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Applied and Environmental Microbiology Dec 2022The exchange of bacterial extracellular vesicles facilitates molecular exchange between cells, including the horizontal transfer of genetic material. Given the...
The exchange of bacterial extracellular vesicles facilitates molecular exchange between cells, including the horizontal transfer of genetic material. Given the implications of such transfer events on cell physiology and adaptation, some bacterial cells have likely evolved mechanisms to regulate vesicle exchange. Past work has identified mechanisms that influence the formation of extracellular vesicles, including the production of small molecules that modulate membrane structure; however, whether these mechanisms also modulate vesicle uptake and have an overall impact on the rate of vesicle exchange is unknown. Here, we show that membrane-binding molecules produced by microbes influence both the formation and uptake of extracellular vesicles and have the overall impact of increasing the vesicle exchange rate within a bacterial coculture. In effect, production of compounds that increase vesicle exchange rates encourage gene exchange between neighboring cells. The ability of several membrane-binding compounds to increase vesicle exchange was demonstrated. Three of these compounds, nisin, colistin, and polymyxin B, are antimicrobial peptides added at sub-inhibitory concentrations. These results suggest that a potential function of exogenous compounds that bind to membranes may be the regulation of vesicle exchange between cells. The exchange of bacterial extracellular vesicles is one route of gene transfer between bacteria, although it was unclear if bacteria developed strategies to modulate the rate of gene transfer within vesicles. In eukaryotes, there are many examples of specialized molecules that have evolved to facilitate the production, loading, and uptake of vesicles. Recent work with bacteria has shown that some small molecules influence membrane curvature and induce vesicle formation. Here, we show that similar compounds facilitate vesicle uptake, thereby increasing the overall rate of vesicle exchange within bacterial populations. The addition of membrane-binding compounds, several of them antibiotics at subinhibitory concentrations, to a bacterial coculture increased the rate of horizontal gene transfer via vesicle exchange.
Topics: Bacteria; Gene Transfer, Horizontal; Extracellular Vesicles; Membranes; Eukaryota
PubMed: 36342184
DOI: 10.1128/aem.01346-22 -
Biophysical Journal Sep 2017Fusion between two lipid bilayers is one of the central processes in cell biology, playing a key role in endocytosis, exocytosis, and vesicle transport. We have...
Fusion between two lipid bilayers is one of the central processes in cell biology, playing a key role in endocytosis, exocytosis, and vesicle transport. We have previously developed a model system that uses the hybridization of complementary DNA strands to model the formation of the SNARE four-helix bundle that mediates synaptic vesicle fusion and used it to study vesicle fusion to a tethered lipid bilayer. Using single vesicle assays, 70% of observed fusion events in the DNA-lipid system are arrested at the hemifusion stage, whereas only 5% eventually go to full fusion. This may be because the diglycerol ether that anchors the DNA in the membrane spans only half the bilayer: upon hemifusion and mixing of the outer leaflets, the DNA-lipid is free to diffuse into the target membrane and away from the vesicle. Here, we test the hypothesis that the length of the membrane anchor may impact the outcome by comparing single leaflet-spanning DNA-lipid mediated vesicle fusion with fusion mediated by DNA anchored by solanesol, a C45 isoprenoid of sufficient length to span the bilayer. When the solanesol anchor was present on the incoming vesicles, target membrane, or both, ∼2-3 times as much full fusion was observed as in the DNA-lipid mediated system, as measured by lipid mixing or content transfer. These results indicate that a transmembrane anchor increases the efficiency of full fusion.
Topics: Animals; Chickens; Chromatography, High Pressure Liquid; DNA; Lipid Bilayers; Membrane Fusion; Microscopy, Fluorescence; SNARE Proteins; Terpenes; Transport Vesicles
PubMed: 28647061
DOI: 10.1016/j.bpj.2017.05.034 -
The Journal of Physiology Oct 2018Synaptic transmission relies on the recruitment of neurotransmitter-filled vesicles to presynaptic release sites. Increased intracellular calcium buffering slows the...
