Plasma membrane resident 'fusion complexes' mediate reconstituted exocytosis

Calcium-triggered exocytosis is thought to be mediated by membrane-associated protein complexes. In sea urchin eggs, high concentrations of calcium activate multiple 'fusion complexes' per cortical vesicle-plasma membrane docking site. Some of these fusion complexes are known to reside in the vesicle membrane. It is not known if fusion complexes also reside in the plasma membrane, or if plasma membrane-resident fusion complexes require cognate partners in the vesicle membrane. Using reconstitution, we show that N-ethylmaleimide treatment of either vesicles or plasma membrane fragments prior to reconstitution does not completely inhibit exocytosis. Treatment of both components did result in complete inhibition. Upon reconstitution, cortical vesicles and the early endosomes formed by compensatory endocytosis both contributed, on average, two fusion complexes per reconstituted docking site. The plasma membrane contributed, on average, two fusion complexes per docking site when assembled with cortical vesicles, but only one complex when reconstituted with endosomes. We conclude that there are at least two types of plasma membrane-resident fusion complexes that participate in reconstituted cortical vesicle-plasma membrane fusion. The activity of one of these fusion complexes is target-specific for cortical vesicles, while the second type also supports fusion with endosomes.     

Calcium-dependent exocytosis in an in vitro secretory granule plasma membrane preparation from sea urchin eggs and the effects of some inhibitors of cytoskeletal function

Egg cortical granules remain attached to the egg plasma membrane when the egg is ruptured. We present evidence that demonstrates that, when the cytoplasmic face of the egg plasma membrane is exposed to micromolar calcium concentrations, an exocytosis of the cortical granules occurs which corresponds to the cortical granule exocytosis seen when the egg is fertilized. The calcium sensitivity of the preparation is decreased by an increase in magnesium concentration and increased by a decrease in magnesium concentration. Exocytosis is inhibited by trifluoperazine (half inhibition at 6 microM), a drug that inhibits the action of the calcium-dependent regulatory protein calmodulin. Colchicine, vinblastine, nocodazole, cytochalasin B, phalloidin, N-ethylmaleimide-modified myosin subfragment 1, and antibody to actin are without effect on this in vitro exocytosis at concentrations that far exceed those required to disrupt microtubules and microfilaments. Conditions are such that penetration to the exocytotic site is optimal. It is unlikely, therefore, that either actin or tubulin participate intimately in exocytosis. Our data also exclude on quantitative grounds several other mechanisms postulated to account for the fusion of the secretory granule with the plasma membrane.     

Ionic and permeability requirements for exocytosis in vitro in sea urchin eggs

We study exocytosis in the planar isolated cortex of the egg of the sea urchin Lytechinus pictus. Solutions bathing the exocytotic apparatus need not contain appreciable amounts of ions: fusion follows addition of submicromolar calcium to solutions containing only nonelectrolyte. We examine the effects of altering the granule membrane permeability to small molecules with ionophores and digitonin. Introducing holes in the secretory granule membrane to the extent of allowing free passage of small molecules does not cause secretion in vitro. We add the amphipathic compound digitonin at 12 to 15 microM concentrations and demonstrate that the granule membrane can become permeable to lucifer yellow, yet that granules remain intact. Granules still undergo exocytosis after digitonin treatment at such concentrations upon subsequent addition of calcium. Higher concentrations of digitonin lead to granule content swelling and vesicle bursting. We conclude that cortical granule hydration during exocytosis is not mediated by small ionic channels.     

Ionic and permeability requirements for exocytosis in vitro in sea urchin eggs

Summary We study exocytosis in the planar isolated cortex of the egg of the sea urchinLytechinus pictus. Solutins bathing the exocytotic apparatus need not contain appreciable amounts of ions: fusion follows addition of submicromolar calcium to solutions containing only nonelectrolyte. We examine the effects of altering the granule membrane permeability to small molecules with ionophores and digitonin. Introducing holes in the secretory granule membrane to the extent of allowing free passage of small molecules does not cause seretion in vitro. We add the amphipathic compound digitonin at 12 to 15 M concentrations and demonstrate that the granule membrane can become permeable to lucifer yellow, yet that granules remain intact. Granules still undergo exocytosis after digitonin treatment at such concentrations upon subsequent addition of calcium. Higher concentrations of digitonin lead to granule content swelling and vesicle bursting. We conclude that cortical granule hydration during exocytosis is not mediated by small ionic channels.     

