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Two types of secretory vesicles co-exist at some presynaptic terminals. Clear synaptic vesicles (CSV) release their contents at the synaptic active zone, upon single impulses, while dense-core vesicles (DCV) usually release their contents in the periphery of the terminal upon repetitive stimulation. Part of the transmitter released by DCV diffuses to produce paracrine effects, and part of it reaches the postsynaptic terminal, adding its effect to that of synaptic release. This article presents an analytical method to separate the contribution of CSV and DCV to the postsynaptic responses, based on the kinetics of postsynaptic currents (PSCs). Since stimulation with single presynaptic impulses usually triggers release only from CSV, the kinetics of the resulting PSC can be used as a template to model the postsynaptic response to release from CSV during stimulation trains, accounting for the variations in the amplitude of PSCs due to short-term synaptic plasticity. Subtraction of this model simulation to the total recorded PSC renders the response to DCV peri‑synaptic release, which has slower kinetics. The method can be further simplified by measuring only the amplitudes of the PSC peaks for synaptic release and the integral of the current for peri‑synaptic release.•The postsynaptic current in response to presynaptic release from clear synaptic vesicles is modeled using the kinetics of the PSC in response to single impulses.•The model synaptic response is subtracted from the total recorded PSC to obtain the response to peri‑synaptic release from dense-core vesicles., Graphical abstract Image, graphical abstract

Secretory vesicle swelling has been demonstrated to be a requirement in cell secretion. In the past 25 years, the quest to elucidate the molecular mechanism of secretory vesicle swelling, serendipitously revealed the presence of water channels or aquaporins at the secretory vesicle membrane and their involvement in rapid gaiting of water into secretory vesicles and their swelling during secretion. These studies further provided an understanding of aquaporin regulation at the secretory vesicle membrane. Secretory vesicles within live cells, isolated secretory vesicles, single vesicle electrophysiological patch clamp studies and the swelling complex reconstituted into liposomes, have all been utilized to elucidate the mechanism and regulation of secretory vesicle swelling. Results from these studies collectively demonstrate the involvement of β-adrenergic receptor, heterotrimeric GTP-binding G-proteins such as GαI; PLA2 and potassium and chloride channels, in the regulation of aquaporins at the secretory vesicle membrane.

Programmed cell death (PCD) plays a critical role in the development of plant pigment glands, while H2O2, which is a kind of reactive oxygen species (ROS) produced by the aerobic metabolism of cells, acts as an important signal in this process. Here, we investigated the temporal and spatial dynamics of accumulated H2O2 in pigment glands of Gossypium hirsutum L. with 3,3-diaminobenzidine (DAB) staining, 2',7'-dichlorodihydrofluorescein diacetate (DCFH2)-DA fluorescent labeling and CeCl3 cytochemical localization techniques. The results showed that the pigment glands of G. hirsutum could generate H2O2, and the amount and localization of H2O2 varied at different developmental stages. At the early developmental stage, a small amount of H2O2 accumulated in the vacuole membrane of pigment gland cells. At the intermediate stage, a large number of H2O2 appeared in the vacuole membrane, while cell walls started to accumulate a small amount of H2O2. When pigment gland cell degraded, H2O2 mainly accumulated on the chloroplast envelope membrane of inner sheath cells. With the degradation of the sheath cells, H2O2 was detected in cell wall and the membrane of secretory vesicles which contains the preliminary contents of pigment gland. With the pigment glands completely maturation, H2O2 would disappeared. The accumulation sites of H2O2 are consistent with the process of PCD of individual gland cells, which started from the degradation of intracellular membrane and ended with the degradation of cell walls. Thus H2O2 probably plays an important role in the development of pigment glands. In addition, the development of pigment glands and the generation of H2O2 are not associated with the light, and no H2O2 was detected in the secretions of pigment glands.

Control of cell volume and osmolarity is central to cellular homeostasis in all eukaryotes. It lies at the heart of the century-old problem of how plants regulate turgor, mineral and water transport. Plants use strongly electrogenic H+-ATPases, and the substantial membrane voltages they foster, to drive solute accumulation and generate turgor pressure for cell expansion. Vesicle traffic adds membrane surface and contributes to wall remodelling as the cell grows. Although a balance between vesicle traffic and ion transport is essential for cell turgor and volume control, the mechanisms coordinating these processes have remained obscure. Recent discoveries have now uncovered interactions between conserved subsets of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins that drive the final steps in secretory vesicle traffic and ion channels that mediate in inorganic solute uptake. These findings establish the core of molecular links, previously unanticipated, that coordinate cellular homeostasis and cell expansion., Trends Vesicle trafficking (SNARE) proteins ‘commandeer’ the voltage sensor domains of Kv channels to confer a voltage dependence on secretory traffic for coordination with ion transport during cell expansion. Sec1/Munc18 (SM) protein-mediated regulation of secretion is selective among plasma membrane SNAREs. SM proteins and Kv channels bind the SNARE SYP121 at overlapping sites, implying a sequential interplay between these proteins to coordinate membrane traffic and transport.

The large GTPase dynamin is well known for its actions on budded cellular membranes to generate vesicles, most often, clathrin-coated endocytic vesicles. The scope of cellular processes in which dynamin-mediated vesicle formation occurs, has expanded to include secretory vesicle formation at the Golgi, from other endosomes and nonclathrin structures, such as caveolae, as well as membrane remodeling during exocytosis and vesicle fusion. An intriguing new facet of dynamin's sphere of influence is the cytoskeleton. Cytoskeletal filament networks maintain cell shape, provide cell movement, execute cell division and orchestrate vesicle trafficking. Recent evidence supports the hypothesis that dynamin influences actin filaments and microtubules via mechanisms that are independent of its membrane-remodeling activities. This chapter discusses this emerging evidence and considers possible mechanisms of action.

