Steps Of Exocytosis Research Articles

An extended model of vesicle fusion at the plasma membrane to estimate protein lateral diffusion from TIRF microscopy images

BackgroundCharacterizing membrane dynamics is a key issue to understand cell exchanges with the extra-cellular medium. Total internal reflection fluorescence microscopy (TIRFM) is well suited to focus on the late steps of exocytosis at the plasma membrane. However, it is still a challenging task to quantify (lateral) diffusion and estimate local dynamics of proteins.ResultsA new model was introduced to represent the behavior of cargo transmembrane proteins during the vesicle fusion to the plasma membrane at the end of the exocytosis process. Two biophysical parameters, the diffusion coefficient and the release rate parameter, are automatically estimated from TIRFM image sequences, to account for both the lateral diffusion of molecules at the membrane and the continuous release of the proteins from the vesicle to the plasma membrane. Quantitative evaluation on 300 realistic computer-generated image sequences demonstrated the efficiency and accuracy of the method. The application of our method on 16 real TIRFM image sequences additionally revealed differences in the dynamic behavior of Transferrin Receptor (TfR) and Langerin proteins.ConclusionAn automated method has been designed to simultaneously estimate the diffusion coefficient and the release rate for each individual vesicle fusion event at the plasma membrane in TIRFM image sequences. It can be exploited for further deciphering cell membrane dynamics.

The Trypanosome Exocyst: A Conserved Structure Revealing a New Role in Endocytosis.

Membrane transport is an essential component of pathogenesis for most infectious organisms. In African trypanosomes, transport to and from the plasma membrane is closely coupled to immune evasion and antigenic variation. In mammals and fungi an octameric exocyst complex mediates late steps in exocytosis, but comparative genomics suggested that trypanosomes retain only six canonical subunits, implying mechanistic divergence. We directly determined the composition of the Trypanosoma brucei exocyst by affinity isolation and demonstrate that the parasite complex is nonameric, retaining all eight canonical subunits (albeit highly divergent at the sequence level) plus a novel essential subunit, Exo99. Exo99 and Sec15 knockdowns have remarkably similar phenotypes in terms of viability and impact on morphology and trafficking pathways. Significantly, both Sec15 and Exo99 have a clear function in endocytosis, and global proteomic analysis indicates an important role in maintaining the surface proteome. Taken together these data indicate additional exocyst functions in trypanosomes, which likely include endocytosis, recycling and control of surface composition. Knockdowns in HeLa cells suggest that the role in endocytosis is shared with metazoan cells. We conclude that, whilst the trypanosome exocyst has novel components, overall functionality appears conserved, and suggest that the unique subunit may provide therapeutic opportunities.

Mroh1, a lysosomal regulator localized by WASH-generated actin.

ABSTRACTThe steps leading to constitutive exocytosis are poorly understood. In Dictyostelium WASH complex mutants, exocytosis is blocked, so cells that take up fluorescent dextran from the medium retain it and remain fluorescent. Here, we establish a FACS-based method to select cells that retain fluorescent dextran, allowing identification of mutants with disrupted exocytosis. Screening a pool of random mutants identified members of the WASH complex, as expected, and multiple mutants in the conserved HEAT-repeat-containing protein Mroh1. In mroh1 mutants, endosomes develop normally until the stage where lysosomes neutralize to postlysosomes, but thereafter the WASH complex is recycled inefficiently, and subsequent exocytosis is substantially delayed. Mroh1 protein localizes to lysosomes in mammalian and Dictyostelium cells. In Dictyostelium, it accumulates on lysosomes as they mature and is removed, together with the WASH complex, shortly before the postlysosomes are exocytosed. WASH-generated F-actin is required for correct subcellular localization; in WASH complex mutants, and immediately after latrunculin treatment, Mroh1 relocalizes from the cytoplasm to small vesicles. Thus, Mroh1 is involved in a late and hitherto undefined actin-dependent step in exocytosis.

7th ISN special neurochemistry conference 'Synaptic function and dysfunction in brain diseases'.

This article is part of a mini review series: "Synaptic Function and Dysfunction in Brain Diseases".

