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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 -
Archives of Biochemistry and Biophysics Sep 2021Chemical neurotransmission is the major mechanism of neuronal communication. Neurotransmitters are released from secretory organelles, the synaptic vesicles (SVs) via... (Review)
Review
Chemical neurotransmission is the major mechanism of neuronal communication. Neurotransmitters are released from secretory organelles, the synaptic vesicles (SVs) via exocytosis into the synaptic cleft. Fusion of SVs with the presynaptic plasma membrane is balanced by endocytosis, thus maintaining the presynaptic membrane at steady-state levels. The protein machineries responsible for exo- and endocytosis have been extensively investigated. In contrast, less is known about the role of lipids in synaptic transmission and how the lipid composition of SVs is affected by dynamic exo-endocytotic cycling. Here we summarize the current knowledge about the composition, organization, and function of SV membrane lipids. We also cover lipid biogenesis and maintenance during the synaptic vesicle cycle.
Topics: Animals; Endocytosis; Exocytosis; Humans; Membrane Lipids; Synaptic Membranes; Synaptic Vesicles
PubMed: 34139199
DOI: 10.1016/j.abb.2021.108966 -
The American Journal of Medicine Aug 2023SARS-CoV-2 invades mitochondria of infected cells resulting in disordered metabolism, mitophagy, and abnormal levels of mitochondrial proteins in extracellular vesicles....
BACKGROUND
SARS-CoV-2 invades mitochondria of infected cells resulting in disordered metabolism, mitophagy, and abnormal levels of mitochondrial proteins in extracellular vesicles. Blood extracellular vesicle SARS-CoV-2 proteins and mitochondrial proteins were quantified in COVID-19 to assess possible roles as biomarkers.
METHODS
Total extracellular vesicles were precipitated from blood of age- and gender-matched participants with no infection (n=10), acute COVID-19 (n=16), post-acute sequelae of COVID-19 (PASC or long COVID) (n=30), or post-acute COVID without PASC (n=8) and their extracted proteins quantified by enzyme-linked immunosorbent assays (ELISAs).
RESULTS
Total extracellular vesicle levels of S1 (receptor-binding domain [RBD]) protein were significantly higher in acute infections than in uninfected controls, post-acute infection without PASC, and PASC. Total extracellular vesicle levels of nucleocapsid (N) protein were significantly higher in PASC than in uninfected controls, acute infections, and post-acute infection without PASC. Neither acute levels of S1(RBD) or N proteins predicted progression to PASC. Levels of neither SARS-CoV-2 protein in established PASC correlated with neuropsychiatric manifestations. Significant decreases in total extracellular vesicle levels of the mitochondrial proteins MOTS-c, VDAC-1, and humanin, and elevations of levels of SARM-1 were observed in acutely infected patients who would develop PASC. Significant decreases in total extracellular vesicle levels of MOTS-c and humanin, but not VDAC-1, and elevations of total extracellular vesicle levels of SARM-1 were characteristic of PASC patients with neuropsychiatric manifestations.
CONCLUSIONS
Total extracellular vesicle levels of SARS-CoV-2 proteins in COVID-19 indicate intracellular presence of SARS-CoV-2. Abnormal total extracellular vesicles levels of mitochondrial proteins in acute infections predict a high risk of PASC and later in established PASC are indicative of neuropsychiatric manifestations.
Topics: Humans; COVID-19; SARS-CoV-2; Post-Acute COVID-19 Syndrome; Mitochondrial Proteins; Extracellular Vesicles; Biomarkers; Disease Progression
PubMed: 37072092
DOI: 10.1016/j.amjmed.2023.03.026 -
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 -
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 -
The Journal of Neuroscience : the... Apr 2021Synaptophysin (syp) is a major integral membrane protein of secretory vesicles. Previous work has demonstrated functions for syp in synaptic vesicle cycling,...
