 2 are broadly expressed during mouse development and are essential during kidney and lung development. In the kidney, blockage of tankyrase activity phenocopies the effect of blocking production of all Wnt ligands. Tankyrase inhibition can be rescued by activation of ,-catenin demonstrating its specificity for the Wnt pathway. In addition, treatment with tankyrase inhibitors appears to be completely reversible in some cell types. These studies suggest that the tankyrases are core components of the canonical Wnt pathway and their inhibitors should enjoy broad usage as antagonists of Wnt signaling. Developmental Dynamics 239:2014,2023, 2010 © 2010 Wiley-Liss, Inc. [source] The HUPO Proteomics Standards Initiative , Overcoming the Fragmentation of Proteomics DataPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue S2 2006Henning Hermjakob Proteomics is a key field of modern biomolecular research, with many small and large scale efforts producing a wealth of proteomics data. However, the vast majority of this data is never exploited to its full potential. Even in publicly funded projects, often the raw data generated in a specific context is analysed, conclusions are drawn and published, but little attention is paid to systematic documentation, archiving, and public access to the data supporting the scientific results. It is often difficult to validate the results stated in a particular publication, and even simple global questions like ,In which cellular contexts has my protein of interest been observed?" can currently not be answered with realistic effort, due to a lack of standardised reporting and collection of proteomics data. The Proteomics Standards Initiative (PSI), a work group of the Human Proteome Organisation (HUPO), defines community standards for data representation in proteomics to facilitate systematic data capture, comparison, exchange and verification. In this article we provide an overview of PSI organisational structure, activities, and current results, as well as ways to get involved in the broad-based, open PSI process. [source] Genetic connections of the actin cytoskeleton and beyond,BIOESSAYS, Issue 5 2007Piergiorgio Percipalle Actin is a key protein in numerous cellular functions. One recent study has identified a large set of genes, associated with the actin cytoskeleton, which could be grouped into a wide spectrum of cytoplasmic and nuclear functions, such as protein biosynthesis and gene transcription.1 Deletions of many of the identified genes affected cellular actin organization,1 suggesting a functional link between different actin fractions probably regulated through changes in actin dynamics. The data are very exciting; speculations on the crosstalk between cytoplasmic and nuclear actin fractions in different cellular contexts may help placing the results in perspective to further understand how actin-mediated signalling affects cellular functions, such as gene expression. BioEssays 29:407,411, 2007. © 2007 Wiley Periodicals, Inc. [source] Protein interacting with C , kinase 1 (PICK1) is involved in promoting tumor growth and correlates with poor prognosis of human breast cancerCANCER SCIENCE, Issue 6 2010Bin Zhang Protein interacting with C , kinase 1 (PICK1), which interacts with multiple different proteins in a variety of cellular contexts, is believed to play important roles in diverse pathological conditions including cancer. In this study, we attempted to investigate the correlation of PICK1 with clinicopathological features as well as prognosis of human breast cancer. In addition, we aimed at a better understanding of the biological function of PICK1 in breast cancer cell biology. As judged by semi- quantitative RT-PCR and western blotting, PICK1 was overexpressed in tumor cells as compared to adjacent normal epithelia in breast, lung, gastric, colorectal, and ovarian cancer. As judged by immunostaining breast cancer tissue microarrays, high levels of PICK1 expression correlated with shortened span of overall survival (OS). Protein interacting with C , kinase 1 (PICK1) expression seemed to be specifically associated with reduced OS in lymph node-positive, Her/neu-2 positive, and the basal-like type subgroups, respectively. Consistently, the expression of PICK1 correlated with histological grade, lymph node metastasis, Her-2/neu-positivity, and triple-negative basal-like breast cancer. Protein interacting with C , kinase 1 (PICK1) was not correlated with menopausal status, tumor size, or hormone receptor status. In a complementary study, transfection of MDA-MB-231 cells with PICK1 siRNA decreased cell proliferation and colony formation in vitro and inhibited tumorigenicity in nude mice. Our clinical and experimental evidence supports an oncogenic role of PICK1 in human breast cancer. In particular, our data suggest that PICK1 promotes tumor cell proliferation. Taken together, PICK1 may serve not only as a marker for poor prognosis, but also as a therapeutic target in breast cancer. (Cancer Sci 2010) [source] | |||||||||||||||||||||||||