Clusters Of Vesicles Research Articles

An Atg9-containing compartment that functions in the early steps of autophagosome biogenesis

Eukaryotes use the process of autophagy, in which structures targeted for lysosomal/vacuolar degradation are sequestered into double-membrane autophagosomes, in numerous physiological and pathological situations. The key questions in the field relate to the origin of the membranes as well as the precise nature of the rearrangements that lead to the formation of autophagosomes. We found that yeast Atg9 concentrates in a novel compartment comprising clusters of vesicles and tubules, which are derived from the secretory pathway and are often adjacent to mitochondria. We show that these clusters translocate en bloc next to the vacuole to form the phagophore assembly site (PAS), where they become the autophagosome precursor, the phagophore. In addition, genetic analyses indicate that Atg1, Atg13, and phosphatidylinositol-3-phosphate are involved in the further rearrangement of these initial membranes. Thus, our data reveal that the Atg9-positive compartments are important for the de novo formation of the PAS and the sequestering vesicle that are the hallmarks of autophagy.

Case 1: Vesicular rash in an infant

Afive-month-old term male infant was referred by his family doctor for evaluation of a vesicular rash. The mother first noticed several clusters of vesicles on the baby’s chest five days before presentation. In the subsequent four days, new adjacent vesicles erupted on the chest with clear discharge. He had slightly decreased appetite but normal voiding and stools, with no accompanying fever. A review of the infant’s systems was otherwise unremarkable. The baby was born to a 32-year-old Pakistani-Canadian woman who remained in Pakistan during her pregnancy. Antenatal screening for HIV, hepatitis B and syphilis was negative, and a rubella immunoglobulin G test was positive. The baby was born at 39 weeks’ gestational age by spontaneous vaginal delivery with no perinatal complications. The birth weight was 3.2 kg and the baby was discharged home on the second day of life with a normal physical examination. He had received his two- and four-month routine immunizations uneventfully. There was no family history of immunodeficiency or recurrent infections. A physical examination revealed a thriving, afebrile and well-appearing infant. Multiple crops of vesicles on an erythematous base were present on both the anterior and posterior right hemithorax (Figure 1). There were no satellite lesions. One further detail on history suggested the diagnosis. Case 2: Making a diagnosis: Lest we forget the family A n 11-year-old girl was referred to the paediatric gastroenterology service with symptoms of hoarse voice, central chest discomfort with a frequent acidic taste in the mouth, and mild epigastric pain. An assessment by an otolaryngologist revealed congestion and small nodules on her vocal cords. A diagnosis of gastroesophageal reflux disease (GERD) had been entertained. The patient was otherwise well without any vomiting, dysphagia, alteration in stool pattern or weight loss. Her history was unremarkable, with normal growth and development. During history taking in the outpatient clinic, it was inquired as to whether anyone else in the family had similar problems. The mother reported that her husband had severe, chronic GERD requiring a fundoplication. The physical examination of the patient was normal. She was prescribed ranitidine and a follow-up was arranged. When the patient was seen two months later, there was little improvement in her symptoms. A trial of omeprazole was initiated. A lack of response led to an increase in the dose of the medication, but that was also of no avail. An upper gastrointestinal endoscopy was performed to look for reflux esophagitis or eosinophilic esophagitis. On endoscopy, the esophagus and stomach appeared normal. Inspection of the duodenum revealed the unexpected diagnosis. CliniCian’s Corner

Synapsin regulates vesicle organization and activity-dependent recycling at Drosophila motor boutons

