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Subcellular Locations (subcellular + locations)
Selected AbstractsDiverting a protein from its cellular location by intracellular antibodiesFEBS JOURNAL, Issue 4 2000The case of p21Ras We describe the use of phage libraries to derive new antibodies against p21Ras to be used for intracellular expression in mammalian cells. A panel of single-chain antibody fragments, binding to Ras, were analyzed and characterized for their capacity to interfere in vitro with (a) the intrinsic GTPase activity of Ras and (b) the binding of Ras to its effector Raf, and were found not to neutralize its function, according to these biochemical criteria. When expressed intracellularly in mouse 3T3 K-Ras transformed cells all the anti-Ras single-chain variable fragments (scFv) tested inhibited cell proliferation, as assessed by bromodeoxyuridine incorporation. Double immunofluorescence analysis of transfected cells using confocal microscopy confirmed that anti-Ras antibody fragments colocalize with endogenous Ras, at subcellular locations where the protein Ras is not normally found. These data suggest that the ability of phage-derived anti-Ras scFv fragments to inhibit the function of Ras in vivo is a rather general and frequent property and that the range of antibodies that can be successfully used for intracellular inhibition studies is much greater than anticipated, exploiting the mode of action of diverting protein traffic. [source] Directing traffic in neural cells: determinants of receptor tyrosine kinase localization and cellular responsesJOURNAL OF NEUROCHEMISTRY, Issue 6 2008Robert J. Romanelli Abstract The trafficking of receptor tyrosine kinases (RTKs) to distinct subcellular locations is essential for the specificity and fidelity of signal transduction and biological responses. This is particularly important in the PNS and CNS in which RTKs mediate key events in the development and maintenance of neurons and glia through a wide range of neural processes, including survival, proliferation, differentiation, neurite outgrowth, and synaptogenesis. The mechanisms that regulate the targeting of RTKs to their subcellular destinations for appropriate signal transduction, however, are still elusive. In this review, we discuss evidence for the spatial organization of signaling machinery into distinct subcellular compartments, as well as the role for ligand specificity, receptor sorting signals, and lipid raft microdomains in RTK targeting and the resultant cellular responses in neural cells. [source] Hippocampal N -Methyl- d -Aspartate Receptor Subunit Expression Profiles in a Mouse Model of Prenatal Alcohol ExposureALCOHOLISM, Issue 2 2010Sabrina L. Samudio-Ruiz Background:, Although several reports have been published showing prenatal ethanol exposure is associated with alterations in N -methyl- d- aspartate (NMDA) receptor subunit levels and, in a few cases, subcellular distribution, results of these studies are conflicting. Methods:, We used semi-quantitative immunoblotting techniques to analyze NMDA receptor NR1, NR2A, and NR2B subunit levels in the adult mouse hippocampal formation isolated from offspring of dams who consumed moderate amounts of ethanol throughout pregnancy. We employed subcellular fractionation and immunoprecipitation techniques to isolate synaptosomal membrane- and postsynaptic density protein-95 (PSD-95)-associated pools of receptor subunits. Results:, We found that, compared to control animals, fetal alcohol-exposed (FAE) adult mice had: (i) increased synaptosomal membrane NR1 levels with no change in association of this subunit with PSD-95 and no difference in total NR1 expression in tissue homogenates; (ii) decreased NR2A subunit levels in hippocampal homogenates, but no alterations in synaptosomal membrane NR2A levels and no change in NR2A-PSD-95 association; and (iii) no change in tissue homogenate or synaptosomal membrane NR2B levels but a reduction in PSD-95-associated NR2B subunits. No alterations were found in mRNA levels of NMDA receptor subunits suggesting that prenatal alcohol-associated differences in subunit protein levels are the result of differences in post-transcriptional regulation of subunit localization. Conclusions:, Our results demonstrate that prenatal alcohol exposure induces selective changes in NMDA receptor subunit levels in specific subcellular locations in the adult mouse hippocampal formation. Of particular interest is the finding of decreased PSD-95-associated NR2B levels, suggesting that synaptic NR2B-containing NMDA receptor concentrations are reduced in FAE animals. This result is consistent with various biochemical, physiological, and behavioral findings that have been linked with prenatal alcohol exposure. [source] Two different Pseudomonas aeruginosa chemosensory signal transduction complexes localize to cell poles and form and remould in stationary phaseMOLECULAR MICROBIOLOGY, Issue 1 2006Zehra Tüzün Güvener Summary Pseudomonas aeruginosa has sets of sensory genes designated che and che2. The che genes are required for flagella-mediated chemotaxis. The che2 genes are expressed in the stationary phase of growth and are probably also involved in flagella,mediated behavioural responses. P. aeruginosa also has 26 chemoreceptor genes, six of which are preferentially expressed in stationary phase. Subcellular localization experiments indicated that Che proteins form signal transduction complexes at cell poles throughout growth. Cyan fluorescent protein (CFP)-tagged McpA, a stationary phase-expressed chemoreceptor, appeared and colocalized with yellow fluorescent protein (YFP)-tagged CheA when cells entered stationary phase. This indicates that P. aeruginosa chemotaxis protein complexes are subject to remoulding by chemoreceptor proteins that are expressed when cells stop growing. CheA-CFP and CheY2-YFP tagged proteins that were coexpressed in the same cell had separate subcellular locations, indicating that Che2 proteins do not enter into direct physical interactions with Che proteins. Che2 protein complex formation required McpB, another stationary phase induced chemoreceptor that is predicted to be soluble. This implies that Che2 complexes have a function that depends on just one chemoreceptor. Our results suggest that motile P. aeruginosa cells have signal transduction systems that are adapted to allow non-growing cells to sense and respond to their environment differently from actively growing cells. [source] A recombinant multimeric immunoglobulin expressed in rice shows assembly-dependent subcellular localization in endosperm cellsPLANT BIOTECHNOLOGY JOURNAL, Issue 1 2005Liz Nicholson Summary To investigate the role of subunit assembly in the intracellular deposition of multimeric recombinant proteins, we expressed a partially humanized secretory immunoglobulin in rice endosperm cells and determined the subcellular locations of the assembled protein and its individual components. Transgenic rice plants expressing either individual subunits or all the subunits of the antibody were generated by particle bombardment, and protein localization was determined by immunoelectron microscopy. Assembly of the antibody was confirmed by immunoassay and coimmunoprecipitation. Immunolocalization experiments showed no evidence for secretion of the antibody or any of its components to the apoplast. Rather, the nonassembled light chain, heavy chain and secretory component accumulated predominantly within endoplasmic reticulum-derived protein bodies, while the assembled antibody, with antigen-binding function, accumulated specifically in protein storage vacuoles. These results show that the destination of a complex recombinant protein within the plant cell is influenced by its state of assembly. [source] | ||||||||||