ABSTRACT
ICA512/PTPRN is a receptor tyrosine-like phosphatase implicated in the biogenesis and turnover of the insulin secretory granules (SGs) in pancreatic islet beta cells. Previously we found biophysical evidence that its luminal RESP18 homology domain (RESP18HD) forms a biomolecular condensate and interacts with insulin in vitro at close-to-neutral pH, that is, in conditions resembling those present in the early secretory pathway. Here we provide further evidence for the relevance of these findings by showing that at pH 6.8 RESP18HD interacts also with proinsulin-the physiological insulin precursor found in the early secretory pathway and the major luminal cargo of ß-cell nascent SGs. Our light scattering analyses indicate that RESP18HD and proinsulin, but also insulin, populate nanocondensates ranging in size from 15 to 300 nm and 10e2 to 10e6 molecules. Co-condensation of RESP18HD with proinsulin/insulin transforms the initial nanocondensates into microcondensates (size >1 µm). The intrinsic tendency of proinsulin to self-condensate implies that, in the ER, a chaperoning mechanism must arrest its spontaneous intermolecular condensation to allow for proper intramolecular folding. These data further suggest that proinsulin is an early driver of insulin SG biogenesis, in a process in which its co-condensation with RESP18HD participates in their phase separation from other secretory proteins in transit through the same compartments but destined to other routes. Through the cytosolic tail of ICA512, proinsulin co-condensation with RESP18HD may further orchestrate the recruitment of cytosolic factors involved in membrane budding and fission of transport vesicles and nascent SGs.
Subject(s)
Insulin , Proinsulin , Insulin/chemistry , Proinsulin/analysis , Proinsulin/chemistry , Proinsulin/metabolism , Receptor-Like Protein Tyrosine Phosphatases, Class 8/analysis , Receptor-Like Protein Tyrosine Phosphatases, Class 8/metabolism , Secretory Vesicles/chemistry , Secretory Vesicles/metabolismABSTRACT
Characterizing relationships between Zn2+, insulin, and insulin vesicles is of vital importance to the study of pancreatic beta cells. However, the precise content of Zn2+ and the specific location of insulin inside insulin vesicles are not clear, which hinders a thorough understanding of the insulin secretion process and diseases caused by blood sugar dysregulation. Here, we demonstrated the colocalization of Zn2+ and insulin in both single extracellular insulin vesicles and pancreatic beta cells by using an X-ray scanning coherent diffraction imaging (ptychography) technique. We also analyzed the elemental Zn2+ and Ca2+ contents of insulin vesicles using electron microscopy and energy dispersive spectroscopy (EDS) mapping. We found that the presence of Zn2+ is an important characteristic that can be used to distinguish insulin vesicles from other types of vesicles in pancreatic beta cells and that the content of Zn2+ is proportional to the size of insulin vesicles. By using dual-energy contrast X-ray microscopy and scanning transmission X-ray microscopy (STXM) image stacks, we observed that insulin accumulates in the off-center position of extracellular insulin vesicles. Furthermore, the spatial distribution of insulin vesicles and their colocalization with other organelles inside pancreatic beta cells were demonstrated using three-dimensional (3D) imaging by combining X-ray ptychography and an equally sloped tomography (EST) algorithm. This study describes a powerful method to univocally describe the location and quantitative analysis of intracellular insulin, which will be of great significance to the study of diabetes and other blood sugar diseases.