Lipids implicated in the journey of a secretory granule: from biogenesis to fusion.

The regulated secretory pathway begins with the formation of secretory granules by budding from the Golgi apparatus and ends by their fusion with the plasma membrane leading to the release of their content into the extracellular space, generally following a rise in cytosolic calcium. Generation of these membrane-bound transport carriers can be classified into three steps: (i) cargo sorting that segregates the cargo from resident proteins of the Golgi apparatus, (ii) membrane budding that encloses the cargo and depends on the creation of appropriate membrane curvature, and (iii) membrane fission events allowing the nascent carrier to separate from the donor membrane. These secretory vesicles then mature as they are actively transported along microtubules toward the cortical actin network at the cell periphery. The final stage known as regulated exocytosis involves the docking and the priming of the mature granules, necessary for merging of vesicular and plasma membranes, and the subsequent partial or total release of the secretory vesicle content. Here, we review the latest evidence detailing the functional roles played by lipids during secretory granule biogenesis, recruitment, and exocytosis steps. In this review, we highlight evidence supporting the notion that lipids play important functions in secretory vesicle biogenesis, maturation, recruitment, and membrane fusion steps. These effects include regulating various protein distribution and activity, but also directly modulating membrane topology. The challenges ahead to understand the pleiotropic functions of lipids in a secretory granule's journey are also discussed. This article is part of a mini review series on Chromaffin cells (ISCCB Meeting, 2015).

More Docked Vesicles and Larger Active Zones at Basket Cell-to-Granule Cell Synapses in a Rat Model of Temporal Lobe Epilepsy.

One in 26 people develops epilepsy, and temporal lobe epilepsy is a common form. Up to one-third of patients are resistant to currently available treatments. This study tested a potential underlying mechanism for previously reported impaired inhibition in epileptic animals at basket cell-to-granule cell (BC→GC) synapses, which normally are reliable and strong. Electron microscopy was used to evaluate 3D ultrastructure of BC→GC synapses in a rat model of temporal lobe epilepsy. The hypothesis was that impaired synaptic transmission is attributable to smaller boutons, smaller synapses, and abnormally low numbers of synaptic vesicles. Results revealed the opposite. These findings suggest that impaired transmission at BC→GC synapses in epileptic rats is attributable to later steps in exocytosis following vesicle docking.

The Role of Phospholipase D in Regulated Exocytosis

There are a diversity of interpretations concerning the possible roles of phospholipase D and its biologically active product phosphatidic acid in the late, Ca(2+)-triggered steps of regulated exocytosis. To quantitatively address functional and molecular aspects of the involvement of phospholipase D-derived phosphatidic acid in regulated exocytosis, we used an array of phospholipase D inhibitors for ex vivo and in vitro treatments of sea urchin eggs and isolated cortices and cortical vesicles, respectively, to study late steps of exocytosis, including docking/priming and fusion. The experiments with fluorescent phosphatidylcholine reveal a low level of phospholipase D activity associated with cortical vesicles but a significantly higher activity on the plasma membrane. The effects of phospholipase D activity and its product phosphatidic acid on the Ca(2+) sensitivity and rate of fusion correlate with modulatory upstream roles in docking and priming rather than to direct effects on fusion per se.

Glutamate Release.

Our aim was to review the processes of glutamate release from both biochemical and neurophysiological points of view. A large body of evidence now indicates that glutamate is specifically accumulated into synaptic vesicles, which provides strong support for the concept that glutamate is released from synaptic vesicles and is the major excitatory neurotransmitter. Evidence suggests the notion that synaptic vesicles, in order to sustain the neurotransmitter pool of glutamate, are endowed with an efficient mechanism for vesicular filling of glutamate. Glutamate-loaded vesicles undergo removal of Synapsin I by CaM kinase II-mediated phosphorylation, transforming to the release-ready pool. Vesicle docking to and fusion with the presynaptic plasma membrane are thought to be mediated by the SNARE complex. The Ca(2+)-dependent step in exocytosis is proposed to be mediated by synaptotagmin.

Novel intracellular mediator of adiponectin secretion from adipocytes.

