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Methods in Molecular Biology (Clifton,... 2022Synaptic vesicle exocytosis can be monitored with genetically encoded pH sensors in an in vitro fluorescence microscopy setup. Here, we describe a workflow starting with...
Synaptic vesicle exocytosis can be monitored with genetically encoded pH sensors in an in vitro fluorescence microscopy setup. Here, we describe a workflow starting with preparation of a primary cell culture to eventually estimate synaptic vesicle pool sizes based on electrical current-evoked vesicle release, which is reported by the synaptobrevin 2-EGFP fusion protein synapto-pHluorin (spH) that is expressed inside the synaptic vesicle membrane. The readily releasable pool and the recycling pool of synaptic vesicles are released separately in response to electrical stimulation. As vesicle reacidification is blocked in this experimental design, every released vesicle is counted only once. This spH-based approach offers different information than styryl-dye (FM dyes)-based approaches because the total synaptic pool size is measured by an alkalinization step. This provides a normalization constant for quantifying and comparing the synaptic vesicle pool sizes. In addition to investigation of basic research questions, spH-reported vesicle release is valuable to determine presynaptic effects of, e.g., pharmacological drug treatments.
Topics: Exocytosis; Green Fluorescent Proteins; Microscopy, Fluorescence; Synaptic Transmission; Synaptic Vesicles
PubMed: 35099800
DOI: 10.1007/978-1-0716-1916-2_14 -
The EMBO Journal Nov 2021All bacteria produce secreted vesicles that carry out a variety of important biological functions. These extracellular vesicles can improve adaptation and survival by... (Review)
Review
All bacteria produce secreted vesicles that carry out a variety of important biological functions. These extracellular vesicles can improve adaptation and survival by relieving bacterial stress and eliminating toxic compounds, as well as by facilitating membrane remodeling and ameliorating inhospitable environments. However, vesicle production comes with a price. It is energetically costly and, in the case of colonizing pathogens, it elicits host immune responses, which reduce bacterial viability. This raises an interesting paradox regarding why bacteria produce vesicles and begs the question as to whether the benefits of producing vesicles outweigh their costs. In this review, we discuss the various advantages and disadvantages associated with Gram-negative and Gram-positive bacterial vesicle production and offer perspective on the ultimate score. We also highlight questions needed to advance the field in determining the role for vesicles in bacterial survival, interkingdom communication, and virulence.
Topics: Animals; Extracellular Vesicles; Gene Expression; Gram-Negative Bacteria; Gram-Positive Bacteria; Host-Parasite Interactions; Humans; Immunity, Innate; Microbial Viability; Quorum Sensing; Secretory Vesicles; Virulence; Virulence Factors
PubMed: 34636061
DOI: 10.15252/embj.2021108174 -
FEBS Letters Nov 2018In presynaptic nerve terminals, synaptic vesicles are recycled locally via an evolutionarily conserved process that ensures maintenance of neurotransmission as well as... (Review)
Review
In presynaptic nerve terminals, synaptic vesicles are recycled locally via an evolutionarily conserved process that ensures maintenance of neurotransmission as well as structural integrity of synapses. Temperature is a key environmental factor that impacts critical steps involved in fusion, endocytosis and transport in different vesicle trafficking pathways. In neurons, temperature changes have been shown to impact synaptic vesicle recycling and synaptic efficacy. But contrary to non-neuronal systems, the temperature dependence of the steps involved in fusion, endocytosis and recycling of synaptic vesicles in presynaptic terminals is not completely understood, and the existing data remain highly debated. In this Review, we discuss the implications of biophysical, biochemical and functional findings on temperature dependence of membrane retrieval in multiple systems. We propose that systematic investigation of the temperature dependence of the presynaptic vesicle trafficking process can provide novel insight into poorly understood mechanisms that govern synaptic vesicle trafficking under diverse physiological conditions.
Topics: Animals; Endocytosis; Humans; Neurons; Presynaptic Terminals; Synapses; Synaptic Transmission; Synaptic Vesicles; Temperature; Time Factors
PubMed: 30311950
DOI: 10.1002/1873-3468.13268 -
Biotechnology Advances 2022Bacterial membrane vesicles (BMVs) are cupped-shaped structures formed by bacteria in response to environmental stress, genetic alteration, antibiotic exposure, and... (Review)
Review
Bacterial membrane vesicles (BMVs) are cupped-shaped structures formed by bacteria in response to environmental stress, genetic alteration, antibiotic exposure, and others. Due to the structural similarities shared with the producer organism, they can retain certain characteristics like stimulating immune responses. They are also able to carry molecules for long distances, without changes in the concentration and integrity of the molecule. Bacteria originally secrete membrane vesicles for gene transfer, excretion, cell to cell interaction, pathogenesis, and protection against phages. These functions are unique and have several innovative applications in the pharmaceutical industry that have attracted both scientific and commercial interest.This led to the development of efficient methods to artificially stimulate vesicle production, purification, and manipulation in the lab at nanoscales. Also, for specific applications, engineering methods to impart pathogen antigens against specific diseases or customization as cargo vehicles to deliver payloads to specific cells have been reported. Many applications of BMVs are in cancer drugs, vaccines, and adjuvant development with several candidates in clinical trials showing promising results. Despite this, applications in therapy and commercialization stay timid probably due to some challenges one of which is the poor understanding of biogenesis mechanisms. Nevertheless, so far, BMVs seem to be a reliable and cost-efficient technology with several therapeutic applications. Research toward characterizing more membrane vesicles, genetic engineering, and nanotechnology will enable the scope of applications to widen. This might include solutions to other currently faced medical and healthcare-related challenges.