Subject(s)
Insulin-Secreting Cells , Insulin , Secretory Vesicles , Zinc , Animals , Blood Glucose , Cell Line , Insulin/analysis , Insulin/metabolism , Insulin-Secreting Cells/metabolism , Insulin-Secreting Cells/ultrastructure , Rats , Secretory Vesicles/chemistry , Secretory Vesicles/metabolism , Spectrometry, X-Ray Emission , X-Ray Diffraction , Zinc/analysisABSTRACT
BACKGROUND: As an efficient tumor immunotherapy, PD-1 antibody has been gradually used in clinical tumor treatment, but the low response rate and excessive immune response limit its extensive application. RESULTS: Herein, a therapeutic regime for the reinvigoration and activation of the tumor immune microenvironment is introduced to improve the anti-tumor effect of the PD-1 antibody. To comprehensively improve the effect of the immunotherapy and reduce excessive immune response, a biomimetic cascade targeting nanosystem, siRNA@PLOV, which was fused by photothermal sensitive liposomes (PTSLs) and attenuated Salmonella outer membrane vesicles (OMVs), was administered in the tumor therapy for targeting of tumor tissues and T cells within tumor respectively. The fused PLOVs which not only retained the biological character of the OMVs, but also enhanced the drug loading ability. The results demonstrated that the immunogenicity of OMVs and photothermal effects can obviously increase the infiltration of T cells and the silencing of CD38 can effectively improve the T cell cytotoxicity, especially combining with PD-1 antibody. CONCLUSIONS: Interesting, this study revealed that anti-PD-1 administration on the 5th day after siRNA@PLOV treatment had the best performance in killing tumors compared with other groups. In addition, this new therapeutic regime also presents a novel strategy for inducing "vaccine effects", conclusively highlighting its potential in preventing tumor recurrence and improving prognosis.
Subject(s)
Immunotherapy/methods , Neoplasms/therapy , Secretory Vesicles/chemistry , ADP-ribosyl Cyclase 1/antagonists & inhibitors , ADP-ribosyl Cyclase 1/genetics , ADP-ribosyl Cyclase 1/metabolism , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/therapeutic use , Bacterial Outer Membrane/metabolism , Cell Line, Tumor , Humans , Liposomes/chemistry , Mice , Mice, Inbred BALB C , Mice, Inbred ICR , Neoplasms/drug therapy , Programmed Cell Death 1 Receptor/immunology , RNA, Small Interfering/chemistry , RNA, Small Interfering/therapeutic use , Salmonella/metabolism , T-Lymphocytes/cytology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Transplantation, HeterologousABSTRACT
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.
Subject(s)
Extracellular Vesicles/metabolism , Gram-Negative Bacteria/metabolism , Gram-Positive Bacteria/metabolism , Microbial Viability/genetics , Secretory Vesicles/metabolism , Virulence Factors/genetics , Animals , Extracellular Vesicles/chemistry , Gene Expression , Gram-Negative Bacteria/genetics , Gram-Negative Bacteria/growth & development , Gram-Negative Bacteria/pathogenicity , Gram-Positive Bacteria/genetics , Gram-Positive Bacteria/growth & development , Gram-Positive Bacteria/pathogenicity , Host-Parasite Interactions/genetics , Humans , Immunity, Innate , Quorum Sensing/genetics , Secretory Vesicles/chemistry , Virulence , Virulence Factors/metabolismABSTRACT
We recently showed that synaptophysin (Syph) and synapsin (Syn) can induce liquid-liquid phase separation (LLPS) to cluster small synaptic-like microvesicles in living cells which are highly reminiscent of SV cluster. However, as there is no physical interaction between them, the underlying mechanism for their coacervation remains unknown. Here, we showed that the coacervation between Syph and Syn is primarily governed by multivalent pi-cation electrostatic interactions among tyrosine residues of Syph C-terminal (Ct) and positively charged Syn. We found that Syph Ct is intrinsically disordered and it alone can form liquid droplets by interactions among themselves at high concentration in a crowding environment in vitro or when assisted by additional interactions by tagging with light-sensitive CRY2PHR or subunits of a multimeric protein in living cells. Syph Ct contains 10 repeated sequences, 9 of them start with tyrosine, and mutating 9 tyrosine to serine (9YS) completely abolished the phase separating property of Syph Ct, indicating tyrosine-mediated pi-interactions are critical. We further found that 9YS mutation failed to coacervate with Syn, and since 9YS retains Syph's negative charge, the results indicate that pi-cation interactions rather than simple charge interactions are responsible for their coacervation. In addition to revealing the underlying mechanism of Syph and Syn coacervation, our results also raise the possibility that physiological regulation of pi-cation interactions between Syph and Syn during synaptic activity may contribute to the dynamics of synaptic vesicle clustering.