The cortical cytoskeleton is a dense network of filamentous actin (F-actin) that participates in the events associated with secretion from neuroendocrine cells. This filamentous web traps secretory vesicles, acting as a retention system that blocks the access of vesicles to secretory sites during the resting state, and it mediates their active directional transport during stimulation. The changes in the cortical cytoskeleton that drive this functional transformation have been well documented, particularly in cultured chromaffin cells. At the biochemical level, alterations in F-actin are governed by the activity of molecular motors like myosins II and V and by other calcium-dependent proteins that influence the polymerization and cross-linking of F-actin structures. In addition to modulating vesicle transport, the F-actin cortical network and its associated motor proteins also influence the late phases of the secretory process, including membrane fusion and the release of active substances through the exocytotic fusion pore. Here, we discuss the potential interactions between the F-actin cortical web and proteins such as SNAREs during secretion. We also discuss the role of the cytoskeleton in organizing the molecular elements required to sustain regulated exocytosis, forming a molecular structure that foments the efficient release of neurotransmitters and hormones.

In multicellular organisms, signaling is a necessity and an important mode of communication between cells is mediated by neurotransmitters, hormones, and other chemical messengers that are stored in secretory vesicles. In stimulated conditions secretory vesicles, which are trafficked to be docked at the plasma membrane, enter exocytosis, characterized by vesicle and plasma membrane merger. Due to repulsive forces of negatively charged membrane surfaces, it was long believed that the fusion pore is merely a short lived intermediate state leading irreversibly to a complete merger of both membranes. However, recent results show that the fusion pore is a rather stable structure, which can reversibly reopen to subnanometer diameters; dimensions too narrow to permit the exit of the cargo into the extracellular space. The aim of this chapter is to first review how can such a structure attain stability and compare two models in which membrane constituents are either isotropic or anisotropic in nature. Then we address the molecular nature of such a stable, release-unproductive fusion pore. We conclude that membrane constituents of the stable fusion pore membrane, being made of proteins and/or lipids, very likely consist of architectural elements that exhibit anisotropicity. The dynamics of fusion pore diameter is then determined by the density and architectural properties of these membrane constituents at fusion pore locales.

9 páginas, 9 figuras.-- El pdf del artículo es la versión manuscrita de autor., Secretory vesicles have low pH and have been classically identified as those labelled by a series of acidic fluorescent dyes such as acridine orange or neutral red, which accumulate into the vesicles according to the pH gradient. More recently, several fusion proteins containing enhanced green fluorescent protein (EGFP) and targeted to the secretory vesicles have been engineered. Both targeted fluorescent proteins and acidic dyes have been used, separately or combined, to monitor the dynamics of secretory vesicle movements and their fusion with the plasma membrane. We have now investigated in detail the degree of colocalization of both types of probes using several fusion proteins targeted to the vesicles (synaptobrevin2-EGFP, Cromogranin A-EGFP and neuropeptide Y-EGFP) and several acidic dyes (acridine orange, neutral red and lysotracker red) in chromaffin cells, PC12 cells and GH3 cells. We find that all the acidic dyes labelled the same population of vesicles. However, that population was largely different from the one labelled by the targeted proteins, with very little colocalization among them, in all the cell types studied. Our data show that the vesicles containing the proteins more characteristic of the secretory vesicles are not labelled by the acidic dyes, and vice versa. Peptide glycyl-L-phenylalanine 2-naphthylamide (GPN) produced a rapid and selective disruption of the vesicles labelled by acidic dyes, suggesting that they could be mainly lysosomes. Therefore, these labelling techniques distinguish two clearly different sets of acidic vesicles in neuroendocrine cells. This finding should be taken into account whenever vesicle dynamics is studied using these techniques., This work was supported by grants from Ministerio de Educación y Ciencia (BFU2008-01871) and from Junta de Castilla y León (VA103A08 and GR105). J.S. holds an FPI (Formación de Personal Investigador) fellowship from the Spanish Ministerio de Educación y Ciencia.

Kiss-and-run refers to a prevailing hypothesis on the way in which secretory vesicle fusion and retrieval are coupled. According to the kiss-and-run hypothesis, following fusion with the plasma membrane secretory vesicles retain their curvature and release chemical substances through a restricted fusion pore. During this process, proteins originally resident within the vesicle membrane do not laterally diffuse onto the plasma membrane, thus allowing vesicles to preserve their identity during endocytosis. In this way, vesicles can be rapidly retrieved following fusion in a form of endocytosis that does not require vesicle reassembly. A key aspect of this model is the potential for postfusion regulation of chemical substance diffusion from the vesicle lumen through regulation of fusion pore opening duration and diameter. Kiss-and-run-type exocytosis–endocytosis coupling has been observed in multiple secretory systems that include mast cells, adrenal chromaffin cells, and pancreatic zymogen granules; however, the prevalence of this pathway for synaptic vesicle fusion and neurotransmitter release is a matter of intense debate.

Polarised growth in fungi occurs through the delivery of secretory vesicles along tracks formed by cytoskeletal elements to specific sites on the cell surface where they dock with a multiprotein structure called the exocyst before fusing with the plasmamembrane. The budding yeast, Saccharomyces cerevisiae has provided a useful model to investigate the mechanisms involved and their control. Cortical markers, provided by bud site selection pathways during budding, the septin ring during cytokinesis or the stimulation of the pheromone response receptors during mating, act through upstream signalling pathways to localise Cdc24, the GEF for the rho family GTPase, Cdc42. Cdc42 in its GTP-bound activates a multiprotein protein complex called the polarisome which nucleates actin cables along which the secretory vesicles are transported to the cell surface. Hyphae can elongate at a rate orders of magnitude faster than the extension of a yeast bud, so understanding hyphal growth will require substantial modification of the yeast paradigm. The rapid rate of hyphal growth is driven by a structure called the Spitzenkorper, located just behind the growing tip and which is rich in secretory vesicles. It is thought that secretory vesicles are delivered to the apical region where they accumulate in the Spitzenkorper. The Spitzenkorper then acts as vesicle supply centre in which vesicles exit the Spitzenkorper in all directions, but because of its proximity, the tip receives a greater concentration of vesicles per unit area than subapical regions. There are no obvious equivalents to the bud site selection pathway to provide a spatial landmark for polarised growth in hyphae. However, an emerging model is the way that the site of polarised growth in the fission yeast, Schizosaccharomyces pombe, is marked by delivery of the kelch repeat protein, Tea1, along microtubules. The relationship of the Spitzenkorper to the polarisome and the mechanisms that promote its formation are key questions that form the focus of current research.

