RESUMO
The peripheral nervous system has remarkable regenerative capacities in that it can repair a fully cut nerve. This requires Schwann cells to migrate collectively to guide regrowing axons across a 'bridge' of new tissue, which forms to reconnect a severed nerve. Here we show that blood vessels direct the migrating cords of Schwann cells. This multicellular process is initiated by hypoxia, selectively sensed by macrophages within the bridge, which via VEGF-A secretion induce a polarized vasculature that relieves the hypoxia. Schwann cells then use the blood vessels as "tracks" to cross the bridge taking regrowing axons with them. Importantly, disrupting the organization of the newly formed blood vessels in vivo, either by inhibiting the angiogenic signal or by re-orienting them, compromises Schwann cell directionality resulting in defective nerve repair. This study provides important insights into how the choreography of multiple cell-types is required for the regeneration of an adult tissue.
Assuntos
Vasos Sanguíneos/metabolismo , Macrófagos/metabolismo , Nervos Periféricos/fisiologia , Células de Schwann/metabolismo , Animais , Axônios/metabolismo , Hipóxia Celular , Células Endoteliais/metabolismo , Inflamação/metabolismo , Masculino , Camundongos , Neovascularização Fisiológica , Ratos , Ratos Sprague-Dawley , Regeneração , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Cellular attachment of viruses determines their cell tropism and species specificity. For entry, vaccinia, the prototypic poxvirus, relies on four binding proteins and an eleven-protein entry fusion complex. The contribution of the individual virus binding proteins to virion binding orientation and membrane fusion is unclear. Here, we show that virus binding proteins guide side-on virion binding and promote curvature of the host membrane towards the virus fusion machinery to facilitate fusion. Using a membrane-bleb model system together with super-resolution and electron microscopy we find that side-bound vaccinia virions induce membrane invagination in the presence of low pH. Repression or deletion of individual binding proteins reveals that three of four contribute to binding orientation, amongst which the chondroitin sulfate binding protein, D8, is required for host membrane bending. Consistent with low-pH dependent macropinocytic entry of vaccinia, loss of D8 prevents virion-associated macropinosome membrane bending, disrupts fusion pore formation and infection. Our results show that viral binding proteins are active participants in successful virus membrane fusion and illustrate the importance of virus protein architecture for successful infection.
Assuntos
Poxviridae , Vacínia , Humanos , Sulfatos de Condroitina , Vaccinia virus/metabolismo , Poxviridae/metabolismo , Proteínas Virais/metabolismo , Fusão de Membrana , Proteínas de TransporteRESUMO
All poxviruses contain a set of proteinaceous structures termed lateral bodies (LB) that deliver viral effector proteins into the host cytosol during virus entry. To date, the spatial proteotype of LBs remains unknown. Using the prototypic poxvirus, vaccinia virus (VACV), we employed a quantitative comparative mass spectrometry strategy to determine the poxvirus LB proteome. We identified a large population of candidate cellular proteins, the majority being mitochondrial, and 15 candidate viral LB proteins. Strikingly, one-third of these are VACV redox proteins whose LB residency could be confirmed using super-resolution microscopy. We show that VACV infection exerts an anti-oxidative effect on host cells and that artificial induction of oxidative stress impacts early and late gene expression as well as virion production. Using targeted repression and/or deletion viruses we found that deletion of individual LB-redox proteins was insufficient for host redox modulation suggesting there may be functional redundancy. In addition to defining the spatial proteotype of VACV LBs, these findings implicate poxvirus redox proteins as potential modulators of host oxidative anti-viral responses and provide a solid starting point for future investigations into the role of LB resident proteins in host immunomodulation.