Subject(s)
Secretory Vesicles/chemistry , Synapsins/chemistry , Synaptophysin/chemistry , Amino Acid Substitution , Animals , Buffers , COS Cells , Chlorocebus aethiops , Fluorescence Recovery After Photobleaching , Genes, Reporter , Glycols/pharmacology , Humans , Hydrophobic and Hydrophilic Interactions/drug effects , Ionic Liquids/chemistry , Luminescent Proteins/analysis , Mice , Mutation, Missense , Osmolar Concentration , Phase Transition , Photochemistry , Point Mutation , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/radiation effects , Secretory Vesicles/radiation effects , Static Electricity , Synaptophysin/genetics , Synaptophysin/radiation effects , Time-Lapse Imaging , Tyrosine/chemistry , Red Fluorescent ProteinABSTRACT
Parkinson's disease (PD) is an aging disorder related to vesicle transport dysfunctions and neurotransmitter secretion. Secretory granules (SGs) are large dense-core vesicles for the biosynthesis of neuropeptides and hormones. At present, the involvement of SGs impairment in PD remains unclear. In the current study, we found that the number of SGs in tyrosine hydroxylase-positive neurons and the marker proteins secretogranin III (Scg3) significantly decreased in the substantia nigra and striatum regions of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) exposed mice. Proteomic study of SGs purified from the dopaminergic SH-sy5Y cells under 1-methyl-4-phenylpyridinium (MPP+) treatments (ProteomeXchange PXD023937) identified 536 significantly differentially expressed proteins. The result indicated that disabled lysosome and peroxisome, lipid and energy metabolism disorders are three characteristic features. Protein-protein interaction analysis of 56 secretory proteins and 140 secreted proteins suggested that the peptide processing mediated by chromogranin/secretogranin in SGs was remarkably compromised, accompanied by decreased candidate proteins and peptides neurosecretory protein (VGF), neuropeptide Y, apolipoprotein E, and an increased level of proenkephalin. The current study provided an extensive proteinogram of SGs in PD. It is helpful to understand the molecular mechanisms in the disease.
Subject(s)
Chromogranins/metabolism , Dopaminergic Neurons/metabolism , Parkinson Disease/metabolism , Secretory Vesicles/metabolism , Animals , Apolipoproteins E/genetics , Apolipoproteins E/metabolism , Chromogranins/genetics , Dopaminergic Neurons/chemistry , Humans , Male , Mice , Mice, Inbred C57BL , Neuropeptide Y/genetics , Neuropeptide Y/metabolism , Parkinson Disease/genetics , Proteins/genetics , Proteins/metabolism , Proteomics , Secretory Vesicles/chemistry , Secretory Vesicles/geneticsABSTRACT
The basis of traditional flow cytometry allergy diagnosis is measurement of the expression of basophilic surface activation and/or degranulation markers. Basophils, upon encounter with a specific allergen that cross-links surface FcRI-bound IgE antibodies, not only secrete and release quantifiable bioactive mediators but also upregulate the expression of different markers (e.g., CD63, CD203c) which can be detected by multicolor flow cytometry using specific monoclonal antibodies. Here, we describe a novel technique that relies upon the staining of exteriorized anionic proteoglycans from a basophil granule matrix by cationic fluorescent avidin probes.
Subject(s)
Basophils/immunology , Cell Degranulation , Flow Cytometry/methods , Immunophenotyping/methods , Secretory Vesicles/metabolism , Staining and Labeling/methods , Avidin/chemistry , Basophils/cytology , Basophils/physiology , Biomarkers/analysis , Cells, Cultured , Extracellular Space/chemistry , Extracellular Space/metabolism , Fluoresceins/chemistry , Humans , Proteoglycans/analysis , Secretory Vesicles/chemistryABSTRACT
Complex hierarchical structure governs emergent properties in biopolymeric materials; yet, the material processing involved remains poorly understood. Here, we investigated the multi-scale structure and composition of the mussel byssus cuticle before, during and after formation to gain insight into the processing of this hard, yet extensible metal cross-linked protein composite. Our findings reveal that the granular substructure crucial to the cuticle's function as a wear-resistant coating of an extensible polymer fiber is pre-organized in condensed liquid phase secretory vesicles. These are phase-separated into DOPA-rich proto-granules enveloped in a sulfur-rich proto-matrix which fuses during secretion, forming the sub-structure of the cuticle. Metal ions are added subsequently in a site-specific way, with iron contained in the sulfur-rich matrix and vanadium coordinated by DOPA-catechol in the granule. We posit that this hierarchical structure self-organizes via phase separation of specific amphiphilic proteins within secretory vesicles, resulting in a meso-scale structuring that governs cuticle function.
