A Cell-free System for Regulated Exocytosis in PC12 Cells (original) (raw)
Related papers
Docked Secretory Vesicles Undergo Ca2+-activated Exocytosis in a Cell-free System
Journal of Biological Chemistry, 1997
The Ca 2؉-activated fusion of secretory vesicles with the plasma membrane responsible for regulated neurotransmitter and hormone secretion has previously been studied in permeable neuroendocrine cells, where requirements for ATP and cytosolic proteins were identified. As reported here, Ca 2؉-activated fusion mechanisms are also preserved following cell homogenization. The release of norepinephrine (NE) and other vesicle constituents from a PC12 cell membrane fraction was activated by micromolar Ca 2؉ (EC 50 ϳ 3 M) and exhibited a dependence upon MgATP and cytosol. Ca 2؉-dependent NE release was inhibited by botulinum neurotoxins and by CAPS (Ca 2؉-dependent activator protein for secretion) antibody implying that syntaxin, synaptobrevin, SNAP-25 (synaptosomal-associated protein of 25 kDa), and CAPS are required for regulated exocytosis in this system. The exocytosis-competent membrane fraction consisted of rapidly sedimenting dense core vesicles associated with plasma membrane fragments. Free vesicles did not release NE either in the absence or presence of plasma membranes, indicating that only docked vesicles were competent for exocytosis under the reconstitution conditions used. A cell-free system for Ca 2؉-activated fusion will facilitate studies on the roles of essential proteins such as syntaxin, synaptobrevin, SNAP-25, and CAPS that act at post-docking steps in the regulated exocytotic pathway.
Brain Research Reviews, 2000
Neuroendocrine cells display a similar calcium dependence of release as synapses but a strongly different organization of channels and vesicles. Biophysical and biochemical properties of large dense core vesicle release in neuroendocrine cells suggest that vesicles and channels are dissociated by a distance of 100-300 nm. This distinctive organization relates to the sensitivity of the release process to mobile calcium buffers, the resulting relationship between calcium influx and release and the modulatory mechanisms regulating the efficiency of excitation-release coupling. At distances of 100-300 nm, calcium buffers determine the calcium concentration close to the vesicle. Notably, the concentration and diffusion rate of mobile buffers affect the efficacy of release, but local saturation of buffers, possibly enhanced by diffusion barriers, may limit their effects. Buffer conditions may result in a linear relationship between calcium influx and exocytosis, in spite of the third or fourth power relation between intracellular calcium concentration and release. Modulation of excitation-secretion coupling not only concerns the calcium channels, but also the secretory process. Transmitter regulation mediated by cAMP and PKA, as well as use-dependent regulation involving calcium, primarily stimulates filling of the releasable pool. In addition, direct effects of cAMP on the probability of release have been reported. One mechanism to achieve increased release probability is to decrease the distance between channels and vesicles. GTP may stimulate release independently from calcium. Thus, while in most cases primary inputs triggering these pathways await identification, it is evident that large dense core vesicle release is a highly controlled and flexible process.
Calcium regulation of exocytosis in PC12 cells
Journal of Biological …, 2001
The calcium (Ca 2؉ ) regulation of neurotransmitter release is poorly understood. Here we investigated several aspects of this process in PC12 cells. We first showed that osmotic shock by 1 M sucrose stimulated rapid release of neurotransmitters from intact PC12 cells, indicating that most of the vesicles were docked at the plasma membrane. Second, we further investigated the mechanism of rescue of botulinum neurotoxin E inhibition of release by recombinant SNAP-25 COOH-terminal coil, which is known to be required in the triggering stage. We confirmed here that Ca 2؉ was required simultaneously with the SNAP-25 peptide, with no significant increase in release if either the peptide or Ca 2؉ was present during the priming stage as well as the triggering, suggesting that SNARE (soluble N-ethylmaleimidesensitive fusion protein attachment protein receptor) complex assembly was involved in the final Ca 2؉ -triggered event. Using this rescue system, we also identified a series of acidic surface SNAP-25 residues that rescued better than wild-type when mutated, due to broadened Ca 2؉ sensitivity, suggesting that this charged patch may interact electrostatically with a negative regulator of membrane fusion. Finally, we showed that the previously demonstrated stimulation of exocytosis in this system by calmodulin required calcium binding, since calmodulin mutants defective in Ca 2؉ -binding were not able to enhance release.