Assuntos
Poxviridae , Linhagem Celular , Oxirredução , Poxviridae/genética , Poxviridae/metabolismo , Vaccinia virus/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação ViralRESUMO
Images are at the core of most modern biological experiments and are used as a major source of quantitative information. Numerous algorithms are available to process images and make them more amenable to be measured. Yet the nature of the quantitative output that is useful for a given biological experiment is uniquely dependent upon the question being investigated. Here, we discuss the 3 main types of information that can be extracted from microscopy data: intensity, morphology, and object counts or categorical labels. For each, we describe where they come from, how they can be measured, and what may affect the relevance of these measurements in downstream data analysis. Acknowledging that what makes a measurement 'good' is ultimately down to the biological question being investigated, this review aims at providing readers with a toolkit to challenge how they quantify their own data and be critical of conclusions drawn from quantitative bioimage analysis experiments.
RESUMO
Understanding the molecular mechanisms involved in the assembly of viruses is essential for discerning how viruses transmit from cell to cell and host to host. Although molecular aspects of assembly have been studied for many viruses, we still have little information about these events in real time. Enveloped viruses such as HIV that assemble at, and bud from, the plasma membrane have been studied in some detail using live cell fluorescence imaging techniques; however, these approaches provide little information about the real-time morphological changes that take place as viral components come together to form individual virus particles. Here we used correlative scanning ion conductance microscopy and fluorescence confocal microscopy to measure the topological changes, together with the recruitment of fluorescently labeled viral proteins such as Gag and Vpr, during the assembly and release of individual HIV virus-like particles (VLPs) from the top, nonadherent surfaces of living cells. We show that 1) labeling of viral proteins with green fluorescent protein affects particle formation, 2) the kinetics of particle assembly on different plasma membrane domains can vary, possibly as a consequence of differences in membrane biophysical properties, and 3) VLPs budding from the top, unimpeded surface of cells can reach full size in 20 s and disappear from the budding site in 0.5 to 3 min from the moment curvature is initially detected, significantly faster than has been previously reported.
Assuntos
HIV-1/metabolismo , Vírion/metabolismo , Montagem de Vírus/fisiologia , Linhagem Celular , Membrana Celular/metabolismo , Humanos , Liberação de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Clathrin light chain (CLC) subunits in vertebrates are encoded by paralogous genes CLTA and CLTB, and both gene products are alternatively spliced in neurons. To understand how this CLC diversity influences neuronal clathrin function, we characterized the biophysical properties of clathrin comprising individual CLC variants for correlation with neuronal phenotypes of mice lacking either CLC-encoding gene. CLC splice variants differentially influenced clathrin knee conformation within assemblies, and clathrin with neuronal CLC mixtures was more effective in membrane deformation than clathrin with single neuronal isoforms nCLCa or nCLCb. Correspondingly, electrophysiological recordings revealed that neurons from mice lacking nCLCa or nCLCb were both defective in synaptic vesicle replenishment. Mice with only nCLCb had a reduced synaptic vesicle pool and impaired neurotransmission compared to WT mice, while nCLCa-only mice had increased synaptic vesicle numbers, restoring normal neurotransmission. These findings highlight differences between the CLC isoforms and show that isoform mixing influences tissue-specific clathrin activity in neurons, which requires their functional balance.