In the past 15 years, the dynamics of intracellular membrane-bound secretory vesicles ranging in size from 200 to 1200 nm in pancreatic acinar cells to 30-50 nm in neurons, have been extensively studied, providing for the first time the molecular process involved in vesicular discharge during cell secretion. Live pancreatic acinar cells in near physiological buffer, when imaged using the atomic force microscope (AFM), reveal at nanometer resolution the size of secretory vesicles called zymogen granules (ZGs) lying immediately below the surface of the apical plasma membrane. Within 2.5 min of exposure to a secretory stimulus, majority of ZGs within cells swell, followed by a decrease in ZG size, and a concomitant release of secretory products. These studies directly demonstrated intracellular swelling of secretory vesicles following stimulation of cell secretion in live cells, and vesicle deflation following partial discharge of vesicular contents. Furthermore, a direct estimation of vesicle size dynamics at nm resolution under various experimental conditions, have enabled the determination of the molecular mechanism of secretory vesicle swelling. Atomic force microscopy and photon correlation spectroscopy have been major players in these studies.

The membrane maturation (flow differentiation) model of Golgi apparatus function embodies concepts of saccule formation at one face of the Golgi apparatus from membranes derived from endoplasmic reticulum and utilization of saccules in vesicle formation at the opposite face for delivery to the plasma membrane as existing saccules are displaced from one position within the stack to another. Derivation of the model came almost entirely from light and electron microscopy. Especially important were observations that passage through the Golgi apparatus was accompanied by differentiation of membranes from endoplasmic reticulum-like to plasma membrane-like across the polarity axis of the stacked saccules. The concept of coparticipation of endoplasmic reticulum and/or nuclear envelope, transition, and secretory vesicles and other pre- and post-Golgi apparatus structures through the operation of an integrated endomembrane system was essential to the model. Dynamic aspects confirmed initially by autoradiographing and cell fractionation studies have been corroborated in newer approaches of fluorescent labeling and with living cells.

The elaborate intracellular membrane system of eukaryotic cells participates in vesicle trafficking and represents an important basis exploited in cell-to-cell signaling. Communication between cells involves the release of neurotransmitters, hormones and other chemical messengers that are stored in secretory vesicles and granules. A key event in the release of these primary messengers is exocytosis, consisting of fusion between the vesicle and the plasma membrane. This leads to the formation of a fusion pore through which a diffusional continuum between the vesicle lumen and the extracellular space is established. In the past, in vitro studies of biological membrane fusion considered this an almost impossible process, because large pressures had to be delivered to counteract the electrostatic repulsion owing to negatively charged membrane surfaces. It is only a decade or so that the omnipresent fusion between biological membranes started to be understood in greater detail. Since the SNARE hypothesis was proposed about a decade ago, several proteins have been identified to play a role in exocytosis, and attempts to define minimal molecular machinery for regulated exocytosis have been considered. However, several studies provided evidence for multiple modes of exocytosis, and that exocytosis may not necessarily lead to the release of vesicle cargo. The aim of this chapter is to review the results obtained on pituitary cells, specialized to release a number of important hormones and to highlight that there are multiple mechanisms of exocytosis present in the same cell. Moreover, the goal is to address elementary properties of exocytosis, consisting of the interaction between a single vesicle and the plasma membrane. These studies indicate that the long-thought concept of membrane fusion as an irreversible process will have to be changed. Here we discuss an unusually regular reversible opening of the fusion pore termed “the pulsing pore”.

This chapter discusses the current knowledge of vesicle proteins in neuroendocrine tumors and the practical use of antibodies against vesicle proteins in the diagnosis of neuroendocrine tumors of the gastrointestinal (GI) tract. Neuroendocrine tumors of the GI tract are rare. Most neuroendocrine tumors occur as sporadic lesions, but 5%-20% of the tumors occur in a clinical setting of inherited neoplastic syndrome. Neuroendocrine tumors of the GI tract are thought to originate from the “diffuse neuroendocrine cell system” of the GI mucosa, as the neuroendocrine tumors have morphologic and functional similarities with the cells of it. Neurons and neuroendocrine cells contain two types of secretory vesicles that are crucial for the regulated secretory process and represent cell-specific compartments of these cells. Peptide hormones and neuropeptides are mainly contained within the secretory granules or large dense core vesicles (LDCV). Synaptic vesicles (SV) are present in the majority of neurons, neuroendocrine cells, and neuroendocrine tumors. Antibodies against SV proteins are powerful tools for the study of neuroendocrine differentiation in tumor tissue. New proteins, such as SV2, vesicular monoamine transporter (VMAT) 1 and 2, and neuroendocrine secretory protein 55 (NESP55) are also discussed in this chapter.

Rab3 and Rab27 GTPases control late events in the secretory pathway of mammalian cells including docking and fusion of secretory vesicles with the plasma membrane. The action of Rab3 and Rab27 on the exocytotic process is exerted through the activation of specific effectors. Several proteins with the capacity to interact in a GTP‐dependent manner with Rab3 and Rab27 have been identified. However, for most of these potential Rab effectors a precise function in the secretory process has not yet been attributed. In this chapter we describe a series of approaches that can be applied to assess the properties of Rab3 and Rab27 effectors and their potential role in the regulation of dense core granule release.

This article discusses the molecular biological, cell biological, and biochemical processes concerned with the production of peptide hormones. These begin with the transcriptional readout of the cognate chromosomal genes into mRNAs, which are then translated into protein precursors in specialized neuroendocrine cells. These initial products of translation (preproteins or preproproteins) are then directed through a series of specialized subcellular compartments where they undergo assisted folding and structural organization, as well as addition of carbohydrate side chains and other selected modifications, followed by their transfer into immature dense-core secretory vesicles where they may be cleaved by specialized proteases and then, in some instances, amidated or N-acetylated to yield the mature, biologically active hormones. These are then stored in the mature secretory granules within glandular cells until they are released via exocytosis in response to appropriate stimuli.

