Introduction: Regulated exocytosis

Calcium ions regulate secretory processes in several ways. Most prominently they (i) trigger the release of vesicle contents rapidly and in a highly cooperative way and they (ii) control priming steps, which prepare vesicles for release. The importance of using assays with high time resolution for separating these distinct roles is pointed out here.     

Regulated exocytosis per partes

This Special Issue (SI) of Cell Calcium focuses on regulated exocytosis, a recent evolutionary invention of eukaryotic cells. This essential cellular process consists of several stages: (i) the delivery of membrane bound vesicles to specific plasma membrane sites, (ii) where the merger between the vesicle and the plasma membranes occurs, (iii) leading to the formation of an aqueous channel through which vesicle content starts to be discharged to the cell exterior, (iv) after the full incorporation of the vesicle membrane into the plasma membrane, the added vesicle membrane is retrieved back into the cytoplasm by endocytosis. (v) When a fusion pore opens it may close again, a process known as transient fusion pore opening (also kiss-and-run exocytosis). In some cell types these stages are extremely shortlived, as in some neurons, and thus relatively inaccessible to experimentation. In other cell types the transition between these stages is orders of magnitude slower and can be studied in more detail. However, despite the intense investigations of this critical biological process over the last decades, the molecular mechanisms underlying regulated exocytosis have yet to be fully resolved. We thus still lack a comprehensive physiological insight into the nature of the progressive and coupled stages of exocytosis. Such a molecular-level understanding would help to fully reconstruct this process in vitro, as well as identify potential therapeutic targets for a range of diseases and dysfunctions. There are 18 papers in this SI which have been organized into three sections: Rapid regulated exocytosis and calcium homeostasis with an introduction by Erwin Neher, Molecular mechanisms of regulated exocytosis, and Cell models for regulated exocytosis. Here we briefly outline and integrate the messages of these sections.     

Regulated exocytosis: merging ideas on fusing membranes

Cellular trafficking pathways end with fusion reactions at the target. These reactions have been studied extensively for many decades, but recent studies have been particularly productive in providing new solutions to old problems, especially in some of the most complex fusion reactions, like synaptic vesicle secretion in neurons. Here, we discuss new studies that begin to merge ideas on three central questions: (A) are all releasable vesicles equally likely to undergo fusion, (B) do different fusion modes contribute to synaptic transmission, and (C) which molecular events are 'upstream' and which ones 'downstream' of SNARE complex assembly.     

Regulated exocytosis: novel insights from intravital microscopy

Regulated exocytosis is a fundamental process that every secretory cell uses to deliver molecules to the cell surface and the extracellular space by virtue of membranous carriers. This process has been extensively studied using various approaches such as biochemistry, electrophysiology and electron microscopy. However, recent developments in time-lapse light microscopy have made possible imaging individual exocytic events, hence, advancing our understanding of this process at a molecular level. In this review, we focus on intravital microscopy (IVM), a light microscopy-based approach that enables imaging subcellular structures in live animals, and discuss its recent application to study regulated exocytosis. IVM has revealed differences in regulation and modality of regulated exocytosis between in vitro and in vivo model systems, unraveled novel aspects of this process that can be appreciated only in in vivo settings and provided valuable and novel information on its molecular machinery. In conclusion, we make the case for IVM being a mature technique that can be used to investigate the molecular machinery of several intracellular events under physiological conditions.     

Regulated exocytosis: a novel,widely expressed system

Electrophysiological studies in some secretory and non-secretory cells have identified an extensive form of calcium-induced exocytosis that is rapid (hundreds of milliseconds), insensitive to tetanus toxin and distinct from regulated secretion. We have now identified a marker of the process, desmoyokin-AHNAK, in a clonal derivative of the neuronal cell line, PC12. In resting cells, desmoyokin-AHNAK is localized within the lumen of specific vesicles, but appears on the cell surface during stimulation. Desmoyokin-AHNAK-positive vesicles exist in a variety of cells and tissues and are distinct from the endoplasmic reticulum, Golgi, trans-Golgi, endosomes and lysosomes, and from Glut4 and constitutive secretion vesicles. They seem to be involved in two models of plasmalemma enlargement: differentiation and membrane repair. We therefore propose that these vesicles should be called 'enlargosomes'.     

