I. INTRODUCTION

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The traffic of secretory vesicles to the plasma membrane of eukayrotic cells is essential for normal cellular function. It also forms the basis of intercellular communication in multicellular organisms through the release of a wide array of extracellularly acting molecules. The fusion of secretory vesicles with the plasma membrane occurs in essentially all cells in the form of constitutive exocytosis that is required for the insertion of new cell membrane (107, 377). Some extracellular molecules (e.g., plasma proteins, antibodies, extracellular matrix components, etc.) are also secreted by a constitutive exocytotic pathway. In many cell types, a second pathway also exists in which exocytosis can be tightly regulated to allow the controlled release of vesicle contents or regulated insertion of new membrane components due to fusion of preformed secretory vesicles only in response to a physiological signal. Regulated exocytosis has been extensively studied in synapses where it is the mechanism by which neurotransmitters are very rapidly released in a controlled manner from synaptic vesicles to mediate neurotransmission (378, 874). Certain neurons also possess dense-core granules (otherwise known as large dense-core vesicles, Refs. 53, 769) that can be triggered to undergo exocytosis independently of synaptic vesicles (52, 284, 446, 801, 871). A wide range of nonneuronal cell types contain regulated secretory vesicles identified as dense-core granules or secretory granules, the contents of which serve a diverse range of physiological functions. These include cells specialized to secrete large amounts of secretory products including, for example, neuroendocrine, endocrine, and exocrine cells but also cells where exocytosis serves a more specialized and transitory function including the sperm (134, 488, 650) and the egg (189, 724, 833). The latter examples demonstrate the essential nature of secretory granule exocytosis even for the continued existence of multicellular species.

Work from a large number of laboratories using biochemical, molecular biological, and genetic approaches has, in recent years, identified many of the proteins involved in the mechanism of synaptic vesicle exocytosis (428, 714, 734) and established their in vivo importance in neurotransmission (93, 95, 245, 434, 441, 490, 581). The interactions between these proteins and the way in which a Ca²⁺ signal leads to synaptic vesicle exocytosis are known in outline (428), although the full details of the processes involved still remain to be resolved. Similar molecular events appear to underlie secretory granule exocytosis (103, 121, 258, 260, 297, 307, 356, 408, 500, 506, 610), and in fact, functional studies of granule exocytosis in certain experimentally favorable cell types (e.g., adrenal chromaffin cells, Refs. 121, 124, 126) have been important in the development of the current description of the various stages in regulated exocytosis. Synaptic vesicle exocytosis has been extensively reviewed in recent years (35, 37, 56, 58, 60, 96, 130, 308, 316, 361, 428, 584, 638, 734, 736, 812, 847) and will not be addressed in extensive detail here. It will, however, be used to illustrate some of the advances in our molecular understanding and in comparisons with secretory granule exocytosis. Our main aim in this review is to concentrate on the diversity of dense-core or secretory granule exocytosis. We describe current views on the mechanistic aspects of secretory granule exocytosis and its triggering and regulation. In addition, we discuss its control and cell-specific specializations related to the physiological role of granule exocytosis in the diverse cell types in which it occurs.

	II. THE WIDESPREAD DISTRIBUTION OF SECRETORY GRANULE EXOCYTOSIS

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Secretory granules and their regulated exocytosis have been most extensively studied in a few cell types chosen either as model systems due to certain experimental advantages or due to their crucial physiological or pathophysiological interest. The pathway followed by secretory proteins through the cell was delineated in classical studies by George Palade from the study of pancreatic exocrine cells (578). In more recent years, probably the most studied cell types have been the adrenal chromaffin cell (and its tumor counterpart the PC12 cell line) as a model for both biochemical and electrophysiological investigation (121, 279, 506), the pancreatic -cell (225, 408, 844) due to the importance of insulin secretion, and its dysfunction in forms of diabetes mellitus and hemopoietic cells [mast cells (430), platelets (159, 415, 416), and neutrophils (88, 540, 689)] due to their importance in normal and pathological immune responses (732). Secretory granule exocytosis also occurs, however, in many different neuroendocrine and endocrine cell types for the secretion of peptides and other hormones (53) and in exocrine cells for the secretion of digestive enzymes (104, 596, 826). Specialized secretory cells are found in various predominantly nonsecretory tissues [e.g., in the heart (524), the lung (651), and the kidney (301)]. In addition, granule exocytosis also has specialized functions in other cells such as the sperm (134, 488) and the egg (40, 54, 356, 469).

The wide range of secretory granule-containing cells present in mammalian species is shown in Table 1. The list includes neurons that can possess dense-core granules in addition to synaptic vesicles or may possess dense-core granules alone (769). Certain neuroendocrine and endocrine cells are present in tissues with a purely secretory function such as those in the pituitary, adrenal medulla, or pancreas. Others are present as less abundant cells in tissues specialized for other functions such as the G cells that reside in the gastric mucosa and secrete gastrin (75, 218, 798), the juxtaglomerular cells of the kidney which secrete renin (255, 301, 545), the alveolar II epithelial cells of the lung responsible for secretion of surfactant (304, 305, 651, 841), or goblet cells in the lung and intestine that secrete mucin (718, 799). Cells with other functions such as endothelial cells also have a regulated secretory function (74, 238, 797) for release from their Weibel-Palade bodies. A more specialized form of exocytosis occurs in the egg where cortical granule exocytosis is not to release extracellularly acting signaling molecules but to release components of the fertilization envelope that prevents further sperm entry (833). In the case of the sperm, a single secretory organelle at the head of the sperm, the acrosome, releases enzymes required to enable fertilization (134, 488). Another unusual cell type is the mammary epithelial cell responsible for milk production (120, 127, 413, 433). These cells produce and secrete large amounts of the milk proteins including the caseins and do so by both constitutive and Ca²⁺-regulated exocytosis (788, 789).

	III. BIOGENESIS OF SECRETORY GRANULES

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In order for secretory cells to release material by exocytosis strictly on demand, this material cannot be allowed to enter the constitutive exocytotic pathway. Therefore, some mechanism(s) must exist to selectively exclude regulated secretory proteins from nascent constitutive secretory vesicles. It follows, then, that an alternative organelle capable of stimulus-induced exocytosis is required to accommodate these regulated secretory proteins. In general, secretory granules are used for this purpose. The highly condensed granule matrix, which gives rise to the eponymous dense core visualized by electron microscopy, allows efficient storage of large amounts of secretory material in an osmotically inert form (107). Although in most cell types secretory granules appear to represent an entirely new class of organelle, granules in various hemopoietic cells and certain other cell types share several properties with lysosomes (83, 732). For example, in addition to possessing lysosomal markers, such granules are accessible via the endocytic pathway and dense-core formation can occur in multivesicular bodies (624, 732). This suggests that a convergence of biosynthetic and endocytic membrane traffic analogous to lysosomal biogenesis is required for secretory granule formation in such cells. Indeed, Chediak-Higashi syndrome in humans (and its mouse model, beige) is characterized by giant intracellular lysosomes and granules in hemopoietic cells and melanocytes, whereas granules in other cell types are unaffected (87, 486). Similarly, the acidic hydrolases contained within the acrosome of spermatozoa suggest that this organelle may be a modified lysosome (785). Indeed, as conventional lysosomes can undergo stimulus-induced fusion with the plasma membrane in a wide range of cells (19), it may be that even the classical secretory granules evolved from a lysosomal progenitor.

In contrast to the biogenesis of "secretory lysosomes" found in hemopoietic cells, the granules found in endocrine, exocrine, and neural cells are a product of the biosynthetic pathway alone. Initial formation of immature granules occurs at the trans-Golgi network (TGN), although subsequent maturation steps occur beyond this compartment (28, 774). Unlike transport vesicle formation at earlier stages in the secretory pathway, granule formation does not apparently require a coat-driven budding process. Instead, it is thought that membrane deformation may result from the aggregation of secretory proteins in the TGN. Chromogranin A (CgA) appears to be essential for this process in neuroendocrine cells, as downregulation of CgA in PC12 cells reduces granule number and, strikingly, expression of CgA in fibroblasts induces the formation of dense-cored vesicles of around 100 nm diameter (383). Although the mechanistic basis of this has not been demonstrated, presumably the aggregative properties of CgA, coupled with its propensity to interact with membranes (351), allows wrapping of the TGN membrane around the forming CgA aggregate, analogous to the budding of enveloped viruses. Although CgA alone seems to turn on secretory granule biogenesis, its restricted expression in neural and endocrine tissues means that other as yet unidentified proteins must fulfill this function in other cell types. Interestingly, heterologous expression of pro-von Willebrand factor in endocrine and neuroendocrine cell lines has been shown to cause the biogenesis of tubular structures resembling Weibel-Palade bodies, the organelle in which endogenous von Willebrand factor is normally stored within endothelial cells (814). Similarly, heterologous expression of the melanocyte-specific protein Pmel17 in nonpigmented cells induces the formation of pre-melanosome-like structures (64). Consistent with this finding, inhibition of synthesis of melanin, the content of melanosomes, inhibits melanosome biogenesis (296). Nascent granule buds formed by CgA or its putative functional homologs must subsequently be pinched off to form immature secretory granules. Cholesterol is essential for this process as depletion of cholesterol arrests granule biogenesis at a late stage with dense-cored buds observable at the TGN (821). The role of cholesterol in vesicle scission may be direct, by facilitating negative membrane curvature at the bud neck (821). Alternatively, because cholesterol is a key component of lipid rafts, it may play an indirect role in the recruitment of raft-associated proteins that then drive membrane scission. Dynamin II is an attractive candidate for such a protein, because it has been shown to control granule formation in AtT20 cells (857), and because its homolog, dynamin I, is thought to directly drive endocytic vesicle scission (201).

Once formed, immature secretory granules must be processed and remodeled to form mature secretory granules. In endocrine and neuroendocrine cells, this involves both fusion with other immature secretory granules and removal of missorted material via budding (775). Fusion between immature secretory granules contributes to the increased size and density of mature granules in PC12 cells (773) and occurs via the ubiquitous core machinery for membrane fusion (792) (see sect. V). Perhaps surprisingly, immature granules can undergo regulated exocytosis (773), indicating that the process of granule maturation does not confer plasma membrane fusion competence, but rather inhibits homotypic granule-granule fusion. Intriguingly, immature granule fusion requires syntaxin 6 (830), and this SNARE protein is absent from mature granules, being removed by the budding of AP-1/clathrin-coated vesicles (390). This suggests that the transition from an immature granule capable of homotypic fusion to a mature granule capable only of exocytosis is due to the removal of SNAREs involved in granule-granule fusion. In a variety of cell types, it has been shown that newly synthesized granule contents are preferentially released, suggesting that immature granules may be inherently more fusogenic (see Ref. 283 and references therein). However, studies on AtT20 cells suggest that the process of maturation actually increases stimulus-secretion coupling, as newly formed granules are poorly responsive to secretagogues relative to mature granules (226). Again, this change is likely to be mediated by removal of proteins via coated vesicles. In this case, the presence of synaptotagmin IV on immature granules is thought to inhibit the putative exocytotic Ca²⁺ receptor synaptotagmin I (437), with its removal during maturation enabling normal Ca²⁺-dependent release (226). In addition to altering organelle fusion specificity, budding via coated vesicles serves to reduce excess membrane from the condensing granule and to remove missorted proteins, such as lysosomal proteins (390).

	IV. THE EXOCYTOTIC PATHWAY

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With the use of a range of functional assays, regulated exocytosis has been functionally dissected into a number of stages (121, 123, 279, 462, 585). In general, regulated exocytosis of secretory granules involves exocytosis of a premade pool of stable granules (Fig. 1) that in some cell types have a very long half-life in the absence of stimulation (194). The pool of secretory granules may be fully releasable [as in mast cells for example (430) where a full all-or-nothing exocytotic response occurs (331)] but in many cell types (such as neuroendocrine, endocrine, and exocrine cells), a large reserve pool exists and only a small portion of the granules (~1%) are initially rapidly releasable. In endocrine cells, ongoing secretion eventually requires synthesis of new granules. In many cells, the granules exist in a range of states from being nonreleasable or in a pool that becomes releasable following a recruitment step, through to being part of a so-called "ready-releasable" pool (18, 325, 519, 522, 559, 585, 654, 766, 767, 851, 852) that can undergo exocytosis within tens of milliseconds. These pools have been defined and studied through the use of permeabilized cell assays of exocytosis (109, 110, 342, 396, 430), single cell electrophysiological approaches using patch-clamp capacitance measurement of cell surface area (306, 521, 559), amperometric measurement of the release of single granule contents (418, 838), or by imaging exocytosis through the use of evanescent wave microscopy (729). These techniques have allowed more direct measurement of exocytosis than is generally possible for synapses where usually the small size of the presynaptic terminals precludes direct measurement of exocytosis. The majority of work on synaptic vesicle exocytosis has relied on overall measurement of synaptic transmission based on a simultaneous pre- and postsynaptic recording and functional assessment of the number of released quanta (95, 349, 459). Alternatively, the overall synaptic vesicle cycle within synaptic terminals has been assessed using uptake and release of the lipophilic FM dyes (20, 69) that became fluorescent following insertion into lipid bilayers and thereby, report plasma membrane area and changes due to exocytosis and membrane retrieval (69, 70, 326, 374, 388, 512, 660, 726, 846). This technique has, however, poor time resolution compared with the speed of neurotransmission (662). Patch-clamp capacitance measurement has been possible for a small number of rather specialized giant synaptic terminals (323, 586, 677, 810, 811). In addition, morphological approaches with electron microscopy have provided a static snapshot of granules (603, 604) or synaptic vesicles (293, 294, 330, 817) and their distribution within the cell. More recently, novel imaging techniques using evanescent wave (also known as total internal reflection) microscopy have been used to examine the movement of single secretory vesicles and exocytosis at the level of the light microscope in PC12 and chromaffin cells (365, 550-552, 728, 730), INS-1 cells, (782), pancreatic -cells (548, 549), and in specialized synapses (863). These new approaches have allowed an analysis of the dynamics of granule recuitement, docking at the plasma membrane, and exocytosis in living cells.

View larger version (151K): [in this window] [in a new window]   Fig. 1. Electron micrographs showing the secretory granules in adrenal chromaffin cells. A: a general view of an unstimulated chromaffin cell. The scale bar represents 1 µm. B: a granule core being released by exocytosis from a cell in the hamster adrenal medulla. The scale bar represents 100 nm.

The functionally defined, sequential stages within the exocytotic pathway (Fig. 2) have been shown to involve ATP-dependent priming steps, physical movement of vesicles to the subplasmalemmal region of the cell, tethering and then docking at release sites on the plasma membrane, conversion to a fully releasable state, triggered membrane fusion, release of granule contents, and finally retrieval of the granule membrane (Fig. 1). In the case of Ca²⁺-triggered exocytosis, Ca²⁺ may stimulate multiple stages before membrane fusion as well as fusion itself and also membrane retrieval by endocytosis (111, 235, 458, 522, 579, 703, 765). Description of some of these stages in exocytosis has resulted in some confusion in the literature due to different uses of terminology or due to the limitations of electron microscopic studies. There are significant technical problems with fixation in electron microscopy of dynamic events like exocytosis (330, 603, 604). In particular, chemical fixation acts too slowly (330, 705) to be able to preserve exocytosis in progress (unless special conditions are used, Ref. 524) and, therefore, exocytotic events are rarely seen in conventional electron microscopy even after treatments that stimulate extensive exocytosis but can be captured by use of rapid freezing techniques (399).

View larger version (11K): [in this window] [in a new window]   Fig. 2. Schematic representation of the steps leading to secretory granule exocytosis. This represents the events observed in neuroendocrine cells undergoing Ca2+-triggered exocytosis. In this case, Ca2+ entry leads to disassembly of the cortical actin cytoskeleton. This allows granule recruitment in MgATP-dependent steps to the plasma membrane where tethering and docking of the granule can occur. Fusion and release of granule contents can be triggered by Ca2+ in the absence of MgATP.

