Dense-core vesicles and non-synaptic exocytosis in the central body of the crayfish brain

Summary The central body in the median protocerebrum of the brain of the crayfish Cherax destructor is a distinctive area of dense neuropile, the nerve fibres of which contain three main types of vesicles: electronlucent vesicles (diameter 35 nm), dense-core vesicles (diameter 64 nm), and large structured dense-core vesicles (diameter 98 nm, maximum 170 nm). Different vesicle types were found together in the same neurons. Electronlucent vesicles were seen at presynaptic sites and rarely observed in the state of exocytosis. Exocytosis of densecore and structured dense-core vesicles was a regular feature on non-synaptic release sites either close to, or at some distance from pre- and subsynaptic sites. Non-synaptic exocytotic sites are more often observed than chemical synapses. Different forms of exocytosis seen at non-synaptic sites included the release of single densecore vesicles, packets of dense-core vesicles, and rows of dense-core vesicles lined up along cell membranes and around fibre invaginations. Swelling and the enhanced electron density of extracellular non-synaptic spaces may mark the positions of prior exocytotic events. In vitro treatment of the brain with tannic acid buffer solution followed by conventional double fixation resulted in the augmentation of non-synaptic exocytosis. Electron microscopy of proctolin- and serotonin-immunoreactive nerve fibres shows them to contain dense-core and electron-lucent vesicles and to be surrounded by many unlabelled profiles similarly laden with dense-core vesicles and electron-lucent vesicles, indicating the presence of other, not yet identified, neuroactive compounds.     

Myosin Vb mobilizes recycling endosomes and AMPA receptors for postsynaptic plasticity

Learning-related plasticity at excitatory synapses in the mammalian brain requires the trafficking of AMPA receptors and the growth of dendritic spines. However, the mechanisms that couple plasticity stimuli to the trafficking of postsynaptic cargo are poorly understood. Here we demonstrate that myosin Vb (MyoVb), a Ca2+-sensitive motor, conducts spine trafficking during long-term potentiation (LTP) of synaptic strength. Upon activation of NMDA receptors and corresponding Ca2+ influx, MyoVb associates with recycling endosomes (REs), triggering rapid spine recruitment of endosomes and local exocytosis in spines. Disruption of MyoVb or its interaction with the RE adaptor Rab11-FIP2 abolishes LTP-induced exocytosis from REs and prevents both AMPA receptor insertion and spine growth. Furthermore, induction of tight binding of MyoVb to actin using an acute chemical genetic strategy eradicates LTP in hippocampal slices. Thus, Ca2+-activated MyoVb captures and mobilizes REs for AMPA receptor insertion and spine growth, providing a mechanistic link between the induction and expression of postsynaptic plasticity.     

Drosophila CAPS is an essential gene that regulates dense-core vesicle release and synaptic vesicle fusion

Calcium-activated protein for secretion (CAPS) is proposed to play an essential role in Ca2+-regulated dense-core vesicle exocytosis in vertebrate neuroendocrine cells. Here we report the cloning, mutation, and characterization of the Drosophila ortholog (dCAPS). Null dCAPS mutants display locomotory deficits and complete embryonic lethality. The mutant NMJ reveals a 50% loss in evoked glutamatergic transmission, and an accumulation of synaptic vesicles at active zones. Importantly, dCAPS mutants display a highly specific 3-fold accumulation of dense-core vesicles in synaptic terminals, which was not observed in mutants that completely arrest synaptic vesicle exocytosis. Targeted transgenic CAPS expression in identified motoneurons fails to rescue dCAPS neurotransmission defects, demonstrating a cell nonautonomous role in synaptic vesicle fusion. We conclude that dCAPS is required for dense-core vesicle release and that a dCAPS-dependent mechanism modulates synaptic vesicle release at glutamatergic synapses.     