Exocytosis in neuroendocrine cells: new tasks for actin

Most secretory cells undergoing calcium-regulated exocytosis in response to cell surface receptor stimulation display a dense subplasmalemmal actin network, which is remodeled during the exocytotic process. This review summarizes new insights into the role of the cortical actin cytoskeleton in exocytosis. Many earlier findings support the actin-physical-barrier model whereby transient depolymerization of cortical actin filaments permits vesicles to gain access to their appropriate docking and fusion sites at the plasma membrane. On the other hand, data from our laboratory and others now indicate that actin polymerization also plays a positive role in the exocytotic process. Here, we discuss the potential functions attributed to the actin cytoskeleton at each major step of the exocytotic process, including recruitment, docking and fusion of secretory granules with the plasma membrane. Moreover, we present actin-binding proteins, which are likely to link actin organization to calcium signals along the exocytotic pathway. The results cited in this review are derived primarily from investigations of the adrenal medullary chromaffin cell, a cell model that is since many years a source of information concerning the molecular machinery underlying exocytosis.     

Dissection of stages in exocytosis in the adrenal chromaffin cell with use of trifluoperazine

Stimulation of isolated chromaffin cells with carbamylcholine led to a number of morphological changes, indicative of exocytosis, apparently resulting from translocation of secretory granules to the plasma membrane and their subsequent fusion with the plasma membrane to release their contents. However, stimulation in the presence of trifluoperazine resulted only in the accumulation of secretory granules close to the plasma membrane. Thus exocytosis could be divided into two stages: a trifluoperazine-insensitive stage involving translocation of secretory granules to the plasma membrane and a second trifluoperazine-sensitive stage resulting in granule-plasma membrane fusion.     

Control of local actin assembly by membrane fusion-dependent compartment mixing

Local actin assembly is associated with sites of exocytosis in processes ranging from phagocytosis to compensatory endocytosis. Here, we examine whether the trigger for actin-coat assembly around exocytosing Xenopus egg cortical granules is 'compartment mixing'--the union of the contents of the plasma membrane with that of the secretory granule membrane. Consistent with this model, compartment mixing occurs on cortical granule-plasma membrane fusion and is required for actin assembly. Compartment mixing triggers actin assembly, at least in part, through diacylglycerol (DAG), which incorporates into the cortical granule membranes from the plasma membrane after cortical granule-plasma membrane fusion. DAG, in turn, directs long-term recruitment of protein kinase Cbeta (PKCbeta) to exocytosing cortical granules, where it is required for activation of Cdc42 localized on the cortical granules. The results demonstrate that mixing of two membrane compartments can direct local actin assembly and indicate that this process is harnessed during Xenopus egg cortical granule exocytosis to drive compensatory endocytosis.     

The dynamics of exocytosis in human neutrophils

We have investigated the dynamics of exocytosis in single human neutrophils. The increase of membrane area associated with granule fusion was followed by time-resolved patch-clamp capacitance measurements. Intracellular application of 20 microM guanosine-5'-O(3- thiotriphosphate) (GTP gamma S) in the presence of 2.5 mM ATP stimulated exocytosis and led to an increase of membrane capacitance from 3.0 to integral of 8.4 pF corresponding to a 540 micron 2 increase of membrane area. This capacitance change is very close to the value expected from morphological data if all primary and secondary granules fuse with the plasma membrane. High resolution measurements revealed stepwise capacitance changes corresponding to the fusion of individual granules. GTP gamma S-stimulated exocytosis did not require pretreatment with cytochalasin B and the amplitude was independent of the intracellular-free calcium concentration between 10 nM and integral of 2.5 microM. In the absence of GTP gamma S elevation of intracellular- free calcium concentration to the micromolar range led to the fusion of only a limited number of granules. Degranulation stimulated with GTP gamma S started after a lag phase of 2-7 min and was usually complete within 5-20 min. The time course was affected by the intracellular ATP and calcium concentration. Exocytosis was markedly accelerated by pretreatment with cytochalasin B. Our results demonstrate that the final steps leading to primary and secondary granule fusion are controlled by a guanine nucleotide-binding protein and do not require an elevation of intracellular calcium. Calcium and other factors are, however, involved in the regulation having pronounced effects on the dynamics of exocytosis.     

