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The structure of the cell nucleus of higher organisms has become a major topic of advanced light microscopy. So far, a variety of methods have been applied, including confocal laser scanning fluorescence microscopy, 4Pi, STED and localisation microscopy approaches, as well as different types of patterned illumination microscopy, modulated either laterally (in the object plane) or axially (along the optical axis). Based on our experience, we discuss here some application perspectives of Modulated Illumination Microscopy (MIM) and its combination with single-molecule localisation microscopy (SMLM). For example, spatially modulated illumination microscopy/SMI (illumination modulation along the optical axis) has been used to determine the axial extension (size) of small, optically isolated fluorescent objects between ≤ 200 nm and ≥ 40 nm diameter with a precision down to the few nm range; it also allows the axial positioning of such structures down to the 1 nm scale; combined with laterally structured illumination/SIM, a 3D localisation precision of ≤1 nm is expected using fluorescence yields typical for SMLM applications. Together with the nanosizing capability of SMI, this can be used to analyse macromolecular nuclear complexes with a resolution approaching that of cryoelectron microscopy.
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Since its introduction in 2017, MINFLUX nanoscopy has shown that it can visualise fluorescent molecules with an exceptional localisation precision of a few nanometres. In this overview, we provide a brief insight into technical implementations, fluorescent marker developments and biological studies that have been conducted in connection with MINFLUX imaging and tracking. We also formulate ideas on how MINFLUX nanoscopy and derived technologies could influence bioimaging in the future. This insight is intended as a general starting point for an audience looking for a brief overview of MINFLUX nanoscopy from theory to application.
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The Vps10p-domain (Vps10p-D) receptor family consists of Sortilin, SorLA, SorCS1, SorCS2, and SorCS3. They mediate internalization and intracellular sorting of specific cargo in various cell types, but underlying molecular determinants are incompletely understood. Deciphering the dynamic intracellular itineraries of Vps10p-D receptors is crucial for understanding their role in physiological and cytopathological processes. However, studying their spatial and temporal dynamics by live imaging has been challenging so far, as terminal tagging with fluorophores presumably impedes several of their protein interactions and thus functions. Here, we addressed the lack of appropriate tools and developed functional versions of all family members internally tagged in their ectodomains. We predict folding of the newly designed receptors by bioinformatics and show their exit from the endoplasmic reticulum. We examined their subcellular localization in immortalized cells and primary cultured neurons by immunocytochemistry and live imaging. This was, as far as known, identical to that of wt counterparts. We observed homodimerization of fluorophore-tagged SorCS2 by coimmunoprecipitation and fluorescence lifetime imaging, suggesting functional leucine-rich domains. Through ligand uptake experiments, live imaging and fluorescence lifetime imaging, we show for the first time that all Vps10p-D receptors interact with the neurotrophin brain-derived neurotrophic factor and mediate its uptake, indicating functionality of the Vps10p-Ds. In summary, we developed versions of all Vps10p-D receptors, with internal fluorophore tags that preserve several functions of the cytoplasmic and extracellular domains. These newly developed fluorophore-tagged receptors are likely to serve as powerful functional tools for accurate live studies of the individual cellular functions of Vps10p-D receptors.
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Background: Platelets contain high levels of amyloid ß (Aß) peptides and have been suggested to participate in the deposition of amyloid plaques in Alzheimer's Disease (AD). Objectives: This study aimed to determine whether human platelets release pathogenic Aß peptides Aß1-42 and Aß1-40 and to characterise the mechanisms regulating this phenomenon. Methods and Results: Enzyme-linked immunosorbent assays (ELISAs) revealed that the haemostatic stimulus thrombin and the pro-inflammatory molecule lipopolysaccharide (LPS) induce platelet release of both Aß1-42 and Aß1-40. Notably, LPS preferentially induced the release of Aß1-42, which was potentiated by the reduction of oxygen from atmospheric levels to physiological hypoxia. The selective ß secretase (BACE) inhibitor LY2886721 showed no effect on the release of either Aß1-40 or Aß1-42 in our ELISA experiments. This suggested a store-and-release mechanism that was confirmed in immunostaining experiments showing co-localisation of cleaved Aß peptides with platelet alpha granules. Conclusions: Taken together, our data suggest that human platelets release pathogenic Aß peptides as a result of a store-and-release mechanism rather than a de novo proteolytic event. Although further studies are required to fully characterise this phenomenon, we suggest the possibility of a role for platelets in the deposition of Aß peptides and the formation of amyloid plaques. Interestingly, the combination of hypoxia and inflammation that we simulated in vitro with reduced oxygen tension and LPS may increase the release of fibrillogenic Aß1-42 and, consequently, exacerbate amyloid plaque deposition in the brain of AD patients.