The existence of a labile ATP-dependent process required to maintain exocytosis was shown in various cell types including adrenal chromaffin cells (39) and sea urchin eggs (40). The term priming was originally used to describe an ATP-dependent process, detectable in permeabilized adrenal chromaffin cells that preceded Ca²⁺-triggered fusion and increased the extent of exocytosis due to a Ca²⁺ trigger (340). Priming was itself stimulated by lower levels of Ca²⁺ (78). Priming and fusion were subsequently shown to be stimulated by distinct cytosolic proteins (140, 315, 816). ATP-dependent priming, requiring ATP hydrolysis, encompasses multiple events. These can include reorganization of the actin cytoskeleton (113, 151, 154, 806) to recruit granules to the subplasmalemmal space and modification of the so-called SNARE proteins by -SNAP and the ATPase N-ethylmaleimide-sensitive fusion protein (NSF) (45, 50, 140, 208, 288, 321, 384, 436, 505, 514, 580, 713, 714) (described in detail in sect. VB). In addition, ATP is also required in a house-keeping role to maintain the levels of polyphosphoinositides required for continued exocytosis (227) as illustrated by the identification of phosphatidylinositol transfer protein (PITP) (314) and phosphatidylinositol 5-kinase (313) as cytosolic priming factors that can reconstitute exocytosis after ATP depletion. In the studies of secretory granule exocytosis, priming is a term used to describe any functionally detected ATP-dependent process that occurs before fusion. It is well established that membrane fusion itself does not require ATP hydrolysis (322, 340, 430, 585, 758, 851, 852). Confusion in the literature has come from the term priming also being applied to an ill-defined step in synaptic vesicle maturation that may not be ATP dependent. Within synapses a pool of synaptic vesicles are described as "docked" (129) as they appear to be very close to the presynaptic plasma membrane at the active zone by electron microscopic examination. Based on electrophysiological measurement, only a small proportion (~1-10%) of these docked vesicles is able to undergo exocytosis in response to Ca²⁺ elevation after Ca²⁺ channel opening following a single action potential (282). This pool of docked vesicles can, however, be released experimentally, through an unknown mechanism, by treatment with hypertonic sucrose (654). It has been suggested that the so-called docked vesicles undergo a maturation process after docking to become releasable. Confusingly, this has also been termed priming even though there is no experimental evidence as to whether or not it is an ATP-dependent step. This maturation step is not understood in molecular terms but is likely to involve the assembly of the fusion machinery, following tethering of the vesicles to the membranes, to allow very rapid synaptic vesicle exocytosis and appears to involve the protein unc13 (33).

The term docking is also one that has generated much confusion. As noted above, the concept of docked vesicles initially came from electron microscopic studies of fixed brain samples (293) in which synaptic vesicles close to the plasma membrane (within 10 nm) could be counted. Some of these vesicles remain associated with isolated plasma membrane preparations (153) and so must be tightly bound to the plasma membrane. These vesicles or granules seen by electron microscopy should correctly be described as "morphologically docked." Confusion arose from the assumption that all morphologically docked vesicles are attached to the plasma membrane via the same mechanism (129, 349) and are engaged with the core exocytotic machinery (particularly the SNARE proteins). We now know, from study of various vesicular traffic steps, that multiple mechanisms exist for initial attachment or tethering of vesicles to the target membrane (177, 599, 827). This precedes a true docked interaction in which the vesicles are poised for exocytosis. In general, the morphological distribution of vesicles and granules can be misleading, as this cannot easily be interpreted in relation to protein interactions known mainly from in vitro studies.

The precise events that initiate and complete membrane fusion are not understood, although many of the proteins involved have been identified (discussed in sect. V). It has been established through biophysical measurements, however, that fusion proceeds through the formation of a fusion pore that can open transiently and then potentially rapidly reclose (Fig. 3) (429). The majority of the data on fusion pore characteristics have come from analysis of secretory granule exocytosis. Fusion pores have, however, recently been observed in recordings from posterior pituitary nerve endings that result from fusion of both large dense-core vesicles and from synaptic-like microvesicles (391). Patch-clamp measurements of plasma membrane capacitance in mast cells were the first to allow the detection of changes in cell surface area due to single granule fusion events and also demonstrated a phenomenon known as capacitance flicker, which suggested that reversible fusion events were occurring (241). Later electrophysiological measurements characterized the transient fusion events (91, 493, 542, 543) and determined the current flow through the initial fusion pore (11, 90, 429, 442, 653, 667), which was of a defined magnitude as if the pore operated like a large ion channel or gap junction channel. It appeared that the initial fusion pore could either reclose or alternatively lead to a phase of fusion pore expansion (429) and thereby full exocytosis. Reclosure of the fusion pore can occur even after an expansion phase (12). The rate of fusion pore expansion was found to be a potentially regulated phenomenon and was increased by elevating intracellular Ca²⁺ concentration in rat mast cells (242, 310) or by activating protein kinase C in eosinophils (667). One physiological consequence of fusion through a reversible fusion pore or reclosure after fusion pore expansion is that this could allow control of the amount of release that occurs per secretory granule. It has been speculated that the rate of fusion pore expansion for synaptic vesicles could determine the rate of increase of glutamate concentration in the synaptic cleft and, thereby, determine whether or not synaptic transmission occurs (172). The concentration of glutamate reached is crucial for whether a synapse is active or "silent," and indirect data show that this concentration can be modulated. It seems unlikely that controlling fusion pore expansion would be a mechanism to regulate release of small molecules such as neurotransmitters, however, because these would diffuse too quickly for release to be limited. It could, in contrast, be significant for the release of proteins (579) or larger structures within secretory granules (305).

View larger version (17K): [in this window] [in a new window]   Fig. 3. Comparison of kiss-and-run exocytosis and full fusion. Membrane fusion initially occurs by the formation of a fusion pore. Fusion pore expansion allows release of granule content. This can be limited by rapid reclosure of the fusion pore (kiss-and-run). Alternatively, full emptying of the granule can be followed by a retrieval mechanism involving dynamin and clathrin.

The concept of the fusion pore has been validated using carbon-fiber amperometry, which detected low levels of release of contents from mast cell granules during the initial phase after fusion (during capacitance flickering) and before the postulated fusion pore expansion had occurred (16). Biophysically similar fusion pores have been detected during fusion mediated by the influenza virus hemagglutinin (HA) fusion protein (200, 429, 872). In addition, analysis of fusion mediated by HA mutant constructs has shown that fusion pore formation and subsequent fusion pore expansion are mechanistically distinct events (457). Little is known about the structure of the fusion pore, and in extreme models (15, 143, 494, 495, 515, 872), it has been postulated to be either entirely protein based (as in an ion channel) or purely lipidic but surrounded by a protein scaffold. Two studies of mutant mice have suggested that the fusion pore is a real structure that may eventually be understood through molecular genetic studies. In the first, transient fusion events were found to be more frequent in membrane capacitance recording of mast cells from the ruby-eye mutant mouse (542). It is not yet known, however, which gene contains this mutation. In the second, mice were generated to knockout expression of SCAMP1. This had no major effect on brain development, but in mast cells, again transient, reversible, membrane fusion events were more frequent (243). These studies suggest that modification of protein expression can affect the opening/closure characteristics of the fusion pore, although the molecular basis for the changes in the mast cells from these mice is yet to be determined.

	V. THE CONSERVED CORE MACHINERY FOR REGULATED EXOCYTOSIS

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By the 1970s, the idea that stimulus-release coupling occurred via regulated exocytosis of secretory or synaptic vesicles was generally accepted. Although it was clear that Ca²⁺ was a key trigger of this process (219, 373), the downstream mechanism responsible for membrane fusion was entirely unknown. Indeed, subsequent studies of granule exocytosis in various cell types over many years failed to significantly illuminate this issue. Ironically, arguably the greatest advances in our understanding of the mechanism of regulated exocytosis came from complementary genetic and biochemical studies of constitutive secretion (613, 657). In the genetic approach, yeast cells were mutagenized and then screened for temperature-sensitive mutants able to secrete at 25°C but not at 37°C. This resulted in the isolation of the first secretory (sec) mutant sec1 (539). Subsequently, further mutants were isolated and subdivided into 23 complementation groups, termed sec1-23, each representing at least one mutant allele of a gene required for vesicle formation or consumption at defined steps in the secretory pathway (369, 537, 538, 613). In the biochemical approach, membrane traffic through the mammalian Golgi was reconstituted in vitro using isolated Golgi complexes and cytosol (41). Subsequent protein purification identified several soluble proteins required for efficient intra-Golgi transport (657).

The first protein identified using the biochemical approach was NSF (Table 2, Fig. 4), which was purified based on its ability to reconstitute intra-Golgi transport after blockade by N-ethylmaleimide (NEM) (82). NSF was also implicated in endoplasmic reticulum (ER) to Golgi transport (55) and endocytic vesicle fusion (216, 647) in vitro, suggesting that NSF was required for multiple steps in the constitutive secretory and endocytic pathways (657). When the mammalian NSF gene was cloned (839), its coding sequence was found to be 48% identical to that of the yeast SEC18 gene (224). Like NSF, Sec18 is required for multiple steps in the secretory and endocytic pathways (292, 636), and Sec18 can substitute for NSF in mammalian in vitro assays of endosome fusion (845), intra-Golgi transport (839), and granule exocytosis (722). These observations suggested that NSF and Sec18p contribute similar, if not identical, biochemical functions to an evolutionarily ancient molecular mechanism of membrane fusion operative in all cells.

View larger version (31K): [in this window] [in a new window]   Fig. 4. Major proteins involved in regulated secretory granule exocytosis. The role of many of these proteins was originally identified from studies of neurotransmitter release or exocytosis in neuroendocrine cells such as chromaffin or PC12 cells. In some secretory cells, nonneuronal isoforms of the proteins are likely to be involved. The functional importance of all of the proteins shown has been established by genetic manipulation of their expression.

This notion received a major boost when biochemical protein-protein interaction studies revealed that NSF and its essential cofactor, -soluble NSF attachment protein (-SNAP; Sec17 in yeast), interact with brain membranes via a SNAP receptor (SNARE) complex comprising VAMP/synaptobrevin (Snc1/2 in yeast), syntaxin1 (Sso1/2 in yeast), and SNAP-25 (Sec9 in yeast) (714). These synaptic SNARE proteins are proteolytic substrates for the clostridial neurotoxins (Table 3; reviewed in Ref. 497), which are potent inhibitors of neurotransmission, thus suggesting that regulated exocytosis at the synapse occurred by the same basic process as constitutive membrane fusion. The realization that SNARE homologs were associated with distinct intracellular compartments and that yeast SNARE mutants blocked membrane fusion with these organelles led to the idea that SNARE proteins are essential for all intracellular membrane fusion events. Furthermore, the realization that two additional protein families, namely, the Rabs and the Sec1 homologs, were also involved in multiple vesicular transport processes led to the concept of a universal mechanism of membrane fusion (61, 246, 657). This idea is now generally accepted, although debate still rages over the proposed functions of the individual components of the conserved core machinery and over the precise mechanism of membrane fusion. Here we discuss current ideas of the biochemical functions of SNAREs and their regulatory interacting proteins and review the evidence for the involvement of these proteins in granule exocytosis.

SNAREs are membrane proteins that are localized to the various intracellular organelles. Although originally defined biochemically as SNAP receptors, most SNAREs have actually been identified by possession of a characteristic heptad repeat sequnce known as the SNARE motif. Searching for similar motifs in the human genome reveals 35 SNAREs, compared with 22 in the yeast genome (84, 588). Despite the large number of SNARE proteins, the synaptic SNARE complex first identified by Sollner et al. (714) remains the most intensively studied, and most current ideas of SNARE function are based on this, the archetypal complex. It comprises three proteins: the integral membrane proteins VAMP/synaptobrevin (777) and syntaxin 1 (59) and the membrane associated protein SNAP-25 (573). VAMP is a synaptic vesicle protein, whereas syntaxin and SNAP-25 are most enriched in the presynaptic plasma membrane (Fig. 4). These localizations led to the classification of SNAREs as vesicle (v-) or target (t-)SNAREs, and the notion (known as the SNARE hypothesis) that the fidelity of vesicle docking and fusion is determined by the specificity of v-SNARE:t-SNARE interactions (714). More recently, a rival classification system has been proposed based on whether a conserved glutamine (Q) or arginine (R) is present in the SNARE motif (240) in the so-called zero layer (Fig. 5), although in practice Q-SNAREs are almost always t-SNAREs and R-SNAREs are almost always v-SNAREs. In the synaptic complex, syntaxin (t-SNARE) contributes one Q-containing helix, SNAP-25 (t-SNARE) contributes two Q-containing helices, and VAMP (v-SNARE) contributes one R-containing helix (605, 743) (Fig. 5). Characterized SNARE complexes always contain three Q-helices (collectively, the t-SNARE) and one R helix (the v-SNARE), although these may be contributed from three or four proteins (SNAP-25 homologs typically contribute 2 helices) (240). This code of 3Q:1R helices has been shown to be the essential combination which, in a lock and key fashion, allows specific SNARE pairing and consequent membrane fusion between appropriate membranes in a reconstituted liposome fusion assay (483, 583). The Q and R residues themselves, however, are not essential for membrane fusion (291); rather, mutation of these amino acids impairs later disassembly of the SNARE complex by NSF and SNAPs (see sect. VB).

View larger version (54K): [in this window] [in a new window]   Fig. 5. Structure of the neuronal SNARE complex. The structure of the core complex formed by the cytoplasmic helices of VAMP, syntaxin 1, and SNAP-25 was solved by X-ray crystallography. A: the overall structure of the 4-helix bundle showing the position of the zero layer. B: a cross-sectional view of the interactions in the zero layer involving Arg-56 of VAMP, Gln-226 of syntaxin with Gln-53 and Gln-174 of SNAP-25.

The idea that SNARE complexes are essential for membrane fusion in living cells is well supported by genetic studies in the yeast, Saccharomyces cerevisiae, where deletion of SNAREs is usually lethal, and where conditional SNARE mutants are blocked at predictable membrane traffic steps (277). Similarly, genetic ablation of synaptic SNAREs in Drosophila melanogaster, Caenorhabditis elegans, and Mus musculus abolishes evoked neurotransmission (533, 678, 681, 825). It is interesting to note, however, that the effect of inactivating syntaxins tends to be more dramatic than when other SNAREs are blocked. For example, ablation of VAMP-2 or SNAP-25 in mice and n-syb in Drosophila abolishes evoked neurotransmission, but spontaneous transmitter release events are relatively unaffected (94, 209, 678, 825). In contrast, syntaxin null mutants in Drosophila exhibit a complete block in both spontaneous and evoked neurotransmission (681). Likewise, deletion of both syntaxin1 homologs (Sso1 and Sso2) in yeast prevents exocytosis and so is lethal, whereas strains deleted in both VAMP homologs (Snc1 and Snc2) are viable. Such v-SNARE-independent membrane fusion can result from complexes formed between Sec9 (yeast SNAP-25) and dephosphorylated Sso1/2 (yeast syntaxin) (452), supporting the idea that SNAREs (or at least syntaxins) are essential for intracellular membrane fusion.

In addition to studies in genetically tractable organisms, the use of clostridial neurotoxins has provided important evidence for an essential role of SNAREs in exocytosis. The key discovery in this regard was that tetanus and botulinum B neurotoxin were metalloendoproteases that blocked neurotransmission presynaptically by selectively cleaving VAMP (432, 669). Following this work, it was established that the remaining six botulinum neurotoxins cleaved either VAMP, syntaxin 1, or SNAP-25 (Table 3 and references therein). Clostridial neurotoxin light chains (the proteolytic subunits) were already known to block granule exocytosis in permeabilized adrenal chromaffin cells and PC12 cells (6, 76, 77, 443, 481), indicating that SNAREs were also required for exocytosis in such neuroendocrine cells. To test if this was true for all granule exocytosis, many labs applied clostridial neurotoxins to permeabilized secretory cells. This was shown to block secretion from pancreatic -cells (630, 663), pancreatic acinar cells (269), parotid acinar cells (261), AtT-20 cells (4), eosinophils (336), platelets (249), peptidergic hypothalamic neurons (206, 367), and gastric enterochromaffin-like cells (338) and to block the acrosome reaction in human sperm (772). Although not all granule exocytosis is blocked by clostridial neurotoxins, this should not be interpreted as providing evidence for SNARE-independent membrane fusion. Rather, the most likely explanation is that exocytosis in such cells proceeds via toxin-resistant SNARE isoforms (it is known that even minor changes to recognition and/or cleavage sites can prevent proteolysis by clostridial neurotoxins, Ref. 794). For example, the lack of sensitivity of mast cell secretion to botulinum neurotoxin E reflects the involvement of the nonneuronal SNAP-25 homolog SNAP-23 (297, 793).

