RESUMEN
Elucidating the 3D nanoscale structure of tissues and cells is essential for understanding the complexity of biological processes. Electron microscopy (EM) offers the resolution needed for reliable interpretation, but the limited throughput of electron microscopes has hindered its ability to effectively image large volumes. We report a workflow for volume EM with FAST-EM, a novel multibeam scanning transmission electron microscope that speeds up acquisition by scanning the sample in parallel with 64 electron beams. FAST-EM makes use of optical detection to separate the signals of the individual beams. The acquisition and 3D reconstruction of ultrastructural data from multiple biological samples is demonstrated. The results show that the workflow is capable of producing large reconstructed volumes with high resolution and contrast to address biological research questions within feasible acquisition time frames.
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Commander is a multiprotein complex that orchestrates endosomal recycling of integral cargo proteins and is essential for normal development. While the structure of this complex has recently been described, how cargo proteins are selected for Commander-mediated recycling remains unclear. Here we identify the mechanism through which the unstructured carboxy-terminal tail of the cargo adaptor sorting nexin-17 (SNX17) directly binds to the Retriever sub-complex of Commander. SNX17 adopts an autoinhibited conformation where its carboxy-terminal tail occupies the cargo binding groove. Competitive cargo binding overcomes this autoinhibition, promoting SNX17 endosomal residency and the release of the tail for Retriever association. Furthermore, our study establishes the central importance of SNX17-Retriever association in the handover of integrin and lipoprotein receptor cargoes into pre-existing endosomal retrieval sub-domains. In describing the principal mechanism of cargo entry into the Commander recycling pathway we provide key insight into the function and regulation of this evolutionary conserved sorting pathway.
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Endosomas , Transporte de Proteínas , Nexinas de Clasificación , Endosomas/metabolismo , Nexinas de Clasificación/metabolismo , Nexinas de Clasificación/genética , Humanos , Unión Proteica , Células HeLa , Integrinas/metabolismoRESUMEN
Tissue repair is disturbed in fibrotic diseases like systemic sclerosis (SSc), where the deposition of large amounts of extracellular matrix components such as collagen interferes with organ function. LAIR-1 is an inhibitory collagen receptor highly expressed on tissue immune cells. We questioned whether in SSc, impaired LAIR-1-collagen interaction is contributing to the ongoing inflammation and fibrosis. We found that SSc patients do not have an intrinsic defect in LAIR-1 expression or function. Instead, fibroblasts from healthy controls and SSc patients stimulated by soluble factors that drive inflammation and fibrosis in SSc deposit disorganized collagen products in vitro, which are dysfunctional LAIR-1 ligands. This is dependent of matrix metalloproteinases and platelet-derived growth factor receptor signaling. In support of a non-redundant role of LAIR-1 in the control of fibrosis, we found that LAIR-1-deficient mice have increased skin fibrosis in response to repeated injury and in the bleomycin mouse model for SSc. Thus, LAIR-1 represents an essential control mechanism for tissue repair. In fibrotic disease, excessive collagen degradation may lead to a disturbed feedback loop. The presence of functional LAIR-1 in patients provides a therapeutic opportunity to reactivate this intrinsic negative feedback mechanism in fibrotic diseases.
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Colágeno , Modelos Animales de Enfermedad , Fibroblastos , Fibrosis , Ratones Noqueados , Receptores Inmunológicos , Esclerodermia Sistémica , Animales , Humanos , Esclerodermia Sistémica/inmunología , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patología , Ratones , Receptores Inmunológicos/metabolismo , Receptores Inmunológicos/genética , Colágeno/metabolismo , Fibroblastos/metabolismo , Bleomicina/efectos adversos , Piel/patología , Piel/metabolismo , Piel/inmunología , Transducción de Señal , Masculino , Femenino , Células CultivadasRESUMEN
The multisubunit HOPS tethering complex is a well-established regulator of lysosome fusion with late endosomes and autophagosomes. However, the role of the HOPS complex in other stages of endo-lysosomal trafficking is not well understood. To address this, we made HeLa cells knocked out for the HOPS-specific subunits Vps39 or Vps41, or the HOPS-CORVET-core subunits Vps18 or Vps11. In all four knockout cells, we found that endocytosed cargos were trapped in enlarged endosomes that clustered in the perinuclear area. By correlative light-electron microscopy, these endosomes showed a complex ultrastructure and hybrid molecular composition, displaying markers for early (Rab5, PtdIns3P, EEA1) as well as late (Rab7, CD63, LAMP1) endosomes. These "HOPS bodies" were not acidified, contained enzymatically inactive cathepsins and accumulated endocytosed cargo and cation-independent mannose-6-phosphate receptor (CI-MPR). Consequently, CI-MPR was depleted from the TGN, and secretion of lysosomal enzymes to the extracellular space was enhanced. Strikingly, HOPS bodies also contained the autophagy proteins p62 and LC3, defining them as amphisomes. Together, these findings show that depletion of the lysosomal HOPS complex has a profound impact on the functional organization of the entire endosomal system and suggest the existence of a HOPS-independent mechanism for amphisome formation.
