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Super-resolution optical imaging tools are crucial in microbiology to understand the complex structures and behavior of microorganisms such as bacteria, fungi, and viruses. However, the capabilities of these tools, particularly when it comes to imaging pathogens and infected tissues, remain limited. MicroMagnify (µMagnify) is developed, a nanoscale multiplexed imaging method for pathogens and infected tissues that are derived from an expansion microscopy technique with a universal biomolecular anchor. The combination of heat denaturation and enzyme cocktails essential is found for robust cell wall digestion and expansion of microbial cells and infected tissues without distortion. µMagnify efficiently retains biomolecules suitable for high-plex fluorescence imaging with nanoscale precision. It demonstrates up to eightfold expansion with µMagnify on a broad range of pathogen-containing specimens, including bacterial and fungal biofilms, infected culture cells, fungus-infected mouse tone, and formalin-fixed paraffin-embedded human cornea infected by various pathogens. Additionally, an associated virtual reality tool is developed to facilitate the visualization and navigation of complex 3D images generated by this method in an immersive environment allowing collaborative exploration among researchers worldwide. µMagnify is a valuable imaging platform for studying how microbes interact with their host systems and enables the development of new diagnosis strategies against infectious diseases.
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Bactérias , Microscopia , Humanos , Animais , Camundongos , Microscopia/métodos , Imagem ÓpticaRESUMO
Metabolism and movement, among the critical determinants in the survival and success of an organism, are tightly regulated by the brain and skeletal muscle. At the cellular level, mitochondria -that powers life, and myosin - the molecular motor of the cell, have both evolved to serve this purpose. Although independently, the skeletal muscle and brain have been intensively investigated for over a century, their coordinated involvement in metabolism and movement remains poorly understood. Therefore, a fundamental understanding of the coordinated involvement of the brain and skeletal muscle in metabolism and movement holds great promise in providing a window to a wide range of life processes and in the development of tools and approaches in disease detection and therapy. Recent developments in new tools, technologies and approaches, and advances in computing power and machine learning, provides for the first time the opportunity to establish a new field of study, the 'Science and Engineering of Metabolism and Movement'. This new field of study could provide substantial new insights and breakthrough into how metabolism and movement is governed at the systems level in an organism. The design and approach to accomplish this objective is briefly discussed in this article.
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The biophysical function of myosin in vitro has been extensively investigated in different motility assays, but the study of myosin ATPase properties at the fiber level is insufficiently investigated. In this study, quantum dot (QD) mediated thermometry measurements were optimized to measure the efficiency of myosin extracted from muscle mini bundles. A reduction in fluorescent intensity of QD reflects an increase in temperature caused by the heat released during ATP hydrolysis and denotes the efficiency of the motor protein myosin. The procedure for extracting myosin was similar to the single fiber in vitro motility assay with some small modifications, and the concentration of myosin was represented by the extracted total protein since the ratio of extracted myosin to total protein was constant. Moreover, the efficiencies of myosin extracted from preparations containing different myosin heavy chain isoforms reveal lower efficiency of slow compared to fast myosin isoforms. Specifically, more heat was released in slow myosin enzymatic reaction, resulting in faster decay of QD fluorescence intensity. Hence, the optimized QD mediated thermometry provides a novel and sensitive approach to evaluate efficiency of myosin ATPase obtained from small muscle samples, representing a significant advantage in the clinical evaluation of neuromuscular disorders.
