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Neuron May 2022Presynaptic active zones are molecular machines that control neurotransmitter secretion. They form sites for vesicle docking and priming and couple vesicles to Ca entry...
Presynaptic active zones are molecular machines that control neurotransmitter secretion. They form sites for vesicle docking and priming and couple vesicles to Ca entry for release triggering. The complexity of active zone machinery has made it challenging to determine its mechanisms in release. Simultaneous knockout of the active zone proteins RIM and ELKS disrupts active zone assembly, abolishes vesicle docking, and impairs release. We here rebuild docking, priming, and Ca secretion coupling in these mutants without reinstating active zone networks. Re-expression of RIM zinc fingers recruited Munc13 to undocked vesicles and rendered the vesicles release competent. Action potential triggering of release was reconstituted by docking these primed vesicles to Ca channels through attaching RIM zinc fingers to Caβ4-subunits. Our work identifies an 80-kDa β4-Zn protein that bypasses the need for megadalton-sized secretory machines, establishes that fusion competence and docking are mechanistically separable, and defines RIM zinc finger-Munc13 complexes as hubs for active zone function.
Topics: Action Potentials; Presynaptic Terminals; Synapses; Synaptic Transmission; Synaptic Vesicles
PubMed: 35176221
DOI: 10.1016/j.neuron.2022.01.026 -
ACS Chemical Neuroscience Sep 2019The synaptic vesicle glycoprotein 2 (SV2) family is comprised of three paralogues: SV2A, SV2B, and SV2C. In vertebrates, SV2s are 12-transmembrane proteins present on... (Review)
Review
The synaptic vesicle glycoprotein 2 (SV2) family is comprised of three paralogues: SV2A, SV2B, and SV2C. In vertebrates, SV2s are 12-transmembrane proteins present on every secretory vesicle, including synaptic vesicles, and are critical to neurotransmission. Structural and functional studies suggest that SV2 proteins may play several roles to promote proper vesicular function. Among these roles are their potential to stabilize the transmitter content of vesicles, to maintain and orient the releasable pool of vesicles, and to regulate vesicular calcium sensitivity to ensure efficient, coordinated release of the transmitter. The SV2 family is highly relevant to human health in a number of ways. First, SV2A plays a role in neuronal excitability and as such is the specific target for the antiepileptic drug levetiracetam. SV2 proteins also act as the target by which potent neurotoxins, particularly botulinum, gain access to neurons and exert their toxicity. Both SV2B and SV2C are increasingly implicated in diseases such as Alzheimer's disease and Parkinson's disease. Interestingly, despite decades of intensive research, their exact function remains elusive. Thus, SV2 proteins are intriguing in their potentially diverse roles within the presynaptic terminal, and several recent developments have enhanced our understanding and appreciation of the protein family. Here, we review the structure and function of SV2 proteins as well as their relevance to disease and therapeutic development.
Topics: Animals; Humans; Membrane Glycoproteins; Nerve Tissue Proteins; Nervous System Diseases; Protein Transport; Synaptic Vesicles
PubMed: 31394034
DOI: 10.1021/acschemneuro.9b00351 -
MBio Dec 2022Extracellular vesicles commonly modulate interactions among cellular communities. Recent studies demonstrate that biofilm maturation features, including matrix...
Extracellular vesicles commonly modulate interactions among cellular communities. Recent studies demonstrate that biofilm maturation features, including matrix production, drug resistance, and dispersion, require the delivery of a core protein and carbohydrate vesicle cargo in species. The function of the vesicle cargo for these advanced-phase biofilm characteristics appears to be conserved across species. Mixed-species interactions in mature biofilms indicate that vesicle cargo serves a cooperative role in preserving the community. Here, we define the function of biofilm-associated vesicles for biofilm initiation both within and among five species across the genus. We found similar vesicle cargo functions for several conserved proteins across species, based on the behavior of mutants. Repletion of the adhesion environment with wild-type vesicles returned the community phenotype toward reference levels in intraspecies experiments. However, cross-species vesicle complementation did not restore the wild-type biology and in fact drove the phenotype in the opposite direction for most cross-species interactions. Further study of mixed-species biofilm adhesion and exogenous wild-type vesicle administration similarly demonstrated competitive interactions. Our studies indicate that similar vesicle cargoes contribute to biofilm initiation. However, vesicles from disparate species serve an interference competitive role in mixed- species scenarios. species commonly form mixed-species biofilms with other species and bacteria. In the established biofilm state, vesicle cargo delivers public goods to support the mature community. At biofilm initiation, however, vesicles play a negative role in cross-species interactions, presumably to allow species to gain a survival advantage. These observations and recent reports reveal that vesicle cargo has both cooperative and competitive roles among species, depending on the needs of the community biofilm formation.
