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  • 1
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    Neuronal calcium sensor 1 and phosphatidylinositol 4-OH kinase βinteract in neuronal cells and are translocated to membranes during nucleotide-evoked exocytosis
    The Company of Biologists ; 2002
    In:  Journal of Cell Science Vol. 115, No. 20 ( 2002-10-15), p. 3909-3922
    Details
    In: Journal of Cell Science, The Company of Biologists, Vol. 115, No. 20 ( 2002-10-15), p. 3909-3922
    Abstract: Neuronal calcium sensor 1 (NCS-1) belongs to a family of EF-hand calcium-binding proteins and is mainly expressed in neurons and neuroendocrine cells, where it causes facilitation of neurotransmitter release through unknown mechanisms. The yeast homologue of NCS-1 has been demonstrated to interact with and regulate the activity of yeast phosphatidylinositol 4-OH kinase β (PI4Kβ). However, in neurons and neurosecretory cells NCS-1 has not unequivocally been shown to interact with PI4Kβ. Here we have compared the subcellular distribution of NCS-1 and PI4Kβ and investigated whether they are capable of forming complexes. In neurons, both proteins are widely distributed and are present in perikarya and, to a lesser extent, in nerve terminals. A consistent portion of NCS-1 and PIK4β is cytosolic,whereas a portion of both proteins appears to be associated with the membranes of the endoplasmic reticulum and the Golgi complex. Very small amounts of NCS-1 and PI4Kβ are present in synaptic vesicles. Our results further demonstrate that in neurosecretory cells, endogenous NCS-1 and PIK4βinteract to form a complex that can be immunoisolated from membrane as well as from cytosolic fractions. Moreover, both proteins can be recruited to membranes when cells are treated with nucleotide receptor agonists known to increase polyphosphoinositide turnover and concomitantly induce exocytosis of secretory vesicles. Finally, in PC12 cells overexpressing NCS-1, the amount of PI4Kβ associated with the membranes is increased concomitantly with the increased levels of NCS-1 detected in the same membrane fractions. Together,these findings demonstrate that mammalian NCS-1 and PI4Kβ interact under physiological conditions, which suggest a possible role for NCS-1 in the translocation of PI4Kβ to target membranes.
    Type of Medium: Online Resource
    ISSN: 1477-9137 , 0021-9533
    URL: Article
    DOI: 10.1242/jcs.00072
    Language: English
    Publisher: The Company of Biologists
    Publication Date: 2002
    detail.hit.zdb_id: 219171-4
    detail.hit.zdb_id: 1483099-1
    SSG: 12
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  • 2
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    Syntaxin 1A is delivered to the apical and basolateral domains of epithelial cells: the role of munc-18 proteins
    The Company of Biologists ; 2001
    In:  Journal of Cell Science Vol. 114, No. 18 ( 2001-09-15), p. 3323-3332
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    In: Journal of Cell Science, The Company of Biologists, Vol. 114, No. 18 ( 2001-09-15), p. 3323-3332
    Abstract: SNARE (Soluble N-ethyl-maleimide sensitive factor Attachment protein Receptor) proteins assemble in tight core complexes, which promote fusion of carrier vesicles with target compartments. Members of this class of proteins are expressed in all eukaryotic cells and are distributed in distinct subcellular compartments. The molecular mechanisms underlying sorting of SNAREs to their physiological sites of action are still poorly understood. Here have we analyzed the transport of syntaxin1A in epithelial cells. In line with previous data we found that syntaxin1A is not transported to the plasma membrane, but rather is retained intracellularly when overexpressed in MDCK and Caco-2 cells. Its delivery to the cell surface is recovered after munc-18-1 cotransfection. Furthermore, overexpression of the ubiquitous isoform of munc-18, munc-18-2, is also capable of rescuing the transport of the t-SNARE. The interaction between syntaxin 1A and munc-18 occurs in the biosynthetic pathway and is required to promote the exit of the t-SNARE from the Golgi complex. This enabled us to investigate the targeting of syntaxin1A in polarized cells. Confocal analysis of polarized monolayers demonstrates that syntaxin1A is delivered to both the apical and basolateral domains independently of the munc-18 proteins used in the cotranfection experiments. In search of the mechanisms underlying syntaxin 1A sorting to the cell surface, we found that a portion of the protein is included in non-ionic detergent insoluble complexes. Our results indicate that the munc-18 proteins represent limiting but essential factors in the transport of syntaxin1A from the Golgi complex to the epithelial cell surface. They also suggest the presence of codominant apical and basolateral sorting signals in the syntaxin1A sequence.