Results achieved in the molecular biology of Paramecium have shed new light on its elaborate membrane trafficking system. Paramecium disposes not only of the standard routes (endoplasmic reticulum → Golgi → lysosomes or secretory vesicles; endo- and phagosomes → lysosomes⧸digesting vacuoles), but also of some unique features, e.g. and elaborate phagocytic route with the cytoproct and membrane recycling to the cytopharynx, as well as the osmoregulatory system with multiple membrane fusion sites. Exocytosis sites for trichocysts (dense-core secretory vesicles), parasomal sacs (coated pits), and terminal cisternae (early endosomes) display additional regularly arranged predetermined fusion⧸fission sites, which now can be discussed on a molecular basis. Considering the regular, repetitive arrangements of membrane components, availability of mutants for complementation studies, sensitivity to gene silencing, and so on, Paramecium continues to be a valuable model system for analyzing membrane interactions. This review intends to set a new baseline for ongoing work along these lines.

This chapter coveres stimulus-secretion coupling as it applies to secretagogue stimulation of calcium influx and cyclic AMP elevation leading to calcium sensor activation, protein phosphorylation, and ultimately secretory vesicle fusion with the plasma membrane. The modes of secretagogue signaling have been discussed. These include depolarization via sodium channel activation leading to VSCC opening and calcium influx; modulation of outwardly directed potassium channels leading to altered calcium influx; directfirst-messenger stimulation of VSCC opening; cyclic AMP stimulation of secretory granule exocytosis via interactions with components of the calcium pathway; and cooperative roles of intracellular calcium mobilization and calcium influx in exocytosis. Stimulus-secretion-synthesis coupUng was discussed to illustrate that secretogogue-based regulation involves control of the export economy of the secretory cell, including both the quantity and quality of the neurosecretory quantum of the SSV, the endocrine hormone cocktail of the LDCV, and the zymogen mixture of the exocrine secretory granule. In the future, secretion is likely to be viewed within a more unitary framework for neuronal, endocrine, and exocrine secretion, but with a better molecular understanding of the diversity among cell types and various types of exocytosed cargo.

Pheochromocytoma-derived cell lines such as PC12 cells maintain a differentiated neuroendocrine phenotype and have been widely used as a convenient model system for a wide variety of cell biological studies on neurotrophin action, monoamine biogenesis, protein trafficking, and secretory vesicle dynamics. This chapter reviews a number of methods that are useful for studies of the regulated dense core vesicle secretory pathway. This includes protocols for maintaining cells and preserving their phenotype. A variety of assays are discussed for monitoring secretion in intact or permeable cells and in transfected cells. Specific methods for immunocytochemical studies in permeable cells are discussed. Finally, protocols for high-efficiency PC12 cell transfections and the isolation of stably transfected cell lines are provided.

Publisher Summary This chapter discusses excitation-secretion coupling. The chapter begins with an introduction to the exocytosis or reverse pinocytosis process. It is mentioned that cytoskeleton and Ca2+ ion are the most important players involved in excitation-secretion coupling. Synthesis occurs in the rough endoplasmic reticulum (RER) and sorting occurs in the Golgi complex. The first step in the secretory process for peptide hormones and neurotransmitters involves synthesis, modification, and sorting of the molecules to be secreted. The discussion of physical events associated with the fusion of vesicles to plasma membrane includes capacitance jump, capacitance flickering, fusion pore, membrane tension as a driving force for fusion, and fusion steps. Exocytosis is an all-or-none phenomenon in which Ca2+ plays a pivotal role. Interaction of cytoskeletal structures with transmembrane signaling is an important feature of excitation-secretion coupling. Actin-binding proteins are in large part responsible for cytoskeleton-receptor interactions. The process of secretion is mediated by contractile elements either associated with secretory vesicles or present elsewhere in the cell. Upon stimulation fodrin rearranges into patches beneath the plasma membrane. Scinderin is a cytosolic protein that shortens actin filament length when Ca2+ is present in the medium. Docking of granules on the plasma membrane is an important step in exocytosis. Priming of the granules is also a pivotal step in exocytosis. In regulated exocytosis, fusion of secretory granules with the plasma membrane is triggered by an appropriate signal. Freeze-fracture images of exocytosis reveal the presence of narrow pores formed between the granules and the plasma membrane called fusion pores. Control of exocytosis occurs not only in electrically excitable cells, but also in nonexcitable systems in response to receptor activation. The regulatory pathways that couple stimulation and secretion vary widely among cell types.

An egg-that took weeks to months to make in the adult-can be extraordinarily transformed within minutes during its fertilization. This review will focus on the molecular biology of the specialized secretory vesicles of fertilization, the cortical granules. We will discuss their role in the fertilization process, their contents, how they are made, and the molecular mechanisms that regulate their secretion at fertilization. This population of secretory vesicles has inherent interest for our understanding of the fertilization process. In addition, they have import because they enhance our understanding of the basic processes of secretory vesicle construction and regulation, since oocytes across species utilize this vesicle type. Here, we examine diverse animals in a comparative approach to help us understand how these vesicles function throughout phylogeny and to establish conserved themes of function.

Publisher Summary The genetic and molecular genetic accessibility of Tetrahymena makes possible a wide set of approaches that are unavailable in mammalian cells. The classical pathway for protein secretion begins with translocation of newly synthesized proteins across the endoplasmic reticulum (ER) membrane into the lumen. Such proteins are then transported through a series of membrane-bounded compartments, terminating in secretory vesicles that fuse with the plasma membrane of the cell, releasing the protein to the cell exterior. This process is also required for cell growth, because secretory vesicles provide the membrane that allows for expansion of the cell surface. A single cell may, however, have multiple types of secretory vesicles. A major distinction can be made between vesicles that fuse constitutively with the plasma membrane and vesicles whose fusion is triggered by an extracellular event. Because different vesicles can contain distinct protein constituents as a result of selective sorting, a cell with several vesicle types can modulate its secretory behavior based on environmental conditions.