Topics: Biofilms; Candida; Extracellular Matrix; Extracellular Vesicles; Candida albicans
PubMed: 36377868
DOI: 10.1128/mbio.02988-22 -
Neuroscience May 2016Neurotransmitters released at synapses activate neighboring astrocytes, which in turn, modulate neuronal activity by the release of diverse neuroactive substances that... (Review)
Review
Neurotransmitters released at synapses activate neighboring astrocytes, which in turn, modulate neuronal activity by the release of diverse neuroactive substances that include classical neurotransmitters such as glutamate, GABA or ATP. Neuroactive substances are released from astrocytes through several distinct molecular mechanisms, for example, by diffusion through membrane channels, by translocation via plasmalemmal transporters or by vesicular exocytosis. Vesicular release regulated by a stimulus-mediated increase in cytosolic calcium involves soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptor (SNARE)-dependent merger of the vesicle membrane with the plasmalemma. Up to 25 molecules of synaptobrevin 2 (Sb2), a SNARE complex protein, reside at a single astroglial vesicle; an individual neuronal, i.e. synaptic, vesicle contains ∼70 Sb2 molecules. It is proposed that this paucity of Sb2 molecules in astrocytic vesicles may determine the slow secretion. In the present essay we shall overview multiple aspects of vesicular architecture and types of vesicles based on their cargo and dynamics in astroglial cells.
Topics: Animals; Astrocytes; Exocytosis; Humans; Membrane Transport Proteins; Synaptic Vesicles; Vesicle-Associated Membrane Protein 2
PubMed: 25727638
DOI: 10.1016/j.neuroscience.2015.02.033 -
Developmental Biology Jun 2020Arl13b is a gene known to regulate ciliogenesis. Functional alterations in this gene's activity have been associated with Joubert syndrome. We found that in Arl13 null...
Arl13b is a gene known to regulate ciliogenesis. Functional alterations in this gene's activity have been associated with Joubert syndrome. We found that in Arl13 null mouse embryos the orientation of the optic cup is inverted, such that the lens is abnormally surrounded by an inverted optic cup whose retina pigmented epithelium is oddly facing the surface ectoderm. Loss of Arl13b leads to the disruption of optic vesicle's patterning and expansion of ventral fates. We show that this phenotype is consequence of miss-regulation of Sonic hedgehog (Shh) signaling and demonstrate that the Arl13b eye phenotype can be rescued by deletion of Gli2, a downstream effector of the Shh pathway. This work identified an unexpected role of primary cilia during the morphogenetic movements required for the formation of the eye.
Topics: ADP-Ribosylation Factors; Animals; Body Patterning; Bone Morphogenetic Protein 4; Cilia; Embryonic Development; Eye; Eye Proteins; Gene Expression Regulation, Developmental; Hedgehog Proteins; Homeodomain Proteins; Humans; Lens, Crystalline; Male; Mice; Mice, Knockout; Morphogenesis; Nerve Tissue Proteins; Organogenesis; Retinal Pigment Epithelium; Signal Transduction; Wnt1 Protein; Zinc Finger Protein Gli2; Homeobox Protein SIX3
PubMed: 32169553
DOI: 10.1016/j.ydbio.2020.02.016 -
Current Protein & Peptide Science 2021The fusion of secretory vesicles with the plasma membrane depends on the assembly of v-SNAREs (VAMP2/synaptobrevin2) and t-SNAREs (SNAP25/syntaxin1) into the SNARE... (Review)
Review
The fusion of secretory vesicles with the plasma membrane depends on the assembly of v-SNAREs (VAMP2/synaptobrevin2) and t-SNAREs (SNAP25/syntaxin1) into the SNARE complex. Vesicles go through several upstream steps, referred to as docking and priming, to gain fusion competence. The vesicular protein synaptotagmin-1 (Syt-1) is the principal Ca sensor for fusion in several central nervous system neurons and neuroendocrine cells and part of the docking complex for secretory granules. Syt-1 binds to the acceptor complex such as synaxin1, SNAP-25 on the plasma membrane to facilitate secretory vesicle docking, and upon Ca-influx, it promotes vesicle fusion. This review assesses the role of the Syt-1 protein involved in the secretory vesicle docking, priming, and fusion.