Widespread association of annexin II with intracellular membrane systems and involvement of annexin II in granule--granule fusion in addition to granule--plasma membrane fusion in anterior pituitary cells

The subcellular localization in anterior pituitary secretory cells of annexin II, one of the Ca2+-dependent phospholipid-binding proteins, was examined by immunohistochemistry and immunoelectron microscopy. Annexin II was associated with the plasma membrane, the membranes of secretory granules and cytoplasmic organelles, such as rough endoplasmic reticulum, mitochondria and vesicles, and with the nuclear envelope. Annexin II was frequently detected at the contact sites of secretory granules with other granules and with the plasma membrane. The anterior pituitary and adrenal medulla were treated with Clostridium perfringens enterotoxin, which induces Ca2+ influx, and examined under an electron microscope. The anterior pituitary cells showed multigranular exocytosis, i.e. multiple fusions of secretory granules with each other and with the plasma membrane, but adrenal chromaffin cells, which lack annexin II on the granule membranes, never showed granule--granule fusion and only single granule exocytosis. From these results, we conclude that, in anterior pituitary secretory cells, annexin II is involved in granule--granule fusion in addition to granule--plasma membrane fusion. © 1998 Chapman & Hall     

Phospholipases D1 and D2 regulate different phases of exocytosis in mast cells

The rat mast cell line RBL-2H3 contains both phospholipase D (PLD)1 and PLD2. Previous studies with this cell line indicated that expressed PLD1 and PLD2 are both strongly activated by stimulants of secretion. We now show by use of PLDs tagged with enhanced green fluorescent protein that PLD1, which is largely associated with secretory granules, redistributes to the plasma membrane in stimulated cells by processes reminiscent of exocytosis and fusion of granules with the plasma membrane. These processes and secretion of granules are suppressed by expression of a catalytically inactive mutant of PLD1 or by the presence of 50 mM 1-butanol but not tert-butanol, an indication that these events are dependent on the catalytic activity of PLD1. Of note, cholera toxin induces translocation of PLD1-labeled granules to the plasma membrane but not fusion of granules with plasma membrane or secretion. Subsequent stimulation of calcium influx with Ag or thapsigargin leads to rapid redistribution of PLD1 to the plasma membrane and accelerated secretion. Also of note, PLD1 is recycled from plasma membrane back to granules within 4 h of stimulation. PLD2, in contrast, is largely confined to the plasma membrane, but it too participates in the secretory process, because expression of catalytically inactive PLD2 also blocks secretion. These data indicate a two-step process: translocation of granules to the cell periphery, regulated by granule-associated PLD1, and a calcium-dependent fusion of granules with the plasma membrane, regulated by plasma membrane-associated PLD2 and possibly PLD1.     

Peripheral actin filaments control calcium-mediated catecholamine release from streptolysin-O-permeabilized chromaffin cells