Synapsin is a phosphoprotein reversibly associated with synaptic vesicles. We investigated synapsin function in mediating synaptic activity during intense stimulation at Drosophila motor boutons. Electron microscopy analysis of synapsin(−) boutons demonstrated that synapsin maintains vesicle clustering over the periphery of the bouton. Cyclosporin A pretreatment disrupted peripheral vesicle clustering, presumably due to increasing synapsin phosphorylated state. Labeling recycling vesicles with a fluorescent dye FM1-43 followed by photoconversion of the dye into electron dense product demonstrated that synapsin deficiency does not affect mixing of the reserve and recycling vesicle pools but selectively reduces the size of the reserve pool. Intense stimulation produced a significant increase in vesicle abundance and vesicle redistribution toward the central core of synapsin (+) boutons, while in synapsin (−) boutons the area occupied by vesicles did not change and the increase in vesicle numbers was not as prominent. However, intense stimulation produced an increase in basal release at synapsin(−) but not in synapsin(+) boutons, suggesting that synapsin may direct vesicles to the reserve pool. Finally, synapsin deficiency inhibited an increase in quantal size and formation of endosome-like cisternae, which was activated either by intense electrical stimulation or by high K+ application. Taken together, these results elucidate a novel synapsin function, specifically, promoting vesicle reuptake and reserve pool formation upon intense stimulation.

High- and Low-Mobility Stages in the Synaptic Vesicle Cycle

Synaptic vesicles need to be mobile to reach their release sites during synaptic activity. We investigated vesicle mobility throughout the synaptic vesicle cycle using both conventional and subdiffraction-resolution stimulated emission depletion fluorescence microscopy. Vesicle tracking revealed that recently endocytosed synaptic vesicles are highly mobile for a substantial time period after endocytosis. They later undergo a maturation process and integrate into vesicle clusters where they exhibit little mobility. Despite the differences in mobility, both recently endocytosed and mature vesicles are exchanged between synapses. Electrical stimulation does not seem to affect the mobility of the two types of vesicles. After exocytosis, the vesicle material is mobile in the plasma membrane, although the movement appears to be somewhat limited. Increasing the proportion of fused vesicles (by stimulating exocytosis while simultaneously blocking endocytosis) leads to substantially higher mobility. We conclude that both high- and low-mobility states are characteristic of synaptic vesicle movement.

Nonhomogeneous distribution of filamentous actin in the presynaptic terminals on the spinal motoneurons

Although actin is known to play important roles in synapses, immunocytochemical and structural studies on synaptic actin have resulted in conflicting data, and the presence and precise localization of filamentous actin (F-actin) in the synapses have still not been well clarified. We recently described a phalloidin-based antifluorescein isothiocyanate system, which has been successfully developed for ultrastructural investigations of F-actin in the ependymal microvilli. By this technique, the present study has demonstrated the presence of F-actin in both the pre- and the postsynaptic regions in the synapses of spinal motoneurons. In the presynaptic terminal, F-actin was localized predominantly in the active zones and the adjacent synaptic vesicle clusters, including the vesicles docked at the active zones and a population of recycling vesicles. By contrast, the proximally located vesicle pool was much less intensely stained. In the postsynaptic region, F-actin was concentrated at the postsynaptic densities and stretched some way into the surrounding cytoplasm. Insofar as the axonal terminals analyzed in this study touched on either the cell body or the dendritic shaft, where the postsynaptic regions did not form spine-like specializations, our results cannot shed any light on the distribution of F-actin within spines. However, the present study has provided a hitherto unreported ultrastructural view of the subcellular distribution of F-actin in the synapse, which is thought to be helpful for understanding the roles of the synaptic actin cytoskeleton.

Are Presynaptic Proteins Predisposed to Self-Assemble?

Tight control of synapse formation ensures that neurons connect to appropriate targets. In this issue of Neuron, Klassen et al. identify ARL-8 GTPase as a regulator of presynaptic assembly. Without ARL-8, presynaptic material aggregates en route to its destination, suggesting that ARL-8 acts like a dispersant to prevent premature synaptic assembly in the axon.

