Calcium In Exocytosis Research Articles

Distinct Domains of Complexins Bind SNARE Complexes and Clamp Fusion in Vitro

In regulated exocytosis, the core membrane fusion machinery proteins, the SNARE proteins, are assisted by a group of regulatory factors in order to couple membrane fusion to an increase of intracellular calcium ion (Ca(2+)) concentration. Complexin-I and synaptotagmin-I have been shown to be key elements for this tightly regulated process. Many studies suggest that complexin-I can arrest the fusion reaction and that synaptotagmin-I can release the complexin-I blockage in a calcium-dependent manner. Although the actual molecular mechanism by which they exert their function is still unknown, recent in vivo experiments postulate that domains of complexin-I produce different effects on neurotransmitter release. Herein, by using an in vitro flipped SNARE cell fusion assay, we have identified and characterized the minimal functional domains of complexin-I necessary to couple calcium and synaptotagmin-I to membrane fusion. Moreover, we provide evidence that other isoforms of complexin, complexin-II, -III, and -IV, can also be functionally coupled to synaptotagmin-I and calcium. These correspond closely to results from in vivo experiments, providing further validation of the physiological relevance of the flipped SNARE system.

Role of Calcium in Exocytosis Induced by Hypotonic Swelling

We studied the influence of Ca(2+) on exocytosis induced by hypotonic shock using the fluorescent dyes acridine orange FM1-43 and FM2-10. It was shown using acridine orange that lowering osmolarity to 230 mOsm/L induces exocytosis both in calcium-containing and calcium-free media. By contrast, we were able to demonstrate calcium dependence of exocytosis using styryl dyes. Lowering osmolarity leads to an increase of neurotransmitter release in a calcium-independent manner. Thus, our data suggest that hypotonic swelling induces calcium-independent exocytosis. Calcium influx mediated by stretch channels is able to switch mode of exocytosis from "kiss and run" to full fusion.

Uncoupling secretion and tip growth in lily pollen tubes: evidence for the role of calcium in exocytosis

Cytoplasmic calcium concentration ([Ca2+]i) and extracellular calcium (Ca2+o) influx has been studied in pollen tubes of Lilium longliflorum in which the processes of cell elongation and exocytosis have been uncoupled by use of Yariv phenylglycoside ((beta-D-Glc)3). Growing pollen tubes were pressure injected with the ratio dye fura-2 dextran and imaged after application of (beta-D-Glc)3, which binds arabinogalactan proteins (AGPs). Application of (beta-D-Glc)3 inhibited growth but not secretion. Ratiometric imaging of [Ca2+]i revealed an initial spread in the locus of the apical [Ca2+]i gradient and substantial elevations in basal [Ca2+]i followed by the establishment of new regions of elevated [Ca2+]i on the flanks of the tip region. Areas of elevated [Ca2+]i corresponded to sites of pronounced exocytosis, as evidenced by the formation of wall ingrowths adjacent to the plasma membrane. Ca2+o influx at the tip of (beta-D-Glc)3-treated pollen tubes was not significantly different to that of control tubes. Taken together these data indicate that regions of elevated [Ca2+]i, probably resulting from Ca2+o influx across the plasma membrane, stimulate exocytosis in pollen tubes independent of cell elongation.

Repetitive calcium transients and the role of calcium in exocytosis and cell cycle activation in the mouse egg