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Endocitosis , Endosomas , Humanos , Células HeLa , Endosomas/metabolismo , Membranas Intracelulares , Lisosomas/metabolismoRESUMEN
Fluorescent biosensors are valuable tools to monitor protein activities and the functional state of organelles in live cells. However, the information provided by fluorescent microscopy (FM) is mostly limited in resolution and lacks ultrastructural context information. Protein activities are confined to organelle zones with a distinct membrane morphology, which can only be seen by electron microscopy (EM). EM, however, intrinsically lacks information on protein activities. The lack of methods to integrate these two imaging modalities has hampered understanding the functional organization of cellular organelles. Here we introduce "functional correlative microscopy" (functional CLEM) to directly infer functional information from live cells to EM with nanometer resolution. We label and visualize live cells with fluorescent biosensors after which they are processed for EM and imaged using a volume electron microscopy technique. Within a single dataset we correlate hundreds of fluorescent spots enabling quantitative analysis of the functional-ultrastructural data. We employ our method to monitor essential functional parameters of late endo-lysosomal compartments, i.e., pH, calcium, enzyme activities and cholesterol content. Our data reveal a steep functional difference in enzyme activity between late endosomes and lysosomes and unexpectedly high calcium levels in late endosomes. The presented CLEM workflow is compatible with a large repertoire of probes and paves the way for large scale functional studies of all types of cellular structures.
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Calcio , Microscopía Electrónica de Volumen , Humanos , Células HeLa , Microscopía Electrónica , LisosomasRESUMEN
The visualization of autophagic organelles at the ultrastructural level by electron microscopy (EM) is essential to establish their identity and reveal details that are important for understanding the autophagic process. However, EM methods often lack molecular information, obstructing the correlation of ultrastructural information obtained by EM to fluorescence microscopy-based localization of specific autophagy proteins. Furthermore, the rarity of autophagosomes in unaltered cellular conditions hampers investigation by EM, which requires high magnification, and hence provides a limited field of view. In answer to both challenges, an on-section correlative light-electron microscopy (CLEM) method based on fluorescent labeling was applied to correlate a common autophagosomal marker, LC3, to EM ultrastructure. The method was used to rapidly screen cells in fluorescence microscopy for LC3 labeling in combination with other relevant markers. Subsequently, the underlying ultrastructural features of selected LC3-labeled spots were identified by CLEM. The method was applied to starved cells without adding inhibitors of lysosomal acidification. In these conditions, LC3 was found predominantly on autophagosomes and rarely in autolysosomes, in which LC3 is rapidly degraded. These data show both the feasibility and sensitivity of this approach, demonstrating that CLEM can be used to provide ultrastructural insights on LC3-mediated autophagy in native conditions-without drug treatments or genetic alterations. Overall, this method presents a valuable tool for ultrastructural localization studies of autophagy proteins and other scarce antigens by bridging light microscopy to EM data.
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Autofagia , Lisosomas , Microscopía Electrónica , Microscopía Fluorescente , OrgánulosRESUMEN
Arl8b, an Arf-like GTP-binding protein, regulates cargo trafficking and positioning of lysosomes. However, it is unknown whether Arl8b regulates lysosomal cargo sorting. Here, we report that Arl8b binds to the Rab4 and Rab14 interaction partner, RUN and FYVE domain-containing protein (RUFY) 1, a known regulator of cargo sorting from recycling endosomes. Arl8b determines RUFY1 endosomal localization through regulating its interaction with Rab14. RUFY1 depletion led to a delay in CI-M6PR retrieval from endosomes to the TGN, resulting in impaired delivery of newly synthesized hydrolases to lysosomes. We identified the dynein-dynactin complex as an RUFY1 interaction partner, and similar to a subset of activating dynein adaptors, the coiled-coil region of RUFY1 was required for interaction with dynein and the ability to mediate dynein-dependent organelle clustering. Our findings suggest that Arl8b and RUFY1 play a novel role on recycling endosomes, from where this machinery regulates endosomes to TGN retrieval of CI-M6PR and, consequently, lysosomal cargo sorting.