Mature white fat cells (adipocytes) are unusual terminally differentiated cells because of their large spherical shape, the fact that their presence is not fixed at a particular location in the body, the fact that they can proliferate in various ways and the weird intracellular structures that permit a unilocular lipid droplet to occupy a large part of the intracellular space, allowing only a small crescent-shaped cytoplasmic space in which all cellular events occur (Sugihara et al. 1987). Since the verification of the hypothesis that insulin stimulates the translocation of glucose transporter (GLUT4) vesicles from intracellular storage to the plasma membrane in isolated rat adipocytes, thus increasing the cellular uptake of glucose (Cushman & Wardzala, 1980; Suzuki & Kono, 1980), the mechanism(s) of vesicular trafficking in fat cells have been the subject of many research studies. Over 10 years later, the discoveries of a number of biologically active cytokines derived from adipocytes, generally named adipokines, have changed the viewpoint, with adipocytes now being considered an endocrine organ rather than a simple energy storage organ. Of the various adipokines, adiponectin has attracted the most studies because of its protective roles against obesity-related diseases, including type-2 diabetes and cardiovascular diseases, and the reports that the reduction in the plasma level of adiponectin in obese subjects precedes the reduction in insulin sensitivity and onset of diabetes (Trujillo & Scherer, 2005; Smith & Minson, 2012; Yamauchi & Kadowaki, 2013). Most of the studies concerning adiponectin secretion have therefore been focused on the long-term chronic regulation, whereas the short-term (≤30 min) regulation of adipocyte exocytosis by intracellular mediators is not well understood. In this issue of The Journal of Physiology, Komai et al. (2014) describe a novel mechanism underlying the short-term regulation of adiponectin secretion, in 3T3-L1 adipocytes derived from mice, using a combination of membrane capacitance patch-clamp recordings and measurements of secreted adiponectin. The mechanism regulating adiponectin secretion is fundamentally different from that occurring in other endocrine cells. The authors demonstrate that the final step of adiponectin secretion in mature adipocytes, the release of adipokines from vesicles fused with the plasma membrane, is stimulated by cAMP and an exchange protein activated by cAMP (Epac), without depending on Ca2+ or protein kinase A (PKA). Biochemical measurements showed that adiponectin is the dominant adipokine secreted in a cAMP/Epac-dependent manner, whereas the amounts of other secreted adipokines, such as leptin, resistin and apelin, are very low. Interestingly, Ca2+ was shown to have no apparent effect on the final step of adipokine release, although most neurotransmitter release is considered to be Ca2+ dependent. However, the role of Ca2+ is important in the early steps of exocytosis when the secretion vesicles are recruited and refilled with adiponectin to form ready-to-release vesicles. The regulation of the entire process of adiponectin secretion in adipocytes is thus proposed to be a sequential regulation by Ca2+ and cAMP, and ATP may provide energy and substrates for the phosphorylation of proteins that play a role in exocytosis. The findings thus indicate that all three intracellular mediators, Ca2+, ATP and cAMP, are required to accomplish the short-term secretion of adiponectin, through Ca2+ and ATP, and cAMP regulates different steps of the exocytosis. In human subcutaneous adipocytes, the adiponectin secretion was also shown to be cAMP-dependent, similar to that observed in 3T3-L1 adipocytes. There is increasing evidence that obesity is associated with not only metabolic syndrome and atherosclerosis, but also non-alcoholic steatohepatitis (NASH), cancer and Alzheimer's disease, and adiponectin plays a pleiotropic role in protecting against such diseases (Smith & Minson, 2012; Yamauchi & Kadowaki, 2013). Adiponectin is thus expected to be a valuable target molecule for therapeutics supporting a healthy, long life in humans. The key findings that each step of the short-term regulation of adiponectin secretion and the packaging of vesicles is individually regulated by cAMP, Ca2+ and ATP would provide a pin-point therapeutic approach for obesity-related diseases. In white adipocytes, the binding of insulin to the receptor located on the plasma membrane is a trigger for a number of dynamic cellular events, including translocation of GLUT4 vesicles, the secretion of adiponectin and other adipokines, which are controlled in different ways. It is amazing that these complex mechanisms are well organized in the small crescent-shaped cytoplasmic space of the fat cells.