Publisher Summary In eukaryotic cells, the transport of secretory proteins from the endoplasmic reticulum (ER) to the plasma membrane (PM) is orchestrated by a number of vesicular transport steps. For each of these steps carrier vesicles bud from topologically separate compartments of the secretory pathway and specifically fuse with the membrane of the subsequent compartment. Biochemical approaches largely based on in vitro reconstitution assays led to the discovery of molecules of involved in the budding and fusion reactions along this pathway. Morphological studies on fixed cells provided snapshots of the underlying membranous structures. The availability of the green fluorescent protein (GFP) opened the door for real-time studies and made it possible to monitor distinct vesicular transport steps of the secretory pathway in real time. Transport from the ER to the Golgi was studied using the vesicular stomatitis virus (VSV) G protein tagged with GFP. This chapter focuses on the transport of proteins from the trans-Golgi network (TGN) to the PM. This transport is either constitutive, if TGN-derived secretory vesicles fuse continuously with the PM, or regulated, if the vesicles fuse only in response to a stimulus. The regulated pathway is a special feature of certain cell types such as exocrine, endocrine, and neuronal cells, whereas the constitutive pathway is common to all eukaryotic cells. This chapter describes the methods for monitoring constitutive and regulated protein secretion in living cells, using hCgB tagged with GFP.

The recent deciphering of the protein composition of the synaptic vesicle membrane has led to the unexpected identification of a compartment of electron-lucent microvesicles in neuroendocrine cells which resemble neuronal synaptic vesicles in terms of molecular structure and function. These vesicles are generally referred to as synaptic-like microvesicles (SLMVs) and have been most intensively studied in pancreatic β-cells, chromaffin cells of the adrenal medulla, and pinealocytes of the pineal gland. This chapter focuses on the present knowledge of SLMVs as now well-established constituents of mammalian pinealocytes. I review the results of morphological, immunocytochemical, and biochemical studies that were important for the characterization of this novel population of secretory vesicles in the pineal organ. The emerging concept that SLMVs serve as a device for intercellular communication within the pineal gland is outlined, and unanswered questions such as those pertaining to the physiological function and regulation of pineal SLMVs are discussed.

Publisher Summary Active transport of monoamines by vesicular monoamine transporter (VMAT1) and VMAT2 is performed in permeabilized fibroblasts transiently expressing these proteins by the recombinant T7 vaccinia virus system. Active transport of acetylcholine (ACh) by human vesicular ACh transporter (VAChT) is performed in postnuclear homogenates containing secretory vesicles from stably transfected neuroendocrine PC-12 cells. Neurotransmission depends on the regulated release of transmitter molecules, such as the biogenic amines and ACh. This requires the packaging of these molecules into the specialized secretory vesicles of neurons and neuroendocrine cells, a process mediated by specific vesicular transporters. Active transport of neurotransmitters into secretory organelles requires the presence of a transmembrane H + electrochemical gradient, established and maintained by a vacuolar-type H + -ATPase and a vesicular transporter molecule, which catalyzes the exchange of H + ions for neurotransmitter. A characteristic feature of the VMATs is their broad range of substrate specificity. The chapter describes the development of active transport and binding assay in permeabilized cells.

Publisher Summary This chapter describes the expression of the three mouse multidrug resistance (mdr) gene products in secretory vesicles (SVs) from sec 6-4 mutant strains. The major advantage of SVs over traditionally prepared vesicle fractions from plasma membranes is that SV preparations of high purity can be easily obtained in good yields. In addition, SVs remain fully intact during the isolation procedure and are uniform with respect to their origin, size, and polarity. The chapter describes two different functional assays to demonstrate the utility of these SVs to characterize membrane transport proteins. The first group of functional assay includes drug uptake assays in Mdr-expressing SVs used to show that P-glycoprotein (Pgps) actively mediate drug transport and to clarify several controversial aspects of the underlying mechanisms of Pgp action. The second functional assay is a lipid flippase assay designed to test whether Pgps, in particular Mdr2, are actively translocating phosphatidylcholine within a membrane bilayer and, thus, function as phospholipid flippases.

Excitation-secretion uncoupling peptides (ESUPs) are inhibitors of Ca2+-dependent exocytosis in neural and endocrine cells. Their mechanism of action, however, remains elusive. We report that ESUP-A, a 20-mer peptide patterned after the C terminus of SNAP-25 (synaptosomal associated protein of 25 kDa) and containing the cleavage sequence for botulinum neurotoxin A (BoNT A), abrogates the slow, ATP-dependent component of the exocytotic pathway, without affecting the fast, ATP-independent, Ca2+-mediated fusion event. Ultrastructural analysis indicates that ESUP-A induces a drastic accumulation of dense-core vesicles near the plasma membrane, mimicking the effect of BoNT A. Together, these findings argue in favor of the notion that ESUP-A inhibits ATP-primed exocytosis by blocking vesicle docking. Identification of blocking peptides which mimic sequences that bind to complementary partner domains on interacting proteins of the exocytotic machinery provides new pharmacological tools to dissect the molecular and mechanistic details of neurosecretion. Our findings may assist in developing ESUPs as substitute drugs to BoNTs for the treatment of spasmodic disorders., This work was supported by grants from the Spanish Direccion General de Investigacion Cientifica y Tecnica PM-0110 (to S. V.), Grant DAMD 17-93-C-3100 from the U. S. Army Medical Research and Development Command (to M. M.), and a Postdoctoral Fellowship from the Dystonia Medical Research Foundation (to J. M. C.).

