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Although fluorescence microscopy is ubiquitous in biomedical research, microscopy methods reporting is inconsistent and perhaps undervalued. We emphasize the importance of appropriate microscopy methods reporting and seek to educate researchers about how microscopy metadata impact data interpretation. We provide comprehensive guidelines and resources to enable accurate reporting for the most common fluorescence light microscopy modalities. We aim to improve microscopy reporting, thus improving the quality, rigor and reproducibility of image-based science.
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Investigación Biomédica/métodos , Investigación Biomédica/normas , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Fluorescente/métodos , Microscopía Fluorescente/normas , Convallaria , Escherichia coli/metabolismo , Colorantes Fluorescentes , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Imagenología Tridimensional , Microscopía Confocal/métodos , Reproducibilidad de los Resultados , Proyectos de Investigación , Relación Señal-Ruido , Programas InformáticosRESUMEN
For quality, interpretation, reproducibility and sharing value, microscopy images should be accompanied by detailed descriptions of the conditions that were used to produce them. Micro-Meta App is an intuitive, highly interoperable, open-source software tool that was developed in the context of the 4D Nucleome (4DN) consortium and is designed to facilitate the extraction and collection of relevant microscopy metadata as specified by the recent 4DN-BINA-OME tiered-system of Microscopy Metadata specifications. In addition to substantially lowering the burden of quality assurance, the visual nature of Micro-Meta App makes it particularly suited for training purposes.
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Metadatos , Microscopía Confocal/instrumentación , Microscopía Confocal/métodos , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Aplicaciones Móviles , Lenguajes de Programación , Programas Informáticos , Animales , Línea Celular , Biología Computacional/métodos , Humanos , Procesamiento de Imagen Asistido por Computador , Ratones , Reconocimiento de Normas Patrones Automatizadas , Control de Calidad , Reproducibilidad de los Resultados , Interfaz Usuario-Computador , Flujo de TrabajoRESUMEN
In the dynamic landscape of scientific research, imaging core facilities are vital hubs propelling collaboration and innovation at the technology development and dissemination frontier. Here, we present a collaborative effort led by Global BioImaging (GBI), introducing international recommendations geared towards elevating the careers of Imaging Scientists in core facilities. Despite the critical role of Imaging Scientists in modern research ecosystems, challenges persist in recognising their value, aligning performance metrics and providing avenues for career progression and job security. The challenges encompass a mismatch between classic academic career paths and service-oriented roles, resulting in a lack of understanding regarding the value and impact of Imaging Scientists and core facilities and how to evaluate them properly. They further include challenges around sustainability, dedicated training opportunities and the recruitment and retention of talent. Structured across these interrelated sections, the recommendations within this publication aim to propose globally applicable solutions to navigate these challenges. These recommendations apply equally to colleagues working in other core facilities and research institutions through which access to technologies is facilitated and supported. This publication emphasises the pivotal role of Imaging Scientists in advancing research programs and presents a blueprint for fostering their career progression within institutions all around the world.
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Investigadores , Humanos , Movilidad Laboral , Investigación Biomédica/métodos , Selección de ProfesiónRESUMEN
In September 2023, the two largest bioimaging networks in the Americas, Latin America Bioimaging (LABI) and BioImaging North America (BINA), came together during a 1-week meeting in Mexico. This meeting provided opportunities for participants to interact closely with decision-makers from imaging core facilities across the Americas. The meeting was held in a hybrid format and attended in-person by imaging scientists from across the Americas, including Canada, the United States, Mexico, Colombia, Peru, Argentina, Chile, Brazil and Uruguay. The aims of the meeting were to discuss progress achieved over the past year, to foster networking and collaborative efforts among members of both communities, to bring together key members of the international imaging community to promote the exchange of experience and expertise, to engage with industry partners, and to establish future directions within each individual network, as well as common goals. This meeting report summarises the discussions exchanged, the achievements shared, and the goals set during the LABIxBINA2023: Bioimaging across the Americas meeting.