Adrenal medullary chromaffin cells were permeabilized by treatment with a streptococcal cytotoxin streptolysin O (SLO) which generates pores of macromolecular dimensions in the plasma membrane. SLO did not provoke spontaneous release of catecholamines or chromogranin A, a protein marker of the secretory granule, showing the integrity of the secretory vesicle membrane. However, the addition of micromolar free calcium concentration induced the corelease of noradrenaline and chromogranin A, indicating that secretory products are liberated by exocytosis. Calcium-dependent exocytosis from SLO-permeabilized cells required Mg-ATP and could not occur in the presence of other nucleotides. The pores generated by the toxin were large enough to introduce proteins, e.g., immunoglobulins, but also caused efflux of the cytosolic marker lactate dehydrogenase. Despite this, the cells remained responsive to calcium for up to 30 min after permeabilization, indicating that they retained their secretory machinery. In the search for a functional role of cytoskeletal proteins in the secretory process, we used SLO-permeabilized cells to examine the localization of filamentous actin, using rhodamine-phalloidin, and that of the actin-severing protein, gelsolin, using specific antibodies. It was found that both F-actin and gelsolin were exclusively localized in the subplasmalemmal region of the cell. We examined the relationship between actin disassembly, the elevation of intracellular calcium and secretion in SLO-treated cells. F-Actin destabilizing agents such as cytochalasin D or DNase I were found to potentiate calcium-stimulated release. The maximal effect was observed at low calcium concentrations (1-4 microM) and at the later stages of the secretory response (after 10 min stimulation). In addition, using rhodamine-phalloidin, we observed that calcium provoked simultaneously both cortical actin disassembly and catecholamine release in SLO-permeabilized cells. These results demonstrate that a close relationship exists between the secretory response and actin disassembly and provide further evidence that intracellular calcium controls the subplasmalemmal cytoskeletal actin organization and thereby the access of secretory granules to exocytotic sites.     

Poisson-distributed active fusion complexes underlie the control of the rate and extent of exocytosis by calcium

We have investigated the consequences of having multiple fusion complexes on exocytotic granules, and have identified a new principle for interpreting the calcium dependence of calcium-triggered exocytosis. Strikingly different physiological responses to calcium are expected when active fusion complexes are distributed between granules in a deterministic or probabilistic manner. We have modeled these differences, and compared them with the calcium dependence of sea urchin egg cortical granule exocytosis. From the calcium dependence of cortical granule exocytosis, and from the exposure time and concentration dependence of N-ethylmaleimide inhibition, we determined that cortical granules do have spare active fusion complexes that are randomly distributed as a Poisson process among the population of granules. At high calcium concentrations, docking sites have on average nine active fusion complexes.     

N-ethylmaleimide-sensitive factor is required to organize functional exocytotic microdomains in paramecium

In exocytosis, secretory granules contact plasma membrane at sites where microdomains can be observed, which are sometimes marked by intramembranous particle arrays. Such arrays are particularly obvious when membrane fusion is frozen at a subterminal stage, e.g., in neuromuscular junctions and ciliate exocytotic sites. In Paramecium, a genetic approach has shown that the "rosettes" of intramembranous particles are essential for stimulated exocytosis of secretory granules, the trichocysts. The identification of two genes encoding the N-ethylmaleimide-sensitive factor (NSF), a chaperone ATPase involved in organelle docking, prompted us to analyze its potential role in trichocyst exocytosis using a gene-silencing strategy. Here we show that NSF deprivation strongly interferes with rosette assembly but does not disturb the functioning of exocytotic sites already formed. We conclude that rosette organization involves ubiquitous partners of the fusion machinery and discuss where NSF could intervene in this mechanism.     

In vitro reconstitution of exocytosis from plasma membrane and isolated secretory vesicles