Regulated exocytosis and SNARE function (Review)

The pairing of cognate v- and t-SNAREs between two opposing lipid bilayers drives spontaneous membrane fusion and confers specificity to intracellular membrane trafficking. These fusion events are regulated by a cascade of protein-protein interactions that locally control SNARE activity and complex assembly, determining when and where fusion occurs with high efficiency in vivo. This basic regulation occurs at all transport steps and is mediated by conserved protein families such as Rab proteins and their effectors and Sec1/unc18 proteins. Regulated exocytosis employs auxiliary components that couple the signal (which triggers exocytosis) to the fusion machinery. At the neuronal synapse, munc13 as well as munc18 control SNARE complex assembly. Synaptotagmin and complexin ensure fast synchronous calcium-evoked neurotransmitter release.     

Regulated ATP release from astrocytes through lysosome exocytosis

Release of ATP from astrocytes is required for Ca2+ wave propagation among astrocytes and for feedback modulation of synaptic functions. However, the mechanism of ATP release and the source of ATP in astrocytes are still not known. Here we show that incubation of astrocytes with FM dyes leads to selective labelling of lysosomes. Time-lapse confocal imaging of FM dye-labelled fluorescent puncta, together with extracellular quenching and total-internal-reflection fluorescence microscopy (TIRFM), demonstrated directly that extracellular ATP or glutamate induced partial exocytosis of lysosomes, whereas an ischaemic insult with potassium cyanide induced both partial and full exocytosis of these organelles. We found that lysosomes contain abundant ATP, which could be released in a stimulus-dependent manner. Selective lysis of lysosomes abolished both ATP release and Ca2+ wave propagation among astrocytes, implicating physiological and pathological functions of regulated lysosome exocytosis in these cells.     

Regulated exocytosis and SNARE function (Review)

The pairing of cognate v- and t-SNAREs between two opposing lipid bilayers drives spontaneous membrane fusion and confers specificity to intracellular membrane trafficking. These fusion events are regulated by a cascade of protein-protein interactions that locally control SNARE activity and complex assembly, determining when and where fusion occurs with high efficiency in vivo. This basic regulation occurs at all transport steps and is mediated by conserved protein families such as Rab proteins and their effectors and Sec1/unc18 proteins. Regulated exocytosis employs auxiliary components that couple the signal (which triggers exocytosis) to the fusion machinery. At the neuronal synapse, munc13 as well as munc18 control SNARE complex assembly. Synaptotagmin and complexin ensure fast synchronous calcium-evoked neurotransmitter release.     

Clostridial neurotoxins: new tools for dissecting exocytosis

Tetanus toxin and botulinal toxins are potent inhibitors of neuronal exocytosis. Within the past five years the protein sequences of all eight neurotoxins have been determined, their mode of action as metalloproteases has been established, and their intraneuronal targets have been identified. The toxins act by selectively proteolysing the synaptic vesicle protein synaptobrevin (VAMP) or the presynaptic membrane proteins syntaxin (HPC-1) and SNAP-25. These three proteins form the core of a complex that mediates fusion of carrier vesicles to target membranes. Tetanus and botulinal neurotoxins could serve in the future as tools to study membrane trafficking events, or even higher brain functions such as behaviour and learning.     

Regulated exocytosis of GABA-containing synaptic-like microvesicles in pancreatic beta-cells

We have explored whether gamma-aminobutyric acid (GABA) is released by regulated exocytosis of GABA-containing synaptic-like microvesicles (SLMVs) in insulin-releasing rat pancreatic beta-cells. To this end, beta-cells were engineered to express GABA(A)-receptor Cl(-)-channels at high density using adenoviral infection. Electron microscopy indicated that the average diameter of the SLMVs is 90 nm, that every beta-cell contains approximately 3,500 such vesicles, and that insulin-containing large dense core vesicles exclude GABA. Quantal release of GABA, seen as rapidly activating and deactivating Cl(-)-currents, was observed during membrane depolarizations from -70 mV to voltages beyond -40 mV or when Ca(2+) was dialysed into the cell interior. Depolarization-evoked GABA release was suppressed when Ca(2+) entry was inhibited using Cd(2+). Analysis of the kinetics of GABA release revealed that GABA-containing vesicles can be divided into a readily releasable pool and a reserve pool. Simultaneous measurements of GABA release and cell capacitance indicated that exocytosis of SLMVs contributes approximately 1% of the capacitance signal. Mathematical analysis of the release events suggests that every SLMV contains 0.36 amol of GABA. We conclude that there are two parallel pathways of exocytosis in pancreatic beta-cells and that release of GABA may accordingly be temporally and spatially separated from insulin secretion. This provides a basis for paracrine GABAergic signaling within the islet.     