Topics: Anti-Bacterial Agents; Antineoplastic Agents; Bacteria; Bacterial Physiological Phenomena; Extracellular Vesicles
PubMed: 34793882
DOI: 10.1016/j.biotechadv.2021.107869 -
Current Opinion in Cell Biology Feb 2023In eukaryotic cells, the budding and fusion of intracellular transport vesicles is carefully orchestrated in space and time. Locally, a vesicle's source compartment, its... (Review)
Review
In eukaryotic cells, the budding and fusion of intracellular transport vesicles is carefully orchestrated in space and time. Locally, a vesicle's source compartment, its cargo, and its destination compartment are controlled by dynamic multi-protein specificity modules. Globally, vesicle constituents must be recycled to ensure homeostasis of compartment compositions. The emergence of a novel vesicle pathway therefore requires new specificity modules as well as new recycling routes. Here, we review recent research on local (molecular) constraints on gene module duplication and global (cellular) constraints on intracellular recycling. By studying the evolution of vesicle traffic, we may discover general principles of how complex traits arise via multiple intermediate steps.
Topics: Organelles; Transport Vesicles; Biological Transport; Eukaryotic Cells; Proteins
PubMed: 36610080
DOI: 10.1016/j.ceb.2022.102151 -
The FEBS Journal Jul 2023Migrasomes comprise a recently identified unique type of extracellular vesicle (EV) containing varying numbers of small vesicles. However, the final fate of these small...
Migrasomes comprise a recently identified unique type of extracellular vesicle (EV) containing varying numbers of small vesicles. However, the final fate of these small vesicles is still unclear. Here, we report the discovery of EV-like migrasome-derived nanoparticles (MDNPs) that are produced by migrasomes releasing internal vesicles via self-rupture and through a process similar to cell plasma membrane budding. Our results demonstrate that MDNPs have a membrane structure with a typical round-shaped morphology and have the characteristic markers of migrasomes, but do not present the markers of EVs from the cell culture supernatant. More importantly, we also show that MDNPs are loaded with a large number of microRNAs different from those found in migrasomes and EVs. Our results provide evidence that migrasomes can produce EV-like nanoparticles. These findings have important implications for understanding the unknown biological functions of migrasomes.
Topics: MicroRNAs; Extracellular Vesicles
PubMed: 36808246
DOI: 10.1111/febs.16756 -
Reproduction, Fertility, and Development Mar 2017The literature on extracellular vesicles consists of rapidly expanding and often contradictory information. In this paper we attempt to review what is currently known... (Review)
Review
The literature on extracellular vesicles consists of rapidly expanding and often contradictory information. In this paper we attempt to review what is currently known regarding extracellular vesicles released specifically from human placental syncytiotrophoblast cells with a focus on the common but complex pregnancy-associated syndrome pre-eclampsia, where the level of syncytiotrophoblast extracellular vesicle release is significantly increased. We review common methods for syncytiotrophoblast extracellular vesicle derivation and isolation and we discuss the cargo of syncytiotrophoblast extracellular vesicles including proteins, RNA and lipids and their possible functions. A meta-analysis of available trophoblast-derived extracellular vesicle proteomic datasets revealed only three proteins in common: albumin, fibronectin-1 and plasminogen activator inhibitor-1, suggesting some variability in vesicle cargo, most likely reflecting stage and cell type of origin. We discuss the possible sources of variability that may have led to the low number of common markers, which has led us to speculate that markers and density in common use may not be strict criteria for identifying and isolating placenta-derived exosomes.
Topics: Endothelial Cells; Exosomes; Extracellular Vesicles; Female; Humans; Placenta; Pregnancy; Proteomics; Trophoblasts
PubMed: 26411402
DOI: 10.1071/RD15143 -
Soft Matter Mar 2020Lipid vesicles are widely used as model systems to study biological membranes. The self-assembly of such vesicles into vesicle pairs provides further opportunity to...