Publisher Summary The functional polarity of axons is reflected in the polarized distribution of a number of proteins, both membrane and cytoskeletal. Targeting to each domain of the neuron may require a specific signal; alternatively, targeting to only one domain may be specific, while localization to the other may occur by default. In addition to sorting to the proper domain, proteins in neurons must also be sorted to the correct subcellular organelle. For example, synaptic vesicle proteins synthesized in the endoplasmic reticulum (ER) and posttranslationally modified in the Golgi must first be targeted to the axon and then to the nerve terminal. A number of studies suggest that synaptic vesicle proteins are finally assembled into mature synaptic vesicles at the synapse; consequently, synaptic vesicle proteins may contain multiple targeting signals to reach their final destination. Research has been going on investigating the molecular basis for targeting of membrane proteins in neurons and to synaptic vesicles in two different systems. Using dissociated cultures of rat embryonic hippocampal neurons, the mechanism for sorting of proteins to dendrites or axons is examined. In the pheochromocytoma cell line PC12, the sequences that are necessary to target an exogenous protein to synaptic vesicles is being determined. It is found that the final stage of sorting to synaptic vesicles in neurons is mediated by similar processes as in PC12 cells. To determine this, chimeras are made between cDNAs encoding the synaptic vesicle protein SV2 and the glucose transporter GLUT1, in which the cytoplasmic loop between TM6 and TM7 is replaced by the cytoplasmic loop of SV2. The distribution of the chimeras have also been analyzied on sucrose gradients designed to separate large dense-core vesicles from endosomes, synaptic vesicles, and other membranes to determine if the same targeting information is used for both types of regulated secretory vesicles.

Summary The lipid bilayer membranes that separate cells and organelles undergo fusion to permit the exchange or compartmentalization of macromolecules. The fusion events involve contact between cytoplasmic or between extracytoplasmic membrane surfaces. These two types of fusion may depend on very different mechanisms because of the different environments of the membrane surfaces. The membrane fusion process can be broken down into the following steps: (1) translocation, the positioning of membranes adjacent to one another, (2) recognition, initial specific interaction between the appropriate fusion partners, (3) attachment, a close physical sealing between the two bilayers, (4) disruption, alteration of bilayer structure to create an aqueous passageway through the membranes, (5) reorganization, the formation of new bilayer(s) from the disrupted membrane components. Any or all of these steps may be mediated by specific proteins and be subject to regulation. The secretory pathway includes a number of steps that involve membrane fusion: pinching off and fusing of transport vesicles in the endoplasmic reticulum and the Golgi pinching off of secretory vesicles from the Golgi; fusion of secretory vesicles with the plasma membrane during simple exocytosis or with one another during compound exocytosis, and pinching off of endocytic vesicles to recycle secretory vesicle membrane. The molecular basis of these events has been studied using several important model systems that can be subjected to biochemical or genetic analyses. These include the constitutive secretory pathway of yeast, the regulated secretory pathway of the endocrine chromaffin cell, and the action of Clostridial neurotoxins on synaptic transmission. A group of yeast genes (the SEC genes) have been identified that mediate several steps in the secretory pathway. These genes have mammalian counterparts that may play similar roles. The annexins are a group of calcium-dependent, lipid-binding proteins that might mediate the binding of the secretory vesicle to the plasma membrane during regulated secretion. Protein components of the secretory vesicle or plasma membrane that may function in exocytosis are targets for the proteolytic activities of neurotoxins that block exocytosis. These membrane proteins include synaptobrevin, syntaxin, and SNAP25. Genetic analysis of lower eukaryotes indicates another secretory vesicle membrane protein, synaptotagmin, may provide regulation of the exocytosis complex of proteins in response to changes in the intracellular calcium concentration. These newly described protein mediators of membrane fusion in exocytosis are promising targets for the development of novel pharmacological agents to manipulate hormone or neurotransmitter release.

Publisher Summary Membrane fusion processes in cells underlie the structural integrity of the cytoplasm and are crucial to directing synthesis and export of enzymes, hormones, and transmitters. Export is accomplished by a process of exocytosis in which the membranes of secretory vesicles fuse with the plasma membrane. The fusion mechanism may involve a class of calcium-binding proteins termed “annexins.” This chapter discusses the annexin hypothesis for exocytosis. It focuses on function and molecular biology synexin (annexin VII) in secretory cells. Results from several laboratories have also shown that annexins I, II, and VII aggregate chromaffin granules and liposomes in vitro , and that annexin VII drives fusion of granule ghosts and liposomes. These data lend support to the hypothesis that these annexins may be secretory factors in vivo . The above mutagenesis experiments revealed the importance in annexin I of repeat one for aggregation activity and of repeats 2, 3, and 4 for calcium-binding sites. As all annexins bind to phospholipids in a calcium-dependent manner, membranes continue to be a focus for understanding annexin activity. Annexin II was detected in postpartum mammary epithelial cells, when calcium-dependent casein secretion starts and immunogold labeling of annexin VII in chromaffin cells was shown to decrease following treatment with a secretagogue. In addition, annexin II has been reported to insert into artificial membranes in the presence of calcium and to produce voltage-dependent channels.

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Publisher Summary This chapter discusses recent advances in the understanding of how proinsulin and other precursor proteins are processed into their biologically active products. The newly-formed secretory vesicles derived from the golgi complex appear to be the main site of precursor processing in both β cells and peptidergic neurones. The insulin-producing β cells are highly specialized secretory cells that share many properties with other neuroendocrine cells and have a number of additional specialized features attributable to their unique chemosensory functions. The chapter proposes to isolate two complementary DNAs (cDNAs) from a human insulinoma and mouse pituitary AtT20 cells that encode serine proteases (PC2 and PC3) having catalytic domains homologous to those of the bacterial substilisins and Kex2, a yeast proα-factor processing endoprotease. These enzymes appear to be designed to function in an acidic environment enriched in calcium ions as is believed to exist in maturing neuroendocrine secretory vesicles. Available evidence suggests that PC2 and PC3 as well as furin, a trans Golgi associated Kex2-like protease, are members of a hitherto unrecognized and ancient superfamily of subtilisin-related proteinases in eukaryotic cells that are involved in the intracellular processing of precursor proteins.