Quantitative analysis of the distribution of organelles in tobacco pollen tubes: implications for exocytosis and endocytosis

Summary The present study provides the first quantitative analysis on the distribution of organelles in pollen tubes ofNicotiana tabacum L. Organelles were studied on living pollen tubes by means of fluorescence confocal laser scanning microscopy and on cryo-fixed, freeze-substituted and serially sectioned material by electron microscopy. In the tip a 300 nm to 400 nm thick wall was secreted that proximately gradually separated into a wall with an opaque inner side and a more translucent, layered outer side. Tubular endoplasmic reticulum was particularly abundant in the tip of the tube, surrounding the region where secretory vesicles (SV) accumulated. Mitochondria were randomly distributed throughout the cytoplasm, no accumulations were present. Dictyosomes, however, showed an increased abundance at 25–30 m behind the tip. The accumulation of coated pits (CP) in a zone 6–15 m behind the tip identifies this zone as the major site of endocytosis: 50% of all CP occur in this zone. Quantification of exo- and endocytosis showed that only part of the membrane material of the SV can be retrieved after exocytosis. The typical zonation in endocytotic activity may serve to maintain a difference in membrane protein composition between the tip and the tube.     

Regulated exocytosis in neuroendocrine cells: a role for subplasmalemmal Cdc42/N-WASP-induced actin filaments

In neuroendocrine cells, actin reorganization is a prerequisite for regulated exocytosis. Small GTPases, Rho proteins, represent potential candidates coupling actin dynamics to membrane trafficking events. We previously reported that Cdc42 plays an active role in regulated exocytosis in chromaffin cells. The aim of the present work was to dissect the molecular effector pathway integrating Cdc42 to the actin architecture required for the secretory reaction in neuroendocrine cells. Using PC12 cells as a secretory model, we show that Cdc42 is activated at the plasma membrane during exocytosis. Expression of the constitutively active Cdc42(L61) mutant increases the secretory response, recruits neural Wiskott-Aldrich syndrome protein (N-WASP), and enhances actin polymerization in the subplasmalemmal region. Moreover, expression of N-WASP stimulates secretion by a mechanism dependent on its ability to induce actin polymerization at the cell periphery. Finally, we observed that actin-related protein-2/3 (Arp2/3) is associated with secretory granules and that it accompanies granules to the docking sites at the plasma membrane upon cell activation. Our results demonstrate for the first time that secretagogue-evoked stimulation induces the sequential ordering of Cdc42, N-WASP, and Arp2/3 at the interface between granules and the plasma membrane, thereby providing an actin structure that makes the exocytotic machinery more efficient.     

Calcium-induced exocytosis from actomyosin-driven, motile varicosities formed by dynamic clusters of organelles

Varicosities are ubiquitous neuronal structures that appear as local swellings along neurites of invertebrate and vertebrate neurons. Surprisingly little is known about their cell biology. We use here cultured Aplysia neurons and demonstrate that varicosities are motile compartments that contain large clusters of organelles. The content of varicosities propagate along neurites within the plasma membrane “sleeve”, split and merge, or wobble in place. Confocal imaging, retrospective immunolabeling, electron microscopy and pharmacological perturbations reveal that the motility of the varicosities’ organelle content occurs in concert with an actin scaffold and is generated by actomyosin motors. Despite the motility of these organelle clusters within the cytoplasm along the neurites, elevation of the free intracellular calcium concentration within varicosities by trains of action potentials induces exocytosis followed by membrane retrieval. Our observations demonstrate that varicosities formed in the absence of postsynaptic cells behave as “ready to go” prefabricated presynaptic terminals. We suggest that the varicosities’ motility serves to increase the probability of encountering a postsynaptic cell and to rapidly form a functional synapse. Electronic Supplementary Material Supplementary material is available in the online version of this article at These authors contributed equally to the paper.     