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Retroviruses incorporate specific host cell RNAs into virions. In particular, the host noncoding 7SL RNA is highly abundant in all examined retroviruses compared with its cellular levels or relative to common mRNAs such as actin. Using live cell imaging techniques, we have determined that the 7SL RNA does not arrive with the HIV-1 RNA genome. Instead, it is recruited contemporaneously with assembly of the protein HIV-1 Gag at the plasma membrane. Further, we demonstrate that complexes of 7SL RNA and Gag can be immunoprecipitated from both cytosolic and plasma membrane fractions. This indicates that 7SL RNAs likely interact with Gag prior to high-order Gag multimerization at the plasma membrane. Thus, the interactions between Gag and the host RNA 7SL occur independent of the interactions between Gag and the host endosomal sorting complex required for transport (ESCRT) proteins, which are recruited temporarily at late stages of assembly. The interactions of 7SL and Gag are also independent of interactions of Gag and the HIV-1 genome which are seen on the plasma membrane prior to assembly of Gag.
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VIH-1/fisiología , ARN Citoplasmático Pequeño/metabolismo , Partícula de Reconocimiento de Señal/metabolismo , Ensamble de Virus , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Células HeLa , Humanos , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoAsunto(s)
Núcleo Celular/ultraestructura , Cromatina/ultraestructura , Procesamiento de Imagen Asistido por Computador/normas , Informática Médica/estadística & datos numéricos , Metadatos/estadística & datos numéricos , Microscopía/métodos , Programas Informáticos , Humanos , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
Human immunodeficiency virus (HIV)-1 infection and the associated disease AIDS are a major cause of human death worldwide with no vaccine or cure available. The trafficking of HIV-1 RNAs from sites of synthesis in the nucleus, through the cytoplasm, to sites of assembly at the plasma membrane are critical steps in HIV-1 viral replication, but are not well characterized. Here we present a broadly accessible microscopy method that captures multiple focal planes simultaneously, which allows us to image the trafficking of HIV-1 genomic RNAs with high precision. This method utilizes a customization of a commercial multichannel emission splitter that enables high-resolution 3D imaging with single-macromolecule sensitivity. We show with high temporal and spatial resolution that HIV-1 genomic RNAs are most mobile in the cytosol, and undergo confined mobility at sites along the nuclear envelope and in the nucleus and nucleolus. These provide important insights regarding the mechanism by which the HIV-1 RNA genome is transported to the sites of assembly of nascent virions.
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Genoma Viral/genética , Infecciones por VIH/virología , VIH-1/genética , Ensamble de Virus/genética , Ensamble de Virus/fisiología , Línea Celular Tumoral , Núcleo Celular/virología , Citoplasma/virología , Células HeLa , Humanos , Imagenología Tridimensional/métodos , Microscopía Fluorescente/métodos , ARN Viral/genética , Replicación Viral/genéticaRESUMEN
HIV-1 virions assemble at the plasma membrane of mammalian cells and recruit the endosomal sorting complex required for transport (ESCRT) machinery to enable particle release. However, little is known about the temporal and spatial organization of ESCRT protein recruitment. Using multiple-color live-cell total internal reflection fluorescence microscopy, we observed that the ESCRT-I protein Tsg101 is recruited together with Gag to the sites of HIV-1 assembly, whereas later-acting ESCRT proteins (Chmp4b and Vps4A) are recruited sequentially, once Gag assembly is completed. Chmp4b, a protein that is required to mediate particle scission, is recruited to HIV-1 assembly sites â¼10 s before the ATPase Vps4A. Using two-color superresolution imaging, we observed that the ESCRT machinery (Tsg101, Alix, and Chmp4b/c proteins) is positioned at the periphery of the nascent virions, with the Tsg101 assemblages positioned closer to the Gag assemblages than Alix, Chmp4b, or Chmp4c. These results are consistent with the notion that the ESCRT machinery is recruited transiently to the neck of the assembling particle and is thus present at the appropriate time and place to mediate fission between the nascent virus and the plasma membrane.
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Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , VIH-1/fisiología , Liberación del Virus , ATPasas Asociadas con Actividades Celulares Diversas , Células HeLa , Humanos , ATPasas de Translocación de Protón Vacuolares/metabolismo , Ensamble de VirusRESUMEN
Presently, there are few estimates of the number of molecules occupying membrane domains. Using a total internal reflection fluorescence microscopy (TIRFM) imaging approach, based on comparing the intensities of fluorescently labeled microdomains with those of single fluorophores, we measured the occupancy of DC-SIGN, a C-type lectin, in membrane microdomains. DC-SIGN or its mutants were labeled with primary monoclonal antibodies (mAbs) in either dendritic cells (DCs) or NIH3T3 cells, or expressed as GFP fusions in NIH3T3 cells. The number of DC-SIGN molecules per microdomain ranges from only a few to over 20, while microdomain dimensions range from the diffraction limit to > 1 µm. The largest fraction of microdomains, appearing at the diffraction limit, in either immature DCs or 3 T3 cells contains only 4-8 molecules of DC-SIGN, consistent with our preliminary super-resolution Blink microscopy estimates. We further show that these small assemblies are sufficient to bind and efficiently internalize a small (â¼ 50 nm) pathogen, dengue virus, leading to infection of host cells.