KEY POINTS
Synaptic transmission relies on the recruitment of neurotransmitter-filled vesicles to presynaptic release sites. Increased intracellular calcium buffering slows the recovery from synaptic depression, suggesting that vesicle recruitment is a calcium-dependent process. However, the molecular mechanisms of vesicle recruitment have only been investigated at some synapses. We investigate the role of calcium in vesicle recruitment at the cerebellar mossy fibre to granule cell synapse. We find that increased intracellular calcium buffering slows the recovery from depression following physiological stimulation. However, the recovery is largely resistant to perturbation of the molecular pathways previously shown to mediate calcium-dependent vesicle recruitment. Furthermore, we find two pools of vesicles with different recruitment speeds and show that models incorporating two pools of vesicles with different calcium-independent recruitment rates can explain our data. In this framework, increased calcium buffering prevents the release of intrinsically fast-recruited vesicles but does not change the vesicle recruitment rates themselves.
ABSTRACT
During sustained synaptic transmission, recruitment of new transmitter-filled vesicles to the release site counteracts vesicle depletion and thus synaptic depression. An elevated intracellular Ca concentration has been proposed to accelerate the rate of vesicle recruitment at many synapses. This conclusion is often based on the finding that increased intracellular Ca buffering slows the recovery from synaptic depression. However, the molecular mechanisms of the activity-dependent acceleration of vesicle recruitment have only been analysed at some synapses. Using physiological stimulation patterns in postsynaptic recordings and step depolarizations in presynaptic bouton recordings, we investigate vesicle recruitment at cerebellar mossy fibre boutons. We show that increased intracellular Ca buffering slows recovery from depression dramatically. However, pharmacological and genetic interference with calmodulin or the calmodulin-Munc13 pathway, which has been proposed to mediate Ca -dependence of vesicle recruitment, barely affects vesicle recovery from depression. Furthermore, we show that cerebellar mossy fibre boutons have two pools of vesicles: rapidly fusing vesicles that recover slowly and slowly fusing vesicles that recover rapidly. Finally, models adopting such two pools of vesicles with Ca -independent recruitment rates can explain the slowed recovery from depression upon increased Ca buffering. Our data do not rule out the involvement of the calmodulin-Munc13 pathway during stronger stimuli or other molecular pathways mediating Ca -dependent vesicle recruitment at cerebellar mossy fibre boutons. However, we show that well-established two-pool models predict an apparent Ca -dependence of vesicle recruitment. Thus, previous conclusions of Ca -dependent vesicle recruitment based solely on increased intracellular Ca buffering should be considered with caution.
Topics: Action Potentials; Animals; Calcium; Calmodulin; Cerebellar Cortex; Excitatory Postsynaptic Potentials; Female; Male; Mice; Mice, Inbred C57BL; Nerve Fibers; Presynaptic Terminals; Synapses; Synaptic Transmission; Synaptic Vesicles
PubMed: 29928766
DOI: 10.1113/JP275911 -
PLoS Computational Biology Feb 2021Biomineralization is the process by which organisms use minerals to harden their tissues and provide them with physical support. Biomineralizing cells concentrate the...