    Type of Medium: Online Resource
    ISSN: 1477-9137 , 0021-9533
    URL: Article
    DOI: 10.1242/jcs.114.18.3323
    Language: English
    Publisher: The Company of Biologists
    Publication Date: 2001
    detail.hit.zdb_id: 219171-4
    detail.hit.zdb_id: 1483099-1
    SSG: 12
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  • 3
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    Neuronal calcium sensor-1 binds to regulated secretory organelles and functions in basal and stimulated exocytosis in PC12 cells
    The Company of Biologists ; 2002
    In:  Journal of Cell Science Vol. 115, No. 11 ( 2002-06-01), p. 2399-2412
    Details
    In: Journal of Cell Science, The Company of Biologists, Vol. 115, No. 11 ( 2002-06-01), p. 2399-2412
    Abstract: Neuronal calcium sensor-1 (NCS-1) and its non-mammalian homologue,frequenin, have been implicated in a spectrum of cellular processes, including regulation of stimulated exocytosis of synaptic vesicles and secretory granules (SGs) in neurons and neuroendocrine cells and regulation of phosphatidylinositol 4-kinase beta activity in yeast. However, apart from these intriguing putative functions, NCS-1 and frequenin are relatively poorly understood. Here, the distribution, dynamics and function of NCS-1 were studied using PC12 cells that stably express NCS-1-EYFP (NCS-1 fused to enhanced yellow fluorescent protein) or that stably overexpress NCS-1. Fluorescence and electron microscopies show that NCS-1-EYFP is absent from SGs but is present on small clear organelles, some of which are just below the plasma membrane. Total internal reflection fluorescence microscopy shows that NCS-1-EYFP is associated with synaptic-like microvesicles (SLMVs) in growth cones. Overexpression studies show that NCS-1 enhances exocytosis of synaptotagmin-labeled regulated secretory organelles (RSOs) under basal conditions and during stimulation by UTP. Significantly, these studies implicate NCS-1 in the enhancement of both basal and stimulated phosphoinositide-dependent exocytosis of RSOs in PC12 cells, and they show that NCS-1 is distributed strategically to interact with putative targets on the plasma membrane and on SLMVs. These studies also reveal that SLMVs undergo both fast directed motion and highly hindered diffusive motion in growth cones, suggesting that cytoskeletal constituents can both facilitate and hinder SLMV motion. These results also reveal interesting similarities and differences between transport organelles in differentiated neuroendocrine cells and neurons.
