Submaximal Responses in Calcium-triggered Exocytosis Are Explained by Differences in the Calcium Sensitivity of Individual Secretory Vesicles (original) (raw)
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The Journal of General Physiology, 1998
Differences in the calcium sensitivity of individual secretory vesicles can explain a defining feature of calcium-regulated exocytosis, a graded response to calcium. The role of the time dependence of calcium delivery in defining the observed differences in the calcium sensitivity of sea urchin egg secretory vesicles in vitro was examined. The calcium sensitivity of individual secretory vesicles (i.e., the distribution of calcium thresholds) is invariant over a range of calcium delivery rates from faster than micromolar per millisecond to slower than micromolar per second. Any specific calcium concentration above threshold triggers subpopulations of vesicles to fuse, and the size of these subpopulations is independent of the time course required to reach that calcium concentration. All evidence supports the hypothesis that the magnitude of the free calcium is the single controlling variable that determines the fraction of vesicles that fuse, and that this fraction is established before the application of calcium. Submaximal responses to calcium cannot be attributed to alterations in the calcium sensitivity of individual secretory vesicles arising from the temporal properties of the calcium delivery. Models that attempt to explain the cessation of fusion using changes in the distribution of calcium thresholds arising from the rate of calcium delivery and/or adaptation are not applicable to this system, and thus cannot be general.
A Kinetic Analysis of Calcium-triggered Exocytosis
The Journal of General Physiology, 2001
Although the relationship between exocytosis and calcium is fundamental both to synaptic and nonneuronal secretory function, analysis is problematic because of the temporal and spatial properties of calcium, and the fact that vesicle transport, priming, retrieval, and recycling are coupled. By analyzing the kinetics of sea urchin egg secretory vesicle exocytosis in vitro , the final steps of exocytosis are resolved. These steps are modeled as a three-state system: activated, committed, and fused, where interstate transitions are given by the probabilities that an active fusion complex commits ( ␣ ) and that a committed fusion complex results in fusion, p . The number of committed complexes per vesicle docking site is Poisson distributed with mean . Experimentally, p and increase with increasing calcium, whereas ␣ and the ratio remain constant, reducing the kinetic description to only one calcium-dependent, controlling variable, . On average, the calcium dependence of the maximum rate (R max ) and the time to reach R max (T peak ) are described by the calcium dependence of . Thus, the nonlinear relationship between the free calcium concentration and the rate of exocytosis can be explained solely by the calcium dependence of the distribution of fusion complexes at vesicle docking sites.
Proteins on exocytic vesicles mediate calcium-triggered fusion
Proceedings of the National Academy of Sciences, 1992
In many exocytic systems, micromolar concentrations of intracellular Ca2' trigger fusion. We find that aggregates of secretory granules isolated from sea urchin eggs fuse together when perfused with >10 FM free Ca2+. Mixing of membrane components was demonstrated by transfer of fluorescent lipophilic dye, and melding of granule contents was seen with differential interference microscopy. A technique based upon light scattering was developed to conveniently detect fusion. Two protein modifiers, trypsin and N-ethylmaleimide, inhibit granule-granule fusion at concentrations similar to those that inhibit granule-plasma membrane fusion. We suggest that molecular machinery sufficient for Ca2+-triggered fusion resides on secretory granules as purified and that at least some of these essential components are proteinaceous.
High calcium concentrations shift the mode of exocytosis to the kiss-and-run mechanism
Nature cell biology, 1999
Exocytosis, the fusion of secretory vesicles with the plasma membrane to allow release of the contents of the vesicles into the extracellular environment, and endocytosis, the internalization of these vesicles to allow another round of secretion, are coupled. It is, however, uncertain whether exocytosis and endocytosis are tightly coupled, such that secretory vesicles fuse only transiently with the plasma membrane before being internalized (the 'kiss-and-run' mechanism), or whether endocytosis occurs by an independent process following complete incorporation of the secretory vesicle into the plasma membrane. Here we investigate the fate of single secretory vesicles after fusion with the plasma membrane by measuring capacitance changes and transmitter release in rat chromaffin cells using the cell-attached patch-amperometry technique. We show that raised concentrations of extracellular calcium ions shift the preferred mode of exocytosis to the kiss-and-run mechanism in a calc...
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.
Bioscience Reports, 1988
Micromolar calcium ion concentrations stimulate exocytosis in a reconstituted system made by recombining in the plasma membrane and cortical secretory granules of the sea urchin egg. The isolated cortical granules are unaffected by calcium concentrations up to 1 mM, nor do granule aggregates undergo any mutual fusion at this concentration. Both isolated plasma membrane and cortical granules can be pretreated with 1 mM Ca before reconstitution without affecting the subsequent exocytosis of the reconstituted system in response to micromolar calcium concentrations. On reconstitution, aggregated cortical granules will fuse with one another in response to micromolar calcium provided that one of their number is in contact with the plasma membrane. If exocytosis involves the generation of lipid fusogens, then these results suggest that the calcium-stimulated production of a fusogen can occur only when contiguity exists between cortical granules and plasma membrane. They also suggest that a...
How calcium may cause exocytosis in sea urchin eggs
Bioscience Reports, 1987
The process of secretory granule-plasma membrane fusion can be studied in sea urchin eggs. Micromolar calcium concentrations are all that is required to bring about exocytosis in vitro. I discuss recent experiments with sea urchin eggs that concentrate on the biophysical aspects of granule-membrane fusion. The backbone of biological membranes is the lipid bilayer. Sea urchin egg membrane lipids have negatively charged head groups that give rise to an electrical potential at the bilayer-water interface. We have found that this surface potential can affect the calcium required for exocytosis. Effects on the surface potential may also explain why drugs like trifluoperazine and tetracaine inhibit exocytosis: they absorb to the bilayer and reduce the surface potential. The membrane lipids may also be crucial to the formation of the exocytotic pore through which the secretory granule contents are released. We have measured calcium-induced production of the lipid, diacylglycerol. This lipi...
Biochimie, 2000
Despite groundbreaking work to identify numerous proteins and to focus attention on molecular interactions, the mechanism of calcium-triggered membrane fusion remains unresolved. A major difficulty in such research has been the many overlapping and interacting membrane trafficking steps in the secretory pathway, including those of membrane retrieval. Identifying the specific role(s) of a given protein, beyond its general involvement in exocytosis, has therefore proven problematic. Furthermore, the power of time-resolved optical and electrophysiological assays can be best applied to testing the function of known proteins rather than to the identification of unknown, critical membrane components. The identification of essential membrane constituents requires combined biochemical (molecular) and functional (physiological) analyses. A fully functional, stage-specific physiological membrane preparation would be one direct approach to dissecting the calcium-triggered fusion steps of regulated exocytosis. Herein we review our use of specific minimal membrane preparations consisting of fully primed and docked secretory vesicles, or the isolated vesicles themselves, and characterize the late events of exocytosis, with an aim towards identification of essential molecular components. We have established a functional definition of the fusion complex and its activation by calcium, based on our kinetic analyses. Together with a variety of biochemical and alternate functional assays, we have tested whether the SNARE core complex that is present in our vesicle membranes satisfies the criteria of the functionally defined fusion complex. Rather than a direct fusogenic role, the SNARE complex may promote the calcium sensitivity of fusion, possibly by defining or delimiting a localized, focal membrane fusion site that ensures rapid and efficient exocytosis in vivo. © 2000 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS
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.