We describe the reconstitution of exocytotic function through recombination of purified cortical secretory vesicles (CVs) and plasma membrane from sea urchin eggs. CVs were dislodged from a cell surface complex preparation by gentle homogenization in an isotonic dissociation buffer, and purified by differential centrifugation. CV-free plasma membrane fragments were obtained by mechanically dislodging CVs from cortical lawn (CL) preparations with a jet of CL isolation buffer. This procedure produced a "plasma membrane lawn" preparation, consisting of plasma membrane fragments attached via their vitelline layer (an extracellular glycocalyx) to a polylysine-coated microscope slide. When freshly prepared CVs were incubated with plasma membrane lawns, CVs reassociated with the cytoplasmic face of the plasma membrane, forming an exocytotically competent, reconstituted cortical lawn (RL). Exocytosis in RLs was monitored by phase-contrast microscopy, and quantitated with a sensitive microphotometric assay. Half-maximal exocytosis in RLs occurred at 18.5 microM free Ca2+; half-maximal exocytosis in control lawns occurred at 5.7 microM free Ca2+. Greater than 90% of the purified CVs that were not attached to a plasma membrane lawn remained intact when bathed in a buffer containing millimolar Ca2+. This result excluded the possibility that Ca2+-triggered CV lysis was responsible for our observations, and confirmed that the association of CVs with the plasma membrane was required for exocytosis in RLs. Evidence that the Ca2+-stimulated release of CV contents in CLs and RLs is the in vitro equivalent of exocytosis was obtained with an immunofluorescence-based vectorial transport assay, using an antiserum directed against a CV content protein: stimulation of RLs or partially CV-depleted CLs with Ca2+ resulted in fusion of the CV and plasma membranes, and the vectorial transport of CV contents from the cytoplasmic to the extracytoplasmic face of the egg plasma membrane.     

Evidence that the ability to respond to a calcium stimulus in exocytosis is determined by the secretory granule membrane: Comparison of exocytosis of injected bovine chromaffin granule membranes and endogenous cortical granules inXenopus laevis oocytes

Summary 1. To understand better the mechanisms which govern the sensitivity of secretory vesicles to a calcium stimulus, we compared the abilities of injected chromaffin granule membranes and of endogenous cortical granules to undergo exocytosis inXenopus laevis oocytes and eggs in response to cytosolic Ca²⁺. Exocytosis of chromaffin granule membranes was detected by the appearance of dopamine--hydroxylase of the chromaffin granule membrane in the oocyte or egg plasma membrane. Cortical granule exocytosis was detected by release of cortical granule lectin, a soluble constituent of cortical granules, from individual cells.2. Injected chromaffin granule membranes undergo exocytosis equally well in frog oocytes and eggs in response to a rise in cytosolic Ca²⁺ induced by incubation with ionomycin.3. Elevated Ca²⁺ triggered cortical granule exocytosis in eggs but not in oocytes.4. Injected chromaffin granule membranes do not contribute factors to the oocyte that allow calcium-dependent exocytosis of the endogenous cortical granules.5. Protein kinase C activation by phorbol esters stimulates cortical granule exocytosis in bothXenopus laevis oocytes andX. laevis eggs (Bement, W. M., and Capco, D. G.,J. Cell Biol. 108, 885–892, 1989). Activation of protein kinase C by phorbol ester also stimulated chromaffin granule membrane exocytosis in oocytes, indicating that although cortical granules and chromaffin granule membranes differ in calcium responsiveness, PKC activation is an effective secretory stimulus for both.6. These results suggest that structural or biochemical characteristics of the chromaffin granule membrane result in its ability to respond to a Ca²⁺ stimulus. In the oocytes, cortical granule components necessary for Ca²⁺-dependent exocytosis may be missing, nonfunctional, or unable to couple to the Ca²⁺ stimulus and downstream events.     

Membrane hemifusion is a stable intermediate of exocytosis

Membrane fusion during exocytosis requires that two initially distinct bilayers pass through a hemifused intermediate in which the proximal monolayers are shared. Passage through this intermediate is an essential step in the process of secretion, but is difficult to observe directly in vivo. Here we study membrane fusion in the sea urchin egg, in which thousands of homogeneous cortical granules are associated with the plasma membrane prior to fertilization. Using fluorescence redistribution after photobleaching, we find that these granules are stably hemifused to the plasma membrane, sharing a cytoplasmic-facing monolayer. Furthermore, we find that the proteins implicated in the fusion process-the vesicle-associated proteins VAMP/synaptobrevin, synaptotagmin, and Rab3-are each immobile within the granule membrane. Thus, these secretory granules are tethered to their target plasma membrane by a static, catalytic fusion complex that maintains a hemifused membrane intermediate.     

High calcium concentrations shift the mode of exocytosis to the kiss-and-run mechanism

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 calcium-concentration-dependent manner. We propose that, during secretion of neurotransmitters at synapses, the mode of exocytosis is modulated by calcium to attain optimal conditions for coupled exocytosis and endocytosis according to synaptic activity.     