Biomineralization is the process by which organisms use minerals to harden their tissues and provide them with physical support. Biomineralizing cells concentrate the mineral in vesicles that they secret into a dedicated compartment where crystallization occurs. The dynamics of vesicle motion and the molecular mechanisms that control it, are not well understood. Sea urchin larval skeletogenesis provides an excellent platform for investigating the kinetics of mineral-bearing vesicles. Here we used lattice light-sheet microscopy to study the three-dimensional (3D) dynamics of calcium-bearing vesicles in the cells of normal sea urchin embryos and of embryos where skeletogenesis is blocked through the inhibition of Vascular Endothelial Growth Factor Receptor (VEGFR). We developed computational tools for displaying 3D-volumetric movies and for automatically quantifying vesicle dynamics. Our findings imply that calcium vesicles perform an active diffusion motion in both, calcifying (skeletogenic) and non-calcifying (ectodermal) cells of the embryo. The diffusion coefficient and vesicle speed are larger in the mesenchymal skeletogenic cells compared to the epithelial ectodermal cells. These differences are possibly due to the distinct mechanical properties of the two tissues, demonstrated by the enhanced f-actin accumulation and myosinII activity in the ectodermal cells compared to the skeletogenic cells. Vesicle motion is not directed toward the biomineralization compartment, but the vesicles slow down when they approach it, and probably bind for mineral deposition. VEGFR inhibition leads to an increase of vesicle volume but hardly changes vesicle kinetics and doesn't affect f-actin accumulation and myosinII activity. Thus, calcium vesicles perform an active diffusion motion in the cells of the sea urchin embryo, with diffusion length and speed that inversely correlate with the strength of the actomyosin network. Overall, our studies provide an unprecedented view of calcium vesicle 3D-dynamics and point toward cytoskeleton remodeling as an important effector of the motion of mineral-bearing vesicles.
Topics: Actomyosin; Animals; Biomineralization; Calcium; Computational Biology; Cytoskeleton; Developmental Biology; Diffusion; Ectoderm; Embryo, Nonmammalian; Endocytosis; Fluoresceins; Gene Expression Regulation, Developmental; Kinetics; Motion; Receptors, Vascular Endothelial Growth Factor; Sea Urchins
PubMed: 33617532
DOI: 10.1371/journal.pcbi.1008780 -
Frontiers in Cellular Neuroscience 2014The trigger for synaptic vesicle exocytosis is Ca(2+), which enters the synaptic bouton following action potential stimulation. However, spontaneous release of... (Review)
Review
The trigger for synaptic vesicle exocytosis is Ca(2+), which enters the synaptic bouton following action potential stimulation. However, spontaneous release of neurotransmitter also occurs in the absence of stimulation in virtually all synaptic boutons. It has long been thought that this represents exocytosis driven by fluctuations in local Ca(2+) levels. The vesicles responding to these fluctuations are thought to be the same ones that release upon stimulation, albeit potentially triggered by different Ca(2+) sensors. This view has been challenged by several recent works, which have suggested that spontaneous release is driven by a separate pool of synaptic vesicles. Numerous articles appeared during the last few years in support of each of these hypotheses, and it has been challenging to bring them into accord. We speculate here on the origins of this controversy, and propose a solution that is related to developmental effects. Constitutive membrane traffic, needed for the biogenesis of vesicles and synapses, is responsible for high levels of spontaneous membrane fusion in young neurons, probably independent of Ca(2+). The vesicles releasing spontaneously in such neurons are not related to other synaptic vesicle pools and may represent constitutively releasing vesicles (CRVs) rather than bona fide synaptic vesicles. In mature neurons, constitutive traffic is much dampened, and the few remaining spontaneous release events probably represent bona fide spontaneously releasing synaptic vesicles (SRSVs) responding to Ca(2+) fluctuations, along with a handful of CRVs that participate in synaptic vesicle turnover.
PubMed: 25538561
DOI: 10.3389/fncel.2014.00409 -
FEBS Letters May 2007During vesicular transport, the assembly of the coat complexes and the selection of cargo proteins must be coordinated with the subsequent translocation of vesicles from... (Review)
Review
During vesicular transport, the assembly of the coat complexes and the selection of cargo proteins must be coordinated with the subsequent translocation of vesicles from the donor to an acceptor compartment. Here, we review recent progress toward uncovering the molecular mechanisms that connect transport vesicles to the protein machinery responsible for cytoskeleton-mediated motility. An emerging theme is that vesicle cargo proteins, either directly or through binding interactions with coat proteins, are able to influence cytoskeletal dynamics and motor protein function. Hence, a vesicle's cargo composition may help direct its intracellular motility and targeting.