    Type of Medium: Online Resource
    ISSN: 1477-9137 , 0021-9533
    URL: Article
    DOI: 10.1242/jcs.115.11.2399
    Language: English
    Publisher: The Company of Biologists
    Publication Date: 2002
    detail.hit.zdb_id: 219171-4
    detail.hit.zdb_id: 1483099-1
    SSG: 12
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  • 4
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    Blockade of membrane transport and disassembly of the Golgi complex by expression of syntaxin 1A in neurosecretion-incompetent cells: prevention by rbSEC1
    The Company of Biologists ; 1999
    In:  Journal of Cell Science Vol. 112, No. 12 ( 1999-06-15), p. 1865-1877
    Details
    In: Journal of Cell Science, The Company of Biologists, Vol. 112, No. 12 ( 1999-06-15), p. 1865-1877
    Abstract: The t-SNAREs syntaxin1A and SNAP-25, i.e. the members of the complex involved in regulated exocytosis at synapses and neurosecretory cells, are delivered to their physiological site, the plasma membrane, when transfected into neurosecretion-competent cells, such as PC12 and AtT20. In contrast, when transfection is made into cells incompetent for neurosecretion, such as those of a defective PC12 clone and the NRK fibroblasts, which have no endogenous expression of these t-SNAREs, syntaxin1A (but neither two other syntaxin family members nor SNAP-25) remains stuck in the Golgi-TGN area with profound consequences to the cell: blockade of both membrane (SNAP-25, GAT-1) and secretory (chromogranin B) protein transport to the cell surface; progressive disassembly of the Golgi complex and TGN; ultimate disappearance of the latter structures, with intermixing of their markers (mannosidase II; TGN-38) with those of the endoplasmic reticulum (calreticulin) and with syntaxin1A itself. When, however, syntaxin 1A is transfected together with rbSec1, a protein known to participate in neurosecretory exocytosis via its dynamic interaction with the t-SNARE, neither the blockade nor the alterations of the Golgi complex take place. Our results demonstrate that syntaxin1A, in addition to its role in exocytosis at the cell surface, possesses a specific potential to interfere with intracellular membrane transport and that its interaction with rbSec1 is instrumental to its physiological function not only at the plasma membrane but also within the cell. At the latter site, the rbSec1-induced conversion of syntaxin1A into a form that can be transported and protects the cell from the development of severe structural and membrane traffic alterations.
    Type of Medium: Online Resource
    ISSN: 0021-9533 , 1477-9137
    URL: Article
    DOI: 10.1242/jcs.112.12.1865
    Language: English
    Publisher: The Company of Biologists
    Publication Date: 1999
    detail.hit.zdb_id: 219171-4
    detail.hit.zdb_id: 1483099-1
    SSG: 12
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  • 5
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    Cholesterol reduction impairs exocytosis of synaptic vesicles
    The Company of Biologists ; 2010
    In:  Journal of Cell Science Vol. 123, No. 4 ( 2010-02-15), p. 595-605
    Details
    In: Journal of Cell Science, The Company of Biologists, Vol. 123, No. 4 ( 2010-02-15), p. 595-605
    Abstract: Cholesterol and sphingolipids are abundant in neuronal membranes, where they help the organisation of the membrane microdomains involved in major roles such as axonal and dendritic growth, and synapse and spine stability. The aim of this study was to analyse their roles in presynaptic physiology. We first confirmed the presence of proteins of the exocytic machinery (SNARES and Cav2.1 channels) in the lipid microdomains of cultured neurons, and then incubated the neurons with fumonisin B (an inhibitor of sphingolipid synthesis), or with mevastatin or zaragozic acid (two compounds that affect the synthesis of cholesterol by inhibiting HMG-CoA reductase or squalene synthase). The results demonstrate that fumonisin B and zaragozic acid efficiently decrease sphingolipid and cholesterol levels without greatly affecting the viability of neurons or the expression of synaptic proteins. Electron microscopy showed that the morphology and number of synaptic vesicles in the presynaptic boutons of cholesterol-depleted neurons were similar to those observed in control neurons. Zaragozic acid (but not fumonisin B) treatment impaired synaptic vesicle uptake of the lipophilic dye FM1-43 and an antibody directed against the luminal epitope of synaptotagmin-1, effects that depended on the reduction in cholesterol because they were reversed by cholesterol reloading. The time-lapse confocal imaging of neurons transfected with ecliptic SynaptopHluorin showed that cholesterol depletion affects the post-depolarisation increase in fluorescence intensity. Taken together, these findings show that reduced cholesterol levels impair synaptic vesicle exocytosis in cultured neurons.
    Type of Medium: Online Resource
    ISSN: 1477-9137 , 0021-9533
    URL: Article
    DOI: 10.1242/jcs.060681
    Language: English
    Publisher: The Company of Biologists
    Publication Date: 2010
    detail.hit.zdb_id: 219171-4
    detail.hit.zdb_id: 1483099-1
    SSG: 12
    Location Call Number Limitation Availability
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