Subnuclear organelles: new insights into form and function

The cell nucleus is a complex and highly dynamic environment with many functionally specialized regions of substructure that form and maintain themselves in the absence of membranes. Relatively little is known about the basic physical properties of the nuclear interior or how domains within the nucleus are structurally and functionally organized and interrelated. Here, we summarize recent data that shed light on the structural and functional properties of three prominent subnuclear organelles--nucleoli, Cajal bodies (CBs) and speckles. We discuss how these findings impact our understanding of the guiding principles of nuclear organization and various types of human disease.     

A novel membrane complex is required for docking and regulated exocytosis of lysosome-related organelles in Tetrahymena thermophila

In the ciliate Tetrahymena thermophila, lysosome-related organelles called mucocysts accumulate at the cell periphery where they secrete their contents in response to extracellular events, a phenomenon called regulated exocytosis. The molecular bases underlying regulated exocytosis have been extensively described in animals but it is not clear whether similar mechanisms exist in ciliates or their sister lineage, the Apicomplexan parasites, which together belong to the ecologically and medically important superphylum Alveolata. Beginning with a T. thermophila mutant in mucocyst exocytosis, we used a forward genetic approach to uncover MDL1 (Mucocyst Discharge with a LamG domain), a novel gene that is essential for regulated exocytosis of mucocysts. Mdl1p is a 40 kDa membrane glycoprotein that localizes to mucocysts, and specifically to a tip domain that contacts the plasma membrane when the mucocyst is docked. This sub-localization of Mdl1p, which occurs prior to docking, underscores a functional asymmetry in mucocysts that is strikingly similar to that of highly polarized secretory organelles in other Alveolates. A mis-sense mutation in the LamG domain results in mucocysts that dock but only undergo inefficient exocytosis. In contrast, complete knockout of MDL1 largely prevents mucocyst docking itself. Mdl1p is physically associated with 9 other proteins, all of them novel and largely restricted to Alveolates, and sedimentation analysis supports the idea that they form a large complex. Analysis of three other members of this putative complex, called MDD (for Mucocyst Docking and Discharge), shows that they also localize to mucocysts. Negative staining of purified MDD complexes revealed distinct particles with a central channel. Our results uncover a novel macromolecular complex whose subunits are conserved within alveolates but not in other lineages, that is essential for regulated exocytosis in T. thermophila.     

Regulated exocytosis contributes to protein kinase C potentiation of vanilloid receptor activity

The vanilloid receptor-1 (TRPV1) plays a key role in the perception of peripheral thermal and inflammatory pain. TRPV1 expression and channel activity are notably up-regulated by proalgesic agents. The transduction pathways involved in TRPV1 sensitization are still elusive. We have used a yeast two-hybrid screen to identify proteins that associate with the N terminus of TRPV1. We report that two vesicular proteins, Snapin and synaptotagmin IX (Syt IX), strongly interact in vitro and in vivo with the TRPV1 N-terminal domain. In primary dorsal root ganglion neurons, TRPV1 co-distributes in vesicles with Syt IX and the vesicular protein synaptobrevin. Neither Snapin nor Syt IX affected channel function, but they notably inhibited protein kinase C (PKC)-induced potentiation of TRPV1 channel activity with a potency that rivaled the blockade evoked by botulinum neurotoxin A, a potent blocker of neuronal exocytosis. Noteworthily, we found that PKC activation induced a rapid delivery of functional TRPV1 channels to the plasma membrane. Botulinum neurotoxin A blocked the TRPV1 membrane translocation induced by PKC that was activated with a phorbol ester or the metabotropic glutamate receptor mGluR5. Therefore, our results indicate that PKC signaling promotes at least in part the SNARE-dependent exocytosis of TRPV1 to the cell surface. Taken together, these findings imply that activity-dependent delivery of channels to the neuronal surface may contribute to the buildup and maintenance of thermal inflammatory hyperalgesia in peripheral nociceptor terminals.     