Subject(s)
Coated Materials, Biocompatible/chemistry , Metalloproteins/chemistry , Mytilus edulis/chemistry , Animal Structures/anatomy & histology , Animal Structures/chemistry , Animal Structures/ultrastructure , Animals , Dihydroxyphenylalanine/chemistry , Imaging, Three-Dimensional , Microscopy, Electron, Scanning , Microscopy, Electron, Transmission , Mytilus edulis/anatomy & histology , Mytilus edulis/ultrastructure , Nanostructures/chemistry , Nanostructures/ultrastructure , Secretory Vesicles/chemistry , Secretory Vesicles/ultrastructureABSTRACT
Synaptic vesicles (SV) contain high concentrations of specific proteins. How these proteins are transported from soma to synapses, and how they become concentrated at SV clusters at presynaptic terminals were examined by immunogold electron microscopy in dissociated rat hippocampal neurons at 3-6 days in culture, a developmental stage when axonal transport of SV proteins is robust. In neuronal somas, labels for the SV integral membrane proteins (synaptophysin, SV2, VAMP/synaptobrevin, and synaptotagmin) were localized at Golgi complexes and other membranous structures that were dispersed in the cytoplasm as individual vesicle/vacuoles. These vesicles/vacuoles became aggregated in axons, with the size of aggregates ranging from 0.2 to 2 µm in length. Pleomorphic vesicle/vacuoles within the aggregate were typically larger (50-300 nm) than SVs, which were uniform in size at ~ 40 nm. These pleomorphic vesicles/vacuoles are probably transport cargos carrying SV integral membrane proteins from the soma, and then are preferentially sorted into axons at early developmental stages. Serial thin sections of young axons indicated that many labeled aggregates were not synaptic, and in fact, some of these axons were without dendritic contacts. In contrast, labels for two SV-associated proteins, synapsin I and α-synuclein, were not localized at the Golgi complexes or associated with membranous structures in the soma, but were dispersed in the cytoplasm. However, these SV-associated proteins became highly concentrated on clusters of SV-like vesicles in axons, and such clusters were already distinctive in axons as early as 3 days in culture. These clusters consisted of ~ 4-30 vesicles in single thin sections, and the vesicles were of a uniform size (~ 40 nm). Serial sectioning analysis showed that these clusters could be part of nascent synapses or exist in axons without any dendritic contact. Importantly, the vesicles were intensely labeled for SV integral membrane proteins as well as SV-associated proteins. Thus, these EM observations reveal that the two groups of proteins, SV integral membrane and SV-associated, proceed through different routes of biosynthesis and axon transport, and are only sorted into the same final compartment, SV clusters, when they are in the axons.
Subject(s)
Hippocampus/cytology , Immunohistochemistry , Nerve Tissue Proteins/analysis , Neurons/chemistry , Synaptic Vesicles/chemistry , Animals , Axonal Transport , Axons/chemistry , Axons/ultrastructure , Cells, Cultured , Golgi Apparatus/chemistry , Golgi Apparatus/ultrastructure , Hippocampus/embryology , Membrane Proteins/analysis , Microscopy, Electron , Neurons/ultrastructure , Protein Transport , Rats , Secretory Vesicles/chemistry , Secretory Vesicles/ultrastructure , Synaptic Vesicles/ultrastructure , Synaptosomal-Associated Protein 25/analysis , Vacuoles/chemistry , Vacuoles/ultrastructureABSTRACT
A fifteen-components model membrane that reflected the 80 % of phospholipids present in Insulin Secretory Granules was obtained and thermodynamic exploitation was performed, through micro-DSC, in order to assess the synergic contributions to the stability of a mixed complex system very close to real membranes. Simpler systems were also stepwise investigated, to complete a previous preliminary study and to highlight a hierarchy of interactions that can be now summarized as phospholipid tail unsaturation > phospholipid tail length > phospholipid headgroup > membrane curvature. In particular, Small Unilamellar Vesicles (SUVs) that consisted in phospholipids with different headgroups (choline, ethanolamine and serine), was step by step considered, following inclusion of sphingomyelins and lysophosphatidylcholines together with a more complete fatty acids distribution characterizing the phospholipid bilayer of the Insulin Secretory Granules. The inclusion of cholesterol was finally considered and the influence of three FFAs (stearic, oleic and elaidic acids) was investigated in comparison with simpler systems, highlighting the magnitude of the effects on such a detailed membrane in the frame of Type 2 Diabetes Mellitus alterations.