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Factores de Ribosilacion-ADP , Proteínas Adaptadoras Transductoras de Señales , Dineínas , Endosomas , Lisosomas , Proteínas de Unión al GTP rab , Humanos , Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/metabolismo , Complejo Dinactina/metabolismo , Dineínas/metabolismo , Endosomas/metabolismo , Células HeLa , Lisosomas/metabolismo , Transporte de Proteínas , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismoRESUMEN
We present a bimodal endocytic tracer, fluorescent BSA-gold (fBSA-Au), as a fiducial marker for 2D and 3D correlative light and electron microscopy (CLEM) applications. fBSA-Au consists of colloidal gold (Au) particles stabilized with fluorescent BSA. The conjugate is efficiently endocytosed and distributed throughout the 3D endolysosomal network of cells and has an excellent visibility in both fluorescence microscopy (FM) and electron microscopy (EM). We demonstrate that fBSA-Au facilitates rapid registration in several 2D and 3D CLEM applications using Tokuyasu cryosections, resin-embedded material, and cryoelectron microscopy (cryo-EM). Endocytosed fBSA-Au benefits from a homogeneous 3D distribution throughout the endosomal system within the cell, does not obscure any cellular ultrastructure, and enables accurate (50-150 nm) correlation of fluorescence to EM data. The broad applicability and visibility in both modalities makes fBSA-Au an excellent endocytic fiducial marker for 2D and 3D (cryo)CLEM applications.
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Crioultramicrotomía , Microscopía por Crioelectrón/métodos , Microscopía Electrónica , Microscopía Fluorescente/métodos , Crioultramicrotomía/métodosRESUMEN
Elevated expression of non-receptor tyrosine kinase FER is an independent prognosticator that correlates with poor survival of high-grade and basal/triple-negative breast cancer (TNBC) patients. Here, we show that high FER levels are also associated with improved outcomes after adjuvant taxane-based combination chemotherapy in high-risk, HER2-negative patients. In TNBC cells, we observe a causal relation between high FER levels and sensitivity to taxanes. Proteomics and mechanistic studies demonstrate that FER regulates endosomal recycling, a microtubule-dependent process that underpins breast cancer cell invasion. Using chemical genetics, we identify DCTN2 as a FER substrate. Our work indicates that the DCTN2 tyrosine 6 is essential for the development of tubular recycling domains in early endosomes and subsequent propagation of TNBC cell invasion in 3D. In conclusion, we show that high FER expression promotes endosomal recycling and represents a candidate predictive marker for the benefit of adjuvant taxane-containing chemotherapy in high-risk patients, including TNBC patients.
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Neoplasias de la Mama , Neoplasias de la Mama Triple Negativas , Neoplasias de la Mama/metabolismo , Hidrocarburos Aromáticos con Puentes/farmacología , Hidrocarburos Aromáticos con Puentes/uso terapéutico , Endosomas/metabolismo , Femenino , Humanos , Taxoides/farmacología , Taxoides/uso terapéutico , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
Intracellular processes depend on a strict spatial and temporal organization of proteins and organelles. Therefore, directly linking molecular to nanoscale ultrastructural information is crucial in understanding cellular physiology. Volume or three-dimensional (3D) correlative light and electron microscopy (volume-CLEM) holds unique potential to explore cellular physiology at high-resolution ultrastructural detail across cell volumes. However, the application of volume-CLEM is hampered by limitations in throughput and 3D correlation efficiency. In order to address these limitations, we describe a novel pipeline for volume-CLEM that provides high-precision (<100 nm) registration between 3D fluorescence microscopy (FM) and 3D electron microscopy (EM) datasets with significantly increased throughput. Using multi-modal fiducial nanoparticles that remain fluorescent in epoxy resins and a 3D confocal fluorescence microscope integrated into a Focused Ion Beam Scanning Electron Microscope (FIB.SEM), our approach uses FM to target extremely small volumes of even single organelles for imaging in volume EM and obviates the need for post-correlation of big 3D datasets. We extend our targeted volume-CLEM approach to include live-cell imaging, adding information on the motility of intracellular membranes selected for volume-CLEM. We demonstrate the power of our approach by targeted imaging of rare and transient contact sites between the endoplasmic reticulum (ER) and lysosomes within hours rather than days. Our data suggest that extensive ER-lysosome and mitochondria-lysosome interactions restrict lysosome motility, highlighting the unique capabilities of our integrated CLEM pipeline for linking molecular dynamic data to high-resolution ultrastructural detail in 3D.