There's been a Flaw in Our Thinking.

There's been a Flaw in Our Thinking.

Dynamic process of phagocytosis and forms of macrophage cell death induced by ingestion of apoptotic neutrophils.

Clearance of apoptotic neutrophils by macrophages is important for both the successful resolution of acute inflammation and homeostasis. However, the dynamic process of phagocytosis of apoptotic neutrophils by macrophages and the fate of macrophages after the ingestion of apoptotic neutrophils has not been well documented. In the present study, we staged the recognition and tethering, internalization, digestion and exocytosis steps of phagocytosis of apoptotic neutrophils. Furthermore, we found that after the ingestion of apoptotic cells, a subset of macrophages underwent cell death by autophagy, apoptosis or oncosis as revealed by transmission electron microscopy and confocal microscopy combined with specific dyes. The percentage of autophagic, apoptotic and oncotic macrophages were 8.00%±2.00%, 12.33%±2.08%, and 3.66%±1.50%, respectively. These results indicated that after ingestion of apoptotic neutrophils, a subset of macrophages undergoes autophagy and apoptosis. We propose that autophagy of macrophages after the ingestion of apoptotic cells may be a new mechanism present in the resolution of inflammation.

Distinct fusion properties of synaptotagmin-1 and synaptotagmin-7 bearing dense core granules.

Adrenal chromaffin cells release hormones and neuropeptides that are essential for physiological homeostasis. During this process, secretory granules fuse with the plasma membrane and deliver their cargo to the extracellular space. It was once believed that fusion was the final regulated step in exocytosis, resulting in uniform and total release of granule cargo. Recent evidence argues for nonuniform outcomes after fusion, in which cargo is released with variable kinetics and selectivity. The goal of this study was to identify factors that contribute to the different outcomes, with a focus on the Ca(2+)-sensing synaptotagmin (Syt) proteins. Two Syt isoforms are expressed in chromaffin cells: Syt-1 and Syt-7. We find that overexpressed and endogenous Syt isoforms are usually sorted to separate secretory granules and are differentially activated by depolarizing stimuli. In addition, overexpressed Syt-1 and Syt-7 impose distinct effects on fusion pore expansion and granule cargo release. Syt-7 pores usually fail to expand (or reseal), slowing the dispersal of lumenal cargo proteins and granule membrane proteins. On the other hand, Syt-1 diffuses from fusion sites and promotes the release of lumenal cargo proteins. These findings suggest one way in which chromaffin cells may regulate cargo release is via differential activation of synaptotagmin isoforms.

Renin release: role of SNAREs.

Little is known about the molecular mechanism mediating renin granule exocytosis and the identity of proteins involved. We previously showed that soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNAREs), a family of proteins required for exocytosis, mediate the stimulated release of renin from juxtaglomerular cells. This minireview focuses on the current knowledge of the proteins that facilitate renin-granule exocytosis. We discuss the identity of potential candidates that mediate the signaling and final steps of exocytosis of the renin granule.

The regulated secretory pathway in neuroendocrine cells.