As an alternative to the use of clostridial neurotoxins, various reports have shown that SNARE-directed antisera or peptides inhibit granule exocytosis. When applied to well-studied model systems such as chromaffin cells, this serves as independent confirmation of neurotoxin data (299, 300). However, such approaches can also reveal new information on SNARE function in exocytosis. In platelets, for example, exocytosis of -granules, dense-core granules, and lysosomes is inhibited by antisera to syntaxin 2 and SNAP-23, but dense-core granule exocytosis is unique in being unaffected by syntaxin 4 antibody (159, 160, 416). Similarly, in neutrophils, while syntaxin 6 antibody prevents exocytosis of both specific and azurophilic granules, SNAP-23 antibody inhibits only specific granule exocytosis (465). This suggests that independent exocytosis of distinct organelles within the same cell may be controlled, at least in part, by distinct SNARE combinations, in keeping with observations on reconstituted liposome fusion (483, 583).

Clearly, SNAREs are critical for granule exocytosis, but what is their role in this process? Several lines of evidence suggest that SNAREs themselves may directly drive membrane fusion. First, tetanus toxin application results in the accumulation of docked vesicles at the synapse, suggesting that SNAREs function after morphological vesicle docking/tethering (see sect. IV). Second, formation of the SNARE complex is very energetically favorable; assembled SNARE complexes are resistant to SDS and have extremely high thermal stability (239, 317). Third, the structure of the synaptic SNARE complex is such that formation of its coiled-coil bundle (Fig. 5) would bring the transmembrane anchors of VAMP and syntaxin (and hence the vesicle and plasma membrane) into close apposition (309, 337, 605, 744). Fourth, and most compelling, membrane fusion between artificial liposomes has been reconstituted using recombinant SNAREs alone (483-485, 829). Taken together, these observations support a model (Fig. 6) where a conformational change in syntaxin exposes its H3 helix, which then serves as a nucleation site for interaction with the VAMP coiled-coil domain and the two helices of SNAP-25. Formation of the full complex then proceeds down its energy gradient, unleashing sufficient energy to overcome the hydration barrier and initiate bilayer fusion.

View larger version (28K): [in this window] [in a new window]   Fig. 6. Schematic scheme for membrane fusion driven by the SNAREs. A: syntaxin is initially unavailable for assembly into the SNARE complex due to its association with munc-18. B: dissociation of munc18 frees syntaxin which adopts an "open" conformation. C: the SNAREs interact and assemble the four-helix bundle. D: as the SNARE complex is fully assembled, the lipid bilayers of the plasma membrane and vesicle are brought close enough for bilayer fusion to occur.

Studies of granule exocytosis have been important in testing if this model applies to physiological membrane fusion. One ingenious approach (629) has been to engineer mutant SNARE proteins that are resistant to clostridial neurotoxin cleavage but are otherwise fully functional. Transfection of such constructs into neurotoxin-treated chromaffin cells and -cell lines restores exocytosis (287, 571, 629, 823), enabling the functional analysis of other mutations to be examined. This has revealed an essential role for the coiled coil domain of VAMP (629), supporting the view that VAMP's function in exocytosis requires incorporation into the SNARE complex. Analogous studies with SNAP-25 have revealed a novel role for the palmitoylated linker domain between its two Q helices in SNARE complex disassembly and exocytosis (824). Furthermore, this system has been used to demonstrate that the conserved glutamine residues that define SNAP-25's two Q helices (Fig. 5) are dispensible for granule exocytosis, suggesting that formation of a fully stable SNARE complex may not be essential for membrane fusion (291). A modification of this approach using soluble fragments of SNAP-25 has further shown that reconstitution of exocytosis in botulinum E-treated PC12 cells requires only the COOH-terminal helix and that this selectively stimulates the late Ca²⁺-induced triggering stage (164, 165), again supporting an active role for SNARE complex formation in driving membrane fusion (166). Furthermore, studies using SNARE-specific antibodies suggest that SNAREs exist in loosely complexed states, with full winding of the complex occurring at a late stage in granule exocytosis (167, 853), in keeping with the model proposed above. However, some data argue against the idea that SNARE complex formation causes membrane fusion. For example, in vitro exocytosis of sea urchin cortical granules can proceed in the absence of detectable SNARE assembly (189, 748). In addition, work on the yeast vacuolar fusion system has suggested that SNARE complex formation acts upstream of membrane fusion (791), with fusion ultimately being catalyzed by vacuolar ATPase subunits (594). Further work is needed to finally define the function of SNAREs in membrane fusion, and studies of granule exocytosis will no doubt be central to such future endeavours.

NSF and -SNAP are soluble proteins that act to disassemble SNARE complexes. Owing to the extreme stability of the SNARE complex (see sect. VA), disassembly requires significant energy input in the form of ATP hydrolysis (714), and this is provided by the homohexameric ATPase NSF. Each identical subunit of NSF comprises an NH₂-terminal domain (N) required for interactions with -SNAP, followed by two AAA⁺ ATPase domains (D1 and D2) with distinct, essential functions (836). The ability to hydrolyze ATP is unique to the D1 domain in both NSF and its yeast ortholog, Sec18 (507, 722, 740, 747, 835, 854), whereas the function of the catalytically inactive D2 domain is to establish the homohexameric conformation (417). These domains are arranged such that the SNARE complex is positioned inside the NSF hexamer, with three -SNAP molecules connecting the SNARE complex to three projecting N domains (309, 337, 836). -SNAP not only recruits NSF to the SNARE complex but also stimulates the ATPase activity of the D1 domain (318, 475, 507, 723), and this SNAP stimulation of ATPase activity is essential for SNARE complex disassembly (50). It is thought that the conformational changes in NSF induced by ATP hydrolysis are transduced through -SNAP to unwind the four helices of the SNARE complex. This process may be mechanistically similar to the unwinding of nucleic acid duplexes by DNA/RNA helicases, to which NSF shares some structural similarity (836). Although it is not yet clear where unraveling of the four helices of the SNARE complex is initiated, the conserved Q residue contributed by syntaxin is essential for SNARE disassembly (666), suggesting that NSF/-SNAP-induced hydration of the zero layer enables unwinding of the helices.

In vivo evidence for a requirement of NSF and SNAPs in membrane fusion comes from the comatose (NSF) mutants in Drosophila melanogaster (580, 700) and the sec18 (NSF) and sec17 (-SNAP) mutants in Saccharomyces cerevisiae (224, 292, 295, 538, 839). These mutants are characterized by temperature-sensitive blocks in presynaptic neurotransmission and membrane traffic through the biosynthetic pathway, respectively. In all cases, the mutant organisms accumulate SNARE complexes at the restrictive temperature (436, 712, 771), confirming that NSF and -SNAP are required to disassemble SNAREs in living cells. Recently, NSF has also been shown to be required for the assembly of exocytotic sites for trichocyst exocytosis in Paramecium (256). Despite ready acceptance of this notion, questions remained over the precise point in the membrane fusion process at which NSF and -SNAP functioned (504). Studies of granule exocytosis have been important in this regard (see below), but rigorous tests have also been performed using yeast and flies. Analysis of in vitro yeast vacuole-vacuole fusion indicated that Sec17 and Sec18 acted in an ATP hydrolysis-dependent stage that preceded membrane contact (476). Although these findings continue to be cited as evidence for a postfusion role of NSF/-SNAP (168), it is nevertheless clear that they strongly support a predocking, priming role. Analysis of comatose mutants has proven controversial, with different groups concluding both pre- and postfusion actions of NSF (376, 435, 436, 771). However, it is important to note that continued neurotransmission requires cycles of exo- and endocytosis, so it can be difficult to define in such recycling systems exactly when postfusion becomes predocking.

No such problems affect analysis of granule exocytosis, where vesicles are preformed and display very long half-lives (194), and where assaying release of endogenous granule contents precludes artefactual effects of vesicle formation/recycling. Functional evidence supports a role for -SNAP and/or NSF in granule exocytosis in chromaffin cells (505), PC12 cells (45), pancreatic -cells (384, 514), spermatozoa (488), and platelets (159, 416). The use of both stage-specific exocytosis assays in permeabilized cells and high-resolution electrophysiological approaches have established that -SNAP/NSF acts in an early ATP-dependent priming reaction that precedes the later Ca²⁺-dependent fusion step (45, 140, 851) (see sect. IV). Furthermore, this priming reaction is likely to be the disassembly of SNARE complexes, since mutant -SNAPs unable to stimulate NSF ATPase activity and consequent SNARE disassembly cannot support exocytosis (50, 288, 513, 851). Because trans-SNARE complexes formed between opposing membranes would be inaccessible to the large NSF hexamer, and because NSF function in vacuole fusion is complete even before membrane tethering, it seems certain that the substrates in the priming reaction are cis-SNARE complexes formed within the same membrane. Indeed, although predominantly plasma membrane proteins, significant levels of syntaxin and SNAP-25 are found on both synaptic vesicles (815) and chromaffin granules (339, 745, 746), and SNARE complexes formed on synaptic vesicles are disassembled by -SNAP/NSF (572). -SNAP and NSF are themselves present on chromaffin granule membranes (44, 128). Taken together, these various observations suggest that -SNAP/NSF act as molecular chaperones, continually disassembling cis-SNARE complexes to release SNARE proteins to engage in trans and hence function in further membrane fusion events (121).

Despite the widespread acceptance of this model, SNARE complex disassembly is not the only function of NSF and SNAPs. In recent years, NSF has been shown to bind to several different receptors or their interacting proteins and to regulate their insertion/removal from the plasma membrane. These include the AMPA receptor (531, 565, 715), the ₂-adrenergic receptor (187), -arrestin (478), and the GABA_A receptor-associated protein (387). This suggests a second function for NSF in membrane traffic that is independent of SNAREs. However, another novel NSF binding protein, GATE-16, interacts with a Golgi v-SNARE, stimulates NSF ATPase activity, and stimulates intra-Golgi transport (664). These properties are similar to the yeast LMA1 protein complex, which binds a vacuolar t-SNARE and stimulates Sec18 ATPase activity (854). This suggests that GATE-16 may be a general factor regulating membrane fusion, but no data are available on whether GATE-16 acts in granule exocytosis. In addition to the ubiquitous -SNAP, two other isoforms have been described (181, 182). -SNAP is brain specific (529, 834) and, despite sharing 83% amino acid identity with -SNAP, displays significant differences in some assays (181, 186), but not others (737). In chromaffin cells, both - and -SNAPs enhance exocytosis induced by micromolar Ca²⁺ (505, 737, 850), but only -SNAP does so at submicromolar Ca²⁺ levels (850). The reason for this is not clear, but it may conceivably involve the putative Ca²⁺ sensor synaptotagmin, which has been claimed to specifically bind -SNAP and thereby recruit NSF (670). The function of -SNAP, which displays only 18% amino acid identity with -SNAP, remains unclear. Although it is effective at binding and stimulating NSF ATPase activity (181, 507, 737), it is inactive in ER-Golgi transport (593) and has no reproducible effect on granule exocytosis in chromaffin cells (505).

Although much attention has focused on NSF, SNAPs, and SNAREs, these are not the only evolutionarily conserved proteins required for intracellular membrane traffic. Genetic studies have established a general requirement for Sec1-related genes in vesicle fusion in a wide variety of organisms, including yeast (SEC1, SLY1, VPS33, VPS45), plants (KEULE), nematodes (UNC18), flies (ROP), and mice (munc18-1) (32, 303, 800). The founding member of this family is yeast Sec1, the original secretory mutant isolated by the classic screen of Novick and Schekman, which is essential for exocytosis in yeast (3, 229, 537-539). The realization that similar proteins were required for exocytosis across phyla led to the identification of Sec1 homologs in mammals (variously named nSec1, munc18, mSec1, or rbSec1) (303). Three putative mammalian Sec1 orthologs have been described: the neuronal/endocrine munc18-1/nSec1 protein and the ubiquitous munc18-2 and munc18-3 isoforms. Although most work on the Sec1 family has focused on exocytosis, their general role in membrane fusion is illustrated by the requirement for Sec1 homologs in ER-Golgi transport (Sly1) and transport to the vacuole (Vps33/Slp1, Vps45) in yeast (1, 196, 564, 813). Sec1 family members from a variety of organisms have been shown to interact with syntaxin homologs, suggesting that the conserved function of Sec1 proteins in membrane fusion may be mediated via syntaxins (360). Indeed, the yeast syntaxins, Sso1 and Sso2, act as multicopy suppressors of sec1-1 mutants (2), and the inhibition of neurotransmitter release in Drosophila caused by overexpression of ROP is relieved by co-overexpression of syntaxin (848), thus supporting this notion. Neuronal Sec1/munc18 binds to syntaxin1a with nanomolar affinity in vitro to form a complex that precludes syntaxin binding to synaptic SNAREs (598). These observations have led to the suggestion that Sec1 proteins act as syntaxin chaperones, preventing SNARE complex assembly until signaled to release syntaxin (360). Although the precise nature of this signal is unknown, various observations suggest that it is linked to the completion of Rab-dependent tethering (see below).

Sec1 proteins have been implicated in granule exocytosis in pancreatic -cell lines (867), platelets (625), pancreatic acinar cells (268), PC12 cells (221), and chromaffin cells (248, 320, 335, 809). In -cell lines, nSec1/munc18 interacts with syntaxin 1, and nSec1/munc18 peptides or antisera increase insulin release (867). Although this supports the above-proposed role of Sec1 proteins as negative regulators of syntaxin function, other studies suggest that this idea is too simplistic. Indeed, loss of Sec1 family members generally results in a complete block in membrane traffic, indicating an additional positive essential role. Studies of nSec1/munc18 in PC12 cells and Vps45 in yeast suggest that, rather than merely preventing syntaxin entry into the core complex, the syntaxin-Sec1 interaction is an essential intermediate that enables activation of syntaxin for efficient incorporation into the SNARE complex (105, 221). This notion is consistent with recent studies in chromaffin cells on the effects of a mutant form of nSec1/munc18 (R39C; Fig. 7) with a reduced affinity for syntaxin. Here, it was found that the mutant resulted in an increased rate of fusion pore expansion (248), which could result from the increased release of activated syntaxin into the fusion machinery. Alternatively, these data may indicate a much later role for nSec1/munc18 in the membrane fusion process than previously appreciated, as suggested by recent studies in yeast. Unlike neuronal Sec1, yeast Sec1 will not bind efficiently to its cognate syntaxin (Sso1/2) in isolation, but only when it is assembled into an exocytotic SNARE complex (132). Clearly, this observation is difficult to reconcile with the proposed function of Sec1 proteins as syntaxin chaperones that regulate subsequent SNARE complex formation; rather, this finding suggests that Sec1 functions at the heart of the membrane fusion process, acting at or after SNARE assembly. In contrast to both of these models, studies of chromaffin cells from munc18-1 knock-out mice have revealed a major defect in granule docking (809), suggesting a much earlier role for this protein. Perhaps nSec1/munc18 has multiple functions, with both predocking and perifusion roles. However, the normal complement of docked vesicles at the synapse in munc18-1 knock-out mice, despite a complete block in membrane fusion (800), argues that there must be additional synaptic docking/tethering factors for this to be true. Clearly, Sec1 proteins have essential and possibly multiple functions in physiological membrane fusion, but much more work is required to understand these in molecular terms.

View larger version (111K): [in this window] [in a new window]   Fig. 7. Structure of the syntaxin/munc18 complex. The structure was solved by use of X-ray crystallography (491). Syntaxin forms a tight bundle seen in green at the bottom which is clasped by Munc18. Residue Arg-39, the mutation of which modifies fusion pore kinetics (248), is shown in space-fill mode in the Munc18 structure.