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Microglia are the major innate immune cells in the brain and are essential for maintaining homeostasis in a neuronal microenvironment. Currently, a genetic tool to modify microglial gene expression in specific brain regions is not available. In this report, we introduce a tailor-designed method that uses lipid and polymer hybridized nanoparticles (LPNPs) for the local delivery of small interfering RNAs (siRNAs), allowing the silencing of specific microglial genes in the hypothalamus. Our physical characterization proved that this LPNP-siRNA was uniform and stable. We demonstrated that, due to their natural phagocytic behavior, microglial cells are the dominant cell type taking up these LPNPs in the hypothalamus of rats. We then tested the silencing efficiency of LPNPs carrying a cluster of differentiation molecule 11b (CD11b) or Toll-like receptor 4 (TLR4) siRNA using different in vivo and in vitro approaches. In cultured microglial cells treated with LPNP-CD11b siRNA or LPNP-TLR4 siRNA, we found a silencing efficiency at protein expression levels of 65 or 77%, respectively. In line with this finding, immunohistochemistry and western blotting results from in vivo experiments showed that LPNP-CD11b siRNA significantly inhibited microglial CD11b protein expression in the hypothalamus. Furthermore, following lipopolysaccharide (LPS) stimulation of cultured microglial cells, gene expression of the TLR4 downstream signaling component myeloid differentiation factor 88 and its associated cytokines was significantly inhibited in LPNP-TLR4 siRNA-treated microglial cells compared with cells treated with LPNP-scrambled siRNA. Finally, after LPNP-TLR4 siRNA injection into the rat hypothalamus, we observed a significant reduction in microglial activation in response to LPS compared with the control rats injected with LPNP-scrambled siRNA. Our results indicate that LPNP-siRNA is a promising tool to manipulate microglial activity locally in the brain and may serve as a prophylactic approach to prevent microglial dysfunction-associated diseases.
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Portadores de Fármacos/química , Expresión Génica/efectos de los fármacos , Hipotálamo/efectos de los fármacos , Microglía/efectos de los fármacos , Nanopartículas/química , ARN Interferente Pequeño/farmacología , Animales , Antígeno CD11b/antagonistas & inhibidores , Antígeno CD11b/genética , Lípidos/química , Masculino , Poliésteres/química , Polietilenglicoles/química , Ratas Wistar , Receptor Toll-Like 4/antagonistas & inhibidores , Receptor Toll-Like 4/genéticaRESUMEN
The key endosomal regulators Rab5, EEA1, and APPL1 are frequently applied in fluorescence microscopy to mark early endosomes, whereas Rab7 is used as a marker for late endosomes and lysosomes. However, endogenous levels of these proteins localize poorly in immuno-EM, and systematic studies on their native ultrastructural distributions are lacking. To address this gap, we here present a quantitative, on-section correlative light and electron microscopy (CLEM) approach. Using the sensitivity of fluorescence microscopy, we label hundreds of organelles that are subsequently visualized by EM and classified by ultrastructure. We show that Rab5 predominantly marks small, endocytic vesicles and early endosomes. EEA1 colocalizes with Rab5 on early endosomes, but unexpectedly also labels Rab5-negative late endosomes, which are positive for PI(3)P but lack Rab7. APPL1 is restricted to small Rab5-positive, tubulo-vesicular profiles. Rab7 primarily labels late endosomes and lysosomes. These data increase our understanding of the structural-functional organization of the endosomal system and introduce quantitative CLEM as a sensitive alternative for immuno-EM.