The regulated secretory pathway shared by excitable cells, including neurons and neuroendocrine cells is an intricate process that comprises multiple, tightly regulated steps. After their synthesis in the endoplasmic reticulum, hormones and neuropeptides have to be sorted and packed into large dense core vesicles (also named secretory granules) in the Golgi apparatus. Granules are transported toward the plasma membrane in a cytoskeleton-dependent manner and mature into competent organelles for secretagogue-induced exocytosis. Granules are then tethered to the plasma membrane, docked, and primed, before finally releasing their contents after fusing with the plasma membrane. To ensure that neurotransmission and neuroendocrine secretion operate correctly, all these steps must be tightly regulated and coordinated both spatially and temporally. Currently, when the field of intracellular trafficking has been honored by the 2013 Nobel Prize in Physiology or Medicine (awarded to James Rothman, Randy Schekman, and Thomas Sudhof for their pioneering works on vesicular transport), this issue of Frontiers in Neuroendocrine Science is aimed to providing an up-to-date overview of the cellular and molecular mechanisms governing the regulated secretory pathway in neuroendocrine cells. Reviews presented here are widely covering this topic, from the architecture of the organelle involved in secretory cargo processing and sorting, the biogenesis of secretory granules, their specific transport toward the plasma membrane to the late steps of exocytosis, and the secretory granule membrane recapture. Early stages of the secretory pathway have been discussed by two groups. Emma Martinez-Alonso and colleagues discuss the Golgi complex architecture, as well as the regulatory proteins that govern extra- and intra-Golgi transport, and the still controversial, but not mutually exclusive, theoretical models proposed to explain cargo progression through the Golgi stack (1). The group of Richard Mains discusses the specific roles of cytosolic adaptor proteins such as AP-1A, PACS-1, and GGAs in the assembly and maturation of secretory granules (2). Closer to the cell surface, several groups discuss the molecular mechanisms regulating the late stage of exocytosis. The group of Frederic Meunier reviews recent insights of the role of the cortical acto-myosin network (3), whereas the role of different tethering and priming factors such as CAPS, Munc13, and Doc2 proteins is described by the groups of Ury Ashery and Tom Martin (4, 5). Paanteha Moghadam and Meyer Jackson review how various synaptotagmins regulate fusion pore kinetics and control the mode of release (6). Lipids have emerged as key players of the regulated exocytosis and the group of Nicolas Vitale presents an overview on the diverse roles that lipids play in defining exocytotic sites, both by affecting membrane topology and by regulating secretory vesicle priming and fusion (7). Finally, exocytosis cannot exist without a compensatory membrane intake process (i.e., endocytosis), which allows recycling of granule components and maintains organelle integrity. The groups of Stephane Gasman and Alla Rynditch discuss the mechanisms that coordinate clathrin-mediated compensatory endocytosis with exocytosis, highlighting the specific role of the intersectin family of scaffold proteins in exocytosis and endocytosis (8, 9). The group of Ana-Maria Cardenas reviews the pleiotropic role of the mechano-GTPase dynamin-2, on intracellular membrane fission and fusion events, vesicle traffic, and cytoskeleton dynamics, as well as the impact of dynamin-2 mutations on the correct functioning of the secretory pathway (10). On a more physiological point of view, Maria-Luisa Duran-Pasten and Tatiana Fiordelisio present an example of how pituitary gonadotrophs receive and transduce extracellular signals to promote luteinizing (LH) and follicle-stimulating (FSH) secretion, highlighting the tremendous plasticity of the system for adapting to different physiological demands (11). Wei-Jye Lin and Stephen Salton report that single nucleotide polymorphisms in genes encoding secreted proteins are associated with neuropsychiatric or endocrine/metabolic disorders (12). Finally, Jennifer Fitch-Tewfik and Robert Flaumenhaft demonstrate how the regulated secretory pathway is similar in mast cells compared to neuroendocrine cells from the adrenal gland (13), and Burton Dickey’s group describes the regulatory mechanism of mucin secretion in a non-neuroendocrine cell model (14). On a more technical point of view, recent improvements in detection technologies, especially in optical microscopy, continually push the limits of sensitivity and resolution. The groups of Colin Rickman and Ute Becherer discuss how advances over the last decade in fluorescence microscopy provided spatial and temporal details on the subcellular organization of the molecular machinery governing the regulated secretory pathway (15, 16), whereas the group of Rory Duncan describes how the combination of new imaging approaches with super-resolution microscopy and novel calcium indicators is appropriate for accurate study of voltage-gated calcium channel locations, interactions, dynamics, and composition in living cells (17). Collectively, this compilation of reviews intends to illustrate the recent progress made to understand the complex regulation of the granule secretory pathways in neuroendocrine cells. We are grateful to all the authors who have contributed to this Research Topic and to the dedicated reviewers who helped us reaching the highest quality standards.