Morphological docking of secretory vesicles

Calcium-dependent secretion of neurotransmitters and hormones is essential for brain function and neuroendocrine-signaling. Prior to exocytosis, neurotransmitter-containing vesicles dock to the target membrane. In electron micrographs of neurons and neuroendocrine cells, like chromaffin cells many synaptic vesicles (SVs) and large dense-core vesicles (LDCVs) are docked. For many years the molecular identity of the morphologically docked state was unknown. Recently, we resolved the minimal docking machinery in adrenal medullary chromaffin cells using embryonic mouse model systems together with electron-microscopic analyses and also found that docking is controlled by the sub-membrane filamentous (F-)actin. Currently it is unclear if the same docking machinery operates in synapses. Here, I will review our docking assay that led to the identification of the LDCV docking machinery in chromaffin cells and also discuss whether identical docking proteins are required for SV docking in synapses.     

Synaptotagmin V is targeted to dense-core vesicles that undergo calcium-dependent exocytosis in PC12 cells

Synaptotagmins (Syts) III, V, VI, and X are classified as a subclass of Syt, based on their sequence similarities and biochemical properties (Ibata, K., Fukuda, M., and Mikoshiba, K. (1998) J. Biol. Chem. 273, 12267-12273; Fukuda, M., Kanno, E., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 31421-31427). Although they have been suggested to be involved in vesicular trafficking, as in the role of the Syt I isoform in synaptic vesicle exocytosis, their exact functions remain to be clarified, and even their precise subcellular localization is still a matter of controversy. In this study, we established rat pheochromocytoma (PC12) cell lines that stably express Syts III-, V-, VI-, and X-GFP (green fluorescence protein) fusion proteins, respectively, to determine their precise subcellular localizations. Surprisingly, Syts III-, V-, VI-, and X-GFP proteins were found to be targeted to specific organelles: Syt III-GFP to near the plasma membrane, Syt V-GFP to dense-core vesicles, Syt VI-GFP to endoplasmic reticulum-like structures, and Syt X-GFP to vesicles (other than dense-core vesicles) present in cytoplasm. We showed that Syt V-containing vesicles at the neurites of PC12 cells were processed to exocytosis in a Ca2+-dependent manner. Immunohistochemical analysis further showed that endogenous Syt V was also localized on dense-core vesicles in the mouse brain and specifically expressed in glucagon-positive alpha-cells in mouse pancreatic islets, but not in beta- or delta-cells. Based on these results, we propose that Syt V is a dense-core vesicle-specific Syt isoform that controls a specific type of Ca2+-regulated secretion.     

Symposium 4: Dynamics of the Neuronal Cytoskeleton. Actin‐based vesicle transport in nerve cell extracts: implications for neurodegenerative diseases

Neurodegenerative diseases may result in part from defects in motor‐driven vesicle transport in neuronal cells. Myosin‐V, an actin‐based motor that is highly enriched in the brain, mediates the movement of vesicles on cortical actin filaments. Recent evidence suggests that the globular tail of myosin‐V interacts with the microtubule‐based motor, kinesin, to form a ‘hetero‐motor’ complex on vesicles. The complex of these two motors, one microtubule‐based and the other actin‐based, facilitates the movement of vesicles from microtubules to actin filaments. Based on our studies of vesicle transport by these two motors in extracts of squid neurons, we hypothesize that one of the functions of the tail–tail interaction is to provide feedback between the two proteins to allow seamless transition of vesicles from microtubules to actin filaments. To study the interactions of the globular tail domain of myosin‐V to kinesin and to neuronal vesicles, we used a GST‐tagged globular tail fragment in motility assays. The MyoV tail fragment inhibited vesicle transport by 81–91% and thereby exhibited a dominant negative effect. These data show that the recombinant protein blocked the activity of native myosin‐V presumably by binding to vesicles and competing away the native myosin‐V motors. The GST‐MyoV‐tail fragment pulled down kinesin by immunoprecipitation from squid brain homogenates and therefore it exhibited binding properties of native myosin‐V. These data show that the headless myosin‐V fragment is an effective inhibitor of vesicle transport in cell extracts. These studies support the hypothesis that tail–tail interactions may be a mechanism for feedback between myosin‐V and kinesin to allow transition of vesicles from microtubules to actin filaments. Acknowledgements: Supported by NSF grant MCB9974709.     