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Bone fragility is a profound complication of type 1 diabetes mellitus (T1DM), increasing patient morbidity. Within the mineralized bone matrix, osteocytes build a mechanosensitive network that orchestrates bone remodeling; thus, osteocyte viability is crucial for maintaining bone homeostasis. In human cortical bone specimens from individuals with T1DM, we found signs of accelerated osteocyte apoptosis and local mineralization of osteocyte lacunae (micropetrosis) compared with samples from age-matched controls. Such morphological changes were seen in the relatively young osteonal bone matrix on the periosteal side, and micropetrosis coincided with microdamage accumulation, implying that T1DM drives local skeletal aging and thereby impairs the biomechanical competence of the bone tissue. The consequent dysfunction of the osteocyte network hampers bone remodeling and decreases bone repair mechanisms, potentially contributing to the enhanced fracture risk seen in individuals with T1DM. STATEMENT OF SIGNIFICANCE: Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease that causes hyperglycemia. Increased bone fragility is one of the complications associated with T1DM. Our latest study on T1DM-affected human cortical bone identified the viability of osteocytes, the primary bone cells, as a potentially critical factor in T1DM-bone disease. We linked T1DM with increased osteocyte apoptosis and local accumulation of mineralized lacunar spaces and microdamage. Such structural changes in bone tissue suggest that T1DM speeds up the adverse effects of aging, leading to the premature death of osteocytes and potentially contributing to diabetes-related bone fragility.
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Diabetes Mellitus Tipo 1 , Osteocitos , Humanos , Envejecimiento , Huesos , ApoptosisRESUMEN
The resolution achievable with the established super-resolution fluorescence nanoscopy methods, such as STORM or STED, is in general not sufficient to resolve protein complexes or even individual proteins. Recently, minimal photon flux (MINFLUX) nanoscopy has been introduced that combines the strengths of STED and STORM nanoscopy and can achieve a localization precision of less than 5 nm. We established a generally applicable workflow for MINFLUX imaging and applied it for the first time to a bacterial molecular machinein situ, i.e., the injectisome of the enteropathogenY. enterocolitica. We demonstrate with a pore protein of the injectisome that MINFLUX can achieve a resolution down to the single molecule levelin situ. By imaging a sorting platform protein using 3D-MINFLUX, insights into the precise localization and distribution of an injectisome component in a bacterial cell could be accomplished. MINFLUX nanoscopy has the potential to revolutionize super-resolution imaging of dynamic molecular processes in bacteria and eukaryotes.
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Bacterias , Microscopía Fluorescente/métodosRESUMEN
Postsynaptic scaffold proteins such as Shank, PSD-95, Homer and SAPAP/GKAP family members establish the postsynaptic density of glutamatergic synapses through a dense network of molecular interactions. Mutations in SHANK genes are associated with neurodevelopmental disorders including autism and intellectual disability. However, no SHANK missense mutations have been described which interfere with the key functions of Shank proteins believed to be central for synapse formation, such as GKAP binding via the PDZ domain, or Zn2+-dependent multimerization of the SAM domain. We identify two individuals with a neurodevelopmental disorder carrying de novo missense mutations in SHANK2. The p.G643R variant distorts the binding pocket for GKAP in the Shank2 PDZ domain and prevents interaction with Thr(-2) in the canonical PDZ ligand motif of GKAP. The p.L1800W variant severely delays the kinetics of Zn2+-dependent polymerization of the Shank2-SAM domain. Structural analysis shows that Trp1800 dislodges one histidine crucial for Zn2+ binding. The resulting conformational changes block the stacking of helical polymers of SAM domains into sheets through side-by-side contacts, which is a hallmark of Shank proteins, thereby disrupting the highly cooperative assembly process induced by Zn2+. Both variants reduce the postsynaptic targeting of Shank2 in primary cultured neurons and alter glutamatergic synaptic transmission. Super-resolution microscopy shows that both mutants interfere with the formation of postsynaptic nanoclusters. Our data indicate that both the PDZ- and the SAM-mediated interactions of Shank2 contribute to the compaction of postsynaptic protein complexes into nanoclusters, and that deficiencies in this process interfere with normal brain development in humans.
