MECHANISMS OF EXOCYTOSIS
Overview: Exocytosis Continues to Amaze Us Nina Vardjana,b and Robert Zorecb,a a
Celica Biomedical Center, LCI, Technology Park 24, 1000 Ljubljana, Slovenia b
Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloˇska 4, 1000 Ljubljana, Slovenia
organizational make-up. An important reason for this is that signaling and communication within the relatively large eukaryotic cell volume could no longer be supported mainly by diffusion-based processes; hence, the development of subcellular organelles represents a solution to the problem. The evolution of subcellular organelles played a further, particularly important, role in the development of eumetazoa (multicellular organisms). In this category of organisms, some types of subcellular organelles gained special function in rapid communication among cells. For example, in nucleated cells chemical messengers are stored in secretory organelles at high concentration. These signaling molecules get discharged swiftly and locally from membrane-bound vesicles following a trigger. The function of these secretory organelles is the focus of research, since they mediate a process that exhibits, at least in neurons, one of the fastest biological reactions known. Moreover, not only in specialized cells, but from the 200 cell types present in the human body, the majority of these perform exocytosis in their repertoire of functions. Therefore, exocytosis represents an important, yet unresolved, topic in cell biology, physiology, biophysics, biochemistry, and nanoengineering, among other disciplines. The understanding of this rather complex process is essential for the understanding of the normal function of unicellular and multicellular organisms in the animal and plant kingdoms and in pathological conditions as well.
This volume summarizes the proceedings of the international meeting entitled Mechanisms of Exocytosis 2008, held on May 22–25, 2008 at the Slovenian Academy of Sciences and Arts in Ljubljana, during the period when Slovenia held the Presidency of the EU Council. Leading scientists in the field addressed the physiological process of exocytosis, which consists of the merger between the vesicle and the plasma membranes. Despite several decades of intense investigation of this biological process, exocytosis continues to amaze us. The book is divided into three parts: (1) vesicle dynamics and cell types, (2) molecular mechanisms of exocytosis, and (3) intracellular messengers and exocytosis. Exocytosis is a universal process, an invention of eukaryotic cells. The defining membrane-bound structure that differentiates eukaryotic from prokaryotic cells is the nucleus. However, eukaryotic cells contain other membrane-bound organelles, such as mitochondria, chloroplasts, Golgi bodies, and secretory vesicles. When nucleated cells evolved from a prokaryotic precursor presumably by fusion of prokaryotic cells 1000 to 2000 million years ago, this process was associated with a cell volume increase of 3 to 4 orders in magnitude. The increased cell size dictated a new
Address for correspondence: Robert Zorec, Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, School of Medicine, Zaloˇska 4, 1001 Ljubljana, Slovenija. Voice: +386 (1) 543 70 80; fax: +386 (1) 543 70 36. [email protected]
Mechanisms of Exocytosis: Ann. N.Y. Acad. Sci. 1152: ix–xiii (2009). C 2009 New York Academy of Sciences. doi: 10.1111/j.1749-6632.2008.04321.x
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Figure 1. The number of papers published per year addressing exocytosis over the last 40 years.
During the last three decades the number of research papers published addressing this topic has rapidly increased. Since the term exocytosis was first mentioned in the PubMed list of journals, almost 14,000 papers have been published, averaging about 800 papers published every year (Figure 1). Vesicle Dynamics and Cell Types To release stored molecules, the membrane of the secretory organelle fuses with the plasma membrane, which leads to the formation of a fusion pore, a water-filled channel mediating the export of secretions. Given the critical role of compartmentalization in eukaryotic cells and that high-energy barriers are involved in the merger of biological membranes, it is perhaps not unexpected that the mechanism of membrane fusion appears to be highly conserved. Therefore, the use of different cell types and in vitro model systems enables a more direct analysis of the nature of the single secretory organelle fusion with the plasma membrane. In the first section several cell models are presented from animals and plants. Gerhard Thiel and colleagues describe that single vesicle fusion and fission events are observed in plant cells using the electrophysiological technique of monitoring membrane capacitance at high resolution. These fusion/fission
events appear rhythmically as previously observed in some animal cells, and the authors suggest that molecular mechanisms underlying exocytosis in plant and animal cells are universal. Robert Chow’s group describes experiments in chromaffin cells in which the mechanism of exocytosis is studied by optical techniques where single vesicles are labeled by the green fluorescent protein (GFP). In their study they demonstrate that the cell in which a construct is expressed also affects the kinetics of fluorescence change at exocytosis. Mateja Erdani Kreft and colleagues present an interesting review paper about the apical plasma membrane traffic in superficial urothelial cells. The authors discuss the role of exocytosis and endocytosis in the formation and maintenance of the urothelial permeability barrier and the clinical significance of the apical plasma membrane traffic in the urinary tract. The next cells