Tight coupling of the t-SNARE and calcium channel microdomains in adrenomedullary slices and not in cultured chromaffin cells

Regulated exocytosis involves calcium-dependent fusion of secretory vesicles with the plasma membrane with three SNARE proteins playing a central role: the vesicular synaptobrevin and the plasma membrane syntaxin1 and SNAP-25. Cultured bovine chromaffin cells possess defined plasma membrane microdomains that are specifically enriched in both syntaxin1 and SNAP-25. We now show that in both isolated cells and adrenal medulla slices these target SNARE (t-SNARE) patches quantitatively coincide with single vesicle secretory spots as detected by exposure of the intravesicular dopamine beta-hydroxylase onto the plasmalemma. During exocytosis, neither area nor density of the syntaxin1/SNAP-25 microdomains changes on the plasma membrane of both preparations confirming that preexisting clusters act as the sites for vesicle fusion. Our analysis reveals a high level of colocalization of L, N and P/Q type calcium channel clusters with SNAREs in adrenal slices; this close association is altered in individual cultured cells. Therefore, microdomains carrying syntaxin1/SNAP-25 and different types of calcium channels act as the sites for physiological granule fusion in "in situ" chromaffin cells. In the case of isolated cells, it is the t-SNAREs microdomains rather than calcium channels that define the sites of exocytosis.     

Digitonin-Permeabilized Cells Are Exocytosis Competent

Release of norepinephrine from PC12 cells can be stimulated by free Ca2+ in micromolar concentrations after permeabilization with 10 micrograms/ml of digitonin. This release is time and temperature dependent, half-maximal at 0.3 microM Ca2+, and, after washing out of endogenous ATP, half-maximal at about 0.5 mM MgATP when exogenously added. Similar results were obtained with bovine adrenal chromaffin cells using the same protocol. Support for the idea that the mechanism of release from both permeabilized cell types is still exocytosis is demonstrated at the electron microscopic level by immunolabeling chromaffin granule membrane antigens that were introduced into the plasma membrane following stimulation. Electron micrographs furthermore demonstrate that chromaffin granules retain typical dense cores after permeabilization, indicating that leakiness of catecholamines from the granules was not a major factor. Pores, formed by digitonin in the plasma membranes, were utilized to introduce antibodies into such exocytosis-competent cells. Anti-actin and anti-chromaffin granule membrane antibodies show a staining pattern similar to conventionally fixed and stained preparations. Our results demonstrate that pores formed by digitonin do not impair the process of exocytosis although they are big enough to allow macromolecules to pass in both directions. The digitonin-permeabilized cell is therefore an ideal in vitro system with which to study the fusion process between chromaffin granules and the plasma membrane.     

Localized exocytosis of primary (lysosomal) granules during phagocytosis: role of Ca2+-dependent tyrosine phosphorylation and microtubules

The uptake and killing of bacteria by human neutrophils are dependent on the fusion of secretory granules with forming phagosomes. The earliest component of exocytosis was found to precede phagosome closure, so that granular membrane constituents were detectable on the plasmalemma. We show that during phagocytosis of IgG-opsonized particles, this early secretory response is highly polarized in the case of primary granules, but less so for specific granules. The vectorial discharge of primary granules was dependent on calcium, but no evidence was found that calcium is involved in determining the polarity of exocytosis. In particular, a redistribution of endomembrane calcium stores toward forming phagosomes could not be detected. Polarized granule exocytosis was accompanied by focal tyrosine phosphorylation and actin polymerization, although the latter was not required for the response. Instead, microtubules seemed to contribute to the vectorial nature of the response. During particle ingestion, the microtubule-organizing center relocated toward forming phagosomes, and colchicine treatment altered the pattern of exocytosis, reducing its directionality. We hypothesize that the focal activation of tyrosine kinases generates localized signals that induce exocytosis in a calcium-dependent manner, and that reorientation of microtubules facilitates preferential delivery of granules toward the forming phagosome.