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Staphylococcus epidermidis is a major causative agent of prosthetic joint infections (PJI). The ability to form biofilms supports this highly selective pathogenic potential. In vitro studies essentially relying on phenotypic assays and genetic approaches have provided a detailed picture of the molecular events contributing to biofilm assembly. A major limitation in these studies is the use of synthetic growth media, which significantly differs from the environmental conditions S. epidermidis encounters during host invasion. Building on evidence showing that growth in serum substantially affects S. epidermidis gene expression profiles and phenotypes, the major aim of this study was to develop and characterize a growth medium mimicking synovial fluid, thereby facilitating research addressing specific aspects related to PJI. Using fresh human plasma, a protocol was established allowing for the large-scale production of a medium that by biochemical analysis matches key characteristics of synovial fluid and therefore is referred to as artificial synovial fluid (ASF). By analysis of biofilm-positive, polysaccharide intercellular adhesion (PIA)-producing S. epidermidis 1457 and its isogenic, PIA- and biofilm-negative mutant 1457-M10, evidence is provided that the presence of ASF induces cluster formation in S. epidermidis 1457 and mutant 1457-M10. Consistent with the aggregative properties, both strains formed multilayered biofilms when analyzed by confocal laser scanning microscopy. In parallel to the phenotypic findings, expression analysis after growth in ASF found upregulation of genes encoding for intercellular adhesins (icaA, aap, and embp) as well as atlE, encoding for the major cell wall autolysin being responsible for eDNA release. In contrast, growth in ASF was associated with reduced expression of the master regulator agr. Collectively, these results indicate that ASF induces expression profiles that are able to support intercellular adhesion in both PIA-positive and PIA-negative S. epidermidis. Given the observation that ASF overall induced biofilm formation in a collection of S. epidermidis isolates from PJI, the results strongly support the idea of using growth media mimicking host environments. ASF may play an important role in future studies related to the pathogenesis of S. epidermidis PJI.
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Staphylococcus epidermidis , Líquido Sinovial , Adhesinas Bacterianas/metabolismo , Biopelículas , Humanos , Polisacáridos Bacterianos/metabolismo , Staphylococcus epidermidis/genética , Líquido Sinovial/metabolismoRESUMEN
Stress granules (SGs) are membrane-less condensates composed of RNA and protein that assemble in response to stress stimuli and disassemble when stress is lifted. Both assembly and disassembly are tightly controlled processes, yet, it remains elusive whether mRNAs in SGs completely recover for translation following stress relief. Using RNA-seq of translating fractions in human cell line, we found that higher fraction of the m6A-modified mRNAs recovered for translation compared to unmodified mRNAs, i.e. 95% vs 84%, respectively. Considering structural mRNA analysis, we found that the m6A modification enhances structuring at nucleotides in its close vicinity. Our results suggest that SG-sequestered mRNAs disassemble nearly completely from SGs and the m6A modification may display some advantage to the mRNAs in their recovery for translation likely by m6A-driven structural stabilization.
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Gránulos Citoplasmáticos , Gránulos de Estrés , Línea Celular , Gránulos Citoplasmáticos/metabolismo , Humanos , ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Dissociation of hyper-phosphorylated Tau from neuronal microtubules and its pathological aggregates, are hallmarks in the etiology of tauopathies. The Tau-microtubule interface is subject to polyglutamylation, a reversible posttranslational modification, increasing negative charge at tubulin C-terminal tails. Here, we asked whether tubulin polyglutamylation may contribute to Tau pathology in vivo. Since polyglutamylases modify various proteins other than tubulin, we generated a knock-in mouse carrying gene mutations to abolish Tuba4a polyglutamylation in a substrate-specific manner. We found that Tuba4a lacking C-terminal polyglutamylation prevents the binding of Tau and GSK3 kinase to neuronal microtubules, thereby strongly reducing phospho-Tau levels. Notably, crossbreeding of the Tuba4a knock-in mouse with the hTau tauopathy model, expressing a human Tau transgene, reversed hyper-phosphorylation and oligomerization of Tau and normalized microglia activation in brain. Our data highlight tubulin polyglutamylation as a potential therapeutic strategy in fighting tauopathies.