Subject(s)
Cell Membrane/chemistry , Insulin/chemistry , Secretory Vesicles/chemistry , Thermodynamics , Cell Membrane/metabolism , Fatty Acids/chemistry , Fatty Acids/metabolism , Insulin/metabolism , Liposomes/chemical synthesis , Liposomes/chemistry , Liposomes/metabolism , Particle Size , Phospholipids/chemistry , Phospholipids/metabolism , Secretory Vesicles/metabolism , Surface PropertiesABSTRACT
Electrochemical techniques with disk and nano-tip electrodes, together with calcium imaging, were used to examine the effect of short-interval repetitive stimuli on both exocytosis and vesicular content in a model cell line. We show that the number of events decreases markedly with repeated stimuli suggesting a depletion of exocytosis machinery. However, repetitive stimuli induce a more stable fusion pore, leading to an increased amount of neurotransmitter release. In contrast, the total neurotransmitter content inside the vesicles decreases after repetitive stimuli, resulting in a higher average release fraction from each event. We suggest a possible mechanism regarding a link between activity-induced plasticity and fraction of release.
Subject(s)
Exocytosis , Neurotransmitter Agents/metabolism , Secretory Vesicles/metabolism , Animals , Biological Transport , Electrochemical Techniques , Neuronal Plasticity , Neurons/chemistry , Neurons/cytology , Neurons/metabolism , PC12 Cells , Rats , Secretory Vesicles/chemistryABSTRACT
Exocytosis involves interactions between secretory vesicles and the plasma membrane. Studying the membrane response is thus critical to understand this important cellular process and to differentiate different mediator release patterns. Here we introduce a label-free optical imaging method to detect the vesicle-membrane-interaction-induced membrane deformation associated with single exocytosis in mast cells. We show that the plasma membrane expands by a few tens of nanometers accompanying each vesicle-release event, but the dynamics of the membrane deformation varies from cell to cell, which reflect different exocytosis processes. Combining the temporal and spatial information allows us to resolve complex vesicle-release processes, such as two vesicle-release events that occur closely in time and location. Simultaneous following a vesicle release with fluorescence and membrane deformation tracking further allows us to determine the propagation speed of the vesicle-release-induced membrane deformation along the cell surface, which has an average value of 5.2 ± 1.8 µm/s.
Subject(s)
Cell Membrane/chemistry , Nanoparticles/chemistry , Optical Imaging , Secretory Vesicles/chemistry , Animals , Exocytosis , Particle Size , Rats , Tumor Cells, CulturedABSTRACT
Outer membrane vesicles (OMVs) are nanostructures of 20-200 nm diameter deriving from the surface of several Gram-negative bacteria. OMVs are emerging as shuttles involved in several mechanisms of communication and environmental adaptation. In this work, OMVs were isolated and characterized from Novosphingobium sp. PP1Y, a Gram-negative non-pathogenic microorganism lacking LPS on the outer membrane surface and whose genome was sequenced and annotated. Scanning electron microscopy performed on samples obtained from a culture in minimal medium highlighted the presence of PP1Y cells embedded in an extracellular matrix rich in vesicular structures. OMVs were collected from the exhausted growth medium during the mid-exponential phase, and purified by ultracentrifugation on a sucrose gradient. Atomic force microscopy, dynamic light scattering and nanoparticle tracking analysis showed that purified PP1Y OMVs had a spherical morphology with a diameter of ca. 150 nm and were homogenous in size and shape. Moreover, proteomic and fatty acid analysis of purified OMVs revealed a specific biochemical "fingerprint", suggesting interesting details concerning their biogenesis and physiological role. Moreover, these extracellular nanostructures do not appear to be cytotoxic on HaCaT cell line, thus paving the way to their future use as novel drug delivery systems.
Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Cell Membrane/metabolism , Secretory Vesicles/chemistry , Secretory Vesicles/enzymology , Sphingomonadaceae/metabolism , Bacterial Outer Membrane Proteins/isolation & purification , Cell Line , Cell Survival/drug effects , Exocytosis , Fatty Acids/analysis , Humans , Keratinocytes/drug effects , Microscopy, Electron, Scanning , Nanoparticles , Peptide Hydrolases/metabolism , Proteomics/methods , Sphingomonadaceae/cytologyABSTRACT
The secretory granules of pancreatic beta cells are specialized organelles responsible for the packaging, storage and secretion of the vital hormone insulin. The insulin secretory granules also contain more than 100 other proteins including the proteases involved in proinsulin-to insulin conversion, other precursor proteins, minor co-secreted peptides, membrane proteins involved in cell trafficking and ion translocation proteins essential for regulation of the intragranular environment. The synthesis, transport and packaging of these proteins into nascent granules must be carried out in a co-ordinated manner to ensure correct functioning of the granule. The process is regulated by many circulating nutrients such as glucose and can change under different physiological states. This chapter discusses the various processes involved in insulin granule biogenesis with a focus on the granule composition in health and disease.
Subject(s)
Cytoplasmic Granules/chemistry , Insulin-Secreting Cells/cytology , Insulin/chemistry , Secretory Vesicles/chemistry , Humans , Proinsulin/chemistryABSTRACT
Electrochemical measurements of exocytosis combined with intracellular vesicle impact electrochemical cytometry have been used to evaluate the effect of an anticancer drug, tamoxifen, on catecholamine release at the single-cell level. Tamoxifen has been used for over 40 years to treat estrogen receptor-positive breast cancers during both early stages of the disease and in the adjuvant setting. Tamoxifen causes memory and cognitive dysfunction, but the reasons for the cognitive impairment and memory problems induced by this anticancer drug are not well-known. We show that tamoxifen, through a nongenomic mechanism, can modulate both exocytosis and vesicle catecholamine storage in a model cell line. The results indicate that exocytosis is inhibited at high concentrations of tamoxifen and is stimulated at low levels. Tamoxifen also elicits a significant concentration-dependent change in total catecholamine content of single vesicles, while sub-nanomolar concentrations of the drug have stimulatory activity on the catecholamine content of vesicles. In addition, it has profound effects on storage at higher concentrations. Tamoxifen also reduces the intracellular free Ca2+ but only at micromolar concentration, by acting on voltage-gated Ca2+ channels, which likely affects neurotransmitter secretion.
Subject(s)
Antineoplastic Agents/pharmacology , Catecholamines/metabolism , Chromaffin Cells/metabolism , Secretory Vesicles/metabolism , Tamoxifen/pharmacology , Animals , Catecholamines/analysis , Chromaffin Cells/chemistry , Chromaffin Cells/drug effects , Dose-Response Relationship, Drug , Exocytosis/drug effects , Exocytosis/physiology , PC12 Cells , Rats , Secretory Vesicles/chemistryABSTRACT
Pore-spanning membranes (PSMs) composed of supported membrane parts as well as freestanding membrane parts are shown to be very versatile to investigate SNARE-mediated fusion on the single-particle level. They provide a planar geometry readily accessible by confocal fluorescence microscopy, which enabled us for the first time, to our knowledge, to investigate the fusion of individual natural secretory granules (i.e., chromaffin granules (CGs)) on the single-particle level by two-color fluorescence microscopy in a time-resolved manner. The t-SNARE acceptor complex ΔN49 was reconstituted into PSMs containing 2 mol % 1,2-dipalmitoyl-sn-glycero-3-phosphatidylinositol-4,5-bisphosphate and Atto488-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, and CGs were fluorescently labeled with 2-((1E,3E)-5-((Z)-3,3-dimethyl-1-octadecylindolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-1-octadecyl-3H-indol-1-ium perchlorate. We compared the dynamics of docked and hemifused CGs as well as their fusion efficacy and kinetics with the results obtained for synthetic synaptobrevin 2-doped vesicles fusing with PSMs of the same composition. Whereas the synthetic vesicles were fully immobile on supported PSMs, docked as well as hemifused CGs were mobile on both PSM parts, which suggests that this system resembles more closely the natural situation. The fusion process of CGs proceeded through three-dimensional post-lipid-mixing structures, which were readily resolved on the gold-covered pore rims of the PSMs and which are discussed in the context of intermediate states observed in live cells.