Essential cooperation of N-cadherin and neuroligin-1 in the transsynaptic control of vesicle accumulation

Cell adhesion molecules are key players in transsynaptic communication, precisely coordinating presynaptic differentiation with postsynaptic specialization. At glutamatergic synapses, their retrograde signaling has been proposed to control presynaptic vesicle clustering at active zones. However, how the different types of cell adhesion molecules act together during this decisive step of synapse maturation is largely unexplored. Using a knockout approach, we show that two synaptic adhesion systems, N-cadherin and neuroligin-1, cooperate to control vesicle clustering at nascent synapses. Live cell imaging and fluorescence recovery after photobleaching experiments at individual synaptic boutons revealed a strong impairment of vesicle accumulation in the absence of N-cadherin, whereas the formation of active zones was largely unaffected. Strikingly, also the clustering of synaptic vesicles triggered by neuroligin-1 overexpression required the presence of N-cadherin in cultured neurons. Mechanistically, we found that N-cadherin acts by postsynaptically accumulating neuroligin-1 and activating its function via the scaffolding molecule S-SCAM, leading, in turn, to presynaptic vesicle clustering. A similar cooperation of N-cadherin and neuroligin-1 was observed in immature CA3 pyramidal neurons in an organotypic hippocampal network. Moreover, at mature synapses, N-cadherin was required for the increase in release probability and miniature EPSC frequency induced by expressed neuroligin-1. This cooperation of two cell adhesion systems provides a mechanism for coupling bidirectional synapse maturation mediated by neuroligin-1 to cell type recognition processes mediated by classical cadherins.

Piccolo and bassoon maintain synaptic vesicle clustering without directly participating in vesicle exocytosis

Piccolo and bassoon are highly homologous multidomain proteins of the presynaptic cytomatrix whose function is unclear. Here, we generated piccolo knockin/knockout mice that either contain wild-type levels of mutant piccolo unable to bind Ca(2+) (knockin), approximately 60% decreased levels of piccolo that is C-terminally truncated (partial knockout), or <5% levels of piccolo (knockout). All piccolo mutant mice were viable and fertile, but piccolo knockout mice exhibited increased postnatal mortality. Unexpectedly, electrophysiology and electron microscopy of piccolo-deficient synapses failed to uncover a major phenotype either in acute hippocampal slices or in cultured cortical neurons. To unmask potentially redundant functions of piccolo and bassoon, we thus acutely knocked down expression of bassoon in wild-type and piccolo knockout neurons. Despite a nearly complete loss of piccolo and bassoon, however, we still did not detect an electrophysiological phenotype in cultured piccolo- and bassoon-deficient neurons in either GABAergic or glutamatergic synaptic transmission. In contrast, electron microscopy revealed a significant reduction in synaptic vesicle clustering in double bassoon/piccolo-deficient synapses. Thus, we propose that piccolo and bassoon play a redundant role in synaptic vesicle clustering in nerve terminals without directly participating in neurotransmitter release.

Targeted three‐dimensional immunohistochemistry reveals localization of presynaptic proteins Bassoon and Piccolo in the rat calyx of Held before and after the onset of hearing

Bassoon and Piccolo contribute to the cytomatrix of active zones (AZ), the sites of neurotransmitter release in nerve terminals. Here, we examined the 3D localization of Bassoon and Piccolo in the rat calyx of Held between postnatal days 9 and 21, the period of hearing onset characterized by pronounced structural and functional changes. Bassoon and Piccolo were identified by immunohistochemistry (IHC) on slices of the brainstem harboring calyces labeled with membrane-anchored green fluorescent protein (mGFP). By using confocal microscopy and 3D reconstructions, we examined the distribution of Bassoon and Piccolo in calyces delineated by mGFP. This allowed us to discriminate calyceal IHC signals from noncalyceal signals located in the spaces between the calyceal stalks, which could mimic a calyx-like distribution. We found that both proteins were arranged in clusters resembling the size of AZs. These clusters were located along the presynaptic membrane facing the principal cell, close to or overlapping with synaptic vesicle (SV) clusters. Only about 60% of Bassoon and Piccolo clusters overlapped, whereas the remaining clusters contained predominantly Bassoon or Piccolo, suggesting differential targeting of these proteins within a single nerve terminal and potentially heterogeneous AZs functional properties. The total number of Bassoon and Piccolo clusters, which may approximate the number of AZs, was 405 +/- 35 at P9 and 601 +/- 45 at P21 (mean +/- SEM, n = 12). Normalized to calyx volume at P9 and P21, the density of clusters was similar, suggesting that the absolute number of clusters, not density, may contribute to the functional maturation associated with hearing onset.