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Endosomas/ultraestructura , Microscopía Electrónica , Proteínas de Transporte Vesicular/ultraestructura , Antígenos/metabolismo , Línea Celular Tumoral , Endosomas/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Lisosomas/metabolismo , Lisosomas/ultraestructura , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Inborn errors of metabolism (IEMs) comprise a diverse group of individually rare monogenic disorders that affect metabolic pathways. Mutations lead to enzymatic deficiency or dysfunction, which results in intermediate metabolite accumulation or deficit leading to disease phenotypes. Currently, treatment options for many IEMs are insufficient. Rarity of individual IEMs hampers therapy development and phenotypic and genetic heterogeneity suggest beneficial effects of personalized approaches. Recently, cultures of patient-own liver-derived intrahepatic cholangiocyte organoids (ICOs) have been established. Since most metabolic genes are expressed in the liver, patient-derived ICOs represent exciting possibilities for in vitro modeling and personalized drug testing for IEMs. However, the exact application range of ICOs remains unclear. To address this, we examined which metabolic pathways can be studied with ICOs and what the potential and limitations of patient-derived ICOs are to model metabolic functions. We present functional assays in patient ICOs with defects in branched-chain amino acid metabolism (methylmalonic acidemia), copper metabolism (Wilson disease), and transporter defects (cystic fibrosis). We discuss the broad range of functional assays that can be applied to ICOs, but also address the limitations of these patient-specific cell models. In doing so, we aim to guide the selection of the appropriate cell model for studies of a specific disease or metabolic process.
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Errores Innatos del Metabolismo de los Aminoácidos , Organoides , Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Humanos , Hígado/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Redes y Vías Metabólicas , Organoides/metabolismoRESUMEN
Malignant rhabdoid tumors (MRTs) represent one of the most aggressive childhood malignancies. No effective treatment options are available, and prognosis is, therefore, dismal. Previous studies have demonstrated that tumor organoids capture the heterogeneity of patient tumors and can be used to predict patient response to therapy. Here, we perform drug screening on patient-derived normal and tumor organoids to identify MRT-specific therapeutic vulnerabilities. We identify neddylation inhibitor MLN4924 as a potential therapeutic agent. Mechanistically, we find increased neddylation in MRT organoids and tissues and show that MLN4924 induces a cytotoxic response via upregulation of the unfolded protein response. Lastly, we demonstrate in vivo efficacy in an MRT PDX mouse model, in which single-agent MLN4924 treatment significantly extends survival. Our study demonstrates that organoids can be used to find drugs selectively targeting tumor cells while leaving healthy cells unharmed and proposes neddylation inhibition as a therapeutic strategy in MRT.
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Ciclopentanos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Organoides/metabolismo , Pirimidinas/farmacología , Tumor Rabdoide , Respuesta de Proteína Desplegada/efectos de los fármacos , Animales , Línea Celular Tumoral , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Tumor Rabdoide/tratamiento farmacológico , Tumor Rabdoide/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The accumulation of α-synuclein (α-syn) aggregates in specific brain regions is a hallmark of synucleinopathies including Parkinson disease (PD). α-Syn aggregates propagate in a "prion-like" manner and can be transferred inside lysosomes to recipient cells through tunneling nanotubes (TNTs). However, how lysosomes participate in the spreading of α-syn aggregates is unclear. Here, by using super-resolution (SR) and electron microscopy (EM), we find that α-syn fibrils affect the morphology of lysosomes and impair their function in neuronal cells. In addition, we demonstrate that α-syn fibrils induce peripheral redistribution of lysosomes, likely mediated by transcription factor EB (TFEB), increasing the efficiency of α-syn fibrils' transfer to neighboring cells. We also show that lysosomal membrane permeabilization (LMP) allows the seeding of soluble α-syn in cells that have taken up α-syn fibrils from the culture medium, and, more importantly, in healthy cells in coculture, following lysosome-mediated transfer of the fibrils. Moreover, we demonstrate that seeding occurs mainly at lysosomes in both donor and acceptor cells, after uptake of α-syn fibrils from the medium and following their transfer, respectively. Finally, by using a heterotypic coculture system, we determine the origin and nature of the lysosomes transferred between cells, and we show that donor cells bearing α-syn fibrils transfer damaged lysosomes to acceptor cells, while also receiving healthy lysosomes from them. These findings thus contribute to the elucidation of the mechanism by which α-syn fibrils spread through TNTs, while also revealing the crucial role of lysosomes, working as a Trojan horse for both seeding and propagation of disease pathology.