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Tauopatías , Tubulina (Proteína) , Animales , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Ratones , Microglía/metabolismo , Microtúbulos/metabolismo , Fosforilación , Tauopatías/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
Shank proteins are major scaffolds of the postsynaptic density of excitatory synapses. Mutations in SHANK genes are associated with autism and intellectual disability. The effects of missense mutations on Shank3 function, and therefore the pathomechanisms are unclear. Several missense mutations in SHANK3 affect the N-terminal region, consisting of the Shank/ProSAP N-terminal (SPN) domain and a set of Ankyrin (Ank) repeats. Here we identify a novel SHANK3 missense mutation (p.L270M) in the Ankyrin repeats in patients with an ADHD-like phenotype. We functionally analysed this and a series of other mutations, using biochemical and biophysical techniques. We observe two major effects: (1) a loss of binding to δ-catenin (e.g. in the p.L270M variant), and (2) interference with the intramolecular interaction between N-terminal SPN domain and the Ank repeats. This also interferes with binding to the α-subunit of the calcium-/calmodulin dependent kinase II (αCaMKII), and appears to be associated with a more severe neurodevelopmental pathology.
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SinapsisRESUMEN
Keratins are the main identification markers of circulating tumor cells (CTCs); however, whether their deregulation is associated with the metastatic process is largely unknown. Previously we have shown by in silico analysis that keratin 16 (KRT16) mRNA upregulation might be associated with more aggressive cancer. Therefore, in this study, we investigated the biological role and the clinical relevance of K16 in metastatic breast cancer. By performing RT-qPCR, western blot, and immunocytochemistry, we investigated the expression patterns of K16 in metastatic breast cancer cell lines and evaluated the clinical relevance of K16 expression in CTCs of 20 metastatic breast cancer patients. High K16 protein expression was associated with an intermediate mesenchymal phenotype. Functional studies showed that K16 has a regulatory effect on EMT and overexpression of K16 significantly enhanced cell motility (p < 0.001). In metastatic breast cancer patients, 64.7% of the detected CTCs expressed K16, which was associated with shorter relapse-free survival (p = 0.0042). Our findings imply that K16 is a metastasis-associated protein that promotes EMT and acts as a positive regulator of cellular motility. Furthermore, determining K16 status in CTCs provides prognostic information that helps to identify patients whose tumors are more prone to metastasize.
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S. epidermidis is a substantial component of the human skin microbiota, but also one of the major causes of nosocomial infection in the context of implanted medical devices. We here aimed to advance the understanding of S. epidermidis genotypes and phenotypes conducive to infection establishment. Furthermore, we investigate the adaptation of individual clonal lines to the infection lifestyle based on the detailed analysis of individual S. epidermidis populations of 23 patients suffering from prosthetic joint infection. Analysis of invasive and colonizing S. epidermidis provided evidence that invasive S. epidermidis are characterized by infection-supporting phenotypes (e.g. increased biofilm formation, growth in nutrient poor media and antibiotic resistance), as well as specific genetic traits. The discriminating gene loci were almost exclusively assigned to the mobilome. Here, in addition to IS256 and SCCmec, chromosomally integrated phages was identified for the first time. These phenotypic and genotypic features were more likely present in isolates belonging to sequence type (ST) 2. By comparing seven patient-matched nasal and invasive S. epidermidis isolates belonging to identical genetic lineages, infection-associated phenotypic and genotypic changes were documented. Besides increased biofilm production, the invasive isolates were characterized by better growth in nutrient-poor media and reduced hemolysis. By examining several colonies grown in parallel from each infection, evidence for genetic within-host population heterogeneity was obtained. Importantly, subpopulations carrying IS insertions in agrC, mutations in the acetate kinase (AckA) and deletions in the SCCmec element emerged in several infections. In summary, these results shed light on the multifactorial processes of infection adaptation and demonstrate how S. epidermidis is able to flexibly repurpose and edit factors important for colonization to facilitate survival in hostile infection environments.