Lipid vesicles are widely used as model systems to study biological membranes. The self-assembly of such vesicles into vesicle pairs provides further opportunity to study interactions between membranes. However, formation of vesicle pairs, while subsequently keeping their colloidal stability intact, is challenging. Here, we report on three strategies that lead to stable finite-sized aggregates of phospholipid vesicles: (i) vesicles containing biotinylated lipids are coupled together with streptavidin, (ii) bridging attraction is exploited by adding cationic polymers (polylysine) to negatively charged vesicles, and (iii) temperature as a control parameter is used for the aggregation of vesicles mixed with a thermo-sensitive surfactant. While each strategy has its own advantages and disadvantages for vesicle pair formation, the latter strategy additionally shows reversible limited aggregation: above the LCST of pNIPAm, vesicle pairs are formed, while below the LCST, single vesicles prevail. Mixing protocols were assessed by dynamic and static light scattering as well as fluorescence correlation spectroscopy to determine under which conditions vesicle pairs dominate the aggregate size distribution. We have strong indications that without subsequent perturbation, the individual vesicles remain intact and no fusion or leakage between vesicles occurs after vesicle pairs have formed.
Topics: Diffusion; Kinetics; Liposomes; Phospholipids; Polylysine
PubMed: 32064491
DOI: 10.1039/c9sm01692a -
Frontiers in Endocrinology 2017MicroRNAs (miRNAs) are short non-coding RNAs that posttranscriptionally regulate gene expression inside the cell. Extracellular circulating miRNAs are also observed... (Review)
Review
MicroRNAs (miRNAs) are short non-coding RNAs that posttranscriptionally regulate gene expression inside the cell. Extracellular circulating miRNAs are also observed outside the cell, but their origin is poorly understood. Recently, miRNA has been shown to be exocytosed by vesicle fusion; this observation demonstrates that vesicle-free miRNAs are secreted from neuroendocrine cells, in a manner similar to hormone secretion. miRNAs are stored in large dense-core vesicles together with catecholamines, then released by vesicle fusion in response to stimulation; in this way, vesicle-free miRNA may regulate cell-to-cell communication including the regulation of gene expression and cellular signaling. Therefore, miRNA has been suggested to function as a hormone; i.e., a ribomone (ribonucleotide + hormone). This review focuses on the mechanisms by which vesicle-free miRNAs are secreted from neuroendocrine cells and will discuss potential functions of vesicle-free miRNAs and how vesicle-free miRNAs regulate cell-to-cell communication.
PubMed: 29312145
DOI: 10.3389/fendo.2017.00355 -
Advances in Experimental Medicine and... 2020Primary diseases of the seminal vesicles (SV) are very rare entities.Nonneoplastic lesions of the seminal vesicles include amyloidosis, inflammation, calcification and...
Primary diseases of the seminal vesicles (SV) are very rare entities.Nonneoplastic lesions of the seminal vesicles include amyloidosis, inflammation, calcification and calculi, radiation-induced changes, and basal cell proliferation.Seminal vesicles are frequently involved by tumors originating elsewhere, in particular by prostatic adenocarcinoma, urothelial carcinoma, and rectal adenocarcinoma. On the contrary, primary tumors of the seminal vesicles are rare. Among these, the most common is seminal vesicle adenocarcinoma. To date, less than 100 cases have been reported in literature. Morphologically, primary SV adenocarcinoma is described as a papillary or sheetlike growth architecture, with trabecular and glandular patterns, composed by hobnail tumor cells, frequently with mucinous differentiation. On the contrary, mesenchymal tumors include benign lesions such as leiomyoma, schwannoma, fibroma, paraganglioma, solitary fibrous tumor, cystadenoma, and mixed epithelial and stromal tumors (MEST).Cystadenoma is a rare benign tumor, while MESTs are biphasic tumors with stromal and benign epithelial components. Histological features such as stromal atypia, mitotic activity, nuclear pleomorphism, and tumor necrosis distinct MEST in low-, intermediate-, and high-grade tumors.In recent years, multiple studies reported a link between tumorigenesis and tumor microenvironment. In this regard, the molecular mechanisms connecting prostate cancer (PCa) progression and the host microenvironment have been described and include extracellular matrix (ECM), myofibroblasts, cancer-associated fibroblasts (CAFs), neuroendocrine cells, adipose tissue, and the immune-modulatory cells. Of note, only one study evaluated the influence of seminal vesicle's tumor microenvironment (SVME) on prostate cancer cells so far. Besides, in vivo experiments in NOD/SCID mice clarified the influence of SVME on PCa progression. As such, the injection of PC3 cells into the prostate or the SV resulted in different tumor aggressiveness, and the incidence of retroperitoneal lymph node metastases was significantly higher in mice models receiving SV injection. These findings demonstrated that SVs (rather than the prostate) offer a stimulating tumor microenvironment for growth and invasion of prostate cancer cells.
Topics: Animals; Carcinoma, Transitional Cell; Humans; Male; Mice; Mice, Inbred NOD; Mice, SCID; Prostatic Neoplasms; Seminal Vesicles; Tumor Microenvironment; Urinary Bladder Neoplasms
PubMed: 34185301
DOI: 10.1007/978-3-030-59038-3_19