Rab proteins comprise the largest family within the Ras superfamily of small GTPases. Individual Rab proteins are localized to distinct, characteristic organelles, suggesting that each regulates a particular membrane traffic step. Rabs are thought to act as molecular switches, with the time spent in the active GTP-bound "on" state determining how long vesicles remain competent for docking/fusion, before GTP hydrolysis switches this state off (661). The initial findings implicating these GTPases in membrane traffic came from the identification of Sec4 and Ypt1, mutations in which bock exocytosis and ER-Golgi transport, respectively (665, 687). Interestingly, whereas the number of different SNAREs, SNAPs, NSF, and Sec1 family members is roughly similar from yeast to human, yeast contains only 11 Rab proteins, compared with 60 in humans (84). As the increased complexity during evolution does not apparently require significant amplification of the general fusion machinery, presumably the greatly increased number of Rab proteins reflects a different, regulatory role(s). The general consensus is that rabs are major determinants of the compartmental specificity of membrane transport and achieve this by selective interactions with distinct effector proteins (686, 864). The best-characterized Rab effectors are involved in the initial tethering of vesicles to target membranes. These include the exocyst complex (Sec4 effector in exocytosis) (760), the HOPS protein complex (Ypt7 effector in transport to the vacuole) (685), p115 (Rab1 effector in Golgi transport) (13), and EEA1 (Rab5 effector in endocytosis) (177). Another class of Rab effectors are motor proteins that act to control organelle movement within cells. For example, Rab5 controls movement of endosomes on microtubules and Rab6 binds to the kinesin-like protein rabkinesin-6 (228, 527). In addition to microtubule-based motors, Rabs have also been implicated in actin-based motility by virtue of functional interactions with class V myosins (207). Finally, genetic (and in some cases, physical) interactions between various Sec1 homologs and Rabs (303, 686) suggest that the Sec1 family may represent a third distinct class of Rab effectors. However, a variety of other proteins that do not clearly segregate into these classes have also been proposed as Rab effectors (864), emphasizing the potential complexity of Rab function(s) in the cell.

Rab3 exists as four isoforms A-D in mammals and is the major Rab implicated in regulated exocytosis in neurons and neuroendocrine cells. Rab3A is not an essential protein in mammals but may regulate neurosecretion by limiting the recruitment of synaptic vesicles into the releasable pool at the plasma membrane for subsequent fusion events (272, 274). Rab3 mutants in C. elegans are also viable but have less synaptic vesicles close to the active zone (534), suggesting a role for Rab3 in vesicle tethering in common with other Rab proteins (177, 597).

Overexpression of active forms of Rab3A, or other Rab3 isoforms, has been found to inhibit exocytosis in chromaffin cells (341), PC12 cells (178, 363, 364), rat basophilic leukemia (rbl) cells (640, 704, 790), insulin-secreting cells (628), pancreatic exocrine cells (162), and the cholecystokinin (CCK)-secreting STC-1 enteroendocrine cell line (278). These forms of Rab3A are also inhibitory when injected into neurons (220) consistent with an inhibitory function of Rab3A in regulated exocytosis. Several Rab3A effector proteins are known including RIM (820) and rabphilin 3A (696). Both RIM (353, 742, 820) and rabphilin 3A (25, 180, 469) are involved in secretory granule exocytosis in chromaffin, PC12, insulin-secreting cells, and mouse eggs. Analysis of specific mutations that disrupt interaction of Rab3 with these effectors has excluded them, however, as the inhibitory effectors of Rab3 (178, 193, 362, 676). The inhibitory effects of Rab3 may instead be exerted via the effector Noc2 (319), a protein expressed predominantly, if not exclusively, in neuroendocrine cells (403). Mutations were made in Noc2 based on its sequence homology with the NH₂ terminus of rabphilin 3A and the structure of the complex of Rab3 with rabphilin 3A (contact regions A and B, Fig. 8) (566). These mutations disrupted both binding to Rab3A and the ability of Noc2 to inhibit exocytosis in PC12 cells (319).

View larger version (70K): [in this window] [in a new window]   Fig. 8. Structure of the complex between Rab3A and the NH2-terminal domain of rabphilin 3A. The structure was solved by use of X-ray crystallography (566), and this figure shows Rab3A in its GTP-bound forms (ribbon structure with GTP in space fill) bound to the rabphilin 3A NH2-terminal domain. The two regions of contact between the two proteins are indicated. Contact A involves a helix from rabphilin 3A formed from residues close to the NH2 terminus of rabphilin 3A. Contact B involves the conserved SGAWFF motif of rabphilin 3A.

Other studies have suggested that certain Rab3 isoforms may, in contrast, exert positive effects on exocytosis in some cell types. Evidence for a positive role of Rab3 isoforms in granule exocytosis includes an inhibitory effect of treatment with Rab-GDI, which depletes rabs, on pepsinogen secretion from permeabilized gastric chief cells (619). In addition, depletion of Rab3B, using antisense oligonucleotides in anterior pituitary cells inhibited exocytosis (439) and overexpression of rab3D in pancreatic acini of transgenic mice enhanced exocytosis in response to secretagogues (555). Furthermore, expression of a GTP-binding deficient mutant of Rab3D (rab3A NI35I) in AtT-20 cells reduced the number of docked secretory granules and impaired exocytosis consistent with a positive role for rab3D in promoting granule docking (42). Confusingly, expression of this mutant also reduced the number of docked granules in PC12 cells (461), and conversely, expression of the wild-type Rab3A produced an increase in docked granules. These data on PC12 cells are difficult to reconcile with other data showing that overexpression of wild-type Rab3A in PC12 cells inhibited exocytosis (178, 828). It is possible, however, that Rab3A has multiple roles in exocytosis. Despite the suggestions that different Rab3 isoforms have distinct functions, a direct test of this possibility shows that the four Rab3A-D isoforms all had the same inhibitory effect after overexpression in PC12, chromaffin (178) and insulin-secreting cell lines (354).

Evidence implicating Rab3A or other Rab3 isoforms in exocytosis has also been described for the sea urchin egg (188), the mouse egg (468), parotid gland cells (620), parathyroid gland chief cells (348), anterior pituitary gonadotrophs (757), and for the acrosome reaction in mouse (822), rat (355), and human sperm (862).

Functional data have also implicated Rab4 in the regulated exocytosis of -granules in platelets (695). In addition, more recent work has established an important role for Rab27 isoform in melanocytes, cytotoxic T lymphocytes, and dense-core granule exocytosis in insulin-secreting and PC12 cells. Melanocytes synthesize the pigment melanin, which is transported to the cell periphery in melanosomes and released by exocytosis. A mutation in mice (ashen) that results in a pigmentation defect was found to reside in Rab 27a (840), and mutations in this gene have also been found in a human disorder known as Griscelli syndrome (487). Rab27a interacts with myosin V to regulate melanosome transport (207) via the Rab27a effector melanophilin (263). Interestingly, mice and patients with these mutations in Rab27a also lack the ability to secrete from the lytic granules of cytotoxic T lymphocytes, but this effect is independent of myosin V function, suggesting an alternative function for Rab27a in T lymphocytes (302, 731). These studies are an interesting example of how analysis of mutations and clinical observations can lead to new molecular insights into granule exocytosis.

Rab27a and Rab27b have also been implicated in regulated exocytosis. Overexpression of Rab27a increased evoked insulin secretion from MIN6 cells (858) and exocytosis in PC12 cells (262). In addition, expression of an inactive mutant of Rab27b inhibited secretion from AtT20 cells (869), implicating these Rabs in a positive function. The granuphilins (which are related to melanophilin) (819) have been suggested to be effectors for Rab27, although their overexpression was inhibitory (192, 858) (262). Intriguingly, granuphilins also bind directly to syntaxin 1A (776) and to Munc18 (192). Granuphilins have a restricted tissue distribution, being absent from brain suggesting a specific role in secretory granule exocytosis. They are, however, members of a family of so-called synaptotagmin-like proteins (264, 406, 819) that have not all been characterized in detail. Overexpression of granuphilin-a has also been shown to inhibit exocytosis in PC12 cells (262), despite conflicting data on whether it is expressed endogenously by these cells (192). It is clear, therefore, that much more information will be needed to fully resolve the functions of these Rab proteins in regulated exocytosis.

The near universal role of Ca²⁺ as the trigger for granule exocytosis predicts the existence of a conserved protein(s) capable of activating the fusion machinery upon binding Ca²⁺. Several different candidate proteins have been suggested to play such a role (122), but synaptotagmin has attracted most attention as the putative Ca²⁺ sensor for regulated exocytosis. Originally discovered over 20 years ago as a synaptic vesicle membrane protein, p65 (474), interest in this protein increased dramatically with the demonstration that it was a Ca²⁺-dependent phospholipid-binding protein (98). This property of synaptotagmin is endowed by its two C₂ domains, C₂A and C₂B, which are homologous to the C₂ domain in protein kinase C (591). Preceding the tandem C2 domains is a variable linker region connecting to the transmembrane anchor and a short intravesicular or extracellular NH₂ terminus (590, 784). This domain structure is common to all 13 synaptotagmins described to date, although not all synaptotagmins are able to bind Ca²⁺ and phospholipids (423, 735). Among those that do, considerable variation in the Ca²⁺ dependence of phospholipid binding is exhibited, with the plasma membrane synaptotagmins 3 and 7 displaying much higher Ca²⁺ affinity (EC₅₀ 1-2 µM) than the vesicular synaptotagmins 1 and 2 (EC₅₀ 10-20 µM) (739). In addition, synaptotagmins have been shown to interact with a variety of proteins implicated in granule exocytosis, including Ca²⁺ channels (419), the SNARE complex (713), -SNAP (670), and calmodulin (253). These interactions occur in the absence of Ca²⁺, but binding of synaptotagmin 1 to isolated t-/Q-SNAREs (868) or the SNARE complex is greatly stimulated by Ca²⁺ (204, 276, 423). Synaptotagmin I can accelerate reconstituted liposome fusion mediated by SNAREs, but in a Ca²⁺-independent manner (451). As with phospholipid binding, individual synaptotagmins display marked differences in the Ca²⁺ dependency of syntaxin binding, with >200 µM Ca²⁺ required for maximal binding by synaptotagmins 1 and 2, in contrast to <10 µM Ca²⁺ for synaptotagmins 3 and 7 (423).

The ability to interact with both membranes and the fusion machinery in response to the spectrum of Ca²⁺ levels that drive exocytosis suggests that synaptotagmins may be ubiquitous exocytotic Ca²⁺ sensors. Evidence to support this view comes from genetic studies in Drosophila, C. elegans, and mice, where synaptotagmin mutants display impaired synaptic transmission (214, 273, 438, 532). In addition, mice with a point mutation reducing synaptotagmin 1's affinity for Ca²⁺ display a similar decrease in the Ca²⁺ affinity of synaptic vesicle exocytosis (244). This strongly suggests that synaptotagmin 1 is a major Ca²⁺ sensor for synaptic vesicle exocytosis, but does this also hold for granule exocytosis? Analysis of chromaffin cells from synaptotagmin 1 knock-out mice reveals a selective impairment in the fast exocytotic burst, but little effect on the slower sustained component (808), a remarkably similar phenotype to that seen for synaptic vesicle exocytosis from the same animals (273). Furthermore, introduction of antisera to synaptotagmins 1 and 2 or recombinant C₂ domains inhibits exocytosis in PC12 cells (212, 230, 762), chromaffin cells (547), and -cell lines (409). This inhibitory effect is specific for the Ca²⁺-activated triggering stage, suggesting that synaptotagmin I controls a late phase of the membrane fusion process (212, 547). Indeed, overexpression of synaptotagmin 1 has been shown to prolong the transition from fusion pore formation to pore expansion (818). Nevertheless, there are some problems with the notion that synaptotagmins 1 and 2 are vesicular Ca²⁺ sensors in granule exocytosis. For example, PC12 cell variants lacking these proteins exhibit normal catecholamine release (699), and the slow phase of exocytosis in chromaffin cells proceeds with normal Ca²⁺ dependency in synaptotagmin 1 knock-out mice (808). Furthermore, the relatively low Ca²⁺ affinities of both phospholipid and syntaxin binding observed for synaptotagmins 1 and 2 are difficult to reconcile with the Ca²⁺ dependency of granule exocytosis in most cells; rather, the higher Ca²⁺ affinity of synaptotagmins 3, 5, 7, and 10 makes these more attractive potential Ca²⁺ sensors for granule exocytosis. Indeed, recombinant C₂ domains from synaptotagmins 3 and 7 inhibit Ca²⁺-activated exocytosis in permeabilized PC12 cells (738, 739), and those from synaptotagmins 5 and 7 inhibit exocytosis in -cells (298). Such effects of recombinant C₂ domains may be complicated, however, since even C₂ domains unable to bind Ca²⁺ have been reported to inhibit exocytosis, possibly by hetero-oligomerization with endogenous synaptotagmins (298, 763). In the cell, the various pairwise combinations of multiple synaptotagmin isoforms could potentially enable a very sophisticated fine-tuning of the Ca²⁺-sensing machinery for exocytosis (437). Evidence that synaptotagmin isoforms may act as Ca²⁺ sensors in nonneuronal exocytosis has also come from the study of synaptotagmin in insulin-secreting cells (102, 271, 298, 409), synaptotagmin VII in lysosome exocytosis (464), synaptotagmin I and II in rat basophilic leukemia cells (47, 48), and synaptotagmin VIII in acrosome exocytosis (350). It has been suggested that synaptotagmin 1 mediates granule docking in AtT-20 cells (171).

It is a possibility that the distinct Ca²⁺ dependency of granule exocytosis is determined by entirely different classes of Ca²⁺ sensors. The C₂ domains present in synaptotagmin are also shared by protein kinase C, rabphilin 3A (696), Doc2 (563) , RIM (820), and munc13 (92, 97), all of which have been suggested to play a role in exocytosis. Protein kinase C has long been implicated as a regulator of granule exocytosis in many cell types and is discussed extensively in section VIIIB. Rabphilin 3A is a soluble protein containing two tandem C₂ domains that mediate binding to phospholipids (especially phosphatidylinositol 4,5-bisphosphate) at low micromolar Ca²⁺ levels (179, 696, 855). Evidence for a role of this protein in granule exocytosis comes from work on chromaffin cells (179, 180), PC12 cells (401), -cell lines (25, 362), and mouse eggs (469). Although originally isolated as a Rab3A binding protein, rabphilin 3A may function independently of Rab3 in various systems (178, 193, 362, 676, 697, 720). However, the molecular function of rabphilin 3A remains unclear. Doc2 is a soluble protein that, as its name (double C₂) implies, also contains two C₂ domains (563). Evidence for the involvement of Doc2 in granule exocytosis is restricted to the observation that overexpression increases secretion from PC12 cells (562). The mechanism underlying this stimulation is unknown, although Doc2 has been shown to interact with both munc18/nSec1 and another C₂ domain-containing protein, munc13 (222, 561, 802). Munc13 (three isoforms: 1, 2, and 3) is the mammalian homolog of C. elegans UNC-13 and contains domains homologous to both the C₁ and C₂ domains of protein kinase C (97, 511). As predicted by possession of the C₁ domain, UNC-13 proteins are phorbol ester/diacylglycerol binding proteins (5, 66, 511), and this property may contribute to the enhancement of exocytosis by these drugs (66, 633) (see also sect. VII). Genetic studies in mice (33), C. elegans (634), and Drosophila (22) strongly support a role for UNC-13 proteins in synaptic vesicle exocytosis. However, the only evidence for a function in granule exocytosis is that overexpression of munc13-1 in chromaffin cells increases the magnitude of both the exocytotic burst and the subsequent slower phase of release, suggesting a role in priming (31). UNC-13 proteins are known to bind several proteins implicated in exocytosis (99), but the interaction with syntaxin (67) appears critical, as a mutant syntaxin allele can bypass the requirement for UNC-13 in C. elegans (635). The final C₂ domain-containing protein implicated in exocytosis is RIM (Rab3 interacting molecule), a soluble protein localized to the active zone at presynaptic terminals (820). Evidence for a role of RIM in granule exocytosis comes from work on PC12 cells (574, 820), -cells (372), -cell lines (353, 574), and chromaffin cells (742). As with rabphilin 3A, the function of RIM in exocytosis may be unrelated to Rab3 (742), and alternative binding partners implicated in exocytosis are munc13 (68) and camp-GEFII (372, 574). The link to munc13 is particularly intriguing, as both proteins are involved in priming (31, 742) and as the requirement for either protein in C. elegans can be bypassed by a mutant syntaxin locked in an open conformation (404).