Assuntos
Cadeias Leves de Clatrina , Vesículas Sinápticas/química , Vesículas Sinápticas/metabolismo , Animais , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/metabolismo , Células Cultivadas , Cadeias Leves de Clatrina/química , Cadeias Leves de Clatrina/genética , Cadeias Leves de Clatrina/metabolismo , Camundongos , Camundongos Knockout , Neurônios/citologia , Neurônios/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismoRESUMO
BACKGROUND: In vertebrate cells, the Golgi functional subunits, mini-stacks, are linked into a tri-dimensional network. How this "ribbon" architecture relates to Golgi functions remains unclear. Are all connections between mini-stacks equal? Is the local structure of the ribbon of functional importance? These are difficult questions to address, without a quantifiable readout of the output of ribbon-embedded mini-stacks. Endothelial cells produce secretory granules, the Weibel-Palade bodies (WPB), whose von Willebrand Factor (VWF) cargo is central to hemostasis. The Golgi apparatus controls WPB size at both mini-stack and ribbon levels. Mini-stack dimensions delimit the size of VWF "boluses" whilst the ribbon architecture allows their linear co-packaging, thereby generating WPBs of different lengths. This Golgi/WPB size relationship suits mathematical analysis. RESULTS: WPB lengths were quantized as multiples of the bolus size and mathematical modeling simulated the effects of different Golgi ribbon organizations on WPB size, to be compared with the ground truth of experimental data. An initial simple model, with the Golgi as a single long ribbon composed of linearly interlinked mini-stacks, was refined to a collection of mini-ribbons and then to a mixture of mini-stack dimers plus long ribbon segments. Complementing these models with cell culture experiments led to novel findings. Firstly, one-bolus sized WPBs are secreted faster than larger secretory granules. Secondly, microtubule depolymerization unlinks the Golgi into equal proportions of mini-stack monomers and dimers. Kinetics of binding/unbinding of mini-stack monomers underpinning the presence of stable dimers was then simulated. Assuming that stable mini-stack dimers and monomers persist within the ribbon resulted in a final model that predicts a "breathing" arrangement of the Golgi, where monomer and dimer mini-stacks within longer structures undergo continuous linking/unlinking, consistent with experimentally observed WPB size distributions. CONCLUSIONS: Hypothetical Golgi organizations were validated against a quantifiable secretory output. The best-fitting Golgi model, accounting for stable mini-stack dimers, is consistent with a highly dynamic ribbon structure, capable of rapid rearrangement. Our modeling exercise therefore predicts that at the fine-grained level the Golgi ribbon is more complex than generally thought. Future experiments will confirm whether such a ribbon organization is endothelial-specific or a general feature of vertebrate cells.
Assuntos
Células Endoteliais , Fator de von Willebrand , Células Cultivadas , Exocitose , Complexo de Golgi , Corpos de Weibel-Palade/fisiologia , Fator de von Willebrand/farmacologia , Fator de von Willebrand/fisiologiaRESUMO
The processes of life take place in multiple dimensions, but imaging these processes in even three dimensions is challenging. Here, we describe a workflow for 3D correlative light and electron microscopy (CLEM) of cell monolayers using fluorescence microscopy to identify and follow biological events, combined with serial blockface scanning electron microscopy to analyse the underlying ultrastructure. The workflow encompasses all steps from cell culture to sample processing, imaging strategy, and 3D image processing and analysis. We demonstrate successful application of the workflow to three studies, each aiming to better understand complex and dynamic biological processes, including bacterial and viral infections of cultured cells and formation of entotic cell-in-cell structures commonly observed in tumours. Our workflow revealed new insight into the replicative niche of Mycobacterium tuberculosis in primary human lymphatic endothelial cells, HIV-1 in human monocyte-derived macrophages, and the composition of the entotic vacuole. The broad application of this 3D CLEM technique will make it a useful addition to the correlative imaging toolbox for biomedical research.
Assuntos
Células Endoteliais/ultraestrutura , Imageamento Tridimensional , Macrófagos/ultraestrutura , Microscopia Eletrônica de Varredura/métodos , Sobrevivência Celular , Células Cultivadas , Células Endoteliais/microbiologia , Entose , HIV/ultraestrutura , Humanos , Espaço Intracelular/microbiologia , Macrófagos/virologia , Monócitos/citologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/ultraestruturaRESUMO
The outer segments of vertebrate rod photoreceptors are renewed every 10 d. Outer segment components are transported from the site of synthesis in the inner segment through the connecting cilium, followed by assembly of the highly ordered discs. Two models of assembly of discrete discs involving either successive fusion events between intracellular rhodopsin-bearing vesicles or the evagination of the plasma membrane followed by fusion of adjacent evaginations have been proposed. Here we use immuno-electron microscopy and electron tomography to show that rhodopsin is transported from the inner to the outer segment via the ciliary plasma membrane, subsequently forming successive evaginations that "zipper" up proximally, but at their leading edges are free to make junctions containing the protocadherin, PCDH21, with the inner segment plasma membrane. Given the physical dimensions of the evaginations, coupled with likely instability of the membrane cortex at the distal end of the connecting cilium, we propose that the evagination occurs via a process akin to blebbing and is not driven by actin polymerization. Disassembly of these junctions is accompanied by fusion of the leading edges of successive evaginations to form discrete discs. This fusion is topologically different to that mediated by the membrane fusion proteins, SNAREs, as initial fusion is between exoplasmic leaflets, and is accompanied by gain of the tetraspanin rim protein, peripherin.