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Pontos Quânticos , Termometria , Músculo Esquelético/metabolismo , Cadeias Pesadas de Miosina , Miosinas/metabolismo , Isoformas de Proteínas/metabolismoRESUMO
Exosomes are a class of small, secreted extracellular vesicles (EV) that have recently gained considerable attention for their role in normal cellular function, disease processes and potential as biomarkers. Exosomes serve as intercellular messengers and carry molecular cargo that can alter gene expression and the phenotype of recipient cells. Here, we investigated alterations of microRNA cargo in exosomes secreted by epileptogenic tissue in tuberous sclerosis complex (TSC), a multi-system genetic disorder that includes brain lesions known as tubers. Approximately 90% of TSC patients suffer from seizures that originate from tubers, and ~60% are resistant to antiseizure drugs. It is unknown why some tubers cause seizures while others do not, and the molecular basis of drug-resistant epilepsy is not well understood. It is believed that neuroinflammation is involved, and characterization of this mechanism may be key to disrupting the "vicious cycle" between seizures, neuroinflammation, and increased seizure susceptibility. We isolated exosomes from epileptogenic and non-epileptogenic TSC tubers, and we identified differences in their microRNA cargo using small RNA-seq. We identified 12 microRNAs (including miR-142-3p, miR-223-3p and miR-21-5p) that are significantly increased in epileptogenic tubers and contain nucleic acid motifs that activate toll-like receptors (TLR7/8), initiating a neuroinflammatory cascade. Exosomes from epileptogenic tissue caused induction of key pathways in cultured cells, including innate immune signaling (TLR), inflammatory response and key signaling nodes SQSTM1 (p62) and CDKN1A (p21). Genes induced in vitro were also significantly upregulated in epileptogenic tissue. These results provide new evidence on the role of exosomes and non-coding RNA cargo in the neuroinflammatory cascade of epilepsy and may help advance the development of novel biomarkers and therapeutic approaches for the treatment of drug-resistant epilepsy.
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OBJECTIVE: The endoplasmic reticulum (ER)-resident E3 ligase HRD1 and its co-activator Sel1L are major components of ER-associated degradation (ERAD) machinery. Here, we investigated the molecular mechanism and functional significance underlying the circadian regulation of HRD1/Sel1L-mediated protein degradation program in hepatic energy metabolism. METHODS: Genetically engineered animal models as well as gain- and loss-of-function studies were employed to address the circadian regulatory mechanism and functional significance. Gene expression, transcriptional activation, protein-protein interaction, and animal metabolic phenotyping analyses were performed to dissect the molecular network and metabolic pathways. RESULTS: Hepatic HRD1 and Sel1L expression exhibits circadian rhythmicity that is controlled by the ER-tethered transcriptional activator CREBH, the nuclear receptor peroxisome proliferator-activated receptor α (PPARα), and the core clock oscillator BMAL1 in mouse livers. HRD1/Sel1L mediates polyubiquitination and degradation of the CREBH protein across the circadian cycle to modulate rhythmic expression of the genes encoding the rate-limiting enzymes or regulators in fatty acid (FA) oxidation, triglyceride (TG) lipolysis, lipophagy, and gluconeogenesis. HRD1 liver-specific knockout (LKO) mice displayed increased expression of the genes involved in lipid and glucose metabolism and impaired circadian profiles of circulating TG, FA, and glucose due to overproduction of CREBH. The circadian metabolic activities of HRD1 LKO mice were inversely correlated with those of CREBH KO mice. Suppressing CREBH overproduction in the livers of HRD1 LKO mice restored the diurnal levels of circulating TG and FA of HRD1 LKO mice. CONCLUSION: Our work revealed a key circadian-regulated molecular network through which the E3 ubiquitin ligase HRD1 and its co-activator Sel1L regulate hepatic circadian metabolism.