Topics: Actins; Animals; Clathrin; Cytoskeleton; Endocytosis; Golgi Apparatus; Humans; Kinesins; Microtubules; Molecular Motor Proteins; Myosins; Transport Vesicles
PubMed: 17335816
DOI: 10.1016/j.febslet.2007.01.094 -
Journal of Neurochemistry Dec 1999The recycling of synaptic vesicles in nerve terminals involves multiple steps, underlies all aspects of synaptic transmission, and is a key to understanding the basis of... (Review)
Review
The recycling of synaptic vesicles in nerve terminals involves multiple steps, underlies all aspects of synaptic transmission, and is a key to understanding the basis of synaptic plasticity. The development of styryl dyes as fluorescent molecules that label recycling synaptic vesicles has revolutionized the way in which synaptic vesicle recycling can be investigated, by allowing an examination of processes in neurons that have long been inaccessible. In this review, we evaluate the major aspects of synaptic vesicle recycling that have been revealed and advanced by studies with styryl dyes, focussing upon synaptic vesicle fusion, retrieval, and trafficking. The greatest impact of styryl dyes has been to allow the routine visualization of endocytosis in central nerve terminals for the first time. This has revealed the kinetics of endocytosis, its underlying sequential steps, and its regulation by Ca2+. In studies of exocytosis, styryl dyes have helped distinguish between different modes of vesicle fusion, provided direct support for the quantal nature of exocytosis and endocytosis, and revealed how the probability of exocytosis varies enormously from one nerve terminal to another. Synaptic vesicle labelling with styryl dyes has helped our understanding of vesicle trafficking by allowing better understanding of different synaptic vesicle pools within the nerve terminal, vesicle intermixing, and vesicle clustering at release sites. Finally, the dyes are now being used in innovative ways to reveal further insights into synaptic plasticity.
Topics: Animals; Biological Transport; Calcium Signaling; Endocytosis; Exocytosis; Fluorescent Dyes; Humans; Kinetics; Long-Term Potentiation; Membrane Fusion; Models, Neurological; Nerve Tissue Proteins; Neurotransmitter Agents; Pyridinium Compounds; Quaternary Ammonium Compounds; Synaptic Vesicles
PubMed: 10582580
DOI: 10.1046/j.1471-4159.1999.0732227.x -
Neuroscience Jan 2009Expression of the integral and associated proteins of synaptic vesicles is subject to regulation over time, by region, and in response to activity. The process by which... (Review)
Review
Expression of the integral and associated proteins of synaptic vesicles is subject to regulation over time, by region, and in response to activity. The process by which changes in protein levels and isoforms result in different properties of neurotransmitter release involves protein trafficking to the synaptic vesicle. How newly synthesized proteins are incorporated into synaptic vesicles at the presynaptic bouton is poorly understood. During synaptogenesis, synaptic vesicle proteins sort through the secretory pathway and are transported down the axon in precursor vesicles that undergo maturation to form synaptic vesicles. Changes in protein content of synaptic vesicles could involve the formation of new vesicles that either mix with the previous complement of vesicles or replace them, presumably by their degradation or inactivation. Alternatively, new proteins could individually incorporate into existing synaptic vesicles, changing their functional properties. Glutamatergic vesicles likely express many of the same integral membrane proteins and share certain common mechanisms of biogenesis, recycling, and degradation with other synaptic vesicles. However, glutamatergic vesicles are defined by their ability to package glutamate for release, a property conferred by the expression of a vesicular glutamate transporter (VGLUT). VGLUTs are subject to regional, developmental, and activity-dependent changes in expression. In addition, VGLUT isoforms differ in their trafficking, which may target them to different pathways during biogenesis or after recycling, which may in turn sort them to different vesicle pools. Emerging data indicate that differences in the association of VGLUTs and other synaptic vesicle proteins with endocytic adaptors may influence their trafficking. These observations indicate that independent regulation of synaptic vesicle protein trafficking has the potential to influence synaptic vesicle protein composition, the maintenance of synaptic vesicle pools, and the release of glutamate in response to changing physiological requirements.
