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To study biosynthetic transport through the constitutive and regulated secretory pathways, we have designed a semi-intact mammalian cell system that restores the transport of secretory proteins from the trans-Golgi/trans-Golgi network (TGN) to the cell surface. The mouse pituitary AtT-20 cell line is a suitable model to biochemically analyze molecular sorting in the secretory pathway. The prohormone...
A current major challenge in the study of regulated exocytosis is the identification of essential proteins that mediate the transit of secretory vesicles through trafficking stages such as recruitment, docking, and fusion. Defining the physiological roles and mechanisms of action of these essential proteins is paramount. The reconstitution of stages of regulated exocytosis in cell-free systems provides...
Neuroendocrine PC12 cells contain small microvesicles that closely resemble synaptic vesicles in their physical and chemical properties. Two defining characteristics of synaptic vesicles are their homogeneous size and their unique protein composition. Since synaptic vesicles arise by endocytosis from the plasma membrane, nerve terminals and PC12 cells must contain the molecular machinery to sort synaptic...
To investigate the mechanism of secretory granule biogenesis in endocrine cells, our laboratory used rat anterior pituitary GH3 cells which secrete growth hormone and prolactin. Here we describe a simple and rapid procedure for generating permeabilized cells to dissect molecular mechanisms involved in nascent secretory vesicle budding from the trans-Golgi network (TGN). Using this system, we demonstrate...
While investigating the localization of synaptophysin in PC12 cells using immunofluorescence microscopy, we noticed a striking difference in its apparent subcellular distribution depending on whether digitonin or Triton X-100 was used as permeabilization agent of paraformaldehyde (PFA)-fixed cells. We found that this difference was due to epitope inaccessibility in the digitonin-treated cells combined...
The process of membrane fusion has been profitably studied by fusing cells that express fusion proteins on their surfaces to the membranes of target cells. Primary methods for monitoring the occurrence of fusion between cells are measurement of formation of heterokaryons, measurement of activation of reporter genes, measurement of transfer of lipidic and aqueous fluorescent dyes, and electrophysiological...
We have established a system that reconstitutes the biogenesis of synaptic-like microvesicles (SLMVs) in perforated cells of the rat neuroendocrine cell line PC12. The system is based on the biotinylation of synaptophysin, a marker of synaptic vesicles and SLMVs. Biotinylation is performed at 18°C, a temperature at which formation of SLMVs is blocked and biotinylated synaptophysin accumulates in the...
We have developed a secretion assay composed of semi-intact synaptosomes from which transmitter release is optimally evoked by micromolar Ca 2+ in the presence of cytosol. Transmitter release from this preparation reconstitutes known characteristics of regulated exocytosis and is accompanied by a marked decrease in synaptic vesicles. The assay is useful in characterizing the components known...
The reconstitution of a membrane fusion event in a cell-free system makes possible a biochemical investigation of the molecular mechanisms underlying it. We have developed anin vitroassay for the fusion of pancreatic zymogen granules with the plasma membrane. The lipid-soluble fluorescent probe octadecylrhodamine is loaded into the granule membrane, and the granules are then incubated with unlabeled...
Neurons and endocrine cells release peptides stored in a limited pool of secretory granules. Although it has been possible to measure secretion or exocytosis, studying events within cells that influence the size and speed of secretory responses has been difficult. Here we describe how green fluorescent protein (GFP)-tagged hormones can be used to measure release and granule mobility. Epifluorescence...
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