Several Ca²⁺ binding proteins that lack C₂ domains have also been implicated in granule exocytosis. Calmodulin binds Ca²⁺ via its four EF-hand domains and was one of the first proteins suggested to be involved in exocytosis (379, 380, 724). More recently, Ca²⁺-calmodulin has been invoked as a universal requirement for the membrane fusion process, based on observations from several constitutive membrane fusion events (185, 595, 608). However, because granule exocytosis is not blocked by calmodulin antagonists and as calmodulin, unlike synaptotagmins, cannot bind Ba²⁺ or Pb²⁺ (both effective Ca²⁺ surrogates for exocytosis), an essential role for this protein in granule exocytosis seems unlikely. Nevertheless, this does not preclude a regulatory role, and indeed, exogenous calmodulin has been shown to enhance granule exocytosis from chromaffin cells (140, 381, 556) and PC12 cells (163). Although calmodulin is known to act in the late Ca²⁺-activated triggering of exocytosis (140), its mechanism of action is unclear as it binds to several proteins implicated in exocytosis, including synaptotagmin 1 (253), Rab3 (582), VAMP (615, 616), and the SNARE complex (312). Intriguingly, the crystal structure of the synaptic SNARE complex contains bound strontium ions (240, 744), suggesting that this could represent an unsuspected site of action for Ca²⁺ in exocytosis. Indeed, recent work has shown that mutation of residues in SNAP-25 involved in coordination of one Sr²⁺ reduces the Ca²⁺ cooperativity of chromaffin granule exocytosis (717).

In contrast to the aforementioned putative Ca²⁺ sensors that are proposed to act in both synaptic vesicle and granule exocytosis, p145/CAPS has emerged as a granule exocytosis-specific Ca²⁺ sensor. Originally identified as a cytosolic factor that reconstituted secretion from GH₃ cells (463) and PC12 cells (816), CAPS has since been shown to regulate dense-core vesicle exocytosis in nerve terminals (65, 755), chromaffin cells (231), and pituitary melanotrophs (659). Although CAPS mutants in C. elegans (UNC-31) and Drosophila (dCAPS) show impaired fast neurotransmission, in Drosophila, at least, these effects are indirect and result from a primary impairment of dense-core vesicle release (21, 631). This fits with work on mammalian synaptosomes, where CAPS has no effect on glutamate release, despite regulating norepinephrine release in the same preparation (65, 755). CAPS is known to act in the late Ca²⁺-activated triggering stage (21, 315), but its mechanism of action is unknown. In particular, its Ca²⁺ affinity (K_d of 270 µM) appears too low to explain its effects on granule exocytosis (21).

	VI. FUSION PORES AND KISS-AND-RUN EXOCYTOSIS

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Related to fusion pore formation and its possible reversibility is the idea of "kiss-and-run" fusion originally suggested for synaptic vesicle exocytosis (Fig. 3). It was postulated a number of years ago that neurotransmitter release from a synaptic vesicle could occur very rapidly without the need for full fusion and flattening of the vesicle membrane into the plasma membrane but instead by a reversible process that would rapidly retrieve the vesicle membrane for reuse after a transient fusion event (139). This hypothetical process was termed "kiss-and-run" (247). It has been predicted (16) that the contents of a synaptic vesicle could be released on a submillisecond time scale through the sort of fusion pore detected in mast cells (90) and described subsequently in other secretory cell types including chromaffin cells (11), eosinophils (667), and neutrophils (442). For synaptic vesicle exocytosis, biophysical measurement of kiss-and-run has been technically difficult, but evidence in its favor has come from analysis of the kinetics of loss of different lipophilic FM-dyes from the vesicle membrane. These appear to be differentially retained during neurotransmitter release under certain conditions (195, 374, 388, 727). These data are consistent with the occurrence of transient fusion events. One study has been able to resolve individual fusion events due to synaptic vesicle exocytosis by averaging many thousands of single vesicle fusion events (741). The data in this study showed that at low stimulus levels exo/endocytosis was completed on a very short time scale, with retrieval having an average time constant of 56 ms. This would be consistent with the existence of kiss-and-run occurring under these conditions. The significance of kiss-and-run for the regulation of neurotransmitter release remains a controversial issue (112, 622). Catecholamine release by kiss-and-run has been directly shown to occur in chromaffin cells using combined capacitance and amperometric recording, albeit in the presence of artificially high external Ca²⁺ concentrations (11, 12). Also, kiss-and-run fusion has been demonstrated based on the trapping of extracellular markers after exocytosis in intracellular vesicles that still retain the characteristics of chromaffin granules including the presence of a dense core (327). These independent data are fully consistent with partial release from chromaffin granules. Evidence from study of insulin-secreting INS-1 cells, based on imaging of fluorescent secretory granule contents or membrane proteins, has suggested that granules may move after fusion in a so-called "kiss-and-glide" phenomenon (782). This phenomenon may be related to the finding, from use of two-photon imaging of fluid-phase or membrane tracers, that the fusion pore in pancreactic -cells appears to remain stably open (mean lifetime of 1.8 s) for much longer than in other cell types such as adrenal chromaffin cells (mean lifetime of 41 ms) (751).

One view of kiss-and-run exocytosis is that it is simply a reflection of the ability of the fusion pore to rapidly reclose after its initial expansion. There is evidence accumulating to suggest that this may be a regulated process, and this would allow exocytosis to be switched from full fusion to a predominance of kiss-and-run fusion under certain conditions (116, 117). While kiss-and-run fusion is likely to allow complete emptying of the synaptic vesicle, dense-core granules may be only partially emptied through a transient fusion pore, allowing regulation of the amount of release (quantal size) per fusion event (112, 232). This could be particularly important for larger secreted molecules or peptides that have been shown to be released slowly after membrane fusion (49). Evidence has been obtained for even partial emptying of catecholamine from secretory granules in adrenal chromaffin cells under normal conditions (679, 849) and that this can be increased. For example, reducing medium osmolality increased the amount of release per vesicle compared with isosmotic conditions (679). Regulation of the amount of granule content released per fusion event, without acutally modifying the granule content, has been demonstrated to occur, so far, only in chromaffin cells (288, 289) and in pancreatic -cells (706). A variety of conditions were found to modify the amount released per granule (quantal size) from chromaffin cells and result in changes in the kinetics of single secretory events consistent with alterations in fusion pore dynamics. These effects have been seen after phobol ester treatment (289), modification of intracellular Ca²⁺ levels (232), in cells overexpressing cysteine string protein (Csp) (288) expressing a Munc18 mutant (R39C) (248) or a mutated form of SNAP-25 (199), after introduction of anti-CAPs antibodies (231), after treatment with nitric oxide (448), or after elevation of cAMP levels (447). In addition, changes in release kinetics, consistent with premature closure of the fusion pore, have been seen in cells overexpressing complexin (23). This protein interacts only with the assembled SNARE complex (482, 575), and mutation of a residue involved in this interaction (89, 161) prevented the effects on release kinetics (23). Further work will be needed, however, to explain in detail how fusion pore dynamics and, thereby, quantal size are controlled.

A major mechanistic issue for fusion pore reversibility and, thereby, kiss-and-run exocytosis is how fusion pore closure occurs. There are two extreme possibilities (Fig. 3). In the first model, it would be necessary to propose that the exocytotic machinery that produces fusion can act in a fully reversible fashion. This idea does not fit well with current ideas of fusion driven by the formation of highly stable SNARE complex (see sect. V) (308, 744) only able to be disassembled after fusion by the slow action of -SNAP and NSF (713). This scenario cannot be ruled out, but experimental data argue against a requirement for a stable SNARE complex for normal release kinetics to be maintained (291). An argument in favor of the first model involving a reversible fusion machinery is the apparent "flickering" behavior of the biophysically detected fusion pore (241) which suggest the movement between open and closed states normally associated with ion channels. This would be more consistent with the existence of a reversible fusion machinery. In the second model, the fission machinery responsible for pinching off clathrin-coated endocytotic vesicles (based on the action of mechanoenzyme dynamin, Ref. 690) would be capable of functioning on a millisecond time scale to pinch-off fused vesicles. The second explanation may be a possibility as studies in a Drosophila mutant with a temperature-sensitive defect in dynamin (Shibire) showed impairment in neurotransmission consistent with a reduction in vesicle retrieval within an onset in milliseconds (375). It may be difficult to imagine that there would be time for the assembly of the proteins (198, 460, 837) required for clathrin coating and conventional endocytosis at the site of fusion. Perhaps vesicle scission is brought about by dynamin in a clathrin-independent manner (26). As well as a rapid retrieval mechanism for secretory granule membrane (26, 111, 764), there is evidence for multiple pathways of endocytosis in secretory cells (587, 703). One possibility is that the clathrin-dependent pathway is used only for slower membrane retrieval after full fusion or after high extensive exocytosis (579). A role for dynamin may be supported by its presence on chromaffin secretory granules (270). The importance of dynamin in the retrieval of secretory granule membrane has been examined in very few cell types so far (27). Analysis of the effects of disrupting dynamin function on the kinetics of amperometric spikes suggests the existence of both dynamin-dependent and dynamin-independent mechanisms for fusion pore closure during kiss-and-run exocytosis (290). It is clear, however, that much more remains to be learned about the physiological significance and molecular basis of kiss-and-run exocytosis.

	VII. SIGNALING PATHWAYS THAT TRIGGER SECRETORY GRANULE EXOCYTOSIS

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In cell types where a rise in intracellular Ca²⁺ is the main (or the only trigger) for the initiation of regulated exocytosis, Ca²⁺ can be derived from entry of external Ca²⁺, by Ca²⁺ release from intracellular Ca²⁺ stores, or both. The relative use and importance of these two Ca²⁺ sources varies considerably between cell types. This ranges from cells in which Ca²⁺ entry is the only effective stimulus for exocytosis to cells in which Ca²⁺ stores are the predominant source. At one extreme is the adrenal chromaffin cell where release of Ca²⁺ from intracellular stores triggers only a very low level of secretion (150, 155, 158, 568, 569) and that may in part be due to capacitative (store-operated) Ca²⁺ entry that follows store emptying (252, 641). In contrast, Ca²⁺ release from the ER after inositol 1,4,5-trisphosphate (IP₃) generation is crucial for exocytosis in, for example, pancreatic exocrine cells (596, 826) and in anterior pituitary gonadotrophs (623, 778, 780, 781). In some cell types, such as pituitary corticotrophs (334, 779), the situation is intermediate where both Ca²⁺ entry and Ca²⁺ release can initiate exocytosis, but neither alone gives maximal responses.

Elevation of cytosolic Ca²⁺ concentration from a resting level of 0.1 µM to the levels required to trigger exocytosis can occur via a variety of entry mechanisms including second messenger-operated (SMOC), voltage-operated (VOC), receptor-activated (ROC), and Ca²⁺ release-activated (CRAC) Ca²⁺ channels. In addition, many other features can modify the extent and duration of an effective Ca²⁺ signal for exocytosis including buffering via Ca²⁺ uptake into mitochondria (280, 498, 770). Other factors that affect Ca²⁺-regulated exocytosis in particular cell types include the spatial distribution of channels (688), Ca²⁺ entry pathways (156), or stores (157, 569, 596) in relation to secretory granules (688). Also important is the local action of Ca²⁺ due to limited diffusion (experimentally shown to occur at ~10-13 µm²/s, Ref. 14) and the Ca²⁺ dependency of the exocytotic machinery in a particular cell type (Table 4). The limited diffusion of Ca²⁺ is due to high levels of Ca²⁺ buffering within cells (389, 456, 520) so that the sequestration of Ca²⁺ by high-capacity immobile Ca²⁺ buffers limits the distance over which Ca²⁺ can diffuse and exert its effects. Spatial distribution may result in particular types of Ca²⁺ channels being associated with exocytosis (137, 138, 831, 832), whereas others are ineffective and this may be enhanced by physical association of vesicles with channel proteins as is believed to occur for P/Q- and N-type channels in synapses (71, 72, 146, 419, 420, 466, 684, 692, 693, 842, 843, 870). The relative importance of Ca²⁺ stores in pancreatic exocrine cells and pituitary gonadotrophs appears to be determined by the spatial localization of their ER Ca²⁺ stores. In pancreatic exocrine cells, fingers of the exceptionally dense ER network present in the basal region of these cells extend into the granular domain in the apical region of the cell (492, 596, 770) where the zymogen granules are concentrated (826) and where exocytosis occurs (Fig. 9). In contrast, gonadotrophs are endowed with abundant subplasmalemmal ER cisternae located close to secretory granules and attached to the plasma membrane (781).

View larger version (47K): [in this window] [in a new window]   Fig. 9. Schematic representation of the organization of a pancreatic acinar cell. The endoplasmic reticulum (ER), which is the internal major Ca2+ store in these cells, is found mainly in the basal pole but extends up into the apical pole where the zymogen granules are located. Exocytosis of the zymogen granules occurs at the apical membrane. With low levels of stimulation, Ca2+ rises are initiated and potentially confined to the granule-containing region.

Considerable effort has been expended in the determination of the Ca²⁺ sensitivity of the exocytotic machinery in a wide range of cell types. This information is required for the interpretation of the relationship of the observed physiological Ca²⁺ signals to the efficiency of activation of exocytosis and also provide clues as to the expected properties of physiologically relevant Ca²⁺ sensor proteins in exocytosis (122). Two major technical approaches have been used: 1) cell population measurements of granule release following cell permeabilization with direct addition of Ca²⁺ to the cell interior and 2) single-cell patch-clamp capacitance recording to follow the kinetics of exocytosis. Early work by Katz and Miledi (373) established the importance of Ca²⁺ by showing a requirement for external Ca²⁺ for the release process at the neuromuscular junction. This was subsequently extended by the work of Douglas (219) to chromaffin cells and posterior pituitary neurosecretory terminals. In addition, activation of exocytosis using Ca²⁺ ionophores (57) and by measurement of Ca²⁺ signals generated in secretory cells by physiological cell stimulation (148, 149) reinforced the importance of Ca²⁺ in stimulus-secretion coupling. Assessment of the Ca²⁺ dependency of exocytosis required, however, the means to control the internal Ca²⁺ concentration at defined levels. This was first achieved by the use of electropermeabilization of sea urchin eggs (40) and adrenal chromaffin cells (39) and application of buffered Ca²⁺ solutions to the leaky cells. This revealed that granule exocytosis in these cells was half-maximally activated at 1 µM free Ca²⁺. These pioneering studies were the first direct measurement of the Ca²⁺ requirement for regulated exocytosis in any cell type. The similar Ca²⁺ dependency of fusion of cortical granules of the sea urchin egg and granules in chromaffin cells demonstrated a potential commonality of Ca²⁺ sensor mechanism in exocytosis. Subsequently, a variety of cell permeabilization techniques have been successfully applied to many different cell types. These studies have not only established the Ca²⁺ requirement for exocytosis in diverse cells (and conversely a lack of requirement in others) but also demonstrated a general requirement for MgATP (39, 40) in an early priming step (340) but not fusion (322, 340, 430, 585, 758, 851, 852), and allowed assay of the role in exocytosis of many proteins added as cytosolic proteins or soluble recombinant protein constructs.

In the majority of cases, the use of permeabilized cells has shown a Ca²⁺ requirement for triggering of exocytosis in the low micromolar range (Table 4). It became apparent, however, that multiple Ca²⁺-dependent steps exist in the exocytotic pathway. In addition, permeabilized cell assays have poor kinetic resolution and essentially provide information on the extent of release over a comparatively long time period. The Ca²⁺-triggered output in these population assays may not, therefore, reflect the true Ca²⁺ dependence of the final exocytotic step, but the average Ca²⁺ requirement to maintain sustained release over the period (several minutes) usually used in these assays.