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Proteínas Bacterianas/genética , Biopelículas/crecimiento & desarrollo , Infección Hospitalaria/microbiología , Mutación , Mucosa Nasal/microbiología , Infecciones Estafilocócicas/microbiología , Staphylococcus epidermidis/genética , Anciano , Anciano de 80 o más Años , Proteínas Bacterianas/metabolismo , Infección Hospitalaria/genética , Infección Hospitalaria/metabolismo , Femenino , Genotipo , Hemólisis , Humanos , Secuencias Repetitivas Esparcidas , Masculino , Persona de Mediana Edad , Mucosa Nasal/metabolismo , Fenotipo , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/metabolismo , Staphylococcus epidermidis/clasificación , Staphylococcus epidermidis/crecimiento & desarrollo , Staphylococcus epidermidis/aislamiento & purificaciónRESUMEN
Extracellular vesicles (EVs) are important means of intercellular communication and a potent tool for regenerative therapy. In ischaemic stroke, transient blockage of a brain artery leads to a lack of glucose and oxygen in the affected brain tissue, provoking neuronal death by necrosis in the core of the ischaemic region. The fate of neurons in the surrounding penumbra region depends on the stimuli, including EVs, received during the following hours. A detailed characterization of such stimuli is crucial not only for understanding stroke pathophysiology but also for new therapeutic interventions. In the present study, we characterize the EVs in mouse brain under physiological conditions and 24 h after induction of transient ischaemia in mice. We show that, in steady-state conditions, microglia are the main source of small EVs (sEVs), whereas after ischaemia the main sEV population originates from astrocytes. Brain sEVs presented high amounts of the prion protein (PrP), which were further increased after stroke. Moreover, EVs were enriched in a proteolytically truncated PrP fragment (PrP-C1). Because of similarities between PrP-C1 and certain viral surface proteins, we studied the cellular uptake of brain-derived sEVs from mice lacking (PrP-KO) or expressing PrP (WT). We show that PrP-KO-sEVs are taken up significantly faster and more efficiently than WT-EVs by primary neurons. Furthermore, microglia and astrocytes engulf PrP-KO-sEVs more readily than WT-sEVs. Our results provide novel information on the relative contribution of brain cell types to the sEV pool in murine brain and indicate that increased release of sEVs by astrocytes together with elevated levels of PrP in sEVs may play a role in intercellular communication at early stages after stroke. In addition, amounts of PrP (and probably PrP-C1) in brain sEVs seem to contribute to regulating their cellular uptake.
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The capture of circulating tumor cells (CTCs) is still a challenging application for microfluidic chips, as these cells are rare and hidden in a huge background of blood cells. Here, different microfluidic ceiling designs in regard to their capture efficiency for CTCs in model experiments and more realistic conditions of blood samples spiked with a clinically relevant amount of tumor cells are evaluated. An optimized design for the capture platform that allows highly efficient recovery of CTCs from size-based pre-enriched samples under realistic conditions is obtained. Furthermore, the viability of captured tumor cells as well as single cell recovery for downstream genomic analysis is demonstrated. Additionally, the authors' findings underline the importance of evaluating rational design rules for microfluidic devices based on theoretical models by application-specific experiments.