Assuntos
Caderinas/metabolismo , Membrana Celular/metabolismo , Células Fotorreceptoras/metabolismo , Segmento Interno das Células Fotorreceptoras da Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Animais , Proteínas Relacionadas a Caderinas , Membrana Celular/ultraestrutura , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Olho/metabolismo , Olho/ultraestrutura , Proteínas do Olho/metabolismo , Camundongos Endogâmicos C57BL , Microscopia Imunoeletrônica , Proteínas Munc18/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células Fotorreceptoras/ultraestrutura , Proteínas Qa-SNARE/metabolismo , Segmento Interno das Células Fotorreceptoras da Retina/ultraestrutura , Células Fotorreceptoras Retinianas Bastonetes/ultraestrutura , Rodopsina/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Segmento Externo da Célula Bastonete/ultraestruturaRESUMO
BACKGROUND & AIMS: In the normal liver, hepatocytes form a uniquely polarised cell layer that enables movement of solutes from sinusoidal blood to canalicular bile. Whilst several cholestatic liver diseases with defects of hepatocyte polarity have been identified, the molecular mechanisms of pathogenesis are not well defined. One example is arthrogryposis, renal dysfunction and cholestasis syndrome, which in most patients is caused by VPS33B mutations. VPS33B is a protein involved in membrane trafficking that interacts with RAB11A at recycling endosomes. To understand the pathways that regulate hepatocyte polarity better, we investigated VPS33B deficiency using a novel mouse model with a liver-specific Vps33b deletion. METHODS: To assess functional polarity, plasma and bile samples were collected from Vps33b liver knockout (Vps33bfl/fl-AlfpCre) and control (Vps33bfl/fl) mice; bile components or injected substrates were quantitated by mass spectrometry or fluorometry. For structural analysis, livers underwent light and transmission electron microscopy. Apical membrane and tight junction protein localisation was assessed by immunostaining. Adeno-associated virus vectors were used for in vivo gene rescue experiments. RESULTS: Like patients, Vps33bfl/fl-AlfpCre mice showed mislocalisation of ATP-binding cassette proteins that are specifically trafficked to the apical membrane via Rab11a-positive recycling endosomes. This was associated with retention of bile components in blood. Loss of functional tight junction integrity and depletion of apical microvilli were seen in knockout animals. Gene transfer partially rescued these defects. CONCLUSIONS: Vps33b has a key role in establishing structural and functional aspects of hepatocyte polarity and may be a target for gene replacement therapy. LAY SUMMARY: Hepatocytes are liver cells with tops and bottoms; that is, they are polarised. At their bottoms they absorb substances from blood. They then, at their tops, secrete these substances and their metabolites into bile. When polarity is lost, this directional flow of substances from blood to bile is disrupted and liver disease follows. In this study, using a new mouse model with a liver-specific mutation of Vps33b, the mouse version of a gene that is mutated in most patients with arthrogryposis, renal dysfunction and cholestasis (ARC) syndrome, we investigated how the Vps33b gene product contributes to establishing hepatocyte polarity. We identified in these mice abnormalities similar to those in children with ARC syndrome. Gene transfer could partly reverse the mouse abnormalities. Our work contributes to the understanding of VPS33B disease and hepatocyte polarity in general, and may point towards gene transfer mediated treatment of ARC liver disease.