A rigorous value for the Ca²⁺ dependency of the final fusion step has subsequently been determined by high-resolution patch-clamp measurement of plasma membrane capacitance. The application of whole cell patch-clamp recording to the measurement of cell surface area was developed by Neher and Marty (521) in studies on adrenal chromaffin cells. Its power to resolve single granule fusion events was subsequently exploited in studies on exocytosis in mast cells that have much larger secretory granules, allowing single fusion events to be resolved in a stepwise manner. Membrane capacitance is the inherent property of a lipid bilayer to become charged and subsequently release that charge when a voltage difference is applied across the bilayer. Because capacitance is directly proportional to the area of the bilayer (the capacitance of a biological bilayer is ~10 fF/µm²), this technique allows quantitative assay of membrane insertion (exocytosis) or membrane removal (endocytosis) from the cell surface based on knowledge of the surface area of secretory granules. Initially in these studies, exocytosis was triggered by depolarization to open voltage-gated Ca²⁺ channels (521) or by infusion of Ca²⁺ buffers from the recording micropipette by intracellular dialysis (36, 118). The latter experiments showed exocytosis to have similar Ca²⁺ dependencies to those determined in cell permeabilization assays. Accurate determination of the Ca²⁺ requirement of the final fusion step was achieved by using flash photolysis of caged Ca²⁺ compounds coupled with high-resolution capacitance recording (522, 767). The caged Ca²⁺ compounds (DM-nitrophen or NP-EGTA) are introduced via the patch pipette with Ca²⁺ tightly bound. An intense flash of light breaks down the caged compound to liberate free Ca²⁺ almost instantaneously and homogeneously throughout the cytoplasm. This approach allows an assessment of the relationship between free Ca²⁺ concentration and the initial rate of exocytosis from the so-called ready-releasable pool. Such experiments, carried out in chromaffin cells (324, 325, 522), pituitary melanotrophs (659, 764-767), and later in -cells (233, 749, 750), PC12 cells (371), and pancreatic acinar cells (358) showed the existence of a limited pool of ready-releasable secretory granules which could undergo rapid exocytosis (following a lag of ~20-50 ms) and with a Ca²⁺ dependency in the 2-30 µM range (Table 4). A similar Ca²⁺ dependency was demonstrated for the exocytosis of dense-core granules in dorsal root ganglion neurons (347). In addition, a recruitment step has also been defined whereby low levels of Ca²⁺ (324, 325, 658), presumably working through a very high-affinity Ca²⁺ sensor, can increase the size of the ready-releasable pool or its replenishment (Fig. 10). The Ca²⁺ dependency of recruitment or pool refilling in adrenal chromaffin cells from these techniques (0. 4 µM) (217, 702) is very close to that for the Ca²⁺ dependency of priming from permeabilization studies (0.3 µM) (78).

View larger version (21K): [in this window] [in a new window]   Fig. 10. Ca2+-dependent steps in regulated granule exocytosis in neuroendocrine cells. Ca2+-dependent stages and the Ca2+ requirement for exocytosis and endocytosis from studies using adrenal chromaffin cells and pituitary cells are shown. The data are derived from experiments using flash photolysis of caged Ca2+ and patch-clamp capacitance measurement.

Within experimental error, the Ca²⁺ dependency of Ca²⁺-triggered granule exocytosis in many cell types is similar (Table 4), suggesting the existence of a common high-affinity Ca²⁺ sensor or members of a closely related family of Ca²⁺-binding proteins with similar properties. There are, however, some notable differences, and in certain cell types [e.g., neutrophils (541, 689)] and also in synapses, there are vesicles or granules with distinct Ca²⁺ dependencies allowing differential control of their release (801). Application of the flash photolysis/capacitance approach to a specialized neuron, the retinal bipolar neuron (323), suggested that synaptic vesicle exocytosis involved a distinct low-affinity Ca²⁺ sensor. In this giant synapse, exocytosis was not triggered until the Ca²⁺ concentration was >10 µM (810) and was half-maximal at 190 µM free Ca²⁺ (323), a 10-fold higher Ca²⁺ concentration than for granule exocytosis. The large difference in Ca²⁺ dependency of exocytosis could be important for the differential regulation of exocytosis of synaptic vesicles and large dense-core vesicles (LDVs) present in the same nerve terminals. Synaptic vesicles anchored at the active zone would respond to high Ca²⁺ concentrations at the mouth of the Ca²⁺ channels. In contrast, LDVs are found away from the active zone, and their exocytosis at other sites would be triggered at lower Ca²⁺ concentrations following Ca²⁺ diffusion across the synapse. Others have suggested, however, that a continuous vesicle cycle runs in goldfish retinal bipolar cells even at submicromolar levels of Ca²⁺ (407), indicating that even in these synapses a high-affinity system can operate for synaptic vesicle exocytosis.

More recently, measurement of neurotransmission at a central synapse (again a specialized type of synapse known as the calyx of Held) has determined a Ca²⁺ dependency in the low micromolar range (2-5 µM) (86, 677) comparable to that for secretory granule exocytosis. These findings were interpreted as suggesting that the earlier value found in retinal bipolar neurons might not reflect the general situation in mammalian central nervous system (CNS) synapses. Earlier data in support of a low-affinity receptor for synaptic vesicle exocytosis in the squid giant synapse included the demonstration of microdomains of very high Ca²⁺ during neurotransmitter release (440). In addition, exocytosis in this synapse was inhibited by BAPTA but not by the slower Ca²⁺ chelator EGTA, suggesting that the sensing of Ca²⁺ by the fusion machinery must occur close to the Ca²⁺ channels (34, 708). Studies on release from populations of synapses (synaptosomes) where Ca²⁺ concentration was modified using the Ca²⁺ ionophore ionomycin (801) or by permeabilization (755) have also suggested the presence of a low-affinity Ca²⁺ sensor for glutamate release. It is possible that it is the calyx of Held synaptic terminals are unusual in their Ca²⁺ requirements, but blockade of transmission by EGTA in cortical synapses favors more widespread use of a low-affinity process (546). It is of significance that a direct comparison of the Ca²⁺ dependency of synaptic vesicle and dense-core granule exocytosis in permeabilized brain synaptosomes (755) showed that these differed by two orders of magnitude. This study used assay of glutamate release for synaptic vesicle exocytosis. Because glutamate is the predominant excitatory neurotransmitter in the CNS, this may give a more general insight into the Ca²⁺ requirement in central synapses. In addition, this study gives very direct support to the original idea that vesicle and dense-core granule exocytosis use distinct Ca²⁺ sensors. One high-affinity Ca²⁺ sensor implicated as specific for dense-core granule exocytosis in this study was the protein CAPS (755). The debate of low- versus high-affinity Ca²⁺ sensors in vesicle exocytosis is still to be resolved and will continue until accurate Ca²⁺ dependencies are determined for more neuronal and nonneuronal cell types.

The significance of the debate on Ca²⁺ sensitivity of synaptic exocytosis is its relevance to the identification of synaptotagmin I (and family members) as a major Ca²⁺ sensor for exocytosis. Synaptotagmin I knock-out mice (273) and mutants in Drosophila (214, 215, 438) and C. elegans (366) show a major impairment in neurotransmission. The low-affinity Ca²⁺-dependent interaction (K_d ~100-200 µM free Ca²⁺) of synaptotagmin with syntaxin in vitro in certain assays (98, 145, 205, 423, 591) (however, see the higher affinity reported more recently, Ref. 244) resulted in synaptotagmin I becoming the prime candidate for the low-affinity Ca²⁺ sensor in synaptic vesicle exocytosis (736). The problem with this interpretation is that synaptotagmin I and synaptotagmin II (which have similar biochemical properties) also appear to act in the high Ca²⁺-affinity secretory granule exocytosis in adrenal chromaffin cells (547) and insulin-secreting cells (298, 409). It has also been suggested that synaptotagmins, including synaptotagmin I, can regulate exocytosis in what were described as "mast cells" (47, 48). It should be noted, however, that these studies were not actually carried out on mast cells but instead on the rat basophilic leukemia cell line in which Ca²⁺ alone can trigger exocytosis with high affinity. This differs from the situation in mast cell where exocytosis requires activation of GTP-binding proteins (see sect. VIC). A recent examination of the ion selectivity of exocytosis in PC12 cells has suggested that the ion selectivity of secretory granule exocytosis is consistent with synaptotagmin I as the Ca²⁺ sensor (385). The data were suggested to be in accord with the ion selectivity of synaptotagmin-phospholipid interaction (422). PC12 cells also show a form of exocytosis, believed to be due to synaptic-like microvesicles (371), which is of lower Ca²⁺ affinity. In this case, the ion selectivity, particularly the lack of effectiveness of Ba²⁺, was not compatible with synaptotagmin as the Ca²⁺ sensor but more likely an EF-hand containing Ca²⁺-binding protein. These data can only be partially reconciled with other work on a knock-in mouse where native synaptotagmin I was replaced by a form with the mutation R233Q in the C2A domain (244). This mutation reduced the affinity of synaptotagmin I for Ca²⁺-dependent binding to phospholipid and lowered the Ca²⁺ sensitivity of the evoked exocytosis of synaptic vesicles, suggesting that synaptotagmin is the Ca²⁺ sensor for synaptic vesicle exocytosis. These two sets of data would be easier to reconcile if secretory granule and synaptic vesicle exocytosis did not have such different apparent Ca²⁺ dependencies. Recent work has examined Ca²⁺-triggered exocytosis in adrenal chromaffin cells from the synaptotagmin I knock-out mice. The extent of exocytosis was reduced in chromaffin cells from these mice, with a major defect in the fast exocytic burst but not the slow component of exocytosis (808), a phenotype comparable to that seen for synaptic release (273). The data suggested that synaptotagmin I was either the Ca²⁺ sensor for only the ready-releasable pool of granules or alternatively controlled the equilibrium between the slowly and ready-releasable pools. It was clear, however, that additional Ca²⁺ sensors for exocytosis must operate in these cells. A further aspect of synaptotagmin that has emerged is its ability to influence the kinetics of single granule release events in PC12 cells, suggesting that it may function during membrane fusion at the level of the fusion pore (818). The significance of Ca²⁺ sensing by either the C2A or C2B domains of synaptotagmin has been controversial (144, 783). As noted above, the knock-in study on the R233Q mutant in mice suggested the importance of the C2A domain for the Ca²⁺ sensitivity of exocytosis. Detailed genetic analyses in Drosophila have shown, however, that Ca²⁺ binding to the C2A domain is dispensable for neurotransmission and survival (643) but that Ca²⁺ binding to the C2B domain is crucial (449, 450).

A further issue in understanding the relationship between Ca²⁺ and exocytosis is the spatial distribution of secretory granules and the source of Ca²⁺ (519, 688). As noted above, this can determine the effectiveness of Ca²⁺ release from internal Ca²⁺ stores in activating exocytosis. In addition, much attention has been given to the question of how close fusion occurs to Ca²⁺ channels and the degree of coupling between Ca²⁺ entry and exocytosis. For synaptic vesicles, it is generally agreed that the rapid speed of neurotransmission [usually over a submillisecond time course and as fast as 60 µs in central synapses (662)] must mean that the vesicles that undergo fusion sense Ca²⁺ that has diffused no further than the mouth of the open Ca²⁺ channel (708). This must also be the case if synaptic vesicle exocytosis involves a low-affinity Ca²⁺ sensor as suggested for goldfish retinal bipolar neurons (323). Molecular interactions that have been demonstrated between proteins of the exocytosis machinery including syntaxin and synaptotagmin and Ca²⁺ channel subunits have been interpreted as suggesting that synaptic voltage-gated Ca²⁺ channels involved in neurotransmission are physically part of the exocytotic site (137, 138). A recent study has suggested, however, that syntaxin within a SNARE complex could not interact with N-type Ca²⁺ channels and would only do so after fusion and SNARE complex disassembly (72). The extent of coupling to Ca²⁺ channels has been examined for secretory granule exocytosis in adrenal chromaffin cells. By comparison of the initial responses due to flash photolysis with those resulting from depolarization, it has been established that exocytosis occurs only after a lag of 10-50 ms after Ca²⁺ channel opening (174, 175). This would be sufficient time for Ca²⁺ diffusion around 0.5 µm from the plasma membrane Ca²⁺ channels. Under these conditions, the granules would sense a Ca²⁺ elevation to ~5-10 µM (173, 174). It has been concluded from these experiments, therefore, that a tight physical coupling between Ca²⁺ channels and secretory granules is unlikely to occur in chromaffin cells. This does not rule out the possibility that exocytosis occurs preferentially in certain domains of the cell and that these are also domains rich in voltage-gated Ca²⁺ channels (496, 644). Such domains have indeed been observed experimentally (642, 680). A similar codistribution of exocytotic sites and Ca²⁺ channels has been demonstrated for pancreatic -cells (85). The Ca²⁺-dependent exocytosis of lysosomes in fibroblasts appears to involve the ubiquitously expressed protein synaptotagmin VII (464).

The application of flash photolysis of caged Ca²⁺ compounds and patch-clamp capacitance recording to cultured fibroblastoid cell lines (CHO cells) surprisingly showed that a Ca²⁺-regulated pathway for exocytosis existed even in these cells (191, 528). Coupled with an earlier demonstration that the muscle myoblasts could take up and release acetylcholine in response to stimulation (607), this suggests that all cell types might possess a regulated exocytotic pathway. One possibility is that this pathway involves Ca²⁺-dependent exocytosis of lysosomes (19, 646). The secretory granules in hemopoietic cells are believed to be derived from modified lysosomes (732) (see sect. III). It has been demonstrated, however, that even in nonsecretory cells, conventional lysosomes can be triggered to undergo exocytosis, and studies with permeabilized cells have shown lysosome exocytosis to have a K_d for Ca²⁺ of ~1 µM (464, 646).

The discussion above is related to cells in which Ca²⁺ acts as the trigger to initiate exocytosis. In two cell types in which Ca²⁺ is not the trigger, it has instead been shown to have an additional role in exocytosis. In mast cells and eosinophils, exocytosis can be activated by the intracellular introduction of nonhydrolyzable GTP analogs (discussed in more detail below). Elevation of Ca²⁺ in these cell types to ~1.5 µM did not increase the number of granule fusion events over that seen at very low Ca²⁺ concentrations but affected a very late stage of the fusion process that was reflected in an increase in the rate of fusion pore expansion (242, 310, 667). This indicates that a Ca²⁺-binding protein must act either at the time of fusion or immediately after fusion. It is possible that Ca²⁺ could have a similar action in other cell types, but this cannot be experimentally resolved as easily in the many cell types where Ca²⁺ is essential as the trigger for exocytosis.

An unusual action of Ca²⁺ has been characterized in parathyroid hormone (PTH)-secreting chief cells of the parathyroid gland. The physiological role of these cells is in the control of body Ca²⁺ homeostasis, with extracellular Ca²⁺ concentration being detected by an extracellular plasma membrane Ca²⁺ receptor protein (101). PTH secretion is stimulated by a fall in extracellular Ca²⁺ which leads to a corresponding fall in the intracellular free Ca²⁺ concentration (101, 489). PTH-secreting cells, therefore, have an inverse Ca²⁺ regulation compared with other secretory cell types. It has been demonstrated, using permeabilized cells, that intracellular Ca²⁺ is directly inhibitory, with a K_d of ~1 µM, provided that a nonhydrolyzable GTP analog is present to activate exocytosis (544). It seems, therefore, that Ca²⁺ inhibits a GTP-triggered exocytotic pathway in these PTH-secreting cells. This inverse regulation by Ca²⁺ is not unique to parathyroid cells since a similar process seems to operate to control renin release from the juxtaglomerular cells of the kidney (301). This has been studied primarily in intact cells, but again the control seems to involve interplay between a GTP-binding protein and an inhibitory action of intracellular Ca²⁺. The inverse regulation of Ca²⁺ in these cell types is intriguing, and it would be of great interest to know the identity of the intracellular Ca²⁺ receptor involved and how this interacts with the exocytotic machinery. Little is known, however, about the proteins involved in exocytosis in these cell types.