Huntingtin‐associated protein 1 regulates exocytosis, vesicle docking, readily releasable pool size and fusion pore stability in mouse chromaffin cells

Huntingtin-associated protein 1 (HAP1) was initially established as a neuronal binding partner of huntingtin, mutations in which underlie Huntington's disease. Subcellular localization and protein interaction data indicate that HAP1 may be important in vesicle trafficking and cell signalling. In this study, we establish that HAP1 is important in several steps of exocytosis in adrenal chromaffin cells. Using carbon-fibre amperometry, we measured single vesicle exocytosis in chromaffin cells obtained from HAP1(-/-) and HAP1(+/+) littermate mice. Numbers of Ca(2+)-dependent and Ca(2+)-independent full fusion events in HAP1(-/-) cells are significantly decreased compared with those in HAP1(+/+) cells. We observed no change in the frequency of 'kiss-and-run' fusion events or in Ca(2+) entry. Whereas release per full fusion event is unchanged in HAP1(-/-) cells, early fusion pore duration is prolonged, as indicated by the increased duration of pre-spike foot signals. Kiss-and-run events have a shorter duration, indicating opposing roles for HAP1 in the stabilization of the fusion pore during full fusion and transient fusion, respectively. We use electron microscopy to demonstrate a reduction in the number of vesicles docked at the plasma membrane of HAP1(-/-) cells, where membrane capacitance measurements reveal the readily releasable pool of vesicles to be reduced in size. Our study therefore illustrates that HAP1 regulates exocytosis by influencing the morphological docking of vesicles at the plasma membrane, the ability of vesicles to be released rapidly upon stimulation, and the early stages of fusion pore formation.

Isoform-Specific Roles of Synaptotagmins in Exocytosis

Adrenal chromaffin cells release hormones and neuropeptides that are essential to the physiological homeostasis of higher organisms. During this process, secretory granules fuse with the plasma membrane to deliver their cargo to the extracellular space. It was classically thought that fusion was the final regulated step in exocytosis, resulting in total release of granule cargo in a uniform manner. More recent evidence argues for non-uniform outcomes after fusion, where cargo is released with variable kinetics and selectivity. The goal of this study was to identify potential factors that contribute to these different outcomes, with a focus on the role of the calcium-sensing synaptotagmin (Syt) proteins. Two Syt isoforms are expressed in chromaffin cells: Syt-1 and Syt-7. We find that overexpressed and endogenous Syt isoforms rarely co-localize on secretory granules and are differentially responsive to weak and strong depolarizing stimuli. In addition, overexpressed Syt-1 and Syt-7 impose isoform-specific effects on fusion pore expansion and granule cargo release. Syt-7 fusion pores usually remain narrow (or reseal), slowing the dispersal of lumenal cargo proteins and granule membrane proteins. On the other hand, Syt-1 diffuses rapidly away from fusion sites and promotes the release of lumenal cargo proteins. These findings suggest one way in which regulation over chromaffin cell cargo release may be achieved is via selective activation of synaptotagmin isoforms.

Fucose, Mannose, and β-N-Acetylglucosamine Glycopolymers Initiate the Mouse Sperm Acrosome Reaction through Convergent Signaling Pathways

The sperm acrosome reaction (AR), an essential exocytosis step in mammalian fertilization, is mediated by a species-specific interaction of sperm surface molecules with glycans on the egg. Previous studies indicate that a subset of terminal carbohydrates on the mouse egg zona pellucida (ZP) trigger the AR by cross-linking or aggregating receptors on the sperm membrane. However, the exact role of those carbohydrates in AR has not been identified and the mechanism underlying the AR still needs further investigation. To study this process, a series of glycopolymers was synthesized. The glycopolymers are composed of a multivalent scaffold (norbornene), a functional ligand (previously identified ZP terminal monosaccharides), and a linker connecting the ligand and the scaffold. The polymers were tested for their ability to initiate AR and through which signaling pathways AR induction occurred. Our data demonstrate that mannose, fucose, and β-N-acetylglucosamine 10-mers and 100-mers initiate AR in a dose-dependent manner, and the 100-mers are more potent on a per monomer basis than the 10-mers. Although nearly equipotent in inducing the AR at the optimal concentrations, their AR activation kinetics are not identical. Similar to mouse ZP3, all 100-mer-activated AR are sensitive to guanine-binding regulatory proteins (G-proteins), tyrosine kinase, protein kinase A, protein kinase C, and Ca2+-related antagonists. Thus, the chemotypes of synthetic glycopolymers imitate the physiologic AR-activation agents and provide evidence that occupation of one of at least three different receptor binding sites is sufficient to initiate the AR.