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Moléculas de Adhesión Celular/metabolismo , Lectinas Tipo C/metabolismo , Microdominios de Membrana/ultraestructura , Microscopía Fluorescente/métodos , Receptores de Superficie Celular/metabolismo , Animales , Células Dendríticas/ultraestructura , Células Dendríticas/virología , Virus del Dengue/metabolismo , Humanos , Microdominios de Membrana/metabolismo , Microdominios de Membrana/virología , Ratones , Células 3T3 NIH , Unión Proteica , Internalización del VirusRESUMEN
Dendritic cell-specific intercellular adhesion molecule (ICAM)-3-grabbing non-integrin (DC-SIGN) is a Ca(2+) -dependent transmembrane lectin that binds a large variety of pathogens and facilitates their uptake for subsequent antigen presentation. This receptor is present in cell surface microdomains, but factors involved in microdomain formation and their exceptional stability are not clear. To determine which domain/motif of DC-SIGN facilitates its presence in microdomains, we studied mutations at key locations including truncation of the cytoplasmic tail, and ectodomain mutations that resulted in the removal of the N-linked glycosylation site, the tandem repeats and the carbohydrate recognition domain (CRD), as well as modification of the calcium sites in the CRD required for carbohydrate binding. Confocal imaging and fluorescence recovery after photobleaching measurements showed that the cytoplasmic domain and the N-linked glycosylation site do not affect the ability of DC-SIGN to form stable microdomains. However, truncation of the CRD results in complete loss of visible microdomains and subsequent lateral diffusion of the mutants. Apart from cell adhesions, membrane domains are thought to be localized primarily via the cytoskeleton. By contrast, we propose that interactions between the CRD of DC-SIGN and the extracellular matrix and/or cis interactions with transmembrane scaffolding protein(s) play an essential role in organizing these microdomains.
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Moléculas de Adhesión Celular/química , Matriz Extracelular/metabolismo , Lectinas Tipo C/química , Receptores de Superficie Celular/química , Secuencias de Aminoácidos , Animales , Antígenos CD/metabolismo , Calcio/química , Calcio/metabolismo , Carbohidratos/química , Adhesión Celular , Moléculas de Adhesión Celular/metabolismo , Citoesqueleto/metabolismo , Glicosilación , Humanos , Ratones , Modelos Biológicos , Mutación , Células 3T3 NIH , Estructura Terciaria de Proteína , Transducción de SeñalRESUMEN
Extracellular vesicles (EVs), or exosomes, play important roles in physiological and pathological cellular communication and have gained substantial traction as biological drug carriers. EVs contain both short and long non-coding RNAs that regulate gene expression and epigenetic processes. To fully capitalize on the potential of EVs as drug carriers, it is important to study and understand the intricacies of EV function and EV RNA-based communication. Here we developed a genetically encodable RNA-based biomaterial, termed EXO-Probe, for tracking EV RNAs. The EXO-Probe comprises an EV-loading RNA sequence (EXO-Code), fused to a fluorogenic RNA Mango aptamer for RNA imaging. This fusion construct allowed the visualization and tracking of EV RNA and colocalization with markers of multivesicular bodies; imaging RNA within EVs, and non-destructive quantification of EVs. Overall, the new RNA-based biomaterial provides a useful and versatile means to interrogate the role of EVs in cellular communication via RNA trafficking to EVs and to study cellular sorting decisions. The system will also help lay the foundation to further improve the therapeutic efficacy of EVs as drug carriers.
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Tau-mediated toxicity is associated with cognitive decline and Alzheimer's disease (AD) progression. In particular, tau post-translational modifications (PTMs) are thought to generate aberrant tau species resulting in neuronal dysfunction. Despite being well characterized in postmortem AD brain, it is unclear how caspase-mediated C-terminal tau cleavage promotes neurodegeneration, as few studies have developed the models to dissect this pathogenic mechanism. Here, we show that proteasome impairment results in cleaved tau accumulation at the post-synaptic density (PSD), a process that is modulated by neuronal activity. Cleaved tau (at residue D421) impairs neuronal firing and causes inefficient initiation of network bursts, consistent with reduced excitatory drive. We propose that reduced neuronal activity, or silencing, is coupled to proteasome dysfunction, which drives cleaved tau accumulation at the PSD and subsequent synaptotoxicity. Our study connects three common themes in the progression of AD: impaired proteostasis, caspase-mediated tau cleavage, and synapse degeneration.