Calcium Regulation of Exocytosis in PC12 Cells* and the
The calcium (Ca 2؉ ) regulation of neurotransmitter release is poorly understood. Here we investigated several aspects of this process in PC12 cells. We first showed that osmotic shock by 1 M sucrose stimulated rapid release of neurotransmitters from intact PC12 cells, indicating that most of the vesicles were docked at the plasma membrane. Second, we further investigated the mechanism of rescue of botulinum neurotoxin E inhibition of release by recombinant SNAP-25 COOH-terminal coil, which is known to be required in the triggering stage. We confirmed here that Ca 2؉ was required simultaneously with the SNAP-25 peptide, with no significant increase in release if either the peptide or Ca 2؉ was present during the priming stage as well as the triggering, suggesting that SNARE (soluble N-ethylmaleimidesensitive fusion protein attachment protein receptor) complex assembly was involved in the final Ca 2؉ -triggered event. Using this rescue system, we also identified a series of acidic surface SNAP-25 residues that rescued better than wild-type when mutated, due to broadened Ca 2؉ sensitivity, suggesting that this charged patch may interact electrostatically with a negative regulator of membrane fusion. Finally, we showed that the previously demonstrated stimulation of exocytosis in this system by calmodulin required calcium binding, since calmodulin mutants defective in Ca 2؉ -binding were not able to enhance release.
Journal of Neuroscience, 2004
Many spikes in amperometric records of exocytosis events initially exhibit a prespike feature, or foot, which represents a steady-state flux of neurotransmitter through a stable fusion pore spanning both the vesicle and plasma membranes and connecting the vesicle lumen to the extracellular fluid. Here, we present the first evidence indicating that vesicular volume before secretion is strongly correlated with the characteristics of amperometric foot events. L-3,4-Dihydroxyphenylalanine and reserpine have been used to increase and decrease, respectively, the volume of single pheochromocytoma cell vesicles. Amperometry and transmission electron microscopy have been used to determine that as vesicle size is decreased the frequency with which foot events are observed increases, the amount and duration of neurotransmitter released in the foot portion of the event decreases, and vesicles release a greater percentage of their total contents in the foot portion of the event. This previously unidentified correlation provides new insight into how vesicle volume can modulate the activity of the exocytotic fusion pore.
Proceedings of the National Academy of Sciences, 2003
Classical cell biology teaches that exocytosis causes the membrane of exocytic vesicles to disperse into the cell surface and that a cell must later retrieve by molecular sorting whatever membrane components it wishes to keep inside. We have tested whether this view applies to secretory granules in intact PC-12 cells. Three granule proteins were labeled with fluorescent proteins in different colors, and two-color evanescent-field microscopy was used to view single granules during and after exocytosis. Whereas neuropeptide Y was lost from granules in seconds, tissue plasminogen activator (tPA) and the membrane protein phogrin remained at the granule site for over 1 min, thus providing markers for postexocytic granules. When tPA was imaged simultaneously with cyan fluorescent protein (CFP) as a cytosolic marker, the volume occupied by the granule appeared as a dark spot where it excluded CFP. The spot remained even after tPA reported exocytosis, indicating that granules failed to flatten into the cell surface. Phogrin was labeled with GFP at its luminal end and used to sense the pH in granules. When exocytosis caused the acidic granule interior to neutralize, GFP-phogrin at first brightened and later dimmed again as the interior separated from the extracellular space and reacidified. Reacidification and dimming could be reversed by application of NH 4Cl. We conclude that most granules reseal in <10 s after releasing cargo, and that these empty or partially empty granules are recaptured otherwise intact.
Reconstitution of calcium-mediated exocytosis of dense-core vesicles
Science advances, 2017
Regulated exocytosis is a process by which neurotransmitters, hormones, and secretory proteins are released from the cell in response to elevated levels of calcium. In cells, secretory vesicles are targeted to the plasma membrane, where they dock, undergo priming, and then fuse with the plasma membrane in response to calcium. The specific roles of essential proteins and how calcium regulates progression through these sequential steps are currently incompletely resolved. We have used purified neuroendocrine dense-core vesicles and artificial membranes to reconstruct in vitro the serial events that mimic SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-dependent membrane docking and fusion during exocytosis. Calcium recruits these vesicles to the target membrane aided by the protein CAPS (calcium-dependent activator protein for secretion), whereas synaptotagmin catalyzes calcium-dependent fusion; both processes are dependent on phosphatidylinositol 4,5-bis...