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Lisosomas/metabolismo , Nanotubos , Pliegue de Proteína , alfa-Sinucleína/metabolismo , Animales , Permeabilidad de la Membrana Celular , Técnicas de Cocultivo , Humanos , Lisosomas/ultraestructura , Microscopía ElectrónicaRESUMEN
Neuronal function relies on careful coordination of organelle organization and transport. Kinesin-1 mediates transport of the endoplasmic reticulum (ER) and lysosomes into the axon and it is increasingly recognized that contacts between the ER and lysosomes influence organelle organization. However, it is unclear how organelle organization, inter-organelle communication and transport are linked and how this contributes to local organelle availability in neurons. Here, we show that somatic ER tubules are required for proper lysosome transport into the axon. Somatic ER tubule disruption causes accumulation of enlarged and less motile lysosomes at the soma. ER tubules regulate lysosome size and axonal translocation by promoting lysosome homo-fission. ER tubule - lysosome contacts often occur at a somatic pre-axonal region, where the kinesin-1-binding ER-protein P180 binds microtubules to promote kinesin-1-powered lysosome fission and subsequent axonal translocation. We propose that ER tubule - lysosome contacts at a pre-axonal region finely orchestrate axonal lysosome availability for proper neuronal function.
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Axones/metabolismo , Retículo Endoplásmico/metabolismo , Lisosomas/metabolismo , Neuronas/metabolismo , Animales , Transporte Axonal/fisiología , Células Cultivadas , Femenino , Cinesinas/metabolismo , Microtúbulos/metabolismo , Neuronas/citología , Unión Proteica , Ratas WistarRESUMEN
Vacuolar protein sorting 41 (VPS41) is as part of the Homotypic fusion and Protein Sorting (HOPS) complex required for lysosomal fusion events and, independent of HOPS, for regulated secretion. Here, we report three patients with compound heterozygous mutations in VPS41 (VPS41S285P and VPS41R662* ; VPS41c.1423-2A>G and VPS41R662* ) displaying neurodegeneration with ataxia and dystonia. Cellular consequences were investigated in patient fibroblasts and VPS41-depleted HeLa cells. All mutants prevented formation of a functional HOPS complex, causing delayed lysosomal delivery of endocytic and autophagic cargo. By contrast, VPS41S285P enabled regulated secretion. Strikingly, loss of VPS41 function caused a cytosolic redistribution of mTORC1, continuous nuclear localization of Transcription Factor E3 (TFE3), enhanced levels of LC3II, and a reduced autophagic response to nutrient starvation. Phosphorylation of mTORC1 substrates S6K1 and 4EBP1 was not affected. In a C. elegans model of Parkinson's disease, co-expression of VPS41S285P /VPS41R662* abolished the neuroprotective function of VPS41 against α-synuclein aggregates. We conclude that the VPS41 variants specifically abrogate HOPS function, which interferes with the TFEB/TFE3 axis of mTORC1 signaling, and cause a neurodegenerative disease.
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Enfermedades Neurodegenerativas , Animales , Autofagia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Caenorhabditis elegans/genética , Células HeLa , Humanos , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Enfermedades Neurodegenerativas/genética , Transporte de Proteínas , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Large-scale electron microscopy (EM) allows analysis of both tissues and macromolecules in a semi-automated manner, but acquisition rate forms a bottleneck. We reasoned that a negative bias potential may be used to enhance signal collection, allowing shorter dwell times and thus increasing imaging speed. Negative bias potential has previously been used to tune penetration depth in block-face imaging. However, optimization of negative bias potential for application in thin section imaging will be needed prior to routine use and application in large-scale EM. Here, we present negative bias potential optimized through a combination of simulations and empirical measurements. We find that the use of a negative bias potential generally results in improvement of image quality and signal-to-noise ratio (SNR). The extent of these improvements depends on the presence and strength of a magnetic immersion field. Maintaining other imaging conditions and aiming for the same image quality and SNR, the use of a negative stage bias can allow for a 20-fold decrease in dwell time, thus reducing the time for a week long acquisition to less than 8 h. We further show that negative bias potential can be applied in an integrated correlative light electron microscopy (CLEM) application, allowing fast acquisition of a high precision overlaid LM-EM dataset. Application of negative stage bias potential will thus help to solve the current bottleneck of image acquisition of large fields of view at high resolution in large-scale microscopy.