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Ritmo Circadiano/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Fígado/metabolismo , PPAR alfa/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Fatores de Transcrição ARNTL/metabolismo , Animais , Autofagia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Retículo Endoplasmático/metabolismo , Ácidos Graxos/metabolismo , Gluconeogênese , Metabolismo dos Lipídeos , Lipólise , Camundongos , Camundongos Endogâmicos C57BL , PPAR alfa/genética , Proteólise , Triglicerídeos/metabolismo , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Expensive and time-consuming approaches of immunoelectron microscopy of biopsy tissues continues to serve as the gold-standard for diagnostic pathology. The recent development of the new approach of expansion microscopy (ExM) capable of fourfold lateral expansion of biological specimens for their morphological examination at approximately 70 nm lateral resolution using ordinary diffraction limited optical microscopy, is a major advancement in cellular imaging. Here we report (1) an optimized fixation protocol for retention of cellular morphology while obtaining optimal expansion, (2) an ExM procedure for up to eightfold lateral and over 500-fold volumetric expansion, (3) demonstrate that ExM is anisotropic or differential between tissues, cellular organelles and domains within organelles themselves, and (4) apply image analysis and machine learning (ML) approaches to precisely assess differentially expanded cellular structures. We refer to this enhanced ExM approach combined with ML as differential expansion microscopy (DiExM), applicable to profiling biological specimens at the nanometer scale. DiExM holds great promise for the precise, rapid and inexpensive diagnosis of disease from pathological specimen slides.
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Fígado/citologia , Músculo Esquelético/citologia , Nanopartículas/química , Imagem Óptica , Animais , Humanos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Polímeros/síntese química , Polímeros/química , RatosRESUMO
Secretory vesicle swelling has been demonstrated to be a requirement in cell secretion. In the past 25 years, the quest to elucidate the molecular mechanism of secretory vesicle swelling, serendipitously revealed the presence of water channels or aquaporins at the secretory vesicle membrane and their involvement in rapid gaiting of water into secretory vesicles and their swelling during secretion. These studies further provided an understanding of aquaporin regulation at the secretory vesicle membrane. Secretory vesicles within live cells, isolated secretory vesicles, single vesicle electrophysiological patch clamp studies and the swelling complex reconstituted into liposomes, have all been utilized to elucidate the mechanism and regulation of secretory vesicle swelling. Results from these studies collectively demonstrate the involvement of ß-adrenergic receptor, heterotrimeric GTP-binding G-proteins such as GαI; PLA2 and potassium and chloride channels, in the regulation of aquaporins at the secretory vesicle membrane.
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Aquaporina 2 , Aquaporinas , Vesículas Secretórias , Animais , Aquaporina 2/metabolismo , Aquaporinas/metabolismo , Humanos , Vesículas Secretórias/metabolismoRESUMO
Swelling of secretory vesicles is critical for the regulated release of intra-vesicular contents from cells during secretion. At the secretory vesicle membrane of the exocrine pancreas and neurons, GTP-binding G proteins, vH+-ATPase, potassium channels and AQP water channels, are among the players implicated in vesicle volume regulation. Here we report in the endocrine insulin-secreting MIN6 cells, the similar requirement of vH+-ATPase-mediated intracellular acidification on glucose-stimulated insulin release. MIN6 cells exposed to the vH+-ATPase inhibitor Bafilomycin A show decreased acidification of the cytosolic compartment that include insulin-carrying granules. Additionally, a loss of insulin granules near the cell plasma membrane following Bafilomycin A treatment, suggests impaired transport of insulin granules and consequent decrease in glucose-stimulated insulin secretion and accumulation of intracellular insulin. These results suggest that vH+-ATPase-mediated intracellular acidification is required for insulin secretion in beta cells.