Topics: Biological Transport; Calcium; Cell Membrane; Membrane Fusion; Neurons
PubMed: 33823763
DOI: 10.2174/1389203722666210325110231 -
Membranes Mar 2023Cells produce nanosized lipid membrane-enclosed vesicles which play important roles in intercellular communication. Interestingly, a certain type of extracellular... (Review)
Review
Cells produce nanosized lipid membrane-enclosed vesicles which play important roles in intercellular communication. Interestingly, a certain type of extracellular vesicle, termed exosomes, share physical, chemical, and biological properties with enveloped virus particles. To date, most similarities have been discovered with lentiviral particles, however, other virus species also frequently interact with exosomes. In this review, we will take a closer look at the similarities and differences between exosomes and enveloped viral particles, with a focus on events taking place at the vesicle or virus membrane. Since these structures present an area with an opportunity for interaction with target cells, this is relevant for basic biology as well as any potential research or medical applications.
PubMed: 37103824
DOI: 10.3390/membranes13040397 -
Methods in Molecular Biology (Clifton,... 2023Commensal microbiota has huge impact on the maintenance of human health, its dysregulation being associated with the development of a plethora of diseases. Release of...
Commensal microbiota has huge impact on the maintenance of human health, its dysregulation being associated with the development of a plethora of diseases. Release of bacterial extracellular vesicles (BEVs) is a fundamental mechanism of systemic microbiome influence on the host organism. Nevertheless, due to the technical challenges of isolation methods, BEV composition and functions remain poorly characterized. Hereby, we describe the up-to-date protocol for isolation of BEV-enriched samples from human feces. Fecal extracellular vesicles (EVs) are purified through the orthogonal implementation of filtration, size-exclusion chromatography (SEC), and density gradient ultracentrifugation. EVs are first separated from bacteria, flagella, and cell debris by size. In the next steps, BEVs are separated from host-derived EVs by density. The quality of vesicle preparation is estimated via immuno-TEM (transmission electron microscopy) for the presence of vesicle-like structures expressing EV markers and via NTA (nanoparticle tracking analysis) for assaying particle concentration and size. Distribution of EVs of human origin in gradient fractions is estimated using antibodies against human exosomal markers with Western blot and ExoView R100 imaging platform. The enrichment for BEVs in vesicle preparation is estimated by Western blot for the presence of bacterial OMVs (outer membrane vesicles) marker and OmpA (outer membrane protein A). Taken together, our study describes a detailed protocol for EV preparation with enrichment for BEVs from feces with a purity level suitable for bioactivity functional assays.
Topics: Humans; Extracellular Vesicles; Microscopy, Electron, Transmission; Feces; Nanoparticles; Bacteria; Ultracentrifugation
PubMed: 37140799
DOI: 10.1007/978-1-0716-3203-1_15 -
The Journal of Neuroscience : the... Aug 2020Retrieval of synaptic vesicles via endocytosis is essential for maintaining sustained synaptic transmission, especially for neurons that fire action potentials at high...