Assuntos
Polaridade Celular , Hepatócitos/fisiologia , Proteínas de Transporte Vesicular/fisiologia , Animais , Artrogripose/patologia , Artrogripose/terapia , Ácidos e Sais Biliares/sangue , Colestase/patologia , Colestase/terapia , Colesterol/sangue , Terapia Genética , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Insuficiência Renal/patologia , Insuficiência Renal/terapia , Junções Íntimas/fisiologia , Proteínas de Transporte Vesicular/genéticaRESUMO
Arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome is caused by deficiencies in the trafficking proteins VPS33B or VIPAR, and is associated with a bleeding diathesis and a marked reduction in platelet α-granules. We generated a tamoxifen-inducible mouse model of VPS33B deficiency, Vps33b(fl/fl)-ER(T2), and studied the platelet phenotype and α-granule biogenesis. Ultrastructural analysis of Vps33b(fl/fl)-ER(T2) platelets identified a marked reduction in α-granule count and the presence of small granule-like structures in agreement with the platelet phenotype observed in ARC patients. A reduction of â¼65% to 75% was observed in the α-granule proteins von Willebrand factor and P-selectin. Although platelet aggregation responses were not affected, a defect in δ-granule secretion was observed. Under arteriolar shear conditions, Vps33b(fl/fl)-ER(T2) platelets were unable to form stable aggregates, and tail-bleeding measurement revealed a bleeding diathesis. Analysis of bone marrow-derived megakaryocytes (MKs) by conventional and immuno-electron microscopy from Vps33b(fl/fl)-ER(T2) mice revealed a reduction in mature type-II multivesicular bodies (MVB II) and an accumulation of large vacuoles. Proteins that are normally stored in α-granules were underrepresented in MVB II and proplatelet extensions. These results demonstrate that abnormal protein trafficking and impairment in MVB maturation in MKs underlie the α-granule deficiency in Vps33b(fl/fl)-ER(T2) mouse and ARC patients.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Megacariócitos/metabolismo , Proteínas de Transporte Vesicular/fisiologia , Animais , Artrogripose/genética , Células Cultivadas , Colestase/genética , Síndrome da Plaqueta Cinza/genética , Síndrome da Plaqueta Cinza/metabolismo , Humanos , Megacariócitos/citologia , Megacariócitos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Organelas/metabolismo , Contagem de Plaquetas , Polimorfismo de Nucleotídeo Único , Transporte Proteico/genética , Insuficiência Renal/genética , Proteínas de Transporte Vesicular/genéticaRESUMO
BACKGROUND: In HIV-infected macrophages, newly formed progeny virus particles accumulate in intracellular plasma membrane-connected compartments (IPMCs). Although the virus is usually seen in these compartments, it is unclear whether HIV assembly is specifically targeted to IPMCs or whether some viruses may also form at the cell surface but are not detected, as particles budding from the latter site will be released into the medium. RESULTS: To investigate the fidelity of HIV-1 targeting to IPMCs compared to the cell surface directly, we generated mutants defective in recruitment of the Endosomal Sorting Complexes Required for Transport (ESCRT) proteins required for virus scission. For mutants unable to bind the ESCRT-I component Tsg101, HIV release was inhibited and light and electron microscopy revealed that budding was arrested. When expressed in human monocyte-derived macrophages (MDM), these mutants formed budding-arrested, immature particles at their assembly sites, allowing us to capture virtually all of the virus budding events. A detailed morphological analysis of the distribution of the arrested viruses by immunofluorescence staining and confocal microscopy, and by electron microscopy, demonstrated that HIV assembly in MDMs is targeted primarily to IPMCs, with fewer than 5 % of budding events seen at the cell surface. Morphometric analysis of the relative membrane areas at the cell surface and IPMCs confirmed a large enrichment of virus assembly events in IPMCs. Serial block-face scanning electron microscopy of macrophages infected with a budding-defective HIV mutant revealed high-resolution 3D views of the complex organisation of IPMCs, with in excess of 15,000 associated HIV budding sites, and multiple connections between IPMCs and the cell surface. CONCLUSIONS: Using detailed quantitative analysis, we demonstrate that HIV assembly in MDMs is specifically targeted to IPMCs. Furthermore, 3D analysis shows, for the first time, the detailed ultrastructure of an IPMC within a large cell volume, at a resolution that allowed identification of individual virus assembly events, and potential portals through which virus may be released during cell-cell transfer. These studies provide new insights to the organisation of the HIV assembly compartments in macrophages, and show how HIV particles accumulating in these protected sites may function as a virus reservoir.