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Dendritic cells express DC-SIGN, a C-type lectin (CTL) that binds a variety of pathogens and facilitates their uptake for subsequent antigen presentation. DC-SIGN forms remarkably stable microdomains on the plasma membrane. However, inner leaflet lipid markers are able to diffuse through these microdomains suggesting that, rather than being densely packed with DC-SIGN proteins, an elemental substructure exists. Therefore, a super-resolution imaging technique, Blink Microscopy (Blink), was applied to further investigate the lateral distribution of DC-SIGN. Blink indicates that DC-SIGN, another CTL (CD206), and influenza hemagglutinin (HA) are all localized in small (â¼80 nm in diameter) nanodomains. DC-SIGN and CD206 nanodomains are randomly distributed on the plasma membrane, whereas HA nanodomains cluster on length scales up to several microns. We estimate, as a lower limit, that DC-SIGN and HA nanodomains contain on average two tetramers or two trimers, respectively, whereas CD206 is often nonoligomerized. Two-color Blink determined that different CTLs rarely occupy the same nanodomain, although they appear colocalized using wide-field microscopy. What to our knowledge is a novel domain structure emerges in which elemental nanodomains, potentially capable of binding viruses, are organized in a random fashion; evidently, these nanodomains can be clustered into larger microdomains that act as receptor platforms for larger pathogens like yeasts.
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Moléculas de Adhesión Celular/química , Membrana Celular/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Lectinas Tipo C/química , Microscopía/métodos , Imagen Molecular/métodos , Nanoestructuras , Receptores de Superficie Celular/química , Animales , Moléculas de Adhesión Celular/metabolismo , Membrana Celular/metabolismo , Vidrio/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Humanos , Lectinas Tipo C/metabolismo , Receptor de Manosa , Lectinas de Unión a Manosa/química , Lectinas de Unión a Manosa/metabolismo , Ratones , Células 3T3 NIH , Estructura Terciaria de Proteína , Transporte de Proteínas , Receptores de Superficie Celular/metabolismoRESUMEN
Recombinant adeno-associated virus (rAAV) vectors have been widely used as favored delivery vehicles for the treatment of inherited diseases in clinical trials, including neurological diseases. However, the noninvasive systemic delivery of rAAV to the central nervous system is severely hampered by the blood-brain barrier (BBB). Several approaches have been exploited to enhance AAV vector brain transduction after systemic administration, including genetic modification of AAV capsids and physical methods. However, these approaches are not always predictive of desirable outcomes in humans and induce complications. It is imperative to explore novel strategies to increase the ability of AAV9 to cross the BBB for enhanced brain transduction. Herein, we have conducted a combinatorial in vivo/in vitro phage display library screening in mouse brains and purified AAV9 virions to identify a customized BBB shuttle peptide, designated as PB5-3. The PB5-3 peptide specifically bound to AAV9 virions and enhanced widespread transduction of AAV9 in mouse brains, especially in neuronal cells, after systemic administration. Further study demonstrated that systemic administration of AAV9 vectors encoding IDUA complexed with PB5-3 increased the phenotypic correction in the brains of MPS I mice. Mechanistic studies revealed that the PB5-3 peptide effectively increased AAV9 trafficking and transcytosis efficiency in the human BBB model hCMEC/D3 cell line but did not interfere with AAV9 binding to the receptor terminal N-linked galactosylated glycans. Additionally, the PB5-3 peptide slowed the clearance of AAV9 from blood without hepatic toxicity. This study highlights, for the first time, the potential of this combinatorial approach for the isolation of peptides that interact with specific AAV vectors for enhanced and targeted AAV transduction. This promising approach will open new combined therapeutic avenues and shed light on the potential applications of peptides for the treatment of human diseases in future clinical trials with AAV vector-mediated gene delivery.