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Separación Celular , Técnicas Analíticas Microfluídicas , Células Neoplásicas Circulantes/química , Línea Celular Tumoral , Separación Celular/instrumentación , Separación Celular/métodos , Supervivencia Celular , Diseño de Equipo , Humanos , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodosRESUMEN
The use of disease-specific signatures of microRNAs (miRNAs) in exosomes has become promising for clinical applications, either as biomarkers or direct therapeutic targets. However, a new approach for exosome enrichment and quantification of miRNAs is urgently needed for its clinical application, since the commercial techniques have shortcomings in quantity and quality. To overcome these deficiencies, we developed a new method for purification of exosomes with subsequent miRNA extraction, followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR), and compared our assays with commercial techniques. For the establishment of these methods, numerous reagents, parameters, and combinations thereof were examined. Our new technique for exosome extraction is based on a mannuronate-guluronate polymer (MGP) which avoids co-precipitating plasma proteins. Quality, concentration and biological activity of the isolated exosomes were examined by Western blot, Nanoparticle Tracking Analysis (NTA), and confocal microscopy. A combination of chaotropic and non-chaotropic salts was used to extract miRNAs from plasma, serum, and exosomes, allowing the exclusion of hazardous components, such as phenol/chloroform. The performance of the miRNAs extraction was verified by RT-qPCR. The chemistry and TaqMan probe were also optimized for RT-qPCR. Sensitivity, efficiency, and linearity of RT-qPCR were tested on serial dilutions of synthetic miR-16 and miR-142. Our established procedure covers all steps of miRNA analyses, and measures the levels of either cell-free and exosomal miRNAs in plasma, serum and other body fluids with high performance.
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MicroARN Circulante/aislamiento & purificación , Exosomas/genética , Polímeros/química , Precipitación Química , Perfilación de la Expresión Génica , Ácidos Hexurónicos/química , Humanos , Microscopía Confocal , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Imagen Individual de MoléculaRESUMEN
Although inactivating mutations of PLS3, encoding the actin-bundling protein plastin-3, have been identified to cause X-linked osteoporosis, the cellular and molecular influence of PLS3 on bone remodeling is poorly defined. Moreover, although a previous study has demonstrated moderate osteopenia in 12 week-old Pls3-deficient mice based on µCT scanning, there is no reported analysis of such a model on the basis of undecalcified histology and bone-specific histomorphometry. To fill this knowledge gap we applied a deep phenotyping approach and studied Pls3-deficient mice at different ages. Surprisingly, we did not detect significant differences between wildtype and Pls3-deficient littermates with respect to trabecular bone mass, and the same was the case for all histomorphometric parameters determined at 12 weeks of age. Remarkably however, the cortical thickness in both, tibia and femur, was significantly reduced in Pls3-deficient mice in all age groups. We additionally studied the ex vivo behavior of Pls3-deficient primary osteoblasts, which displayed moderately impaired mineralization capacity. Of note, while most osteoblastogenesis markers were not differentially expressed between wildtype and Pls3-deficient cultures, the expression of Sfrp4 was significantly reduced in the latter, a potentially relevant finding, since Sfrp4 inactivation, in mice and humans, specifically causes cortical thinning. We finally addressed the question, if Pls3-deficiency would impair the osteoanabolic influence of parathyroid hormone (PTH). For this purpose we applied daily injection of PTH into wildtype and Pls3-deficient mice and found a similar response regardless of the genotype. Taken together, our data reveal that Pls3-deficiency in mice only recapitulates the cortical bone phenotype of individuals with X-linked osteoporosis by negatively affecting the early stage of cortical bone acquisition.
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Densidad Ósea , Osteoporosis , Animales , Densidad Ósea/genética , Hueso Cortical/diagnóstico por imagen , Glicoproteínas de Membrana , Ratones , Proteínas de Microfilamentos/genética , Osteoporosis/genéticaRESUMEN
Amphisomes are organelles of the autophagy pathway that result from the fusion of autophagosomes with late endosomes. While biogenesis of autophagosomes and late endosomes occurs continuously at axon terminals, non-degradative roles of autophagy at boutons are barely described. Here, we show that in neurons BDNF/TrkB traffick in amphisomes that signal locally at presynaptic boutons during retrograde transport to the soma. This is orchestrated by the Rap GTPase-activating (RapGAP) protein SIPA1L2, which connects TrkB amphisomes to a dynein motor. The autophagosomal protein LC3 regulates RapGAP activity of SIPA1L2 and controls retrograde trafficking and local signaling of TrkB. Following induction of presynaptic plasticity, amphisomes dissociate from dynein at boutons enabling local signaling and promoting transmitter release. Accordingly, sipa1l2 knockout mice show impaired BDNF-dependent presynaptic plasticity. Taken together, the data suggest that in hippocampal neurons, TrkB-signaling endosomes are in fact amphisomes that during retrograde transport have local signaling capacity in the context of presynaptic plasticity.