Assuntos
Compartimento Celular , Membrana Celular/virologia , HIV-1/fisiologia , Espaço Intracelular/metabolismo , Macrófagos/patologia , Macrófagos/virologia , Montagem de Vírus/fisiologia , Microscopia Crioeletrônica , Células HEK293 , HIV-1/ultraestrutura , Humanos , Imageamento Tridimensional , Monócitos/patologia , Mutação/genética , Provírus/fisiologia , TransfecçãoRESUMO
Tissue slicing is at the core of many approaches to studying biological structures. Among the modern volume electron microscopy (vEM) methods, array tomography (AT) is based on serial ultramicrotomy, section collection onto solid support, imaging via light and/or scanning electron microscopy, and re-assembly of the serial images into a volume for analysis. While AT largely uses standard EM equipment, it provides several advantages, including long-term preservation of the sample and compatibility with multi-scale and multi-modal imaging. Furthermore, the collection of serial ultrathin sections improves axial resolution and provides access for molecular labeling, which is beneficial for light microscopy and immunolabeling, and facilitates correlation with EM. Despite these benefits, AT techniques are underrepresented in imaging facilities and labs, due to their perceived difficulty and lack of training opportunities. Here we point towards novel developments in serial sectioning and image analysis that facilitate the AT pipeline, and solutions to overcome constraints. Because no single vEM technique can serve all needs regarding field of view and resolution, we sketch a decision tree to aid researchers in navigating the plethora of options available. Lastly, we elaborate on the unexplored potential of AT approaches to add valuable insight in diverse biological fields.
RESUMO
Inorganic polyphosphate (polyP) is a ubiquitous polymer that controls fundamental processes. To overcome the absence of a genetically tractable mammalian model, we developed an inducible mammalian cell line expressing Escherichia coli polyphosphate kinase 1 (EcPPK1). Inducing EcPPK1 expression prompted polyP synthesis, enabling validation of polyP analytical methods. Virtually all newly synthesized polyP accumulates within the nucleus, mainly in the nucleolus. The channeled polyP within the nucleolus results in the redistribution of its markers, leading to altered rRNA processing. Ultrastructural analysis reveals electron-dense polyP structures associated with a hyper-condensed nucleolus resulting from an exacerbation of the liquid-liquid phase separation (LLPS) phenomena controlling this membraneless organelle. The selective accumulation of polyP in the nucleoli could be interpreted as an amplification of polyP channeling to where its physiological function takes place. Indeed, quantitative analysis of several mammalian cell lines confirms that endogenous polyP accumulates within the nucleolus.