Expression of the Dominant-Negative Tail of Myosin Va Enhances Exocytosis of Large Dense Core Vesicles in Neurons

Regulated exocytosis of secretory vesicles is a fundamental process in neurotransmission and the release of hormones and growth factors. The F-actin-binding motor protein myosin Va was recently shown to be involved in exocytosis of peptide-containing large dense core vesicles of neuroendocrine cells. It has not previously been discussed whether it plays a similar role in neurons. We performed live-cell imaging of cultured hippocampal neurons to measure the exocytosis of large dense core vesicles containing fluorescently labelled neuropeptide Y. To address the role of myosin Va in this process, neurons were transfected with the dominant-negative tail domain of myosin Va (myosinVa-tail). Under control conditions, about 0.75% of the labelled large dense core vesicles underwent exocytosis during 5 min of stimulation. This value was doubled to 1.80% of the vesicles when myosinVa-tail was expressed. Depolymerization of F-actin using latrunculin B resulted in a similar increase in exocytosis in both control and myosinVa-tail expressing cells. Interestingly, the increase in exocytosis caused by myosinVa-tail expression was completely abolished in the presence of KN-62, an inhibitor of calcium–calmodulin-dependent kinase II. We suggest that myosinVa-tail causes the liberation of large dense core vesicles from the actin cytoskeleton, leading to an increase in exocytosis in the cultured hippocampal neurons. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Vesicle cycle in the presynaptic nerve terminal

Presynaptic nerve terminals contain a great number ofsynaptic vesicles filled with neurotransmitter. The transmission of information in synapses is mediated by release of transmitter from vesicles: exocytosis, after their fusion with presynaptic membrane. At the functioning synapses, the continuous recycling of synaptic vesicles occurs (vesicle cycle), which provides multiple reuse of vesicular membrane material during synaptic activity. Vesicle cycle consists of large number of steps, including vesicle fusion--exocytosis, formation of new vesicles--endocytosis, vesicle sorting, filling of vesicles with transmitter, intraterminal vesicle transport driving the vesicles to different vesicle pools and preparing to next exocytic event. At this paper, I presented the latest literature and our data regarding the steps and mechanisms of vesicle cycle at synapses. Special attention was paid to neuromuscular synapse as the most thoroughly investigated and as my favorite preparation.     

A Role of myosin Vb and Rab11-FIP2 in the aquaporin-2 shuttle

Arginine-vasopressin (AVP) regulates water reabsorption in renal collecting duct principal cells. Its binding to Gs-coupled vasopressin V2 receptors increases cyclic AMP (cAMP) and subsequently elicits the redistribution of the water channel aquaporin-2 (AQP2) from intracellular vesicles into the plasma membrane (AQP2 shuttle), thereby facilitating water reabsorption from primary urine. The AQP2 shuttle is a paradigm for cAMP-dependent exocytic processes. Using sections of rat kidney, the AQP2-expressing cell line CD8, and primary principal cells, we studied the role of the motor protein myosin Vb, its vesicular receptor Rab11, and the myosin Vb- and Rab11-binding protein Rab11-FIP2 in the AQP2 shuttle. Myosin Vb colocalized with AQP2 intracellularly in resting and at the plasma membrane in AVP-treated cells. Rab11 was found on AQP2-bearing vesicles. A dominant-negative myosin Vb tail construct and Rab11-FIP2 lacking the C2 domain (Rab11-FIP2-DeltaC2), which disrupt recycling, caused condensation of AQP2 in a Rab11-positive compartment and abolished the AQP2 shuttle. This effect was dependent on binding of myosin Vb tail and Rab11-FIP2-DeltaC2 to Rab11. In summary, we identified myosin Vb as a motor protein involved in AQP2 recycling and show that myosin Vb- and Rab11-FIP2-dependent recycling of AQP2 is an integral part of the AQP2 shuttle.     

Kinesin I and cytoplasmic dynein orchestrate glucose-stimulated insulin-containing vesicle movements in clonal MIN6 beta-cells

Glucose-stimulated mobilization of large dense-core vesicles (LDCVs) to the plasma membrane is essential for sustained insulin secretion. At present, the cytoskeletal structures and molecular motors involved in vesicle trafficking in beta-cells are poorly defined. Here, we describe simultaneous imaging of enhanced green fluorescent protein (EGFP)-tagged LDCVs and microtubules in beta-cells. Microtubules exist as a tangled array, along which vesicles describe complex directional movements. Whilst LDCVs frequently changed direction, implying the involvement of both plus- and minus-end directed motors, inactivation of the minus-end motor, cytoplasmic dynein, inhibited only a small fraction of all vesicle movements which were involved in vesicle recovery after glucose-stimulated exocytosis. By contrast, selective silencing of the plus-end motor, kinesin I, with small interfering RNAs substantially inhibited all vesicle movements. We conclude that the majority of LDCV transport in beta-cells is mediated by kinesin I, whilst dynein probably contributes to the recovery of vesicles after rapid kiss-and-run exocytosis.     