Retrieval of synaptic vesicles via endocytosis is essential for maintaining sustained synaptic transmission, especially for neurons that fire action potentials at high frequencies. However, how neuronal activity regulates synaptic vesicle recycling is largely unknown. Here we report that Na substantially accumulated in the mouse calyx of Held terminals of either sex during repetitive high-frequency spiking. Elevated presynaptic Na accelerated both slow and rapid forms of endocytosis and facilitated endocytosis overshoot, but did not affect the readily releasable pool size, Ca influx, or exocytosis. To examine whether this facilitation of endocytosis is related to the Na-dependent vesicular content change, we dialyzed glutamate into the presynaptic cytosol or blocked the vesicular glutamate uptake with bafilomycin and found that the rate of endocytosis was not affected by regulating the vesicular glutamate content. Endocytosis is critically dependent on intracellular Ca, and the activity of Na/Ca exchanger (NCX) may be altered when the Na gradient is changed. However, neither NCX inhibitor nor change of extracellular Na concentration affected the endocytosis rate. Moreover, two-photon Ca imaging showed that presynaptic Na did not affect the action potential-evoked intracellular Ca transient and decay. Therefore, we revealed a novel mechanism of cytosolic Na in accelerating vesicle endocytosis. During high-frequency synaptic transmission, when large numbers of synaptic vesicles were fused, the rapid buildup of presynaptic cytosolic Na promoted vesicle recycling and sustained synaptic transmission. High-frequency firing neurons are widely distributed in the CNS. A large number of synaptic vesicles are released during high-frequency synaptic transmission; accordingly, synaptic vesicles need to be recycled rapidly to replenish the vesicle pool. Synaptic vesicle exocytosis and endocytosis are tightly coupled, and their coupling is essential for synaptic function and structural stability. We showed here that intracellular Na concentration at the calyx of Held terminal increased rapidly during spike activity and the increased Na accelerated endocytosis. Thus, when large numbers of synaptic vesicles are released during high-frequency synaptic transmission, Na accumulated in terminals and facilitated vesicle recycling. These findings represent a novel cellular mechanism that supports reliable synaptic transmission at high frequency in the CNS.
Topics: Animals; Calcium; Calcium Signaling; Cytoplasm; Endocytosis; Female; Glutamic Acid; Male; Mice; Mice, Inbred C57BL; Sodium; Sodium-Calcium Exchanger; Synaptic Transmission; Synaptic Vesicles
PubMed: 32605936
DOI: 10.1523/JNEUROSCI.0119-20.2020 -
ACS Measurement Science Au Dec 2021In this work, we introduce a novel method for visualization and quantitative measurement of the vesicle opening process by correlation of vesicle impact electrochemical...
In this work, we introduce a novel method for visualization and quantitative measurement of the vesicle opening process by correlation of vesicle impact electrochemical cytometry (VIEC) with confocal microscopy. We have used a fluorophore conjugated to lipids to label the vesicle membrane and manipulate the membrane properties, which appears to make the membrane more susceptible to electroporation. The neurotransmitters inside the vesicles were visualized by use of a fluorescence false neurotransmitter 511 (FFN 511) through accumulation inside the vesicle via the neuronal vesicular monoamine transporter 2 (VMAT 2). Optical and electrochemical measurements of single vesicle electroporation were carried out using an in-house, disk-shaped, gold-modified ITO (Au/ITO) microelectrode device (5 nm thick, 33 μm diameter), which simultaneously acted as an electrode surface for VIEC and an optically transparent surface for confocal microscopy. As a result, the processes of adsorption, electroporation, and opening of single vesicles followed by neurotransmitter release on the Au/ITO surface have been simultaneously visualized and measured. Three opening patterns of single isolated vesicles were frequently observed. Comparing the vesicle opening patterns with their corresponding VIEC spikes, we propose that the behavior of the vesicular membrane on the electrode surface, including the adsorption time, residence time before vesicle opening, and the retention time after vesicle opening, are closely related to the vesicle content and size. Large vesicles with high content tend to adsorb to the electrode faster with higher frequency, followed by a shorter residence time before releasing their content, and their membrane remains on the electrode surface longer compared to the small vesicles with low content. With this approach, we start to unravel the vesicle opening process and to examine the fundamentals of exocytosis, supporting the proposed mechanism of partial or subquantal release in exocytosis.
PubMed: 34939075
DOI: 10.1021/acsmeasuresciau.1c00021