Synaptotagmin Interaction with SNAP-25 Governs Vesicle Docking, Priming, and Fusion Triggering

SNARE complex assembly constitutes a key step in exocytosis that is rendered Ca(2+)-dependent by interactions with synaptotagmin-1. Two putative sites for synaptotagmin binding have recently been identified in SNAP-25 using biochemical methods: one located around the center and another at the C-terminal end of the SNARE bundle. However, it is still unclear whether and how synaptotagmin-1 × SNARE interactions at these sites are involved in regulating fast neurotransmitter release. Here, we have used electrophysiological techniques with high time-resolution to directly investigate the mechanistic ramifications of proposed SNAP-25 × synaptotagmin-1 interaction in mouse chromaffin cells. We demonstrate that the postulated central binding domain surrounding layer zero covers both SNARE motifs of SNAP-25 and is essential for vesicle docking, priming, and fast fusion-triggering. Mutation of this site caused no further functional alterations in synaptotagmin-1-deficient cells, indicating that the central acidic patch indeed constitutes a mechanistically relevant synaptotagmin-1 interaction site. Moreover, our data show that the C-terminal binding interface only plays a subsidiary role in triggering but is required for the full size of the readily releasable pool. Intriguingly, we also found that mutation of synaptotagmin-1 interaction sites led to more pronounced phenotypes in the context of the adult neuronal isoform SNAP-25B than in the embryonic isoform SNAP-25A. Further experiments demonstrated that stronger synaptotagmin-1 × SNAP-25B interactions allow for the larger primed vesicle pool supported by SNAP-25 isoform B. Thus, synaptotagmin-1 × SNARE interactions are not only required for multiple mechanistic steps en route to fusion but also underlie the developmental control of the releasable vesicle pool.

Dynamin-2 Regulates Fusion Pore Expansion and Quantal Release through a Mechanism that Involves Actin Dynamics in Neuroendocrine Chromaffin Cells

Over the past years, dynamin has been implicated in tuning the amount and nature of transmitter released during exocytosis. However, the mechanism involved remains poorly understood. Here, using bovine adrenal chromaffin cells, we investigated whether this mechanism rely on dynamin’s ability to remodel actin cytoskeleton. According to this idea, inhibition of dynamin GTPase activity suppressed the calcium-dependent de novo cortical actin and altered the cortical actin network. Similarly, expression of a small interfering RNA directed against dynamin-2, an isoform highly expressed in chromaffin cells, changed the cortical actin network pattern. Disruption of dynamin-2 function, as well as the pharmacological inhibition of actin polymerization with cytochalasine-D, slowed down fusion pore expansion and increased the quantal size of individual exocytotic events. The effects of cytochalasine-D and dynamin-2 disruption were not additive indicating that dynamin-2 and F-actin regulate the late steps of exocytosis by a common mechanism. Together our data support a model in which dynamin-2 directs actin polymerization at the exocytosis site where both, in concert, adjust the hormone quantal release to efficiently respond to physiological demands.

Critical Role of Cortical Vesicles in Dissecting Regulated Exocytosis: Overview of Insights Into Fundamental Molecular Mechanisms

Regulated exocytosis is one of the defining features of eukaryotic cells, underlying many conserved and essential functions. Definitively assigning specific roles to proteins and lipids in this fundamental mechanism is most effectively accomplished using a model system in which distinct stages of exocytosis can be effectively separated. Here we discuss the establishment of sea urchin cortical vesicle fusion as a model to study regulated exocytosis-a system in which the docked, release-ready, and late Ca(2+)-triggered steps of exocytosis are isolated and can be quantitatively assessed using the rigorous coupling of functional and molecular assays. We provide an overview of the insights this has provided into conserved molecular mechanisms and how these have led to and integrate with findings from other regulated exocytotic cells.