Topics: Animals; Central Nervous System; Endocytosis; Glutamic Acid; Humans; Membrane Proteins; Presynaptic Terminals; Protein Transport; Synaptic Transmission; Synaptic Vesicles; Vesicular Glutamate Transport Proteins
PubMed: 18472224
DOI: 10.1016/j.neuroscience.2008.03.029 -
Nature Protocols Jan 2013This protocol describes a single vesicle-vesicle microscopy system to study Ca(2+)-triggered vesicle fusion. Donor vesicles contain reconstituted synaptobrevin and...
This protocol describes a single vesicle-vesicle microscopy system to study Ca(2+)-triggered vesicle fusion. Donor vesicles contain reconstituted synaptobrevin and synaptotagmin-1. Acceptor vesicles contain reconstituted syntaxin and synaptosomal-associated protein 25 (SNAP-25), and they are tethered to a PEG-coated glass surface. Donor vesicles are mixed with the tethered acceptor vesicles and incubated for several minutes at a zero-Ca(2+) concentration, resulting in a collection of single interacting vesicle pairs. The donor vesicles also contain two spectrally distinct fluorophores that allow simultaneous monitoring of temporal changes of the content and membrane. Upon Ca(2+) injection into the sample chamber, our system therefore differentiates between hemifusion and complete fusion of interacting vesicle pairs and determines the temporal sequence of these events on a sub-100-millisecond time scale. Other factors such as complexin can be easily added. Our system is unique in that it monitors both content and lipid mixing and starts from a metastable state of interacting vesicle pairs before Ca(2+) injection.
Topics: Calcium; Image Processing, Computer-Assisted; Lipid Metabolism; Membrane Fusion; Microscopy, Fluorescence; R-SNARE Proteins; Synaptic Vesicles; Synaptosomal-Associated Protein 25; Synaptotagmin I; Synaptotagmins
PubMed: 23222454
DOI: 10.1038/nprot.2012.134 -
Sensors (Basel, Switzerland) 2010A single lipid vesicle can be regarded as an autonomous ultra-miniaturised 3D biomimetic "scaffold" (Ø≥13 nm) ideally suited for reconstitution and interrogation of... (Review)
Review
A single lipid vesicle can be regarded as an autonomous ultra-miniaturised 3D biomimetic "scaffold" (Ø≥13 nm) ideally suited for reconstitution and interrogation of biochemical processes. The enclosing lipid bilayer membrane of a vesicle can be applied for studying binding (protein/lipid or receptor/ligand interactions) or transmembrane events (membrane permeability or ion channel activation) while the aqueous vesicle lumen can be used for confining few or single macromolecules and probe, e.g., protein folding, catalytic pathways of enzymes or more complex biochemical reactions, such as signal transduction cascades. Immobilisation (arraying) of single vesicles on a solid support is an extremely useful technique that allows detailed characterisation of vesicle preparations using surface sensitive techniques, in particular fluorescence microscopy. Surface-based single vesicle arrays allow a plethora of prototypic sensing applications in a high throughput format with high spatial and high temporal resolution. In this review we present a series of applications of single vesicle arrays for screening/sensing of: membrane curvature dependent protein-lipid interactions, bilayer tension, reactions triggered in the vesicle lumen, the activity of transmembrane protein channels and biological membrane fusion reactions.
Topics: Animals; Biomimetics; Biosensing Techniques; Cell Membrane; Humans; Lipid Bilayers; Liposomes; Protein Array Analysis; Protein Binding; Protein Interaction Mapping; Proteins; Surface Properties
PubMed: 22163531
DOI: 10.3390/s101211352