Effect of forskolin on synaptotagmin IV protein trafficking in PC12 cells

Synaptotagmin IV (Syt IV) was originally described as an immediate early gene product induced by forskolin or membrane depolarization in PC12 cells; however, nothing is known about the subcellular localization and transport of the newly translated Syt IV protein in PC12 cells. In this study, we investigated the transport mechanism of Syt IV protein induced by forskolin and found that forskolin treatment dramatically increases the Syt IV protein level (approximately 10-fold, to a level comparable to that of Syt IX) and promotes the transport of Syt IV protein from the Golgi to the cell periphery by a microtubule-dependent motor(s). The expression levels and subcellular localizations of two major Syt isoforms (I and IX) in PC12 cells, on the other hand, were unaffected by such treatment. Immunoelectron microscopic analysis showed that some Syt IV signals are clearly associated with dense-core vesicles in forskolin-treated PC12 cells, although the majority of the Syt IV molecules at the cell periphery were present on clear vesicular structures other than dense-core vesicles. An N-terminal antibody-uptake experiment indicated that Syt IV-containing vesicles in forskolin-treated PC12 cells undergo Ca(2+)-dependent exocytosis, because uptake of the anti-Syt IV-N antibody from the culture medium was slightly, but significantly, increased after forskolin treatment. Our results indicate that forskolin (or the increased cAMP level) is important for the transport of the Syt IV protein from the Golgi to the cell periphery, but not sufficient for the sorting of all Syt IV molecules to mature dense-core vesicles.     

Rapid recycling of beta-adrenergic receptors is dependent on the actin cytoskeleton and myosin Vb

For the beta(2)-adrenergic receptor (beta(2)AR), published evidence suggests that an intact actin cytoskeleton is required for the endocytosis of receptors and their proper sorting to the rapid recycling pathway. We have characterized the role of the actin cytoskeleton in the regulation of beta(2)AR trafficking in human embryonic kidney 293 (HEK293) cells using two distinct actin filament disrupting compounds, cytochalasin D and latrunculin B (LB). In cells pretreated with either drug, beta(2)AR internalization into transferrin-positive vesicles was not altered but both agents significantly decreased the rate at which beta(2)ARs recycled to the cell surface. In LB-treated cells, nonrecycled beta(2)ARs were localized to early embryonic antigen 1-positive endosomes and also accumulated in the recycling endosome (RE), but only a small fraction of receptors localized to LAMP-positive late endosomes and lysosomes. Treatment with LB also markedly enhanced the inhibitory effect of rab11 overexpression on receptor recycling. Dissociating receptors from actin by expression of the myosin Vb tail fragment resulted in missorting of beta(2)ARs to the RE, while the expression of various CART fragments or the depletion of actinin-4 had no detectable effect on beta(2)AR sorting. These results indicate that the actin cytoskeleton is required for the efficient recycling of beta(2)ARs, a process that likely is dependent on myosin Vb.     

What is the function of neuronal AP‐3?

Neurotransmission requires the proper organization and rapid recycling of synaptic vesicles. Rapid retrieval has been suggested to occur either by kiss-and-stay or kiss-and-run mechanisms, whereas classical recycling is mediated by clathrin-dependent endocytosis. Molecular coats are key components in the selection of cargos, AP-2 (adaptor protein 2) playing a prominent role in synaptic vesicle endocytosis. Another coat protein, AP-3, has been implicated in synaptic vesicle biogenesis and in the generation of secretory and lysosomal-related organelles. In the present review, we will particularly focus on the recent data concerning the recycling of synaptic vesicles and the function of AP-3 and the v-SNARE (vesicular soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) TI-VAMP (tetanus neurotoxin-insensitive vesicle-associated membrane protein) in these processes. We propose that AP-3 plays an important regulatory role in neurons which contributes to the basal and stimulated exocytosis of synaptic vesicles.     