In many cell types increases in intracellular cAMP concentration and thereby activation of protein kinase A (PKA) have been found to regulate Ca²⁺-triggered exocytosis. An enhancement of exocytosis by cAMP has been shown to occur, for example, in synapses (147), adrenal chromaffin cells (108, 398, 508), melanotrophs (856), PC12 cells (618), pancreatic -cells (17), AtT-20 cells (480), and pancreatic acinar cells (394, 570) as well as for lysosome exocytosis (645). In certain synapses, constitutive activation of PKA seems to be required for continued exocytosis (332). In all of these cell types, however, an elevation of cAMP alone in the absence of a Ca²⁺ rise is not sufficient to trigger exocytosis. In contrast, a limited range of cell types (334), including those of the salivary glands (submandibular and parotid glands) (43, 258, 617, 752) use cAMP as a major trigger for exocytosis. An elevation in cAMP levels can also trigger exocytosis in response to glucagon in pancreatic -cells (17) and in response to gonadotropin-releasing hormone in pituitary cells (203). Elevation of intracellular cAMP levels also triggers exocytosis in renal juxtaglomerular cells (255) and PTH secretion in parathyroid cells (510). The cAMP-dependent pathways coexist with Ca²⁺-dependent pathways for exocytosis in these cell types, and it is likely that they use a common final SNARE-dependent mechanism. The basis of cAMP-triggered exocytosis has been studied most extensively in cells of the rat parotid gland where it requires PKA activity and involves SNARE proteins and other components of the general fusion machinery (258, 259, 752, 754). Experiments using streptolysin O-permeabilized parotid cells have demonstrated a robust level of exocytosis triggered by addition of cAMP even when Ca²⁺ was buffered to very low levels (261). The cAMP-induced exocytosis was inhibited by treatment with botulinum neurotoxin B, indicating a requirement for VAMP-2 whose presence in these cells was also demonstrated (259, 261). The involvement of other SNAREs or other components of the general fusion machinery in cAMP-triggered exocytosis has not yet been investigated. In addition, the molecular mechanism by which cAMP triggers exocytosis remains to be established.

The regulatory role of cAMP in Ca²⁺-triggered exocytosis that has been observed in various cell types may be due to the phosphorylation of one or more of the proteins of the general exocytotic machinery that are PKA substrates (786). A major candidate for this role is cysteine string protein (Csp), which is a PKA substrate both in vitro and in vivo (237). Phosphorylation of Csp reduces its affinity for binding to both syntaxin and synaptotagmin I (236). Another possibility is the action of a non-PKA target for cAMP such as the recently identified cAMP-binding protein cAMP-GEFII, which acts via the rab3 effector RIM (574).

We have discussed the general importance of the Rab family of GTPases in the control of the core fusion machinery and in exocytosis in section V. The exact role of Rabs in regulated exocytosis is unclear, and evidence has been described that supports both inhibitory and stimulatory roles for Rab proteins (202). Various pieces of evidence have suggested that other GTP proteins may regulate exocytosis. Considerable effort has been put into the analysis of regulatory roles of GTP-binding proteins in exocytosis in a wide range of secretory cell types (430). The starting point for these studies was the demonstration that guanosine 5'-O-(3-thiotriphosphate) (GTPS), a nonhydrolyzable analog of GTP, could trigger exocytosis detected by patch-clamp capacitance measurement in mast cells in the absence of an elevation of intracellular Ca²⁺ (241). A Ca²⁺-independent action of GTP analogs was subsequently observed in permeabilized neutrophils (51), and the suggestion was made that nonhydrolyzable GTP analogs initiated exocytosis by activation of a heterotrimeric G protein at the heart of the exocytotic machinery (286). It was subsequently shown that GTPS and other GTP analogs had modulatory effects on exocytosis during patch-clamp recording or after permeabilization in many other cell types (Table 5). The effects of GTP analogs can be complex, however, and potentially may involve multiple GTP-binding proteins. In addition, activation of G proteins only acts as a Ca²⁺-independent trigger for exocytosis in certain cell types. Elsewhere, the effect of activating GTP-binding proteins is to reveal stimulatory or inhibitory regulation of Ca²⁺-triggered exocytosis. The distinction between simple G protein-triggered exocytosis and a more complex regulation of Ca²⁺-triggered exocytosis is highlighted by a comparison of data from mast cells (286, 430) with that from adrenal chromaffin cells (119), where the effects of GTP analogs have been extensively studied as described below.

The possible importance of GTP-binding proteins for exocytosis in mast cells was revealed by an acute loading technique to introduce GTP analogs into the cells, which increased stimulated exocytosis (285). Subsequent work with permeabilized mast cells (184, 331, 343-345, 426, 427), patch-clamp capacitance measurement (241), or microinjection (759) has established that activation of GTP-binding proteins is sufficient to trigger exocytosis in the absence of a rise in Ca²⁺ in mast cells. Triggering of exocytosis in mast cells does require the presence of Ca²⁺, but Ca²⁺ alone is unable to trigger exocytosis (343, 402). The identity of the GTP-binding protein(s) involved in triggering exocytosis (termed G_E) is unclear as both heterotrimeric (24, 602) and monomeric [e.g., rho, rac, and cdc42 (100, 536, 567, 609)] GTP-binding proteins have been implicated. It is not clear, therefore, whether G_E in mast cells is a single GTP-binding protein or whether several GTP-binding proteins work in concert to trigger exocytosis. Despite this difference in the trigger, GTP-dependent exocytosis in mast cells may still use the SNARE machinery (48, 297). The final mechanisms involved in granule fusion in mast cells are, therefore, likely to be essentially the same as that for Ca²⁺-triggered exocytosis in other cell types.

Some of the regulatory effects on exocytosis of activation of G proteins are mediated by the G protein -subunits. Work on both mast cells (602) and insulin-secreting cells (865) has suggested that the -subunits of G proteins are required for exocytosis. This is in addition to regulatory roles of the -subunits seen in the same cell types (410). The data derived from experiments showed that addition of -subunits retarded the run down of secretory activity in mast cells. In addition, reagents that acted as -sponges to sequester -subunits inhibited exocytosis (602, 865).

Adrenal chromaffin cells are a very well-characterized cell type where the key trigger for exocytosis is a rise in cytosolic Ca²⁺ concentration, and elevation of Ca²⁺ is sufficient to stimulate exocytosis (109). Activation of GTP-binding proteins has multiple effects in these cells, with the effect observed being dependent on the specific protocol used and the particular GTP analog tested (10, 79, 119). Some exocytosis can be triggered at low Ca²⁺ by the analog 5'-guanylylimidodiphosphate (GppNHp) when added to permeabilized chromaffin cells (501) or introduced during patch-clamp capacitance recording (118). The extent of exocytosis is, however, considerably lower than that triggered by Ca²⁺. Both GppNHp and GTPS can enhance Ca²⁺-dependent exocytosis in chromaffin cells (8, 79, 125, 392, 501). In addition, preincubation with GTPS leads to an inhibition of Ca²⁺-dependent exocytosis (340, 392, 716). These results suggest the involvement of multiple GTP-binding proteins, and indeed, both monomeric (Rab3 in an inhibitory role, Ref. 341) and heterotrimeric (in inhibitory and stimulatory roles, Ref. 807) GTP-binding proteins have been implicated in controlling exocytosis in chromaffin cells. Ca²⁺-dependent exocytosis in chromaffin cells proceeds through a SNARE-dependent mechanism and is sensitive to Clostridium neurotoxins (76, 77). The Ca²⁺-independent triggering of exocytosis by GTP analogs most likely involves a related final mechanism because it is also inhibited by Botulinum neurotoxins C, D, and E (281) that cleave syntaxin, VAMP, and SNAP-25, respectively. Similar results have been reported concerning the neurotoxin sensitivity of GTPS-triggered exocytosis in PC12 cells (46).

	VIII. REGULATION OF SECRETORY GRANULE EXOCYTOSIS

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A number of unrelated secretory cell types possess multiple secretory granules or secretory vesicles that undergo regulated exocytosis and that can be activated to undergo fusion independently of each other. A classical example comes from the studies of neurotransmitters localized in the same synapses where it was found that varying the stimulation protocol leads to different patterns of release (52, 359, 472). This is a consequence of the distinct localization of neurotransmitters to either synaptic vesicles or to LDVs (871). The differential exocytosis of these two organelles is not due to fundamental differences in the exocytotic fusion machinery (412) but to differences in the Ca²⁺ affinity of the exocytotic process (801) and the localization of the vesicles in the nerve terminals (871). As described above, synaptic vesicle release is mediated by a low-affinity Ca²⁺ sensor and involves vesicles localized close to Ca²⁺ channels. In contrast, LDV release involves a high-affinity Ca²⁺ sensor, but these granules need more intense stimulation because they are localized away from the plasma membrane (801) and therefore can only sense Ca²⁺ as it diffuses deep into the synaptic terminal following prolonged stimulation.

Many cell types, including adrenal chromaffin cells, pancreatic -cells, and PC12 cells contain small vesicles termed synaptic-like microvesicles (SLMVs) in addition to secretory granules (768). The SLMVs were identified due to their possession of synaptic vesicle-specific proteins such as synaptophysin (518). The physiological function of SLMVs and the control of their exocytosis has been relatively little studied, although it is known that the SLMVs in pancreatic -cells contain and release GABA and that this may regulate the surrounding cells in the pancreatic islets (9, 626). From analysis of exocytosis in PC12 cells, it has been suggested that they show more rapid exocytotic kinetics than secretory granules in the same cells (371). The Ca²⁺ dependencies for exocytosis of each secretory organelle appear to be very similar or similar depending on the cell type and assay used (370, 371, 530, 750).

Among cells with multiple types of secretory granules are the neutrophils and platelets. For the neutrophils, clear evidence is available to demonstrate distinct Ca²⁺ dependencies for the exocytosis of each granule type, whereas for platelets, such a difference has not been detected. Neutrophils possess four distinct secretory organelles: three types of granules (azurophil, specific, and gelatinase) and nongranular secretory vesicles (88, 689). These organelles can be separated by fractionation and contain distinct membrane and content proteins (88). The exocytosis from these secretory organelles can be varied so that neutrophils secrete different cocktails of proteins or insert distinct new proteins into the plasma membrane. Studies on intact cells in which intracellular Ca²⁺ concentration has been varied using ionomycin and monitored using quin 2 (421) or fura 2 (689) have demonstrated a hierarchy of Ca²⁺ sensitivities of the secretory organelles for exocytosis. In only one study has exocytosis of all four organelles been assayed, which gave the order of Ca²⁺ sensitivity as secretory vesicle > gelatinase granule > specific granule > azurophil granule (689). Other studies using direct control of Ca²⁺ concentration in permeabilized cells or in whole cell capacitance recording have supported this hierarchy for the specific and azurophil granules (541, 709, 711). There also appears to be differences between the neutrophil granules in their ATP requirements for exocytosis (761). The physiological significance of these differences is not yet clear because they would only become significant under conditions of very low cytosolic ATP concentration. In addition, another distinction between granule types was identified based on the finding that GTPS inhibited exocytosis of specific granules but enhanced the release of azurophil granules (709). It is likely, therefore, that the balance of exocytosis in intact neutrophils is determined by both the intracellular Ca²⁺ concentration and the degree of activation of the relevant GTP-binding proteins.

Platelets possess three types of regulated secretory organelle: the dense granules, the -granules, and lysosomes, which again contain distinct secretory components. The exocytosis of lysosomes is triggered by the same agonists as dense granules in intact cells but requires higher agonist concentrations (393). This is not due, however, to differences in Ca²⁺ sensitivity because the Ca²⁺ dependence of release of markers for these organelles from permeabilized cells was indistinguishable (393). Similarly, the Ca²⁺ dependency of release of markers for dense and -granules from permeabilized platelets is very similar, and GTP analogs had similar stimulatory effects on their exocytosis (190, 577). One difference that was seen was a stimulation of dense granule but not -granule exocytosis by phorbol ester in the presence of low concentrations of Ca²⁺ (190). This difference could provide a mechanism for the differential activation of exocytosis from the dense and -granule populations in platelets. It has also been shown that exocytosis from different secretory organelles (dense granules and -granules) in platelets involves distinct isoforms of VAMP (606).

In almost all secretory cell types, a level of basal secretion can be detected in the absence of cell stimulation. It has long been a matter of debate as to whether this represented uncontrolled release from secretory granules or a true constitutive exocytosis (471). This aspect has been characterized in detail in rat parotid acinar cells (136, 346) and in insulin-secreting cells (30, 787). It appears that in these cell types, some secretory proteins are sorted away from secretory granules during their biogenesis to reach constitutive secretory vesicles that account for basal release (29). In parotid acinar cells, these studies also revealed the existence of an additional regulated pathway (346). Parotid cells release some amylase via a true constitutive secretion. They also package amylase and other secretory proteins (such as parotid secretory protein, PSP) into conventional secretory granules, which undergo exocytosis in response to intense stimulation. At low levels of stimulation (for example, with low concentrations of carbachol), however, secretion is activated from a third pathway. This so-called "minor regulated" pathway leads to the secretion of a cocktail of proteins similar to that seen due to constitutive secretion. The nature of the secretory organelle responsible for the minor pathway is not known, nor is it known whether the minor and granule exocytosis pathways differ in their inherent Ca²⁺ sensitivities for triggering of exocytosis. A further twist on the constitutive versus regulated aspect of exocytosis is demonstrated by the mammary epithelial cell (115). These cells secrete copious amounts of milk constituents including the milk proteins, the caseins, largely by an apparently constitutive route. Study of isolated mouse mammary acini demonstrated, however, that around one-third of the synthesized casein remains in a stored intracellular pool and can be released in response to Ca²⁺ elevation in intact (788) or permeabilized (789) cells. No information is available on the relationship between the constitutive and the regulated casein secretory vesicles or the basis for this differential regulation of their exocytosis. The examples discussed here demonstrate the possibility that, to varying extents, regulated secretory proteins may be diverted into constitutive or alternative regulated exocytotic pathways. It is unlikely that this is limited to the few cell types that have been studied in detail so far and may be a more general phenomenon.

A large number of studies on many different secretory cell types have implicated protein phosphorylation in the control of regulated exocytosis. Many of the older studies were based on the use of cell-permeable inhibitors or activators and suggested roles for Ca²⁺/calmodulin kinase II (526, 682), mitogen-activated protein kinase (197), protein kinase C (PKC) (637, 691), and tyrosine kinases (197). A complication in these approaches is that the effects on exocytosis could be indirect through effects on plasma membrane receptor or ion channel phosphorylation (425). These could, nevertheless, be important mechanisms for the cell-specific regulation of exocytosis. Over more recent years, the application of reagents to permeabilized cells has provided evidence for a more direct regulation of the exocytotic machinery. In addition, biochemical approaches have resulted in the identification of many of the possible protein substrates for the various protein kinases (786). The most general modulators of exocytosis are PKA and PKC, which enhance triggered exocytosis in essentially all cell types examined including neurons (691, 725), adrenal chromaffin cells (398, 503, 508), PC12 cells (7) (698), AtT20 cells (480), pancreatic exocrine cells (570), parotid acinar cells (753), SPOC1 cells (683), neutrophils (710), platelets (861), and mast cells (345, 402). Rapid recruitment of PKC-II to the plasma membrane and secretory granules of pancreatic -cells is also consistent with a role for this kinase in insulin secretion (601).

Information on the substrates for PKA and PKC has begun to provide information for the mechanistic basis for the action of these kinases. The key proteins of the exocytotic machinery that have been identified as kinase substrates in vitro, and to a lesser extent in vivo, are shown in Figure 11 and include the SNARE proteins and their regulators. The physiological significance of the phosphorylations shown is only known for a few examples. Rabphilin3A, for example, is phosphorylated by PKA both in vitro (267) and in vivo (266). It is also phosphorylated after induction of long-term potentiation (LTP) in the CA3 region of the hippocampus where LTP requires the activity of PKA (444). Phosphorylation of rabphilin3A on serine-234 and serine-274 is altered in rat brain slices under a number of physiological conditions (250, 251). Phosphorylated rabphilin3A was found to have a reduced affinity for membranes, indicating that its phosphorylation could regulate its association with synaptic vesicles (250). So far, however, no direct functional effects of rabphilin 3A phosphorylation on exocytosis have been reported. In contrast, two other vesicle proteins have been shown to be PKA substrates and their phosphorylation demonstrated to affect the kinetics of exocytosis. The first of these, Snapin, was originally identified from its binding to SNAP-25 (357). Phosphorylation of Snapin by PKA on serine-50 increased its binding to SNAP-25. Expression of a form of Snapin with serine-50 mutated to aspartate acid (a phosphomimetic residue) resulted in an increase in the releasable pool of vesicles in adrenal chromaffin cells (170). The relevance of this study is questionable because it is unclear that chromaffin cells normally express Snapin. The other vesicle protein, Csp, was found to be phosphorylated by PKA on serine-10 (237), and this reduced its binding to syntaxin (237) and to synaptotagmin. The phosphorylation of Csp at this site affected whether or not Csp overexpression could modify the kinetics of single granule release events in chromaffin cells. Wild-type Csp overexpression slowed release kinetics and increased quantal size (288), whereas Csp harboring an S10A mutation, rendering the protein nonphosphorylatable, had no effect on kinetics. These data suggest that phosphorylation of this residue could regulate late steps during membrane fusion. These data were consistent with the findings that elevation of cAMP in chromaffin cells also resulted in a slowing of release kinetics (447).