The role of calcium in cortical granule exocytosis and activation of the cell cycle at fertilization was examined in the mouse egg using the calcium chelator BAPTA (1,2-bis( o-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid) and the fluorescent calcium indicator fluo-3. BAPTA and fluo-3 were introduced into zona-free mouse eggs by a 30-min incubation with 0.01–50 μ M BAPTA acetoxymethyl ester (AM) and/or 1–20 μ M fluo-3 AM prior to in vitro fertilization. Incubation of eggs in ⩾5.0 μ M BAPTA AM inhibited cortical granule exocytosis in all cases. Introduction of the calcium chelator into the egg blocked second polar body formation at ⩾1.0 μ M BAPTA AM. Sperm entry occurred in all eggs regardless of the BAPTA AM concentration. Sperm induce a large transient increase in calcium lasting 2.3 ± 0.6 min, followed by repetitive transients lasting 0.5 ± 0.1 min and occurring at 3.4 ± 1.4-min intervals. Incubation with ⩾5.0 μ M BAPTA AM inhibited all calcium transients. Introduction of BAPTA also inhibited calcium transients, exocytosis, and the resumption of meiosis following application of the calcium ionophore A23187 or SrCl 2, which activate eggs. These results demonstrate that the calcium increase at fertilization is required for cortical granule exocytosis and resumption of the cell cycle in a mammalian egg.

The role of calcium in exocytosis and endocytosis in plant cells

The role of calcium in the individual cellular events leading to exocytosis is considered. Both vesicle movement processes and vesicle fusion at the cell surface require calcium for completion of specific events in this pathway. Our knowledge of these events is incomplete. In particular the movement of secretory vesicles by the cytoskeleton in response to added calcium is a key event that is beyond our comprehension at present. At the whole cell level, it is shown that external calcium, at the appropriate concentration, is required to elicit secretion at optimal rates. In both plant and animal cells secretion appears to be dependent on, or is triggered by, a rise in the level of internal free calcium ions from about 10‐7 to 10‐6M or even higher. In these eukaryotes internal organelles take up calcium and maintain a low level of calcium in the cell, offsetting the inflow of calcium from the plasma membrane. In some systems the inflow is restricted to a certain part of the plasma membrane, which then acts as a focus for exocytosis and, thereby, establishes a cellular polarity. In plant tissues there appears to be a requirement for some circulation of calcium within the apoplast, to sustain secretion.Recent papers on endocytosis have confirmed its occurrence in plant cells and made significant advances in isolating and characterising the clathrin coats of the coated vesicles involved in the uptake. There is no evidence, at present, for a direct role for calcium in these events. Indirectly, calcium stimulates exocytosis, and hence the delivery of excess membrane to the cell surface, which may be retrieved by an increase in the rate of endocytosis. Quantitative comparisons of the membrane flow occurring in these pathways are not available.Several plant cellular systems have been employed to study secretion and some of these may prove to be superior model systems for the investigation of certain aspects of the control of exocytosis and endocytosis by calcium ions.

Novel role of calcium in exocytosis: mechanism of nematocyst discharge as shown by x-ray microanalysis.

Mature nematocysts of the sea anemones Rhodactis rhodostoma and Anthopleura elegantissima contain a fluid that has a high concentration of solutes and is extraordinarily rich in calcium (ca. 500-600 mmol/kg wet weight); this contrasts with the surrounding cytoplasm which is rich in potassium but poor in calcium. The undischarged capsule is surrounded by a membrane that probably acts as a selective permeability barrier between the cytoplasm and the nematocyst fluid. During discharge the nematocyst moves to the surface of the nematocyte and comes into contact with the external sea water medium. Calcium, which may be bound to proteins in the undischarged state, is rapidly lost from the fluid; at the same time, sea water enters the capsule. In vitro experiments have already shown that calcium loss increases the osmotic pressure of the capsular fluid, causing an influx of water from the external medium; this influx appears to increase the hydrostatic pressure inside the capsule to the point that the thread everts explosively.

Surface electrostatic-charge measurements on islet and zymogen granules: Effect of calcium ions

Particle microeleetrophoresis has been used to measure the surface charge of secretory granules isolated from the exocrine and endocrine pancreas of guineapigs. Zymogen granules were found to have a surface charge density of 2378 esu.cm−2 and islet granules 4177 esu.cm−2. Calcium ions decreased the surface charge density of both types of granule; binding of calcium at the zymogen granule surface was found to fit a Langmuir adsorption isotherm. The results are discussed with respect to the role of calcium in exocytosis.