The Myosin Motor Domain of Fungal Chitin Synthase V Is Dispensable for Vesicle Motility but Required for Virulence of the Maize Pathogen Ustilago maydis

Class V chitin synthases are fungal virulence factors required for plant infection. They consist of a myosin motor domain fused to a membrane-spanning chitin synthase region that participates in fungal cell wall formation. The function of the motor domain is unknown, but it might deliver the myosin chitin synthase-attached vesicles to the growth region. Here, we analyze the importance of both domains in Mcs1, the chitin synthase V of the maize smut fungus Ustilago maydis. By quantitative analysis of disease symptoms, tissue colonization, and single-cell morphogenic parameters, we demonstrate that both domains are required for fungal virulence. Fungi carrying mutations in the chitin synthase domain are rapidly recognized and killed by the plant, whereas fungi carrying a deletion of the motor domain show alterations in cell wall composition but can invade host tissue and cause a moderate plant response. We also show that Mcs1-bound vesicles exhibit long-range movement for up to 20 μm at a velocity of ~1.75 μm/s. Apical Mcs1 localization depends on F-actin and the motor domain, whereas Mcs1 motility requires microtubules and persists when the Mcs1 motor domain is deleted. Our results suggest that the myosin motor domain of ChsV supports exocytosis but not long-range delivery of transport vesicles.     

The COOH-terminal domain of Myo2p, a yeast myosin V, has a direct role in secretory vesicle targeting

MYO2 encodes a type V myosin heavy chain needed for the targeting of vacuoles and secretory vesicles to the growing bud of yeast. Here we describe new myo2 alleles containing conditional lethal mutations in the COOH-terminal tail domain. Within 5 min of shifting to the restrictive temperature, the polarized distribution of secretory vesicles is abolished without affecting the distribution of actin or the mutant Myo2p, showing that the tail has a direct role in vesicle targeting. We also show that the actin cable-dependent translocation of Myo2p to growth sites does not require secretory vesicle cargo. Although a fusion protein containing the Myo2p tail also concentrates at growth sites, this accumulation depends on the polarized delivery of secretory vesicles, implying that the Myo2p tail binds to secretory vesicles. Most of the new mutations alter a region of the Myo2p tail conserved with vertebrate myosin Vs but divergent from Myo4p, the myosin V involved in mRNA transport, and genetic data suggest that the tail interacts with Smy1p, a kinesin homologue, and Sec4p, a vesicle-associated Rab protein. The data support a model in which the Myo2p tail tethers secretory vesicles, and the motor transports them down polarized actin cables to the site of exocytosis.     

Disruption of actin motor function due to MoMyo5 mutation impairs host penetration and pathogenicity in Magnaporthe oryzae

Actin motor myosin proteins are the driving forces behind the active transport of vesicles, and more than 20 classes of myosin have been found to contribute to a wide range of cellular processes, including endocytosis and exocytosis, autophagy, cytokinesis and the actin cytoskeleton. In Saccharomyces cerevisiae, class V myosin Myo2 (ScMyo2p) is important for the transport of distinct sets of cargo to regions of the cell along the cytoskeleton for polarized growth. To study whether myosins play a role in the formation or function of the appressorium (infectious structure) of the rice blast fungus Magnaporthe oryzae, we identified MoMyo5 as an orthologue of ScMyo2p and characterized its function. Targeted gene disruption revealed that MoMyo5 is required for intracellular transport and is essential for hyphal growth and asexual reproduction. Although the ΔMomyo5 mutant could form appressorium‐like structures, the structures were unable to penetrate host cells and were therefore non‐pathogenic. We further found that MoMyo5 moves dynamically from the cytoplasm to the hyphal tip, where it interacts with MoSec4, a Rab GTPase involved in secretory transport, hyphal growth and fungal pathogenicity. Our studies indicate that class V myosin and its translocation are tightly coupled with hyphal growth, asexual reproduction, appressorium function and pathogenicity in the rice blast fungus.     