The highly conserved synapsin domain E mediates synapsin dimerization and phospholipid vesicle clustering

Synapsins are abundant SV (synaptic vesicle)-associated phosphoproteins that regulate synapse formation and function. The highly conserved C-terminal domain E was shown to contribute to several synapsin functions, ranging from formation of the SV reserve pool to regulation of the kinetics of exocytosis and SV cycling, although the molecular mechanisms underlying these effects are unknown. In the present study, we used a synthetic 25-mer peptide encompassing the most conserved region of domain E (Pep-E) to analyse the role of domain E in regulating the interactions between synapsin I and liposomes mimicking the phospholipid composition of SVs (SV-liposomes) and other pre-synaptic protein partners. In affinity-chromatography and cross-linking assays, Pep-E bound to endogenous and purified exogenous synapsin I and strongly inhibited synapsin dimerization, indicating a role in synapsin oligomerization. Consistently, Pep-E (but not its scrambled version) counteracted the ability of holo-synapsin I to bind and coat phospholipid membranes, as analysed by AFM (atomic force microscopy) topographical scanning, and significantly decreased the clustering of SV-liposomes induced by holo-synapsin I in FRET (Förster resonance energy transfer) assays, suggesting a causal relationship between synapsin oligomerization and vesicle clustering. Either Pep-E or a peptide derived from domain C was necessary and sufficient to inhibit both dimerization and vesicle clustering, indicating the participation of both domains in these activities of synapsin I. The results provide a molecular explanation for the effects of domain E in nerve terminal physiology and suggest that its effects on the size and integrity of SV pools are contributed by the regulation of synapsin dimerization and SV clustering.

Synaptic vesicle pools: an update

During the last few decades synaptic vesicles have been assigned to a variety of functional and morphological classes or “pools”. We have argued in the past (Rizzoli and Betz, 2005) that synaptic activity in several preparations is accounted for by the function of three vesicle pools: the readily releasable pool (docked at active zones and ready to go upon stimulation), the recycling pool (scattered throughout the nerve terminals and recycling upon moderate stimulation), and finally the reserve pool (occupying most of the vesicle clusters and only recycling upon strong stimulation). We discuss here the advancements in the vesicle pool field which took place in the ensuing years, focusing on the behavior of different pools under both strong stimulation and physiological activity. Several new findings have enhanced the three-pool model, with, for example, the disparity between recycling and reserve vesicles being underlined by the observation that the former are mobile, while the latter are “fixed”. Finally, a number of altogether new concepts have also evolved such as the current controversy on the identity of the spontaneously recycling vesicle pool.

Relax? Don't do it!—Linking presynaptic vesicle clustering with mechanical tension

The release of transmitter-filled vesicles from presynaptic terminals is a key step of neurotransmission. Prior to release, synaptic vesicles get clustered at a specialized patch of the presynaptic membrane, here referred to as the active zone. So far, mainly biochemical regulations at the active zone were regarded as decisive for synaptic vesicle clustering and release. However, using biophysical approaches, a recent paper [Siechen, et al. (2009). Proc. Natl. Acad. Sci. U.S.A. 106, 12611-12616] indicated also that the micromechanical regulations within axon and terminal could be crucial for proper vesicle clustering. The authors demonstrated that the synaptic vesicle accumulations vanished after axotomy but were restored after the application of physical tension. Furthermore, axons seem to be under an intrinsic tension, which could be perceived and tuned by an axon-internal tension sensing mechanism. Therefore, mechanical force could steer vesicle clustering and consequently synapse function. Here, we review this interdisciplinary study of Siechen, et al. [Proc. Natl. Acad. Sci. U.S.A. 106, 12611-12616 (2009)] and discuss the significance of cellular mechanics on synaptic function.