View larger version (35K): [in this window] [in a new window]   Fig. 11. Protein kinase substrates within the exocytotic machinery. The protein kinases that have been demonstrated to phosphorylate key proteins involved in regulated exocytosis are indicated. Where data are available for in vivo phosphorylation, the kinase is indicated with an asterisk.

PKC has been thought to modulate regulated exocytosis in many neurons and in many different secretory cell types. Much of the data have been based, however, on the use of phorbol esters to activate PKC. It became subsequently realized that phorbol esters also bind to unc-13 (467), and it has recently been suggested that all of the effects of phorbol esters on neurotransmission between hippocampal neurons are in fact due to munc-13 (632). This conclusion was based on the analysis of mice expressing Munc13-1 (H567K). This mutation impairs phorbol ester and diacylglycerol binding by Munc13-1, and hippocampal neurons expressing this mutant protein no longer showed an increase in evoked neurotransmission in response to phorbol ester treatment.

PKC is clearly involved in the regulation of granule exocytosis in a number of cell types. Direct stimulatory roles for PKC-mediated phosphorylation have been demonstrated by addition of purified PKC to permeabilized chromaffin cells (503), PC12 cells (62), pituitary cells (516), and for - and dense-core granule exocytosis in platelets (861). It is not clear what is the substrate(s) responsible for the effects of PKC. Phosphorylation of both SNAP-25 (694) and munc-18 (257) by PKC reduces their affinity for syntaxin. In the case of SNAP-25, this could be most likely to inhibit SNARE complex formation and thereby exocytosis. In contrast, phosphorylation of munc-18 would allow it to dissociate more readily from syntaxin so that syntaxin could participate in SNARE complex assembly. The putative Ca²⁺ sensor synaptotagmin I is also a substrate for PKC both in vitro and in vivo. The physiological significance of the phosphorylation of these proteins is, however, currently unknown.

It appears that changes in the extent of release due to a switch to kiss-and-run exocytosis can be controlled by activation of PKC. Treatment of chromaffin cells with phorbol esters (289) modified the single granule release events so that release was initially faster and also terminated more rapidly. The initial increase in the rate of release was consistent with data derived from independent techniques showing that PKC activation increased the rate of fusion pore expansion for granules in eosinophils (667). The more rapid termination of release is consistent with kiss-and-run exocytosis being activated (289). In addition, use of membrane-associated, lipophilic fluorescent dyes and examination of their dissociation from synaptic vesicle membranes after fusion showed a conversion from predominantly full fusion to predominantly kiss-and-run fusion after phorbol ester treatment of synaptosomes (195). Other work using FM dyes has shown that a subpopulation of synaptic vesicles in cultured hippocampal neurons undergo kiss-and-run fusion even under normal physiological conditions (727). The effect of PKC activation to speed up release kinetics was mimicked by the expression of the R39C (Fig. 7) mutant of Munc18 (248). Because Munc18 is a PKC substrate (257), this implicates Munc18 as a potential target for the control of kiss-and-run exocytosis through PKC-mediated phosphorylation (48a). Synaptotagmin I is also a substrate for PKC (333), and overexpression of this protein has also been shown to modify fusion pore kinetics measured using amperometry in PC12 cells (818).

Most cell types possess actin filaments organized into a variety of structures including a cortical actin network found beneath the plasma membrane (153). The idea that the cortical actin network could form a barrier to exocytosis and, thereby, act as a site of regulation of exocytosis was first proposed for the pancreatic -cell by Orci et al. in 1972 (560). Their work showed the existence of cortical actin (the "cell web") and that treatment with cytochalasin B to disrupt this actin resulted in increased insulin release. Evidence that agonist stimulation leads to disassembly of cortical actin (114, 151) and that this is regulated by second messenger pathways (113, 125, 152) was first described in adrenal chromaffin cells and subsequently characterized in detail (113, 1248, 803, 805, 806, 866). Subsequently, the important role of cortical actin as a barrier and its controlled disassembly/assembly in the regulation of exocytosis has been substantiated for many secretory cell types (Table 6). The alternative idea of an active role for the actin cytoskeleton in transport of secretory granules to the plasma membrane has been discussed in the past but was not supported by direct experimental data. Evidence for a dual role for the actin cytoskeleton has emerged again recently from studies of secretory granule mobility in intact cells (411, 553).

The evidence in support of a general role for the cortical actin network as a barrier to exocytosis has come from the use of actin-stabilizing or disassembling drugs. These have demonstrated that disassembly of cortical actin enhances agonist-induced exocytosis, and its stabilization is inhibitory in many cell types (Table 6). With one exception, all of these studies suggest that cortical actin disassembly modifies the extent of secretion but is not itself sufficient to activate exocytosis (113), which also requires an elevation of cytosolic Ca²⁺ concentration (125). The exception comes from a study of pancreatic exocrine cells where it was claimed that cortical actin disassembly was sufficient to trigger exocytosis in the absence of a rise in intracellular Ca²⁺ concentration (509). These data have not been confirmed, and the conclusions of this study are not consistent with other work on other cell types.

In parallel with the investigations of the effect of actin-specific drugs have been the demonstrations that agonists that trigger exocytosis disassemble cortical actin. This has been demonstrated in numerous cell types (Table 6). Studies of the mechanisms involved in actin disassembly have shown that it is activated by elevation of cytosolic Ca²⁺ concentration (125) and by activation of PKC (125, 382, 805). The molecular basis for the disassembly of cortical actin is not entirely clear. Two mechanisms have been proposed involving either the Ca²⁺-dependent actin-severing protein scinderin (414, 453-455, 648, 649, 804, 803, 866) or the 14-3-3 proteins (140, 655, 656), which may mediate the actions of PKC (502, 503). An alternative PKC substrate MARCKS has also been implicated (234). The roles of these proteins have so far been investigated mainly in adrenal chromaffin cells and for scinderin and MARCKS also in platelets. The accumulated data have suggested that secretory granule exocytosis is regulated by cortical actin in an inhibitory manner, and recently, this has been shown also to be the case for synaptic vesicle exocytosis (499).

Recent studies on the mobility of secretory granules in living cells have made use of two technical advances: 1) the use of green fluorescent protein constructs to label secretory granule proteins (275, 368) and 2) the use of evanescent wave microscopy to image secretory granules in a thin plane close to the plasma membrane (365, 550-553, 728-730, 764). The latter techniques have been particularly important in demonstrating the mobility of secretory granules approaching the plasma membrane, their docking, and subsequent fusion. It appears that some secretory granules are mobile while others show little mobility either because they are "docked" or are restrained by the cytoskeleton. Analysis of the effects of actin-specific drugs using evanescent wave microscopy has shown that intact actin filaments may be required for the transport of secretory granules to exocytotic sites (411, 553). In both PC12 cells (728) and adrenal chromaffin cells (553), the mobility of secretory granules was reduced after treatment with latrunculins to disassemble the cortical actin. This suggests that granule movement is mediated by active transport on actin filaments. It was also found, however, that stabilization of actin following treatment with phalloidin (728) or jasplakinolide (553) inhibited granule movement and reduced the recruitment of granules for exocytosis. It seems likely, therefore, that actin filaments have dual roles in both facilitating and inhibiting secretory granule exocytosis.

	IX. PHYSIOLOGICAL SIGNIFICANCE OF VARIATIONS IN CONTROL MECHANISMS FOR SECRETORY GRANULE EXOCYTOSIS

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Despite considerable similarities in the process of regulated exocytosis in most cell types, differences are evident that reflect the physiological function of the particular cell. These include variations in the rate of exocytosis, the lag time before it begins, its time course, the proportion of vesicles that undergo fusion in response to stimulation, the presence of single or multiple granule types, and the nature of the regulation of exocytosis by second messenger pathways. The nerve terminal has a highly specialized form of regulated exocytosis designed for very fast onset on a submillisecond time scale (662) and to be terminated quickly to allow the transmission of a fast, short-lived signal across the synapse. Some neuroendocrine and endocrine cells can initiate exocytosis within tens of milliseconds (18, 175, 324, 612, 766, 767), but in many other cell types, the initiation is slow (seconds) (241) but exocytosis can persist for prolonged periods. In most cases, release of hormones does not need to be rapid because their effects are systemic following transport around the body in the circulation.

The proportion of the granules that undergo exocytosis in response to stimulation is variable between cell types. In the case of adrenal chromaffin cells, for example, a physiological stimulus (an action potential) to the adrenal medulla results in release of <1 granule/cell on average despite a reserve pool of ~30,000 granules/cell (600). Indeed, massive release from adrenal chromaffin cells would likely be fatal to the organism. In contrast, mast cells function in an all-or-none manner. Within a population, mast cells either do or do not degranulate, and if they do, essentially all granules undergo exocytosis (331). This is presumably physiologically important to allow release of a burst of histamine from a few cells at the correct sites in tissues. A third type of response is seen in pancreatic -cells. These can initiate exocytosis on a millisecond time scale (18, 611, 707) but also continue to secrete for minutes, hours, and even days. The requirement for prolonged regulation of plasma glucose levels means that these cells must respond to chronically elevated glucose by continual secretion of insulin. This occurs by secretion of newly synthesized insulin as the stored pool is depleted. A fourth type of response is seen in the sperm acrosome reaction where a single secretory organelle undergoes a single round of fusion (134). A single episode of fusion also occurs following fertilization in egg cortical granule exocytosis but in this case involving many secretory granules (833).

More complex specialization is apparent in those cells that possess multiple types of distinct granules containing different secretory products such as neutrophils (88, 689) and platelets (393). The complex physiological functions of these cell types require them to be able to selectively secrete different cocktails of bioactive components and to selectively modify their cell surface by insertion of new plasma membrane proteins. This requires the existence of mechanisms, as yet little understood, to control the selective exocytosis of one or other secretory granule population. This appears to involve differences in Ca²⁺ sensitivity of the granule populations or in their sensitivity to activation of GTP-binding proteins or PKC.

A further difference between secretory cell types that may be related to their physiological function is the existence or otherwise of compound (also known as piggy-back) exocytosis. In this form of exocytosis, the fusion of one secretory granule with the plasma membrane is followed by the additional fusion of granules to the originally fused granule membrane. In electron microscopy, this can be seen in images where apparently several granules have fused in a focal manner so that a large invagination enters deep into the cell interior. The existence of this form of exocytosis has been highly controversial for some secretory cell types including the adrenal chromaffin cell (38, 265, 604), where it is likely to be a rare event. In contrast, it may be a significant or even the major form of exocytosis in some cell types.

The major physiological role of eosinophils is to kill parasites by secreting cytotoxic proteins onto them. The restriction of compound exocytosis to a focal site next to the parasite would be an obvious physiological mechanism to direct secretion only onto the invading organism. Compound exocytosis in eosinophils has been visualized by electron microscopy (525), and electrophysiological studies have established that it can be triggered by GTPS introduced during whole cell capacitance recording (311, 668). In eosinophils, compound exocytosis may be preceded by intracellular granule-granule fusion (668) so that a large structure formed from multiple granules fuses with the plasma membrane. In other cell types, compound exocytosis does not appear to be accompanied by prior granule-granule fusion. Cells in which piggy-back exocytosis occurs are able to prevent intracellular granule-granule exocytosis, so the piggy-back fusion must require components from the plasma membrane provided by the first fusion event. Compound exocytosis may also explain the all-or-none exocytosis that occurs in the closely related mast cells so that the fusion of one granule leads to the piggy-back fusion of all of the others in the cell. This is consistent with electron microscopic images of degranulated mast cells, which support the idea of compound exocytosis (142, 430).

Compound exocytosis of the piggy-back type has been visualized and characterized in pancreatic acinar cells (523). It has previously been established that the polarized secretion at the apical membrane of these cells leads to the appearance of structures suggesting compound exocytosis (352, 578, 795). Imaging using two-photon excitation has allowed analysis of the dynamics of fusion to be carried out. This has established that exocytosis in pancreatic acinar cells is sequential (i.e., in a piggy-back fashion) rather than involving intracellular granule-granule fusion events. The sequential fusion events occur in an ordered manner with the same time lag as the initial fusion, and it was suggested that this might reflect the time for diffusion of plasma membrane t-SNAREs (523).

Evidence suggesting the possible existence of compound exocytosis has been described for a number of endocrine and neuroendocrine cells where compound exocytosis would only involve a small proportion of the secretory granule pool. Of particular interest is recent data suggesting that in pituitary lactotrophs the extent of compound exocytosis could be under physiological regulation (183). Compound exocytosis was visualized based on the staining of the granule cores with the fluorescent dye FM1-43. Elevation of cAMP levels increased the extent of compound exocytosis involving sequential fusion of granules with the originally fused granule membrane. In contrast, treatment with phorbol ester not only increased compound exocytotic events but also recruited additional fusion sites around the plasma membrane (183). In the case of pituitary cells, regulation of compound exocytosis could be an alternative mechanism to allow an increase in secretion. It appears, therefore, that compound exocytosis may have different outcomes depending on the cell type: it can lead to extensive focal exocytosis or to complete degranulation or it can be used to turn up the amount of secretion due to a stimulus.

The basic machinery for exocytotic membrane fusion involving the SNAREs and SNARE regulators is undoubtedly the same in all secretory cell types and in many the same cell signal, a rise in cytosolic Ca²⁺ concentration, is used to trigger exocytosis. Each cell type is sensitive, however, to different types of external stimuli that elicit the Ca²⁺ elevation and also differ in regulatory mechanisms. External stimuli that lead to activation of GTP-binding proteins could have quite diverse effects on Ca²⁺-triggered exocytosis or trigger Ca²⁺-independent exocytosis in some cell types. In addition, agents that elevate cAMP concentration or activate PKC could have important cell-type specific effects on the fine-tuning of the exocytotic machinery through phosphorylation of key components.

	X. CONCLUDING REMARKS

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The regulated exocytosis of secretory granules has been studied for many years in a wide range of cell types. This has resulted in extensive characterization of the physiological regulation of secretion and the identification of the intracellular signals involved. For a long time it was suspected that the mechanisms underlying regulated exocytosis would be quite different from cell to cell and, in particular, would be distinct in synapses where exocytosis is so rapid. The last few years have seen a remarkable explosion in our knowledge of the many proteins that are required for exocytosis and that regulate the process. One of the most striking aspects has been the discovery in the early 1990s that very similar proteins playing the same roles function in synaptic exocytosis and in constitutive exocytosis in yeast. Initially searches for the key proteins (such as the SNAREs) in nonneuronal, nonneuroendocrine cell types were not successful. The expression of SNARE isoforms and their regulators has now been catalogued extensively in many types of secretory cells. It is apparent, therefore, that essentially the same core machinery with identical proteins (or closely related isoforms of them) functions in exocytosis in all cell types that have been examined, and these are likely to be universally essential proteins. A similar core machinery also functions in all other intracellular fusion events within the cell but without control by a sensor requiring elevated Ca²⁺ concentrations or changes in other signals (cAMP and activation of GTP-binding proteins). The universality of mechanisms underlying exocytosis has greatly simplified our thinking and means that these need not be studied in detail in all cell types, but future work can concentrate on the analysis of favored model secretory cells.

What are the key issues that remain to be resolved in the study of secretory granule exocytosis? It is still unclear how Ca²⁺ binding to synaptotagmin (if these are the key Ca²⁺ sensors) triggers the steps that lead to membrane fusion. In addition, the link between cAMP and activated GTP-binding proteins and the SNAREs, in those cells where Ca²⁺ is not the trigger, is unknown. Although we know the identity of many of the proteins (perhaps almost all of them) that act in exocytosis, it is still unclear what is the sequence of protein-protein interactions that occur in the steps leading to membrane fusion. We also do not understand how membrane fusion (i.e., bilayer mixing) is actually brought about and exactly which proteins are required during physiological membrane fusion. A final aspect that will certainly be the subject of future investigation is the analysis of the regulation of exocytosis (in extent and its kinetics), which may be cell type specific through the action of protein kinases and other regulators on the exocytotic machinery. It is clear then that despite considerable advances, much still remains to be learned and the field of regulated exocytosis will remain active for many more years.