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Adenosina Trifosfatases/metabolismo , Glucose/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Animais , Células Cultivadas , Glucose/antagonistas & inibidores , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Macrolídeos/farmacologia , CamundongosRESUMO
AIM: Critical illness myopathy (CIM) represents a common consequence of modern intensive care, negatively impacting patient health and significantly increasing health care costs; however, there is no treatment available apart from symptomatic and supportive interventions. The chaperone co-inducer BGP-15 has previously been shown to have a positive effect on the diaphragm in rats exposed to the intensive care unit (ICU) condition. In this study, we aim to explore the effects of BGP-15 on a limb muscle (soleus muscle) in response to the ICU condition. METHODS: Sprague-Dawley rats were subjected to the ICU condition for 5, 8 and 10 days and compared with untreated sham-operated controls. RESULTS: BGP-15 significantly improved soleus muscle fibre force after 5 days exposure to the ICU condition. This improvement was associated with the protection of myosin from post-translational myosin modifications, improved mitochondrial structure/biogenesis and reduced the expression of MuRF1 and Fbxo31 E3 ligases. At longer durations (8 and 10 days), BGP-15 had no protective effect when the hallmark of CIM had become manifest, that is, preferential loss of myosin. Unrelated to the effects on skeletal muscle, BGP-15 had a strong positive effect on survival compared with untreated animals. CONCLUSIONS: BGP-15 treatment improved soleus muscle fibre and motor protein function after 5 days exposure to the ICU condition, but not at longer durations (8 and 10 days) when the preferential loss of myosin was manifest. Thus, long-term CIM interventions targeting limb muscle fibre/myosin force generation capacity need to consider both the post-translational modifications and the loss of myosin.
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Estado Terminal , Unidades de Terapia Intensiva , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiopatologia , Doenças Musculares/tratamento farmacológico , Oximas/farmacologia , Oximas/uso terapêutico , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Animais , Modelos Animais de Doenças , Feminino , Doenças Musculares/fisiopatologia , Ratos , Ratos Sprague-DawleyRESUMO
The cell plasma membrane is a highly dynamic organelle governing a wide range of cellular activities including ion transport, secretion, cell division, growth, and development. The fundamental process involved in the addition of new membranes to pre-existing plasma membranes, however, is unclear. Here, we report, using biophysical, morphological, biochemical, and molecular dynamic simulations, the selective incorporation of proteins and lipids from the cytosol into the cell plasma membrane dictated by membrane stretch and composition. Stretching of the cell membrane as a consequence of volume increase following incubation in a hypotonic solution and results in the incorporation of cytosolic proteins and lipids into the existing plasma membrane. Molecular dynamic simulations further confirm that increased membrane stretch results in the rapid insertion of lipids into the existing plasma membrane. Similarly, depletion of cholesterol from the cell plasma membrane selectively alters the incorporation of lipids into the membrane.
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Proteínas Sanguíneas/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Citosol/metabolismo , Eritrócitos/metabolismo , Insulinoma/metabolismo , Lipídeos de Membrana/metabolismo , Animais , Camundongos , Simulação de Dinâmica Molecular , Neoplasias Pancreáticas/metabolismo , Proteoma/análise , Proteoma/metabolismo , Ratos , Ratos Sprague-Dawley , Células Tumorais CultivadasRESUMO
Objective: To identify whether factors toxic to oligodendrocytes (OLs), released by B cells from patients with MS, are found in extracellular microvesicles enriched in exosomes. Methods: Conditioned medium (Sup) was obtained from cultures of blood B cells of patients with MS and normal controls (NCs). Exosome-enriched (Ex-En) fractions were prepared by solvent precipitation from Sup containing bovine serum and from serum-free Sup by ultracentrifugation (UC) or immunoprecipitation (IP) with antibodies to CD9. Ex-En fractions were diluted 1:4 with OL culture medium and screened for toxic effects on cultured rat OLs as measured by trypan blue uptake. Proteomic analysis was performed on Sup fractions. Results: MS B cell-derived Ex-En fractions prepared from Sup by solvent extraction, UC, or IP induced OL death, whereas corresponding Ex-En fractions from NC showed little toxicity. Proteomic analysis of Sup demonstrated enrichment of proteins characteristic of exosomes from both NC and MS B-cell Sup. Ontology enrichment analysis suggested differences in the types and cargo of exosomes from MS Sup compared with NC, with proteins related to cell surface, extracellular plasma membrane, and gliogenesis enriched in MS. Conclusions: Much of the in vitro toxicity of Sup from B cells of patients with relapsing-remitting MS is found in Ex-En fractions, as confirmed by 3 methods. Proteomic analysis of B-cell Sup indicates multiple differences between MS and NC.