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Barrera Hematoencefálica , Vectores Genéticos , Animales , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Dependovirus/genética , Técnicas de Transferencia de Gen , Ratones , Péptidos/metabolismo , Transducción GenéticaRESUMEN
DC-SIGN, a Ca(2+)-dependent transmembrane lectin, is found assembled in microdomains on the plasma membranes of dendritic cells. These microdomains bind a large variety of pathogens and facilitate their uptake for subsequent antigen presentation. In this study, DC-SIGN dynamics in microdomains were explored with several fluorescence microscopy methods and compared with dynamics for influenza hemagglutinin (HA), which is also found in plasma membrane microdomains. Fluorescence imaging indicated that DC-SIGN microdomains may contain other C-type lectins and that the DC-SIGN cytoplasmic region is not required for microdomain formation. Fluorescence recovery after photobleaching measurements showed that neither full-length nor cytoplasmically truncated DC-SIGN in microdomains appreciably exchanged with like molecules in other microdomains and the membrane surround, whereas HA in microdomains exchanged almost completely. Line-scan fluorescence correlation spectroscopy indicated an essentially undetectable lateral mobility for DC-SIGN but an appreciable mobility for HA within their respective domains. Single-particle tracking with defined-valency quantum dots confirmed that HA has significant mobility within microdomains, whereas DC-SIGN does not. By contrast, fluorescence recovery after photobleaching indicated that inner leaflet lipids are able to move through DC-SIGN microdomains. The surprising stability of DC-SIGN microdomains may reflect structural features that enhance pathogen uptake either by providing high-avidity platforms and/or by protecting against rapid microdomain endocytosis.
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Moléculas de Adhesión Celular/metabolismo , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Lectinas Tipo C/metabolismo , Microdominios de Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , Moléculas de Adhesión Celular/química , Clatrina/metabolismo , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Lectinas Tipo C/química , Receptor de Manosa , Lectinas de Unión a Manosa/metabolismo , Proteínas de la Membrana/metabolismo , Movimiento , Proteínas del Tejido Nervioso/metabolismo , Transporte de Proteínas , Puntos Cuánticos , Receptores de Superficie Celular/químicaRESUMEN
Tauopathies including Alzheimer's disease (AD) are marked by the accumulation of aberrantly modified tau proteins. Acetylated tau, in particular, has recently been implicated in neurodegeneration and cognitive decline. HDAC6 reversibly regulates tau acetylation, but its role in tauopathy progression remains unclear. Here, we identified an HDAC6-chaperone complex that targets aberrantly modified tau. HDAC6 not only deacetylates tau but also suppresses tau hyperphosphorylation within the microtubule-binding region. In neurons and human AD brain, HDAC6 becomes co-aggregated within focal tau swellings and human AD neuritic plaques. Using mass spectrometry, we identify a novel HDAC6-regulated tau acetylation site as a disease specific marker for 3R/4R and 3R tauopathies, supporting uniquely modified tau species in different neurodegenerative disorders. Tau transgenic mice lacking HDAC6 show reduced survival characterized by accelerated tau pathology and cognitive decline. We propose that a HDAC6-dependent surveillance mechanism suppresses toxic tau accumulation, which may protect against the progression of AD and related tauopathies.
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Disfunción Cognitiva/patología , Histona Desacetilasa 6/metabolismo , Tauopatías/patología , Proteínas tau/metabolismo , Acetilación , Anciano , Anciano de 80 o más Años , Animales , Encéfalo/patología , Disfunción Cognitiva/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Histona Desacetilasa 6/genética , Humanos , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Fosforilación , Procesamiento Proteico-Postraduccional , Tauopatías/genética , Proteínas tau/genéticaRESUMEN
Adeno-associated virus (AAV) vectors have become an important tool for delivering therapeutic genes for a wide range of neurological diseases. AAV serotypes possess differential cellular tropism in the central nervous system. Although several AAV serotypes or mutants have been reported to transduce the brain efficiently, conflicting data occur across studies with the use of various rodent strains from different genetic backgrounds. Herein, we performed a systematic comparison of the brain transduction properties among five AAV serotypes (AAV2, 5, 7, 8, and 9) in two common rodent strains (C57BL/6J and FVB/N), following local intrastriatal injection of AAV vectors encoding enhanced green fluorescent protein (EGFP) driven by the CBh promoter. Important differences were found regarding overall cellular tropism and transduction efficiency, including contralateral transduction among the AAV serotypes and between the mouse strains. We have further found loss of NeuN-immunoreactivity and microglial activation from AAV transduction in the different mouse strains. The important strain-specific differences from our study suggest that the genetic background of the mouse may affect AAV serotype transduction properties in the brain. These data can provide valuable information about how to choose an effective AAV vector for clinical application and interpret the data obtained from preclinical studies and clinical trials.