Publisher Summary This chapter provides an overview of the processes of biosynthesis, routing and release of neurochemical messengers in two representative peptidergic systems, one of neuronal, the other of endocrine signature: the caudodorsal cell neurons (CDC) in the central nervous system of the freshwater snail Lymnaea stagnalis and the endocrine melanotrope cells in the pituitary pars intermedia of the clawed toad Xenopus laevis . The data presented in the chapter for the CDC and the melanotrope cells strongly indicate that neuronal and endocrine cell systems have many characteristics in common with respect to their input and output messengers, such as cells of both systems are able to produce more than one type of messenger, of which, the peptides are most conspicuous. Before the peptides are released, various intracellular differentiating processes may take place, such as differential storage of peptides into secretory vesicles, differential acetylation during posttranslational processing, activated final processing as a result of cellular activation, and release of different peptides from different cellular sites via different release mechanisms. The targets of these cells may be complex as well, as they may include neurons, gland cells, endocrine cells, and muscles.

Publisher Summary The cell-free system described reconstitutes the formation of ISGs and CSVs from the TGN, including the sorting events that occur during these processes. The cell-free formed ISGs and CSVs are indistinguishable from those formed in vivo by several criteria, and TGN markers are retained in the TGN during the reaction. To study the formation of CSVs and ISGs from the TGN and the sorting events occurring, these processes have been reconstituted in a cell-free system. This chapter focuses on the experimental details of this cell-free system. The outline of the experimental approach includes (1) Pulse-labeling of intact cells with radioactive sulfate to label marker proteins for the regulated and constitutive secretory pathway selectively in the TGN, (2) preparation of a postnuclear supernatant from the labeled cells, (3) incubation of the postnuclear supernatant under conditions that allow the formation of ISGs and CSVs containing the labeled marker proteins, (4) Separation of ISGs and CSVs from the TGN, and (5) Separation of ISGs and CSVs. In addition, the chapter describes possible modifications of the standard protocol that may be introduced to investigate the mechanism of formation of ISGs and CSVs in the cell-free system.

Publisher Summary This chapter discusses the subcellular distribution of neuropeptides and measurement of their in vitro release. The selection of a method of subcellular fractionation depends on the goals of the research. Subcellular fractionation has been utilized in many experiments. Some examples include defining which subcellular compartment contains what subcellular components, providing an enriched source of a particular substance to simplify its purification, providing a cell-free system to study cellular function, and following intracellular passage of materials. Subcellular fractionation has been used to define what subcellular fractions are enriched in particular neuropeptides and to make a preparation of purified secretory granules as a starting material for purification of putative proneuropeptide-processing enzymes. For both applications, discontinuous sucrose density gradients have been employed. Sucrose has been used traditionally. It is inexpensive and easy to remove by dilution and centrifugation. Some other density media that have been employed include metrizamide, Ficoll, Nycodenz, and Percoli. Continuous density gradients are frequently employed to separate synaptic or secretory vesicles from other subcellular fractions. They are more precise and yield purer subcellular fractions; however, they require more equipment for making and collecting the gradients, and the neuropeptide-containing organelles are spread out over a number of fractions.

Publisher Summary This chapter discusses the techniques used for analysis of polypeptide transit through yeast secretory pathway. Two simple operations, differential centrifugation of membrane fractions and radiolabeling of biosynthetic intermediates are discussed in the chapter to permit a preliminary study of the localization pathway employed for a typical secretory protein. For differential fractionation, certain organelles, such as the nucleus, plasma membrane, vacuole, mitochondria, secretory vesicles, and peroxisomes, can be isolated from appropriate swains. In spite of the lack of experience in achieving complete membrane fractionation, it is usually possible to assign a subcellular location and pathway through the use of monospecific antibody directed against a protein of interest. First, a simple scheme for membrane fractionation is presented that may be used to achieve a preliminary assessment of the location of a protein. Virtually every yeast membrane and luminal space can now be detected by immunoblot of fractions using monospecific antibodies directed against organelle-specific marker proteins. The technique is sensitive, quantifiable, and not dependent on retention of marker enzyme activity. Most applications for immunoblotting are not quantitative. To give reliable data that allow antigen yield and purification to be assessed, antibody must be in excess and the secondary detecting agent 125 I-labeled protein A or horseradish peroxidase secondary antibody must be measured over a linear range of exposure.

A detailed ultrastructural investigation of the assembly, intracellular transport, and secretion of chylomicrons by rat proximal jejunal intestinal cells was performed in rats fed corn oil. Following fat feeding the smooth endoplasmic reticulum of the absorptive cells becomes laden with triglyceride droplets which are transported through channels of the endoplasmic reticulum to the Golgi apparatus. The Golgi zones become extremely prominent due to the accumulation of osmiophilic droplets, similar in size and configuration to chylomicrons, within proliferated Golgi vesicles. Golgi-derived secretory vesicles, containing nascent chylomicrons, migrate towards the lateral cell membrane. The secretory vesicle membranes fuse with the lateral plasmalemma and nascent chylomicrons are then discharged into the intercellular spaces. Alterations of specific domains of the secretory vesicles were prominent, appearing as coated pits. Coated pits were apparent in the lateral plasmalemma in areas of active chylomicron exocytosis suggesting their derivation from secretory vesicle-membrane fusion. Chylomicrons, within the intercellular spaces, pass through the basement membrane that lines the basal surfaces of the epithelial cells, traverse the cellular elements of the lamina propria, and finally gain access to the lymphatics entering these channels through gaps between adjacent endothelial cells. These observations indicate that nascent chylomicrons accumulate within Golgi vesicles as a pre-requisite to secretion and that secretion occurs by exocytosis resulting in the release of nascent chylomicrons from secretory vesicles.

Publisher Summary The apical plasma membrane of secretory cells undergoes considerable transformations during release of the products of secretion. Secretory vesicles derived from Golgi cisternae contact and fuse with the apical plasma membrane with formation of a small bilayer diaphragm, which separates the vacuolar content from the acinar lumen of the gland. Rupture of this transient diaphragm results in the release of the secretory products and incorporation of the secretory vesicle membrane in the apical segment of the plasma membrane. This process has been investigated in different types of cells. In the lactating cells of the mammary alveolus, the spectrum of transformations observed at the apical surface is wider. In addition to the release of proteins and other secretory products by exocytosis, other events occur at the apical membrane during the secretion of fat droplets. Milk fat droplets are released into the alveolar lumen surrounded by a membrane envelope derived from the cell membrane. The process results in the exportation of plasma membrane, which can be easily isolated from milk. This membrane system represents a useful model to study the dynamics of the apical surface of a secretory cell, as well as other membrane events of wider biological significance. The observation of lactating tissues with freeze fracture techniques provides imaging of the process and allows the analysis of the structural rearrangements of these membranes.