Exo- and Endocytotic trafficking of SCAMP2

Exo- and endocytotic membrane trafficking is an essential process for transport of secretory proteins, extracellular glycans, transporters and lipids in plant cells. Using secretory carrier membrane protein 2 (SCAMP2) as a marker for secretory vesicles and tobacco BY-2 cells as a model system, we recently demonstrated that SCAMP2 positive structures containing secretory materials are transported from the Golgi apparatus to the plasma membrane and/or cell plate. This structure is consisted with clustered vesicles and was thus named the secretory vesicle cluster (SVC). Here, we have utilized the reversible photo-switching fluorescent protein Dronpa1 to trace the movement of SCAMP2 on the PM and cell plate. Activated SCAMP2-Dronpa fluorescence on the plasma membrane and cell plate moved into the BY-2 cells within several minutes, but did not spread around plasma membrane. This is consistent with recycling of SCAMP2 among endomembrane compartments such as the TGN, plasma membrane and cell plate. The relationship between SVC-mediated trafficking and exo- and endocytosis of plant cells is discussed taking into account this new data and knowledge provided by recent reports.

LAMELLAR BODY FORMATION AND DARK MULTIVESICULAR BODY POCKETS IN FETAL AND POSTNATAL NORMAL RAT ALVEOLAR TYPE II CELLS: ULTRASTRUCTURAL STUDY

To investigate the osmiophilic lamellar body (LB) formation in the alveolar type II cells, the authors examined lung tissues from fetal (day 19 of gestation) and postnatal normal rat by electron microscopy. Lamellar body formation was identical in fetal and postnatal rat lungs. Immature dark multivesicular body (MVB) showed incomplete formation of the limiting membrane and contained a cluster of vesicles and an electron-dense granule probably originated from Golgi complex. Dark and light MVBs were seen in alveolar type II cells. However, only dark MVB was involved in the production of LB. First, a drumstick-shaped projection arising from the surface of MVB appeared, which partly trapped cytoplasmic materials such as glycogen granules and ribosomes. The projection was ultimately fused with the MVB surface to form an MVB pocket described here for the first time. In prelamellar body, lamellar structures appeared to provide a communicative MVB pocket and even found in a drumstick-shape. Mature LBs showed disappearance of multivesicles, dense matrix, and MVB pocket. Thus, lamellar body formation might not only need MVB materials, but also glycogen granules and ribosomes in the MVB pocket.

Diffuse Clusters of Vesicles on the Face and Extremities of a 10‐Month‐Old Girl

Pediatric DermatologyVolume 26, Issue 6 p. 747-748 Diffuse Clusters of Vesicles on the Face and Extremities of a 10-Month-Old Girl Renee D. Straub M.D., Renee D. Straub M.D. Department of Dermatology, Penn State/Milton S. Hershey Medical Center, Hershey, PennsylvaniaSearch for more papers by this authorHeather L. Salvaggio M.D., Heather L. Salvaggio M.D. Department of Dermatology, Penn State/Milton S. Hershey Medical Center, Hershey, PennsylvaniaSearch for more papers by this authorDavid R. Adams M.D., PharmD., David R. Adams M.D., PharmD. Department of Dermatology, Penn State/Milton S. Hershey Medical Center, Hershey, PennsylvaniaSearch for more papers by this authorAndrea L. Zaenglein M.D., Andrea L. Zaenglein M.D. Departments of Dermatology and Pediatrics, Penn State/Milton S. Hershey Medical Center, Hershey, PennsylvaniaSearch for more papers by this author Renee D. Straub M.D., Renee D. Straub M.D. Department of Dermatology, Penn State/Milton S. Hershey Medical Center, Hershey, PennsylvaniaSearch for more papers by this authorHeather L. Salvaggio M.D., Heather L. Salvaggio M.D. Department of Dermatology, Penn State/Milton S. Hershey Medical Center, Hershey, PennsylvaniaSearch for more papers by this authorDavid R. Adams M.D., PharmD., David R. Adams M.D., PharmD. Department of Dermatology, Penn State/Milton S. Hershey Medical Center, Hershey, PennsylvaniaSearch for more papers by this authorAndrea L. Zaenglein M.D., Andrea L. Zaenglein M.D. Departments of Dermatology and Pediatrics, Penn State/Milton S. Hershey Medical Center, Hershey, PennsylvaniaSearch for more papers by this author First published: 21 December 2009 https://doi.org/10.1111/j.1525-1470.2009.01025.xCitations: 2 Address correspondence to Andrea L. Zaenglein, M.D., Department of Dermatology, HU14, Penn State/Milton S. Hershey Medical Center, 500 University Drive, Hershey, PA 17033, or e-mail: [email protected]. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article.Citing Literature Volume26, Issue6November/December 2009Pages 747-748 RelatedInformation