Assuntos
Nucléolo Celular , Polifosfatos , Polifosfatos/metabolismo , Nucléolo Celular/metabolismo , Humanos , Animais , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Escherichia coli/metabolismo , Linhagem Celular , RNA Ribossômico/metabolismo , Células HeLaRESUMO
Presynaptic terminals are specialized sites for information transmission where vesicles fuse with the plasma membrane and are locally recycled. Recent work has extended this classical view, with the observation that a subset of functional vesicles is dynamically shared between adjacent terminals by lateral axonal transport. Conceptually, such transport would be expected to disrupt vesicle retention around the active zone, yet terminals are characterized by a high-density vesicle cluster, suggesting that counteracting stabilizing mechanisms must operate against this tendency. The synapsins are a family of proteins that associate with synaptic vesicles and determine vesicle numbers at the terminal, but their specific function remains controversial. Here, using multiple quantitative fluorescence-based approaches and electron microscopy, we show that synapsin is instrumental for resisting vesicle dispersion and serves as a regulatory element for controlling lateral vesicle sharing between synapses. Deleting synapsin disrupts the organization of presynaptic vesicle clusters, making their boundaries hard to define. Concurrently, the fraction of vesicles amenable to transport is increased, and more vesicles are translocated to the axon. Importantly, in neurons from synapsin knock-out mice the resting and recycling pools are equally mobile. Synapsin, when present, specifically restricts the mobility of resting pool vesicles without affecting the division of vesicles between these pools. Specific expression of synapsin IIa, the sole isoform affecting synaptic depression, rescues the knock-out phenotype. Together, our results show that synapsin is pivotal for maintaining synaptic vesicle cluster integrity and that it contributes to the regulated sharing of vesicles between terminals.
Assuntos
Hipocampo/citologia , Neurônios/fisiologia , Terminações Pré-Sinápticas/fisiologia , Sinapsinas/metabolismo , Vesículas Sinápticas/fisiologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Antagonistas de Aminoácidos Excitatórios/farmacologia , Recuperação de Fluorescência Após Fotodegradação , Regulação da Expressão Gênica/genética , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/ultraestrutura , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Purinas/farmacologia , Compostos de Piridínio/metabolismo , Compostos de Amônio Quaternário/metabolismo , Roscovitina , Estatísticas não Paramétricas , Sinapsinas/deficiência , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/ultraestrutura , Fatores de Tempo , Transfecção/métodos , Valina/análogos & derivados , Valina/farmacologia , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
The mechanisms that underlie the selection of an inhibitory GABAergic axon's postsynaptic targets and the formation of the first contacts are currently unknown. To determine whether expression of GABAA receptors (GABAA Rs) themselves--the essential functional postsynaptic components of GABAergic synapses--can be sufficient to initiate formation of synaptic contacts, a novel co-culture system was devised. In this system, the presynaptic GABAergic axons originated from embryonic rat basal ganglia medium spiny neurones, whereas their most prevalent postsynaptic targets, i.e., α1/ß2/γ2-GABAA Rs, were expressed constitutively in a stably transfected human embryonic kidney 293 (HEK293) cell line. The first synapse-like contacts in these co-cultures were detected by colocalization of presynaptic and postsynaptic markers within 2 h. The number of contacts reached a plateau at 24 h. These contacts were stable, as assessed by live cell imaging; they were active, as determined by uptake of a fluorescently labelled synaptotagmin vesicle-luminal domain-specific antibody; and they supported spontaneous and action potential-driven postsynaptic GABAergic currents. Ultrastructural analysis confirmed the presence of characteristics typical of active synapses. Synapse formation was not observed with control or N-methyl-d-aspartate receptor-expressing HEK293 cells. A prominent increase in synapse formation and strength was observed when neuroligin-2 was co-expressed with GABAA Rs, suggesting a cooperative relationship between these proteins. Thus, in addition to fulfilling an essential functional role, postsynaptic GABAA Rs can promote the adhesion of inhibitory axons and the development of functional synapses.