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Linfócitos B/metabolismo , Córtex Cerebral , Exossomos/metabolismo , Esclerose Múltipla Recidivante-Remitente/metabolismo , Oligodendroglia , Adulto , Animais , Animais Recém-Nascidos , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Humanos , Esclerose Múltipla Recidivante-Remitente/sangue , Proteômica , RatosRESUMO
The 'Human Cell Atlas' project has been launched to obtain a comprehensive understanding of all cell types, the fundamental living units that constitute the human body. This is a global partnership and effort involving experts from many disciplines, from computer science, engineering to medicine, and is supported by several private and public organizations, among them, the Chan Zuckerberg Foundation, the National Institutes of Health, and Google, that will greatly benefit humanity. Nearly 37 trillion cells of various shapes, sizes, and composition, are precisely organized to constitute the human body. Humans, like all other living organisms, are dynamic, and therefore a comprehensive understanding of different cells in their various dynamic states is required to provide a reference map for the early diagnosis and various preventive approach to disease, and in the development of precision therapeutics. Skeletal muscles being the most abundant tissue and the largest locomotor and metabolic organ in the human body, requires a global understanding of its structure, composition, and function. The objective of creating a 'Human Skeletal Muscle Cell Atlas', necessitates therefore a comprehensive understanding of the emergent properties of skeletal muscle cell growth, development, structure, function and chemistry, under conditions of activity and inactivity. To achieve this objective would require a very precise yet rapid and cost-effective approach of combined multimodal imaging, including our new and novel 'Differential Expansion Microscopy', our 'Nanoscale Thermometry', combined with 'Mass Spectrometry', 'Motor Protein Motility Assay' and 'Machine Learning' tools.
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Microscopia/métodos , Músculo Esquelético/citologia , Músculo Esquelético/ultraestrutura , Anatomia Artística , Atlas como Assunto , Biologia Celular , Humanos , Aprendizado de Máquina , Espectrometria de MassasRESUMO
Current approaches in regenerative medicine to develop human skeletal muscle replicating native tissue for engrafts and high-throughput drug screening and gene therapy are still in their infancy and have not proven to recapitulate the behavior and regulatory processes present in endogenous skeletal muscle tissue. This stems at least in part from the lack of a comprehensive understanding of the emergent properties of in vitro skeletal muscle growth and development. To address this gap in our current knowledge, we have developed a stretchable micropatterned 3D human skeletal muscle platform that recapitulates organized and parallel growth of muscle cells and fibers as opposed to the randomly oriented cells growth on a 2D glass surface. Mass spectrometry of the muscle cells growing on the 3D platform express key myogenic proteins such as myoferlin for myoblast fusion required in the formation of muscle tissue, and proteins involved in mitochondrial health and biogenesis, in contrast to cells growing on 2D glass surface. These results demonstrate that the engineered human muscle cells grown on the 3D platform holds great promise to further establish the emergent properties of in vitro skeletal muscle growth and development for a wide range of biomedical applications.
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Ions greatly influence protein structure-function and are critical to health and disease. A 10,â¯000-fold higher calcium in the sarcoplasmic reticulum (SR) of muscle suggests elevated calcium levels near active calcium channels at the SR membrane and the impact of localized high calcium on the structure-function of the motor protein myosin. In the current study, combined quantum dot (QD)-based nanothermometry and circular dichroism (CD) spectroscopy enabled detection of previously unknown enthalpy changes and associated structural remodeling of myosin, impacting its function following exposure to elevated calcium. Cadmium telluride QDs adhere to myosin, function as thermal sensors, and reveal that exposure of myosin to calcium is exothermic, resulting in lowering of enthalpy, a decrease in alpha helical content measured using CD spectroscopy, and the consequent increase in motor efficiency. Isolated muscle fibers subjected to elevated levels of calcium further demonstrate fiber lengthening and decreased motility of actin filaments on myosin-functionalized substrates. Our results, in addition to providing new insights into our understanding of muscle structure-function, establish a novel approach to understand the enthalpy of protein-ion interactions and the accompanying structural changes that may occur within the protein molecule.