Synaptophysin (syp) is a major integral membrane protein of secretory vesicles. Previous work has demonstrated functions for syp in synaptic vesicle cycling, endocytosis, and synaptic plasticity, but the role of syp in the process of membrane fusion during Ca-triggered exocytosis remains poorly understood. Furthermore, although syp resides on both large dense-core and small synaptic vesicles, its role in dense-core vesicle function has received less attention compared with synaptic vesicle function. To explore the role of syp in membrane fusion and dense-core vesicle function, we used amperometry to measure catecholamine release from single vesicles in male and female mouse chromaffin cells with altered levels of syp and the related tetraspanner protein synaptogyrin (syg). Knocking out syp slightly reduced the frequency of vesicle fusion events below wild-type (WT) levels, but knocking out both syp and syg reduced the frequency 2-fold. Knocking out both proteins stabilized initial fusion pores, promoted fusion pore closure (kiss-and-run), and reduced late-stage fusion pore expansion. Introduction of a syp construct lacking its C-terminal dynamin-binding domain in syp knock-outs (KOs) increased the duration and fraction of kiss-and-run events, increased total catecholamine release per event, and reduced late-stage fusion pore expansion. These results demonstrated that syp and syg regulate dense-core vesicle function at multiple stages to initiate fusion, control the choice of mode between full-fusion and kiss-and-run, and influence the dynamics of both initial and late-stage fusion pores. The transmembrane domain (TMD) influences small initial fusion pores, and the C-terminal domain influences large late-stage fusion pores, possibly through an interaction with dynamin. The secretory vesicle protein synaptophysin (syp) is known to function in synaptic vesicle cycling, but its roles in dense-core vesicle functions, and in controlling membrane fusion during Ca-triggered exocytosis remain unclear. The present study used amperometry recording of catecholamine release from endocrine cells to assess the impact of syp and related proteins on membrane fusion. A detailed analysis of amperometric spikes arising from the exocytosis of single vesicles showed that these proteins influence fusion pores at multiple stages and control the choice between kiss-and-run and full-fusion. Experiments with a syp construct lacking its C terminus indicated that the transmembrane domain (TMD) influences the initial fusion pore, while the C-terminal domain influences later stages after fusion pore expansion.
Topics: Animals; Animals, Newborn; Catecholamines; Chromaffin Cells; Dynamins; Electrophysiological Phenomena; Exocytosis; Female; Membrane Fusion; Mice; Mice, Knockout; Pregnancy; Primary Cell Culture; Synaptic Vesicles; Synaptogyrins; Synaptophysin
PubMed: 33664131
DOI: 10.1523/JNEUROSCI.2833-20.2021 -
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 -
The Journal of Biological Chemistry Jun 2023ELKS proteins play a key role in organizing intracellular vesicle trafficking and targeting in both neurons and non-neuronal cells. While it is known that ELKS interacts...
ELKS proteins play a key role in organizing intracellular vesicle trafficking and targeting in both neurons and non-neuronal cells. While it is known that ELKS interacts with the vesicular traffic regulator, the Rab6 GTPase, the molecular basis governing ELKS-mediated trafficking of Rab6-coated vesicles, has remained unclear. In this study, we solved the Rab6B structure in complex with the Rab6-binding domain of ELKS1, revealing that a C-terminal segment of ELKS1 forms a helical hairpin to recognize Rab6B through a unique binding mode. We further showed that liquid-liquid phase separation (LLPS) of ELKS1 allows it to compete with other Rab6 effectors for binding to Rab6B and accumulate Rab6B-coated liposomes to the protein condensate formed by ELKS1. We also found that the ELKS1 condensate recruits Rab6B-coated vesicles to vesicle-releasing sites and promotes vesicle exocytosis. Together, our structural, biochemical, and cellular analyses suggest that ELKS1, via the LLPS-enhanced interaction with Rab6, captures Rab6-coated vesicles from the cargo transport machine for efficient vesicle release at exocytotic sites. These findings shed new light on the understanding of spatiotemporal regulation of vesicle trafficking through the interplay between membranous structures and membraneless condensates.