presented in this volume are astrocytes, the most abundant glial cell type in the central nervous system. These cells are increasingly viewed as crucial cells supporting and integrating brain function. It is thought that the release of gliotransmitters into the extracellular space by regulated exocytosis supports a significant part of the communication between astrocytes and neurons. Marko Kreft and colleagues present a review of their own research on regulated exocytosis in astrocytes. The endocrine pituitary lactotroph, secreting the hormone prolactin, represents a useful cell model for studying exocytosis at the level of a single vesicle. Nina Vardjan and colleagues expand the previously published results on the transient and repetitive nature of the elementary exocytotic events in lactotrophs by specifically analyzing the extent of compound exocytosis in resting and stimulated endocrine cells. A very interesting cell type in which exocytosis is studied are type II pneumocytes, which secrete surfactant, a lipoprotein-like substance reducing the surface tension in the lung by regulated exocytosis of secretory vesicles, termed lamellar bodies (LBs). Paul Dietl’s group is studying these cells and describes in this volume that
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actin coating of LBs after fusion with the plasma membrane is Ca2+ dependent and is required for exocytotic content release. The authors suggest that actin coating creates a force needed for either extrusion of vesicle content or retrieval and intracellular propulsion. In the last chapter in this section, Marjan Rupnik’s group describes regulated exocytosis in insulin-secreting cells from the endocrine pancreas. In particular they address when an insulin-positive cell becomes functional in vivo and starts to exocytose insulin in a regulated, nutrient-dependent manner. Molecular Mechanisms of Exocytosis The chapters in the second part of this volume focus on the molecular mechanisms of exocytosis. Perhaps one of the most significant advances in the last two decades was the discovery that vesicular fusion is associated with a special class of proteins termed SNARE (soluble N -ethylmaleimide sensitive-factor attachment receptor), including syntaxin 1, SNAP25, and VAMP, which have been identified as mediating spontaneous and stimulated fusion of the vesicle and the plasma membrane.1 In addition to the SNARE proteins, there are others mediating the trigger. In excitable neurons the release of vesicle cargo is evoked by massive Ca2+ influx from the cell exterior and the subsequent binding of Ca2+ to the vesicle proteins synaptotagmin-1 or -2.2–4 Moreover, within the pathway leading to exocytosis, there is an essential requirement for a member of the conserved Sec1/Munc18 (SM) protein family, which in neurotransmitter and neurohormone release in mammalian cells is Munc18–1. Robert Burgoyne and colleagues review the functions of this protein. Current evidence suggests that Munc18–1 acts via distinct modes of interactions with syntaxin 1 and the other SNARE proteins and influences all of the steps leading to exocytosis, including vesicle recruitment, tethering, docking, priming, and membrane fusion.
Lori Feinshreiber and colleagues discuss the idea that K+ channels (here Kv2.1) can affect exoctyosis via a mechanism that is independent of the conductance of the protein. The chapter by Michela Matteoli’s group reviews the involvement of SNAP-25 in different neuropsychiatric and neurological disorders. Yves Dunant and colleagues consider rapid changes in presynaptic membrane structure affecting intramembrane particles linked to a proteolipid mediatophore, a special structure mediating the release of acetylcholine in a Ca2+ -dependent manner. They used a fast cryofixation method to observe these changes in fish, which are exposed to lower body temperatures than mammals. In the first part of their chaper, Vladimir Parpura and Wei Liu review the atomic force microscopy (AFM) techniques. Then they describe the use of AFM in studying SNARE protein interactions within the binary complexes of syntaxin 1A-synaptobrevin-2 and ternary complexes of syntaxin 1A-SNAP25-synaptobrevin2. Measurements of force and extension values for the dissociation of these complexes are described. In addition, calculations for spontaneous dissociation times and interaction energy for SNARE protein–protein interactions are presented. The paper by Jens Coorssen’s group provides a comprehensive overview of the role of proteins and lipids in the Ca2+ -triggered membrane fusion, which is the defining step of regulated exocytosis. They study membrane fusion on a sea urchin cortical vesicle model system. The focus of their discussions is on the contribution of cholesterol molecules to the localized negative curvature of the membrane. On the basis of studies on pituitary cells, the chapter of Robert Zorec’s group discusses the mechanisms regulating the discharge of vesicle cargo. In their overview they highlight recent evidence that shows that the fusion pore is subject to physiological regulation. Fusion pore kinetics and fusion pore conductance may affect vesicle release competence. Christina Bark addresses in her review two variants of SNARE protein SNAP25, SNAP25a and SNAP25b. Both