Subject(s)
Membrane Fusion , Secretory Vesicles/chemistry , Syntaxin 1/chemistry , Vesicle-Associated Membrane Protein 2/chemistry , Animals , Chromaffin Cells/metabolism , Liposomes/chemistry , Molecular Docking Simulation , Protein Domains , Rats , Secretory Vesicles/metabolism , Syntaxin 1/metabolism , Vesicle-Associated Membrane Protein 2/metabolismABSTRACT
A stepwise micro-DSC study of Small, Large and Giant Unilamellar Vesicles prepared as pure and mixed systems of DMPC, DPPC, DSPC and DOPC was performed, achieving the preparation of final model membranes whose phospholipid compositions represent the 75% in terms of the phospholipids tails and the 50% headgroups of the Insulin Secretory Granules (vesicles located in the pancreatic Langerhans ß-cells and which are responsible for insulin and amylin storage and secretion in response to nutrient intake). Moreover, the effect of Free Fatty Acids, whose levels are recurrently altered in diabetic and/or obese subjects, on the thermodynamic stability of the final membranes was eventually investigated. The results allowed to discriminate each single thermodynamic contribution among the main factors that dictate the overall thermodynamic stability of these complex unilamellar systems evidencing mainly entropic effects hierarchically summarized as phospholipid unsaturations > phospholipid tail length > membrane curvature. The effect of the Free Fatty Acids highlighted a strong stabilizing effect on the membranes as well as more pronounced phase segregations in the case of saturated acids (palmitic and stearic), whereas the opposite effect was observed in the case of an unsaturated one (oleic).
Subject(s)
Fatty Acids/chemistry , Insulin/chemistry , Molecular Mimicry , Phosphatidylcholines/chemistry , Secretory Vesicles/chemistry , Thermodynamics , Calorimetry, Differential Scanning , Membranes, Artificial , Models, MolecularABSTRACT
A dual electrofluorescent probe (FFN42) belonging to the fluorescent false neurotransmitter family was rationally designed for investigating cell secretion. This probe, which comprises a coumarin core with one amino and two hydroxy groups, is very promising due to its electroactive and fluorescent properties. The optimal excitation and emission wavelengths (380â nm and 470â nm respectively) make this probe adapted for use in fluorescence microscopy. FFN42 has a quantum yield of 0.18, a molar absorption coefficient of 12000â M-1 cm-1 and pKa values of 5.4 and 6.7 for the hydroxy groups. The electroactivity of FFN42 was evidenced on carbon fiber and ITO electrodes at relatively low oxidation potentials (0.24â V and 0.45â V vs Ag/AgCl respectively). Epifluorescence observations showed that FFN42 accumulated into secretory vesicles of PC12 and N13 cells. Toxicity tests further revealed that FFN42 had no lethal effect on these cells. Amperometric data obtained on carbon fiber electrodes proved that the probe is released by N13 cells.
Subject(s)
Electrochemical Techniques/methods , Fluorescent Dyes/chemistry , Models, Biological , Secretory Vesicles/chemistry , Animals , Cell Line , Coumarins/chemistry , Electrodes , Humans , Microscopy, Fluorescence , Neurotransmitter Agents , PC12 Cells , RatsABSTRACT
During exocytosis, vesicles fuse with the plasma membrane and release their contents. The fusion pore is the initial, nanometer-sized connection between the plasma membrane and the cargo-laden vesicle. A growing body of evidence points toward the fusion pore being a regulator of exocytosis, but the shortcomings of current experimental techniques to investigate single-fusion pores make it difficult to study factors governing pore behavior. Here we describe an assay that fuses v-SNARE-reconstituted nanodiscs with cells ectopically expressing "flipped" t-SNAREs to monitor dynamics of single fusion pores in a biochemically defined system using electrical recordings. We also describe a fluorescence microscopy-based approach to monitor nanodisc-cell fusion that is much simpler to employ, but cannot resolve single pores.