Topics: Adaptor Proteins, Signal Transducing; Coated Vesicles; Exocytosis; Liposomes; Nerve Tissue Proteins; Neurons; rab GTP-Binding Proteins
PubMed: 37172719
DOI: 10.1016/j.jbc.2023.104808 -
Autophagy Apr 2024In neurons, autophagosome biogenesis occurs mainly in distal axons, followed by maturation during retrograde transport. Autophagosomal growth depends on the supply of...
In neurons, autophagosome biogenesis occurs mainly in distal axons, followed by maturation during retrograde transport. Autophagosomal growth depends on the supply of membrane lipids which requires small vesicles containing ATG9, a lipid scramblase essential for macroautophagy/autophagy. Here, we show that ATG9-containing vesicles are enriched in synapses and resemble synaptic vesicles in size and density. The proteome of ATG9-containing vesicles immuno-isolated from nerve terminals showed conspicuously low levels of trafficking proteins except of the AP2-complex and some enzymes involved in endosomal phosphatidylinositol metabolism. Super resolution microscopy of nerve terminals and isolated vesicles revealed that ATG9-containing vesicles represent a distinct vesicle population with limited overlap not only with synaptic vesicles but also other membranes of the secretory pathway, uncovering a surprising heterogeneity in their membrane composition. Our results are compatible with the view that ATG9-containing vesicles function as lipid shuttles that scavenge membrane lipids from various intracellular membranes to support autophagosome biogenesis.: AP: adaptor related protein complex: ATG2: autophagy related 2; ATG9: autophagy related 9; DNA PAINT: DNA-based point accumulation for imaging in nanoscale topography; DyMIN STED: dynamic minimum stimulated emission depletion; EL: endosome and lysosome; ER: endoplasmic reticulum; GA: Golgi apparatus; iBAQ: intensity based absolute quantification; LAMP: lysosomal-associated membrane protein; M6PR: mannose-6-phosphate receptor, cation dependent; Minflux: minimal photon fluxes; Mito: mitochondria; MS: mass spectrometry; PAS: phagophore assembly site; PM: plasma membrane; Px: peroxisome; RAB26: RAB26, member RAS oncogene family; RAB3A: RAB3A, member RAS oncogene family; RAB5A: RAB5A, member RAS oncogene family; SNARE: soluble N-ethylmaleimide-sensitive-factor attachment receptor; SVs: synaptic vesicles; SYP: synaptophysin; TGN: network; TRAPP: transport protein particle; VTI1: vesicle transport through interaction with t-SNAREs.
Topics: Animals; Autophagy-Related Proteins; Presynaptic Terminals; Synaptic Vesicles; Membrane Proteins; Mice; Autophagy; Endosomes; Vesicular Transport Proteins
PubMed: 37881948
DOI: 10.1080/15548627.2023.2274204 -
Biophysical Reviews Apr 2020Membrane potential plays various key roles in live bacterial and eukaryotic cells. So far, the effects of membrane potential on action of antimicrobial peptides (AMPs)... (Review)
Review
Membrane potential plays various key roles in live bacterial and eukaryotic cells. So far, the effects of membrane potential on action of antimicrobial peptides (AMPs) and cell-penetrating peptides (CPPs) have been examined using cells and small lipid vesicles. However, due to the technical drawbacks of these experiments, the effect of membrane potential on the actions of AMPs and CPPs and the elementary processes of interactions of these peptides with cell membranes and vesicle membranes are not well understood. In this short review, we summarize the results of the effect of membrane potential on the action of an AMP, lactoferricin B (LfcinB), and a CPP, transportan 10 (TP10), in vesicle membranes revealed by the single giant unilamellar vesicle (GUV) method. Parts of the actions and their elementary steps of AMPs and CPPs interacting vesicle membranes under membrane potential are clearly revealed using the single GUV method. The experimental methods and their analysis described here can be used to elucidate the effects of membrane potential on various activities of peptides such as AMPs, CPPs, and proteins. Moreover, GUVs with membrane potential are more suitable as a model of cells or artificial cells, as well as GUVs containing small vesicles.
PubMed: 32152921
DOI: 10.1007/s12551-020-00662-z