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Subcellular Domains (subcellular + domain)
Selected AbstractsModulation of calcium signalling by intracellular organelles seen with targeted aequorinsACTA PHYSIOLOGICA, Issue 1 2009M. T. Alonso Abstract The cytosolic Ca2+ signals that trigger cell responses occur either as localized domains of high Ca2+ concentration or as propagating Ca2+ waves. Cytoplasmic organelles, taking up or releasing Ca2+ to the cytosol, shape the cytosolic signals. On the other hand, Ca2+ concentration inside organelles is also important in physiology and pathophysiology. Comprehensive study of these matters requires to measure [Ca2+] inside organelles and at the relevant cytosolic domains. Aequorins, the best-known chemiluminescent Ca2+ probes, are excellent for this end as they do not require stressing illumination, have a large dynamic range and a sharp Ca2+ -dependence, can be targeted to the appropriate location and engineered to have the proper Ca2+ affinity. Using this methodology, we have evidenced the existence in chromaffin cells of functional units composed by three closely interrelated elements: (1) plasma membrane Ca2+ channels, (2) subplasmalemmal endoplasmic reticulum and (3) mitochondria. These Ca2+ -signalling triads optimize Ca2+ microdomains for secretion and prevent propagation of the Ca2+ wave towards the cell core. Oscillatory cytosolic Ca2+ signals originate also oscillations of mitochondrial Ca2+ in several cell types. The nuclear envelope slows down the propagation of the Ca2+ wave to the nucleus and filters high frequencies. On the other hand, inositol-trisphosphate may produce direct release of Ca2+ to the nucleoplasm in GH3 pituitary cells, thus providing mechanisms for selective nuclear signalling. Aequorins emitting at different wavelengths, prepared by fusion either with green or red fluorescent protein, permit simultaneous and independent monitorization of the Ca2+ signals in different subcellular domains within the same cell. [source] Vascular smooth muscle cell phenotypic modulation in culture is associated with reorganisation of contractile and cytoskeletal proteinsCYTOSKELETON, Issue 3 2001Nathalie F. Worth Abstract Smooth muscle cells (SMC) exhibit a functional plasticity, modulating from the mature phenotype in which the primary function is contraction, to a less differentiated state with increased capacities for motility, protein synthesis, and proliferation. The present study determined, using Western analysis, double-label immunofluorescence and confocal microscopy, whether changes in phenotypic expression of rabbit aortic SMC in culture could be correlated with alterations in expression and distribution of structural proteins. "Contractile" state SMC (days 1 and 3 of primary culture) showed distinct sorting of proteins into subcellular domains, consistent with the theory that the SMC structural machinery is compartmentalised within the cell. Proteins specialised for contraction (,-SM actin, SM-MHC, and calponin) were highly expressed in these cells and concentrated in the upper central region of the cell. Vimentin was confined to the body of the cell, providing support for the contractile apparatus but not co-localising with it. In line with its role in cell attachment and motility, ,-NM actin was localised to the cell periphery and basal cortex. The dense body protein ,-actinin was concentrated at the cell periphery, possibly stabilising both contractile and motile apparatus. Vinculin-containing focal adhesions were well developed, indicating the cells' strong adhesion to substrate. In "synthetic" state SMC (passages 2,3 of culture), there was decreased expression of contractile and adhesion (vinculin) proteins with a concomitant increase in cytoskeletal proteins (,-non-muscle [NM] actin and vimentin). These quantitative changes in structural proteins were associated with dramatic changes in their distribution. The distinct compartmentalisation of structural proteins observed in "contractile" state SMC was no longer obvious, with proteins more evenly distributed throughout the cytoplasm to accommodate altered cell function. Thus, SMC phenotypic modulation involves not only quantitative changes in contractile and cytoskeletal proteins, but also reorganisation of these proteins. Since the cytoskeleton acts as a spatial regulator of intracellular signalling, reorganisation of the cytoskeleton may lead to realignment of signalling molecules, which, in turn, may mediate the changes in function associated with SMC phenotypic modulation. Cell Motil. Cytoskeleton 49:130,145, 2001. © 2001 Wiley-Liss, Inc. [source] Cadherin-8 and N-cadherin differentially regulate pre- and postsynaptic development of the hippocampal mossy fiber pathwayHIPPOCAMPUS, Issue 4 2008Iddil H. Bekirov Abstract Cells sort into regions and groups in part by their selective surface expression of particular classic cadherins during development. In the nervous system, cadherin-based sorting can define axon tracts, restrict axonal and