Cryobanking of viable biomaterials: implementation of new strategies for conservation purposes

Cryobanking, the freezing of biological specimens to maintain their integrity for a variety of anticipated and unanticipated uses, offers unique opportunities to advance the basic knowledge of biological systems and their evolution. Notably, cryobanking provides a crucial opportunity to support conservation efforts for endangered species. Historically, cryobanking has been developed mostly in response to human economic and medical needs — these needs must now be extended to biodiversity conservation. Reproduction technologies utilizing cryobanked gametes, embryos and somatic cells are already vital components of endangered species recovery efforts. Advances in modern biological research (e.g. stem cell research, genomics and proteomics) are already drawing heavily on cryobanked specimens, and future needs are anticipated to be immense. The challenges of developing and applying cryobanking for a broader diversity of species were addressed at an international conference held at Trier University (Germany) in June 2008. However, the magnitude of the potential benefits of cryobanking stood in stark contrast to the lack of substantial resources available for this area of strategic interest for biological science — and society at large. The meeting at Trier established a foundation for a strong global incentive to cryobank threatened species. The establishment of an Amphibian Ark cryobanking programme offers the first opportunity for global cooperation to achieve the cryobanking of the threatened species from an entire vertebrate class.     

Intravesicular calcium release mediates the motion and exocytosis of secretory organelles: a study with adrenal chromaffin cells

Secretory vesicles of sympathetic neurons and chromaffin granules maintain a pH gradient toward the cytosol (pH 5.5 versus 7.2) promoted by the V-ATPase activity. This gradient of pH is also responsible for the accumulation of amines and Ca2+ because their transporters use H+ as the counter ion. We have recently shown that alkalinization of secretory vesicles slowed down exocytosis, whereas acidification caused the opposite effect. In this paper, we measure the alkalinization of vesicular pH, caused by the V-ATPase inhibitor bafilomycin A1, by total internal reflection fluorescence microscopy in cells overexpressing the enhanced green fluorescent protein-labeled synaptobrevin (VAMP2-EGFP) protein. The disruption of the vesicular gradient of pH caused the leak of Ca2+, measured with fura-2. Fluorimetric measurements, using the dye Oregon green BAPTA-2, showed that bafilomycin directly released Ca2+ from freshly isolated vesicles. The Ca2+ released from vesicles to the cytosol dramatically increased the granule motion of chromaffin- or PC12-derived granules and triggered exocytosis (measured by amperometry). We conclude that the gradient of pH of secretory vesicles might be involved in the homeostatic regulation of cytosolic Ca2+ and in two of the major functions of secretory cells, vesicle motion and exocytosis.     

Roles of microfilaments in exocytosis: a new hypothesis

We observed the dynamic changes in the localization of microfilaments during the exocytic secretion of rat parotid and submandibular gland acinar cells, and obtained results which led us to propose a new concept of microfilament function in exocytosis. With the electron microscopy, NBD-Phallacidin (NBD-PL) fluorescence technique and immunohistochemistry for myosin, microfilaments consisting of F-actin and myosin were localized mainly underneath the luminal plasma membrane. Microfilaments were not detectable around the secretory granules which were stored in the cytoplasm, but were clearly observed around them whose membranes were continuous with the luminal plasma membrane. When viewed with NBD-PL and myosin fluorescence, the area of fused granule membranes revealed bright fluorescence in association with the luminal border, so that the luminal membrane undergoing exocytosis appeared like a 'bunch of grapes'. When excess exocytosis was stimulated by isoproterenol (IPR), the number of individual 'grapes' increased dramatically, indicating that the secretory granules are surrounded by microfilaments after the fusion with the luminal membrane. Microfilaments thus continuously undercoat the luminal membrane during exocytosis although the exocytic process involves the dilation and subsequent reduction of the luminal membrane due to the addition and removal of secretory granule membranes. This reduction of the dilated luminal membrane following exocytosis was, however, inhibited when the microfilaments were disrupted by cytochalasin D. Following this treatment, the lumina was expanded extraordinarily and the secretory products remained in the enlarged lumina, showing that the release of secretory products is inhibited when the microfilament function is disturbed. These results indicate that 1) microfilaments are localized mainly underneath the luminal plasma membrane and act as an obstacle to exocytosis when cells are at the resting phase and 2) at the secretory phase microfilaments allow exocytosis by disorganizing their barrier system and then, by encircling the discharged secretory granule membranes, provide forces for the extrusion of secretory products through the action of the acto-myosin contractile system.