Publisher Summary This chapter describes a new protein called synexin that induces Ca 2+ -dependent formation of pentalaminar complexes among secretory granule membranes. Synexin has proved distinct from other more conventional candidates for Ca 2+ -effect mediators, such as calmodulin, actomyosin, and tubulin. The chapter also describes the process of membrane breakage or fission that finally results in the release of secretory vesicle contents. Studies comparing the granule lysis reaction with predicted behavior of secreting chromaffin and other cells are briefly reviewed in the chapter. The biochemical data supporting the exocytosis hypothesis are that the storage organelles from different cells can be isolated and therefore shown to be discrete objects and when secretion occurs, the entire organelle contents ranging from small molecules to large proteins can be found in the extracellular medium. Calcium plays a critical role in regulating exocytosis in a number of systems.

Adrenal medullary tissue contains a soluble protein of M.W. 47,000 which specifically binds Ca2+ and, in the presence of Ca2+, induces the fusion of the outer leaflets of chromaffin granule membranes. We have named this protein synexin, from the Greek word “synexis” which means “a meeting”. We suggest synexin may be an intracellular receptor for Ca2+ in the process of exocytosis, acting to promote fusion between secretory vesicles and the plasma membrane, or, in the case of compound exocytosis, between vesicles.

Publisher Summary This chapter discusses hormone secretion by exocytosis with an emphasis on information from the chromaffin cell system. Exocytosis is the general mechanism by which many hormones, transmitters, enzymes, and other special proteins and peptides are secreted from cells. Exocytosis means that the chemical species to be secreted is stored in a secretory vesicle, which, upon the appropriate stimulus, fuses with the cell membrane and deposits the vesicle contents outside the cell. The “handmaiden” of this process is usually calcium. In many cases, the ultimate signal for exocytosis seems to be an increase in the intracellular free calcium ion concentration. For many reasons, the chromaffin cell is the very best vantage point from which to examine the hormone-secretion process. From a biochemical viewpoint, the chromaffin cell has no peer, subcellular fractionation is simple, and the cells are easily cultured. Chromaffin cells in the adrenal medulla are joined and electrically coupled into cords of cells surrounded in the intact gland by an abundant fenestrated capillary bed.

Publisher Summary This chapter discusses gastrointestinal digestion and lipid absorption. It also discusses the various experimental models proposed to study the various aspects of the complex processes involved in the absorption and transport of dietary lipids. The chapter presents the role of microtubules. The importance of microtubules and microfilaments in intestinal lipid absorption has been based on ultrastructural studies and drug studies utilizing agents that are known to interfere with microtubular function. Ultrastructural studies have repeatedly demonstrated the presence of microtubules and microfilaments in the proximity of intracellular structures assumed to be important in the formation of chylomicrons. One theory proposed is that the microtubular system may direct secretory vesicles to specific recognition sites on the cell membrane. The other evidence for the role of microtubules in lipid absorption has been derived from investigations using either colchicine or vincristine, two known inhibitors of microtubular assembly.

Publisher Summary This chapter presents two subcellular-fractionation schemes. One, based on a protocol described by Ruohola and Ferro-Novick, is an organelle separation procedure that gives sufficient resolution to allow the localization of proteins to particular organelles of the secretory pathway. The other scheme is designed for the purification of post-Golgi secretory vesicles from Saccharumyces cerevisiae . For the localization of proteins to particular organelles, the ability to lyse cells osmotically is an important improvement over the glass bead lysis procedure. The shear forces generated during glass bead lysis could potentially remove proteins from the surface of organelles that would otherwise be membrane-attached, causing them to appear soluble. Similarly, because the conditions required for stabilizing the association of a protein with a membrane can be quite variable depending on the lysis buffer, confirmation of localization using alternative schemes is prudent.

Publisher Summary This chapter describes the purification and handling of α-toxin and streptolysin O (SLO) for the poration of cells. In contrast to other permeabilizing procedures, the pores inserted into the plasma membrane with the aid of bacterial toxins are stabilized by a proteinaceous ring like structure. Depending on the aim of an experiment, the cells can be made permeable either for large or for small molecules by selection of a suitable bacterial pore-forming protein. This allows permanent access to the cells' interior to investigate intracellular processes as diverse as fusion of secretory vesicles with the inner surface of the cell membrane, contraction, metabolism of hormones, fluxes of ions, or glucose metabolism. Besides the different secretory cells, poration by α-toxin or SLO was carried out with hepatocytes, rat basophilic leukemia cells, fibroblasts, and smooth muscle cells. This indicates that the novel approach of permeabilization of cells by channel-forming toxins has already become a widely used tool for the investigation of a variety of intracellular processes in situ .

Highly purified secretory vesicles from adrenal medulla, isolated by differential and density gradient centrifugation using isotonic gradient material (Percoll™), contain acetylcholinesterase. The enzyme was latent in isolated secretory vesicles i.e. acetylcholinesterase was inaccessible to added substrate. The enzyme activity became patent after addition of detergent or in hypotonic media. Hypotonic treatment or specific lysis of the vesicles with Mg 2+ /ATP in the presence of a permeant anion resulted in the release of soluble acetylcholinesterase from the vesicular content. Membrane-bound enzyme sedimented with the membranes. Binding of α-bungarotoxin could only be observed when secretory vesicles were lyzed. It is concluded, that the acetylcholine receptor as well as the membrane-bound form a acetylcholinesterase are localized on the inner surface of the secretory vesicle membrane, which becomes the outer surface of the cell membrane during exocytosis. Concomitantly the soluble form of acetylcholinesterase present within secretory vesicles is released into the extracellular fluid.