Assuntos
Axônios/fisiologia , Receptores de GABA-A/metabolismo , Sinapses/fisiologia , Potenciais Sinápticos , Potenciais de Ação , Animais , Axônios/metabolismo , Gânglios da Base/citologia , Gânglios da Base/crescimento & desenvolvimento , Gânglios da Base/fisiologia , Moléculas de Adesão Celular Neuronais/metabolismo , Processos de Crescimento Celular , Neurônios GABAérgicos/metabolismo , Neurônios GABAérgicos/fisiologia , Células HEK293 , Humanos , Proteínas do Tecido Nervoso/metabolismo , Ratos , Ratos Sprague-Dawley , Sinapses/metabolismoRESUMO
The techniques collectively known as volume electron microscopy (vEM) each come with their own advantages and challenges, making them more or less suitable for any specific project. SEM array tomography (SEM-AT) is certainly no different in this respect. Requiring microtomy skills, and involving more data alignment post imaging, SEM-AT presents challenges to its users, nevertheless, as perhaps the most flexible, cost effective and potentially accessible vEM approach to regular EM facilities, it benefits those same users with multiple advantages due to its inherently non-destructive nature. The general principles and advantages/disadvantages of SEM-AT are described here, together with a step-by-step guide to the workflow, from block trimming, sectioning and collection on coverslips, to alignment of the high-resolution 3D dataset. With a suitable SEM/backscatter electron detector setup, and equipment readily found in an electron microscopy lab, it should be possible to begin to acquire 3D ultrastructural data. With the addition of appropriate SEM-AT imaging software, this process can be significantly enhanced to automatically image hundreds, potentially thousands, of sections. Hardware and software advances and future improvements will only make this easier, to the extent that SEM-AT could become a routine vEM technique throughout the world, rather than the privilege of a small number of experts in limited specialist facilities.
Assuntos
Imageamento Tridimensional , Microscopia Eletrônica de Volume , Microscopia Eletrônica de Varredura , Imageamento Tridimensional/métodos , Microtomia/métodos , TomografiaRESUMO
The blood barriers of the nervous system protect neural environments but can hinder therapeutic accessibility. The blood-brain barrier (BBB) is well characterized, consisting of endothelial cells with specialized tight junctions and low levels of transcytosis, properties conferred by contacting pericytes and astrocytes. In contrast, the blood-nerve barrier (BNB) of the peripheral nervous system is poorly defined. Here, we characterize the structure of the mammalian BNB, identify the processes that confer barrier function, and demonstrate how the barrier can be opened in response to injury. The homeostatic BNB is leakier than the BBB, which we show is due to higher levels of transcytosis. However, the barrier is reinforced by macrophages that specifically engulf leaked materials, identifying a role for resident macrophages as an important component of the BNB. Finally, we demonstrate the exploitation of these processes to effectively deliver RNA-targeting therapeutics to peripheral nerves, indicating new treatment approaches for nervous system pathologies.
Assuntos
Barreira Hematoneural , Células Endoteliais , Animais , Barreira Hematoneural/fisiologia , Células Endoteliais/fisiologia , Barreira Hematoencefálica/fisiologia , Macrófagos , Pericitos/fisiologia , MamíferosRESUMO
Alzheimer's disease (AD) is characterized by synaptic loss, which can result from dysfunctional microglial phagocytosis and complement activation. However, what signals drive aberrant microglia-mediated engulfment of synapses in AD is unclear. Here we report that secreted phosphoprotein 1 (SPP1/osteopontin) is upregulated predominantly by perivascular macrophages and, to a lesser extent, by perivascular fibroblasts. Perivascular SPP1 is required for microglia to engulf synapses and upregulate phagocytic markers including C1qa, Grn and Ctsb in presence of amyloid-ß oligomers. Absence of Spp1 expression in AD mouse models results in prevention of synaptic loss. Furthermore, single-cell RNA sequencing and putative cell-cell interaction analyses reveal that perivascular SPP1 induces microglial phagocytic states in the hippocampus of a mouse model of AD. Altogether, we suggest a functional role for SPP1 in perivascular cells-to-microglia crosstalk, whereby SPP1 modulates microglia-mediated synaptic engulfment in mouse models of AD.