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Compostos de Cádmio/química , Cálcio/química , Dicroísmo Circular , Miosinas/química , Pontos Quânticos/química , Telúrio/química , Termometria , Animais , Camundongos , Estrutura Quaternária de Proteína , Relação Estrutura-AtividadeRESUMO
Valproate (VPA), an FDA approved anti-epileptic drug with a half-life of 12-18 h in humans, has been shown to perturb the vacuolar proton pump (vH+-ATPase) function in yeasts by inhibiting myo-inositol phosphate synthase, the first and rate-limiting enzyme in inositol biosynthesis, thereby resulting in inositol depletion. vH+-ATPase transfers protons (H+) across cell membranes, which help maintain pH gradients within cells necessary for various cellular functions including secretion. This proton pump has a membrane (V0) and a soluble cytosolic (V1) domain, with C-subunit associated with V1. In secretory cells such as neurons and insulin-secreting beta cells, vH+-ATPase acidifies vesicles essential for secretion. In this study, we demonstrate that exposure of insulin-secreting Min6 cells to a clinical dose of VPA results in inositol depletion and loss of co-localization of subunit C of vH+-ATPase with insulin-secreting granules. Consequently, a reduction of glucose-stimulated insulin secretion is observed following VPA exposure. These results merit caution and the reassessment of the clinical use of VPA.
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Glucose/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Ácido Valproico/farmacologia , Animais , Secreção de Insulina , Camundongos , Células Tumorais Cultivadas , Ácido Valproico/químicaRESUMO
A molecular model of pancreatic zymogen granule (ZG) is critical for understanding its functions. We have extensively characterized the composition and membrane topology of rat ZG proteins. In this study, we report the development of targeted proteomics approaches to quantify representative mouse and human ZG proteins using LC-SRM and heavy isotope-labeled synthetic peptides. The absolute quantities of mouse Rab3D and VAMP8 were determined as 1242 ± 218 and 2039 ± 151 (mean ± SEM) copies per ZG. The size distribution and the averaged diameter of ZGs 750 ± 23 nm (mean ± SEM) were determined by atomic force microscopy. The absolute quantification of Rab3D was then validated using semi-quantitative Western blotting with purified GST-Rab3D proteins as an internal standard. To extend our proteomics analysis to human pancreas, ZGs were purified using human acini obtained from pancreatic islet transplantation center. One hundred and eighty human ZG proteins were identified for the first time including both the membrane and the content proteins. Furthermore, the copy number per ZG of human Rab3D and VAMP8 were determined to be 1182 ± 45 and 485 ± 15 (mean ± SEM). The comprehensive proteomic analyses of mouse and human pancreatic ZGs have the potential to identify species-specific ZG proteins. The determination of protein copy numbers on pancreatic ZGs represents a significant advance towards building a quantitative molecular model of a prototypical secretory vesicle using targeted proteomics approaches. The identification of human ZG proteins lays a foundation for subsequent studies of altered ZG compositions and secretion in pancreatic diseases.