dendritic arbors to particular regions or layers, and may encode certain aspects of synapse specificity. The underlying model has been that afferents and their targets hold in common the expression of a particular cadherin, thereby providing a recognition code of homophilic cadherin binding. However, most neurons express multiple cadherins, and it is not clear whether multiple cadherins all act similarly in shaping neural circuitry. Here we asked how two such cadherins, cadherin-8 and N-cadherin, influence the guidance and differentiation of hippocampal mossy fibers. Using organotypic hippocampal cultures, we find that cadherin-8 regulates mossy fiber fasciculation and targeting, but has little effect on CA3 dendrites. In contrast, N-cadherin regulates mossy fiber fasciculation, but has little impact on axonal growth and targeting. However, N-cadherin is essential for CA3 dendrite arborization. Both cadherins are required for formation of proper numbers of presynaptic terminals. Mechanistically, such differential actions of these two cadherins could, in theory, reflect coupling to distinct intracellular binding partners. However, we find that both cadherins bind ,-catenin in dentate gyrus (DG). This suggests that cadherins may engage different intracellular signaling cascades downstream of ,-catenin, coopt different extracellular binding partners, or target distinct subcellular domains. Together our findings demonstrate that cadherin-8 and N-cadherin are critical for generating the mossy fiber pathway, but that each contributes differentially to afferent and target differentiation, thereby complementing one another in the assembly of a synaptic circuit. © 2007 Wiley-Liss, Inc. [source] Mechanically Strained Cells of the Osteoblast Lineage Organize Their Extracellular Matrix Through Unique Sites of ,V,3 -Integrin ExpressionJOURNAL OF BONE AND MINERAL RESEARCH, Issue 9 2000Magdalena Wozniak Abstract Bone cells transduce mechanical signals into anabolic biochemical responses. However, the mechanisms of mechanotransduction are unknown. To address this issue, we performed studies in primary cells of the human osteoblast lineage grown on collagen/vitronectin-coated supports. We discovered that mechanical strain stimulated a redistribution of the ,v,3 -integrin to irregular plaque-like areas at the cell-extracellular matrix surface. Proteins involved in integrin-matrix interactions in focal adhesions, vinculin and talin, did not localize to the plaque-like areas of ,v,3 -expression, but signaling molecules such as focal adhesion kinase (FAK) did. Mechanical strain increased the number and size of the plaques defined by surface expression of ,v,3 -integrin. Osteopontin was secreted as a cross-linked macromolecular complex, likely through the action of tissue transglutaminase that also was found in the plaques of ,v,3 -integrin cell-matrix interaction. Mechanical strain increased mineralization of the extracellular matrix that developed in these plaques in ,v,3 -integrin-dependent manner. Because the plaque-like areas of cell-matrix interaction exhibit macromolecular assembly and mineralization, we conclude that they may represent subcellular domains of bone formation and that ,v,3 -integrin activation represents one mechanism by which mechanical strain stimulates bone formation. [source] Altered distribution of mitochondria impairs calcium homeostasis in rat hippocampal neurons in cultureJOURNAL OF NEUROCHEMISTRY, Issue 1 2003Guang Jian Wang Abstract The specificity of Ca2+ signals is conferred in part by limiting changes in cytosolic Ca2+ to subcellular domains. Mitochondria play a major role in regulating Ca2+ in neurons and may participate in its spatial localization. We examined the effects of changes in the distribution of mitochondria on NMDA-induced Ca2+ increases. Hippocampal cultures were treated with the microtubule-destabilizing agent vinblastine, which caused the mitochondria to aggregate and migrate towards one side of the neuron. This treatment did not appear to decrease the energy status of mitochondria, as indicated by a normal membrane potential and pH gradient across the inner membrane. Moreover, electron microscopy showed that vinblastine treatment altered the distribution but not the ultrastructure of mitochondria. NMDA (200 µm, 1 min) evoked a greater increase in cytosolic Ca2+ in vinblastine-treated cells than in untreated cells. This increase did not result from impaired Ca2+ efflux, enhanced Ca2+ influx, opening of the mitochondrial permeability transition pore or altered function of endoplasmic reticulum Ca2+ stores. Ca2+ uptake into mitochondria was reduced by 53% in vinblastine-treated cells, as reported by mitochondrially targeted aequorin. Thus, the distribution of mitochondria maintained by microtubules is critical for buffering Ca2+ influx. A subset of mitochondria close to a Ca2+ source may preferentially regulate Ca2+ microdomains, set the threshold for Ca2+ -induced toxicity and participate in local ATP production. [source] | ||||||||||