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Autofagosomas/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Endosomas/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Plasticidad Neuronal/genética , Neuronas/metabolismo , Terminales Presinápticos/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Animales , Transporte Axonal , Axones/metabolismo , Dineínas/metabolismo , Proteínas Activadoras de GTPasa/genética , Hipocampo , Ratones , Ratones Noqueados , Transporte de ProteínasRESUMEN
The endocannabinoid system (ECS) consists particularly of cannabinoid receptors 1 and 2 (CB1 and CB2), their endogenous ligands, and enzymes that synthesize and degrade their ligands. It acts in a variety of organs and disease states ranging from cancer progression over neuropathic pain to neurodegeneration. Protein components engaged in the signaling, trafficking, and homeostasis machinery of the G-protein coupled CB2, are however largely unknown. It is therefore important to identify further interaction partners to better understand CB2 receptor functions in physiology and pathophysiology. For this purpose, we used an affinity purification and mass spectrometry-based proteomics approach of Strep-HA-CB2 receptor in HEK293 cells. After subtraction of background interactions and protein frequency library assessment we could identify 83 proteins that were classified by the identification of minimally 2 unique peptides as highly probable interactors. A functional protein association network analysis obtained an interaction network with a significant enrichment of proteins functionally involved in protein metabolic process, in endoplasmic reticulum, response to stress but also in lipid metabolism and membrane organization. The network especially contains proteins involved in biosynthesis and trafficking like calnexin, Sec61A, tubulin chains TUBA1C and TUBB2B, TMED2, and TMED10. Six proteins that were only expressed in stable CB2 expressing cells were DHC24, DHRS7, GGT7, HECD3, KIAA2013, and PLS1. To exemplify the validity of our approach, we chose a candidate having a relatively low number of edges in the network to increase the likelihood of a direct protein interaction with CB2 and focused on the scaffold/phagosomal protein p62/SQSTM1. Indeed, we independently confirmed the interaction by co-immunoprecipitation and immunocytochemical colocalization studies. 3D reconstruction of confocal images furthermore showed CB2 localization in close proximity to p62 positive vesicles at the cell membrane. In summary, we provide a comprehensive repository of the CB2 interactome in HEK293 cells identified by a systematic unbiased approach, which can be used in future experiments to decipher the signaling and trafficking complex of this cannabinoid receptor. Future studies will have to analyze the exact mechanism of the p62-CB2 interaction as well as its putative role in disease pathophysiology.
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BACKGROUND: About 3%-5% of adults with membranous nephropathy have autoantibodies directed against thrombospondin type 1 domain-containing 7A (THSD7A), a podocyte-expressed transmembrane protein. However, the temporal and spatial expression of THSD7A and its biologic function for podocytes are unknown, information that is needed to understand the effects of THSD7A autoantibodies in this disease. METHODS: Using a variety of microscopic techniques, we analyzed THSD7A localization in postnatal, adult, and autoantibody-injected mice as well as in human podocytes. We also analyzed THSD7A function in human podocytes using confocal microscopy; Western blotting; and adhesion and migration assays. RESULTS: We found that THSD7A expression begins on glomerular vascularization with slit diaphragm formation in development. THSD7A localizes to the basal aspect of foot processes, closely following the meanders of the slit diaphragm in human and mice. Autoantibodies binding to THSD7A localize to the slit diaphragm. In human podocytes, THSD7A expression is accentuated at filopodia and thin arborized protrusions, an expression pattern associated with decreased membrane activity of cytoskeletal regulators. We also found that, phenotypically, THSD7A expression in human podocytes is associated not only with increases in cell size, enhanced adhesion, and reduced detachment from collagen type IV-coated plates but also, with decreased ability to migrate. CONCLUSIONS: Our findings suggest that THSD7A functions as a foot process protein involved in the stabilization of the slit diaphragm of mature podocytes and that autoantibodies to THSD7A, on the basis of their localization, might structurally and functionally alter the slit diaphragm's permeability to protein.