Physical techniques for the study of exocytosis in isolated cells
Biochimie, 1998
S~mmary ~-Membrane traffic is an fluportant aspect of cell biology which implies shuttle vesicles and multiple binding/fusion event,;. In spite ot' rapid progress at the biochemical level, the mechanism of fusion is still not understood. A detailed physical description of the phenomenon in possible at the level of the plasma membrane where secretory ,,esicles fuse with the cell membrane, a process known as exocytosis. Tiffs process is specially active in neuron~ (release of neurotransmiuer) and in endocrine cells {release of hormones). ~here exocytosis is tightly regulated, Among the biophysical techniques developed, cell membrane capacitance measurernents by the technique of patch-clamp and amperometry of the oxidizable secretory produc|s have resulted m interesting intormation. These techniques llave described the initial fusion pore, its fluctuations, the efflux of material through the pore and its irreversible expansion. Optical teci'|niques, using bioluminescent and fluorescent probes are also in progress. For instance, the dye FM 1-43 binds to but is not ~ranslocated through biological membranes and it has been used to measure membrane surface, as done by capacitance measurement. Evanescent wave fluorescence microscopy has been recently introduced to analyse the behaviour of secretory granules ill the vicinity of the plasma membrane (Q Societ6 franqaise de biochimie el biologic inol6cuhlire / Elsevier, Paris). membrane htsion I exocytosis I patch-clamp / amperometry / evanescent wave microscopy Membrane fusion is an important biological phenomenon Cells are limited by a i~hospholipidic bilaycr, the plasma nlenlbraue, but they contain much more membrane surface inside than around then!, These internal membranes dcl'inc specialised compartments (nucleus. inilochondria, cntioplasmic reliculum, Golgi apparatus, secretory vesicles, cndosomes, peroxisomes, etc) where differetlt chemical reactions can be perlbrmed at the same time under optimal physico-chemical conditions. Several of these comparto ments communicate with each other. This is the case for the compartments composing the secretory pathway (endoplasmic reticulum, Golgi apparatus, trans-Golgi network and secretory vesicles) and the reverse endocytotic pathway (endosomes, lysosomes). It is now clear that the main stream of the corresponding traffic involves shuttle vesicles, budding from the donor compartment and fusing with the accepter compartment. This traffic occurs in all cells and it is absolutely nece,;sary Ik~r them. In tiffs respect, membrane fusion appears to be an universal and important phenomenon. At the present time, our knowledge of tht ° proteins
Exocytosis and Endocytosis in Neuroendocrine Cells: Inseparable Membranes!
Frontiers in Endocrinology, 2013
Although much has been learned concerning the mechanisms of secretory vesicle formation and fusion at donor and acceptor membrane compartments, relatively little attention has been paid toward understanding how cells maintain a homeostatic membrane balance through vesicular trafficking. In neurons and neuroendocrine cells, release of neurotransmitters, neuropeptides, and hormones occurs through calcium-regulated exocytosis at the plasma membrane. To allow recycling of secretory vesicle components and to preserve organelles integrity, cells must initiate and regulate compensatory membrane uptake. This review relates the fate of secretory granule membranes after full fusion exocytosis in neuroendocrine cells. In particular, we focus on the potential role of lipids in preserving and sorting secretory granule membranes after exocytosis and we discuss the potential mechanisms of membrane retrieval.
Cytosolic calcium facilitates release of secretory products after exocytotic vesicle fusion
FEBS Letters, 1995
We monitored single vesicle exocytosis by simultaneous measurements of cell membrane capacitance as an indicator of fusion and amperometric detection of serotonin release. We show here that vesicle-plasma membrane fusion in rat mast cell granules is followed by a variable, exponentially distributed, delay before bulk release. This delay reflects the time required for the expansion of the exocytotic fusion pore, lasting, on average, 231 ms in resting cytosolic calcium, [Ca2+L (50 nM). In the presence of [Ca2+]i in the low micromollar range, the lag between fusion and release was reduced to 123 ms. The characteristics of the amperometric signals were unchanged by [Ca2+]i. These resuits show a novel site of regulation in the exocytotic process, the fusion pore, which may represent a different mechanism facilitating transmitter release.