Filamin A Regulates Caveolae Internalization and Trafficking in Endothelial Cells

Transcytosis via caveolae is critical for maintaining vascular homeostasis by regulating the tissue delivery of macromolecules, hormones, and lipids. In the present study, we test the hypothesis that interactions between F-actin cross-linking protein filamin A and caveolin-1 facilitate the internalization and trafficking of caveolae. Small interfering RNA-mediated knockdown of filamin A, but not filamin B, reduced the uptake and transcytosis of albumin by approximately 35 and 60%, respectively, without altering the actin cytoskeletal structure or cell-cell adherens junctions. Mobility of both intracellular caveolin-1-green fluorescent protein (GFP)-labeled vesicles measured by fluorescence recovery after photobleaching and membrane-associated vesicles measured by total internal reflection-fluorescence microscopy was decreased in cells with reduced filamin A expression. In addition, in melanoma cells that lack filamin A (M2 cells), the majority of caveolin-1-GFP was localized on the plasma membrane, whereas in cells in which filamin A expression was reconstituted (A7 cells and M2 cells transfected with filamin A-RFP), caveolin-1-GFP was concentrated in intracellular vesicles. Filamin A association with caveolin-1 in endothelial cells was confirmed by cofractionation of these proteins in density gradients, as well as by coimmunoprecipitation. Moreover, this interaction was enhanced by Src activation, associated with increased caveolin-1 phosphorylation, and blocked by Src inhibition. Taken together, these data suggest that filamin A association with caveolin-1 promotes caveolae-mediated transport by regulating vesicle internalization, clustering, and trafficking.

Cannabinoid receptor 1 signalling dampens activity and mitochondrial transport in networks of enteric neurones

Cannabinoid (CB) receptors are expressed in the enteric nervous system (ENS) and CB(1) receptor activity slows down motility and delays gastric emptying. This receptor system has become an important target for GI-related drug development such as in obesity treatment. The aim of the study was to investigate how CB(1) ligands and antagonists affect ongoing activity in enteric neurone networks, modulate synaptic vesicle cycling and influence mitochondrial transport in nerve processes. Primary cultures of guinea-pig myenteric neurones were loaded with different fluorescent markers: Fluo-4 to measure network activity, FM1-43 to image synaptic vesicles and Mitotracker green to label mitochondria. Synaptic vesicle cluster density was assessed by immunohistochemistry and expression of CB(1) receptors was confirmed by RT-PCR. Spontaneous network activity, displayed by both excitatory and inhibitory neurones, was significantly increased by CB(1) receptor antagonists (AM-251 and SR141716), abolished by CB(1) activation (methanandamide, mAEA) and reduced by two different inhibitors (arachidonylamide serotonin, AA-5HT and URB597) of fatty acid amide hydrolase. Antagonists reduced the number of synaptic vesicles that were recycled during an electrical stimulus. CB(1) agonists (mAEA and WIN55,212) reduced and antagonists enhanced the fraction of transported mitochondria in enteric nerve fibres. We found immunohistochemical evidence for an enhancement of synaptophysin-positive release sites with SR141716, while WIN55,212 caused a reduction. The opposite effects of agonists and antagonists suggest that enteric nerve signalling is under the permanent control of CB(1) receptor activity. Using inhibitors of the endocannabinoid degrading enzyme, we were able to show there is endogenous production of a CB ligand in the ENS.