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variants are encoded by the same single copy gene and generated in the process of alternative splicing. They are differently expressed during development, and the reason for such strict regulation of expression still needs to be elucidated. In the paper the authors discuss recent studies with the SNAP25 gene–targeted mouse mutants and the possible physiological role of the two alternative SNAP25 variants. Edwin Kwan and Herbert Gaisano provide a balanced overview of the priming role of protein Munc-13 and other priming factors (Munc-18, tomosyn, and CAPS) in the exocytosis of insulin granules. The priming of insulin granules is an important step in the process of exocytosis in which already docked insulin granules become fusion competent. The authors also discuss the cAMP-mediated potentiation of insulin release in connection with Munc13 priming and the involvement of cAMP signaling in the compound fusion of insulin granules. Intracellular Messengers and Exocytosis The classical trigger of regulated exocytosis is an increase in cytosolic calcium activity. However, there are other second messengers that modulate the exocytotic response. In the third part, chapters address the role of several second messenger systems in the regulation of exocytosis. Marjan Rupnik’s group studied the role of phosphatidylinositol-4, 5bisphosphate (PI(4,5)P 2 ) in the priming of large dense-core vesicles in pituitary cells, specifically in the Ca2+ -dependent secretory activity. Using the whole-cell patch-clamp technique in pituitary tissue slices, the authors propose that PI(4,5)P 2 increases the size of the readily releasable vesicle pool by regulating the effectiveness of vesicular mobilization and fusion in an ATP-dependent manner. Stanko Stojilkovic and colleagues describe the role of G proteins in the stimulus-secretion coupling in pituitary cells. They show that G protein–mediated con-
trol of secretory activity affects calcium signaling and importantly also sites downstream of calcium/cAMP signaling, which involves G z protein expression. Matthias Braun, Patrik Rorsman, and colleagues present the exocytic properties of single pancreatic beta cells from humans. The secretory capacitance responses as well as insulin release from intact islets were strongly amplified by elevation of intracellular cAMP levels. Exocytosis was more dependent on Ca2+ -influx through P/Q-type than through L-type Ca2+ channels, reflecting the relative contribution of these channels to the total Ca2+ current. Exocytosis (as monitored by capacitance or amperometric measurements) decreased during repetitive stimulation, due to inactivation of Ca2+ channels as well as depletion of a readily releasable pool of granules. The results reveal both similarities and differences between human and rodent beta cells. Jacopo Meldolesi’s group studied the mechanism by which neurons and neurosecretory cells govern the expression and the exocytic discharge of their clear and dense-core vesicles (DCVs). The studies performed on the neurosecretory cell model, PC12 cells, revealed that these processes are orchestrated by the transcriptional repressor, NRSF/REST. Nicolas Vitale and colleagues discuss the role of fusogenic cone-shaped lipids, such as phosphatidic acid (PA) in membrane fusion during exocytosis. Phospholipase D (PLD) appears to be the main provider of PA at the exocytotic site in neuroendocrine cells. They show that ribosomal S6 kinase 2 (RSK2) stimulates PLD activity through the phosphorylation of Thr147 in the PLD1 amino-terminal Phox-homology domain. In chromaffin and PC12 cells, depletion of RSK2 dramatically prevents PA synthesis at exocytotic sites and inhibits hormone release. The authors suggest that RSK2 is a critical upstream signaling element in the activation of PLD1 to produce the lipids required for exocytosis. St´ephane Gasman and colleagues studied the role of actin cytoskeleton remodeling in
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regulated exocytosis in the late stages of exocytosis. They propose that the neuronal guanine nucleotide exchange factor, intersectin-1L, activates the GTPase Cdc42, which in turn provides de novo actin filaments that are important for calcium-regulated exocytosis in PC12 cells. Joˇze Pungerˇcar’s group presents results about ammodytoxin A (AtxA), a presynaptically neurotoxic secretory phospholipase A 2 from snake venom, with the aim of determining the mechanism of its cytotoxicity. They propose that the cytotoxic- and apoptosis-inducing effects of AtxA were specific for the motoneuronal cells. Future Directions In the last decades we have witnessed remarkable progress in the understanding of the molecular mechanisms underlying regulated vesicle-based release of hormones and neurotransmitters. However, we are still a distance away from understanding the nature of membrane merger at exocytosis. We need to solve many questions, specifically those related to the phenomena of priming, docking, and recruit-
ment of vesicles in the view of membrane fusion intermediates. Are some of these intermediates energetically stable? What is the interplay between lipids and proteins, such as SNAREs, in membrane merger? We need to understand the architecture of the fusion pore and the mechanisms that regulate fusion pore diameter (conductance) and fusion pore kinetics. Since exocytosis is a universal process of eukaryotic cells, which are exposed to different environments, it is of great value to study different model and cell systems, all to find answers to open questions, to solve the engineering riddle of how to reconstitute regulated exocytosis in vitro. References 1. Jahn, R. & R. Scheller. 2006. SNAREs—engines for membrane fusion. Nat. Rev. Mol. Cell Biol. 7: 631–643. 2. Geppert, M. et al. 1994. Synaptotagmin I: A major Ca2+ sensor for transmitter release at a central synapse. Cell 79: 717–727. 3. Kreft, M. et al. 2003. Synaptotagmin I increases the probability of vesicle fusion at low [Ca2+ ] in pituitary cells. Am. J. Physiol. Cell Physiol. 284: C547–554. 4. Pang, Z. et al. 2006. Genetic analysis of synaptotagmin 2 in spontaneous and Ca2+ -triggered neurotransmitter release. EMBO J. 25: 2039–2050.