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A wide range of cellular activities including protein folding and cell secretion, such as neurotransmission or insulin release, are all governed by intracellular pH homeostasis, underscoring the importance of pH on critical life processes. Nano- scale pH measurements of cells and biomolecules therefore hold great promise in understanding a plethora of cellular functions, in addition to disease detection and therapy. In the current study, a novel approach using cadmium telluride quantum dots (CdTeQDs) as pH sensors, combined with fluorescent imaging, spectrofluorimetry, atomic force microscopy (AFM), and Western blot analysis, enabled the study of intracellular pH dynamics at 1 milli-pH sensitivity and 80nm pixel resolution, during insulin secretion. Additionally, the pH-dependent interaction between membrane fusion proteins, also called the soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE), was determined. Glucose stimulation of CdTeQD-loaded insulin secreting Min-6 mouse insulinoma cell line demonstrated the initial (5-6min) intracellular acidification reflected as a loss in QD fluorescence, followed by alkalization and a return to resting pH in 10min. Analysis of the SNARE complex in insulin secreting Min-6 cells demonstrated an initial gain followed by loss of complexed SNAREs in 10min. Stabilization of the SNARE complex at low intracellular pH is further supported by results from studies utilizing both native and AFM measurements of liposome-reconstituted recombinant neuronal SNAREs, providing a molecular understanding of the role of pH during cell secretion.
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Fluorescência , Insulinoma/metabolismo , Insulinoma/patologia , Fusão de Membrana , Microscopia de Força Atômica , Imagem Óptica , Animais , Concentração de Íons de Hidrogênio , Simulação de Dinâmica MolecularRESUMO
In the past 50 years, isolated blood platelets have had restricted use in wound healing, cancer therapy, and organ and tissue transplant, to name a few. The major obstacle for its unrestricted use has been, among others, the presence of ultrahigh concentrations of growth factors and the presence of both pro-angiogenic and anti-angiogenic proteins. To overcome this problem requires the isolation and separation of the membrane bound secretory vesicles containing the different factors. In the current study, high-resolution imaging of isolated secretory vesicles from human platelets using atomic force microscopy (AFM) and mass spectrometry enabled characterization of the remaining vesicles size and composition following their immunoseparation. The remaining vesicles obtained following osmotic lysis, when subjected to immunoseparation employing antibody to different vesicle-associated membrane proteins (VAMPs), demonstrate for the first time that VAMP-3-, VAMP-7-, and VAMP-8-specific vesicles each possesses distinct size range and composition. These results provide a window into our understanding of the heterogeneous population of vesicles in human platelets and their stability following both physical manipulation using AFM and osmotic lysis of the platelet. This study further provides a platform for isolation and the detailed characterization of platelet granules, with promise for their future use in therapy. Additionally, results from the study demonstrate that secretory vesicles of different size found in cells reflect their unique and specialized composition and function.
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Plaquetas/química , Proteoma/isolamento & purificação , Proteínas R-SNARE/isolamento & purificação , Vesículas Secretórias/química , Proteína 3 Associada à Membrana da Vesícula/isolamento & purificação , Plaquetas/metabolismo , Células Cultivadas , Fracionamento Químico/métodos , Humanos , Imunoprecipitação/métodos , Microscopia de Força Atômica , Anotação de Sequência Molecular , Pressão Osmótica , Proteoma/metabolismo , Proteínas R-SNARE/metabolismo , Vesículas Secretórias/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteína 3 Associada à Membrana da Vesícula/metabolismoRESUMO
Synaptic vesicles measuring 30-50 nm in diameter containing neurotransmitters either completely collapse at the presynaptic membrane or dock and transiently fuse at the base of specialized 15 nm cup-shaped lipoprotein structures called porosomes at the presynaptic membrane of synaptosomes to release neurotransmitters. Recent study reports the unique composition of major lipids associated with neuronal porosomes. Given that lipids greatly influence the association and functions of membrane proteins, differences in lipid composition of synaptic vesicle and the synaptosome membrane was hypothesized. To test this hypothesis, the lipidome of isolated synaptosome, synaptosome membrane, and synaptic vesicle preparation were determined by using mass spectrometry in the current study. Results from the study demonstrate the enriched presence of triacyl glycerols and sphingomyelins in synaptic vesicles, as opposed to the enriched presence of phospholipids in the synaptosome membrane fraction, reflecting on the tight regulation of nerve cells in compartmentalization of membrane lipids at the nerve terminal.