Mechanical tension contributes to clustering of neurotransmitter vesicles at presynaptic terminals

Memory and learning in animals are mediated by neurotransmitters that are released from vesicles clustered at the synapse. As a synapse is used more frequently, its neurotransmission efficiency increases, partly because of increased vesicle clustering in the presynaptic neuron. Vesicle clustering has been believed to result primarily from biochemical signaling processes that require the connectivity of the presynaptic terminal with the cell body, the central nervous system, and the postsynaptic cell. Our in vivo experiments on the embryonic Drosophila nervous system show that vesicle clustering at the neuromuscular presynaptic terminal depends on mechanical tension within the axons. Vesicle clustering vanishes upon severing the axon from the cell body, but is restored when mechanical tension is applied to the severed end of the axon. Clustering increases when intact axons are stretched mechanically by pulling the postsynaptic muscle. Using micro mechanical force sensors, we find that embryonic axons that have formed neuromuscular junctions maintain a rest tension of approximately 1 nanonewton. If the rest tension is perturbed mechanically, axons restore the rest tension either by relaxing or by contracting over a period of approximately 15 min. Our results suggest that neuromuscular synapses employ mechanical tension as a signal to modulate vesicle accumulation and synaptic plasticity.

Mouse Norovirus Replication Is Associated with Virus-Induced Vesicle Clusters Originating from Membranes Derived from the Secretory Pathway

Human noroviruses (family Caliciviridae) are the leading cause of nonbacterial gastroenteritis worldwide. Despite the prevalence of these viruses within the community, the study of human norovirus has largely been hindered due to the inability to cultivate the viruses ex vivo and the lack of a small-animal model. In 2003, the discovery of a novel murine norovirus (MNV-1) and the identification of the tropism of MNV-1 for cells of a mononuclear origin led to the establishment of the first norovirus tissue culture system. Like other positive-sense RNA viruses, MNV-1 replication is associated with host membranes, which undergo significant rearrangement during infection. We characterize here the subcellular localization of the MNV-1 open reading frame 1 proteins and viral double-stranded RNA (dsRNA). Over the course of infection, dsRNA and the MNV-1 RNA-dependent RNA polymerase (NS7) were observed to proliferate from punctate foci located in the perinuclear region. All of the MNV-1 open reading frame 1 proteins were observed to colocalize with dsRNA during the course of infection. The MNV-1 replication complex was immunolocalized to virus-induced vesicle clusters formed in the cytoplasm of infected cells. Both dsRNA and MNV-1 NS7 were observed to localize to the limiting membrane of the individual clusters by cryo-immunoelectron microscopy. We show that the MNV-1 replication complex initially associates with membranes derived from the endoplasmic reticulum, trans-Golgi apparatus, and endosomes. In addition, we show that MNV-1 replication is insensitive to the fungal metabolite brefeldin A and consistently does not appear to recruit coatomer protein complex I (COPI) or COPII component proteins during replication. These data provide preliminary insights into key aspects of replication of MNV-1, which will potentially further our understanding of the pathogenesis of noroviruses and aid in the identification of potential targets for drug development.

Scribble Interacts with β-Catenin to Localize Synaptic Vesicles to Synapses

An understanding of how synaptic vesicles are recruited to and maintained at presynaptic compartments is required to discern the molecular mechanisms underlying presynaptic assembly and plasticity. We have previously demonstrated that cadherin-beta-catenin complexes cluster synaptic vesicles at presynaptic sites. Here we show that scribble interacts with the cadherin-beta-catenin complex to coordinate vesicle localization. Scribble and beta-catenin are colocalized at synapses and can be coimmunoprecipitated from neuronal lysates, indicating an interaction between scribble and beta-catenin at the synapse. Using an RNA interference approach, we demonstrate that scribble is important for the clustering of synaptic vesicles at synapses. Indeed, in scribble knockdown cells, there is a diffuse distribution of synaptic vesicles along the axon, and a deficit in vesicle recycling. Despite this, synapse number and the distribution of the presynaptic active zone protein, bassoon, remain unchanged. These effects largely phenocopy those observed after ablation of beta-catenin. In addition, we show that loss of beta-catenin disrupts scribble localization in primary neurons but that the localization of beta-catenin is not dependent on scribble. Our data supports a model by which scribble functions downstream of beta-catenin to cluster synaptic vesicles at developing synapses.