Involvement of neuropeptide mechanisms in the integration of heterotopic dentate fascia grafts and recipient brain

Embryonic dentate fascia was grafted into the somatosensory neocortex of adult rats. Nine months post-grafting, the ultrastructural and morphometric analysis of the giant synapses established between the grafted granular neurons and inappropriate targets in the recipient brain was performed. As compared to the intact synaptic endings in the control hippocampus, differences were found in both the number and distribution of large dense-core synaptic vesicles, which store the neuropeptide co-transmitters. The peptidergic vesicle proportion (of total vesicle pool) within the ectopic giant synapses was 5.8 +/- 0.6% (versus 3.3 +/- 0.6% in the control). Clusters of large dense-core vesicles near the active zones of aberrant connections were observed almost 7.9 times more frequently than that of normal contacts. These data provide evidence that neuropeptide transmitters are critical for the maintenance of synaptic connections between the heterotopic dentate grafts and host brain.     

Plasticity-induced growth of dendritic spines by exocytic trafficking from recycling endosomes

Dendritic spines are micron-sized membrane protrusions receiving most excitatory synaptic inputs in the mammalian brain. Spines form and grow during long-term potentiation (LTP) of synaptic strength. However, the source of membrane for spine formation and enlargement is unknown. Here we report that membrane trafficking from recycling endosomes is required for the growth and maintenance of spines. Using live-cell imaging and serial section electron microscopy, we demonstrate that LTP-inducing stimuli promote the mobilization of recycling endosomes and vesicles into spines. Preventing recycling endosomal transport abolishes LTP-induced spine formation. Using a pH-sensitive recycling cargo, we show that exocytosis from recycling endosomes occurs locally in spines, is triggered by activation of synaptic NMDA receptors, and occurs concurrently with spine enlargement. Thus, recycling endosomes provide membrane for activity-dependent spine growth and remodeling, defining a novel membrane trafficking mechanism for spine morphological plasticity and providing a mechanistic link between structural and functional plasticity during LTP.     

Kinesin- and Myosin-driven Steps of Vesicle Recruitment for Ca2+-regulated Exocytosis

Kinesin and myosin have been proposed to transport intracellular organelles and vesicles to the cell periphery in several cell systems. However, there has been little direct observation of the role of these motor proteins in the delivery of vesicles during regulated exocytosis in intact cells. Using a confocal microscope, we triggered local bursts of Ca²⁺-regulated exocytosis by wounding the cell membrane and visualized the resulting individual exocytotic events in real time. Different temporal phases of the exocytosis burst were distinguished by their sensitivities to reagents targeting different motor proteins. The function blocking antikinesin antibody SUK4 as well as the stalk-tail fragment of kinesin heavy chain specifically inhibited a slow phase, while butanedione monoxime, a myosin ATPase inhibitor, inhibited both the slow and fast phases. The blockage of Ca²⁺/calmodulin-dependent protein kinase II with autoinhibitory peptide also inhibited the slow and fast phases, consistent with disruption of a myosin-actin– dependent step of vesicle recruitment. Membrane resealing after wounding was also inhibited by these reagents. Our direct observations provide evidence that in intact living cells, kinesin and myosin motors may mediate two sequential transport steps that recruit vesicles to the release sites of Ca²⁺-regulated exocytosis, although the identity of the responsible myosin isoform is not yet known. They also indicate the existence of three semistable vesicular pools along this regulated membrane trafficking pathway. In addition, our results provide in vivo evidence for the cargo-binding function of the kinesin heavy chain tail domain.     

Myosin accelerates synaptic vesicle recycling in the motor nerve endings

Neurotransmitter release and exocytosis of synaptic vesicles in the motor nerve endings of the frog cutaneous-pectoris muscle were studied using electrophysiological and optical methods under the conditions of inhibition of the myosin light-chain kinase and non-muscle myosin by the specific inhibitors ML-7 (12 μM) and (–)-blebbistatin (100 μM). At high-frequency stimulation (20 pulses/s), these inhibitors strengthened suppression of transmitter release during the first 20–25 s and slowed down the release of the fluorescent dye FM 1-43. The obtained results indicate that myosin accelerates rapid synaptic vesicle recycling upon high-frequency stimulation.