RESUMEN
Endosymbioses have shaped the evolutionary trajectory of life and remain ecologically important. Investigating oceanic photosymbioses can illuminate how algal endosymbionts are energetically exploited by their heterotrophic hosts and inform on putative initial steps of plastid acquisition in eukaryotes. By combining three-dimensional subcellular imaging with photophysiology, carbon flux imaging, and transcriptomics, we show that cell division of endosymbionts (Phaeocystis) is blocked within hosts (Acantharia) and that their cellular architecture and bioenergetic machinery are radically altered. Transcriptional evidence indicates that a nutrient-independent mechanism prevents symbiont cell division and decouples nuclear and plastid division. As endosymbiont plastids proliferate, the volume of the photosynthetic machinery volume increases 100-fold in correlation with the expansion of a reticular mitochondrial network in close proximity to plastids. Photosynthetic efficiency tends to increase with cell size, and photon propagation modeling indicates that the networked mitochondrial architecture enhances light capture. This is accompanied by 150-fold higher carbon uptake and up-regulation of genes involved in photosynthesis and carbon fixation, which, in conjunction with a ca.15-fold size increase of pyrenoids demonstrates enhanced primary production in symbiosis. Mass spectrometry imaging revealed major carbon allocation to plastids and transfer to the host cell. As in most photosymbioses, microalgae are contained within a host phagosome (symbiosome), but here, the phagosome invaginates into enlarged microalgal cells, perhaps to optimize metabolic exchange. This observation adds evidence that the algal metamorphosis is irreversible. Hosts, therefore, trigger and benefit from major bioenergetic remodeling of symbiotic microalgae with potential consequences for the oceanic carbon cycle. Unlike other photosymbioses, this interaction represents a so-called cytoklepty, which is a putative initial step toward plastid acquisition.
Asunto(s)
Metabolismo Energético , Haptophyta/metabolismo , Plancton/citología , Simbiosis , Ciclo del Carbono , División Celular , Núcleo Celular/metabolismo , Microalgas/citología , Mitocondrias/metabolismo , Fotosíntesis , Plastidios/metabolismoRESUMEN
Live-cell correlative light-electron microscopy (live-cell-CLEM) integrates live movies with the corresponding electron microscopy (EM) image, but a major challenge is to relate the dynamic characteristics of single organelles to their 3-dimensional (3D) ultrastructure. Here, we introduce focused ion beam scanning electron microscopy (FIB-SEM) in a modular live-cell-CLEM pipeline for a single organelle CLEM. We transfected cells with lysosomal-associated membrane protein 1-green fluorescent protein (LAMP-1-GFP), analyzed the dynamics of individual GFP-positive spots, and correlated these to their corresponding fine-architecture and immediate cellular environment. By FIB-SEM we quantitatively assessed morphological characteristics, like number of intraluminal vesicles and contact sites with endoplasmic reticulum and mitochondria. Hence, we present a novel way to integrate multiple parameters of subcellular dynamics and architecture onto a single organelle, which is relevant to address biological questions related to membrane trafficking, organelle biogenesis and positioning. Furthermore, by using CLEM to select regions of interest, our method allows for targeted FIB-SEM, which significantly reduces time required for image acquisition and data processing.
Asunto(s)
Lisosomas/ultraestructura , Biogénesis de Organelos , Tomografía con Microscopio Electrónico/métodos , Células HeLa , Humanos , Proteínas de Membrana de los Lisosomas/metabolismo , Lisosomas/metabolismo , Imagen Óptica/métodosRESUMEN
Intravital microscopy provides dynamic understanding of multiple cell biological processes, but its limited resolution has so far precluded structural analysis. Because it is difficult to capture rare and transient events, only a few attempts have been made to observe specific developmental and pathological processes in animal models using electron microscopy. The multimodal correlative approach that we propose here combines intravital microscopy, microscopic X-ray computed tomography and three-dimensional electron microscopy. It enables a rapid (c.a. 2â weeks) and accurate (<5â µm) correlation of functional imaging to ultrastructural analysis of single cells in a relevant context. We demonstrate the power of our approach by capturing single tumor cells in the vasculature of the cerebral cortex and in subcutaneous tumors, providing unique insights into metastatic events. Providing a significantly improved throughput, our workflow enables multiple sampling, a prerequisite for making correlative imaging a relevant tool to study cell biology in vivo. Owing to the versatility of this workflow, we envision broad applications in various fields of biological research, such as cancer or developmental biology.
Asunto(s)
Rastreo Celular/métodos , Animales , Encéfalo/irrigación sanguínea , Encéfalo/patología , Neoplasias Encefálicas/diagnóstico por imagen , Neoplasias Encefálicas/secundario , Línea Celular Tumoral , Femenino , Microscopía Intravital , Ratones Desnudos , Microscopía Electrónica de Rastreo , Trasplante de Neoplasias , Microambiente Tumoral , Microtomografía por Rayos XRESUMEN
We identified a novel, nontoxic mushroom protein that specifically binds to a complex of sphingomyelin (SM), a major sphingolipid in mammalian cells, and cholesterol (Chol). The purified protein, termed nakanori, labeled cell surface domains in an SM- and Chol-dependent manner and decorated specific lipid domains that colocalized with inner leaflet small GTPase H-Ras, but not K-Ras. The use of nakanori as a lipid-domain-specific probe revealed altered distribution and dynamics of SM/Chol on the cell surface of Niemann-Pick type C fibroblasts, possibly explaining some of the disease phenotype. In addition, that nakanori treatment of epithelial cells after influenza virus infection potently inhibited virus release demonstrates the therapeutic value of targeting specific lipid domains for anti-viral treatment.-Makino, A., Abe, M., Ishitsuka, R., Murate, M., Kishimoto, T., Sakai, S., Hullin-Matsuda, F., Shimada, Y., Inaba, T., Miyatake, H., Tanaka, H., Kurahashi, A., Pack, C.-G., Kasai, R. S., Kubo, S., Schieber, N. L., Dohmae, N., Tochio, N., Hagiwara, K., Sasaki, Y., Aida, Y., Fujimori, F., Kigawa, T., Nishibori, K., Parton, R. G., Kusumi, A., Sako, Y., Anderluh, G., Yamashita, M., Kobayashi, T., Greimel, P., Kobayashi, T. A novel sphingomyelin/cholesterol domain-specific probe reveals the dynamics of the membrane domains during virus release and in Niemann-Pick type C.
Asunto(s)
Colesterol/metabolismo , Proteínas Fúngicas/farmacología , Grifola/química , Microdominios de Membrana/efectos de los fármacos , Enfermedad de Niemann-Pick Tipo C/metabolismo , Esfingomielinas/metabolismo , Sitios de Unión , Células Cultivadas , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Células HeLa , Humanos , Microdominios de Membrana/metabolismo , Microdominios de Membrana/virología , Unión Proteica , Liberación del VirusRESUMEN
Several studies have suggested crosstalk between different clathrin-independent endocytic pathways. However, the molecular mechanisms and functional relevance of these interactions are unclear. Caveolins and cavins are crucial components of caveolae, specialized microdomains that also constitute an endocytic route. Here we show that specific caveolar proteins are independently acting negative regulators of clathrin-independent endocytosis. Cavin-1 and Cavin-3, but not Cavin-2 or Cavin-4, are potent inhibitors of the clathrin-independent carriers/GPI-AP enriched early endosomal compartment (CLIC/GEEC) endocytic pathway, in a process independent of caveola formation. Caveolin-1 (CAV1) and CAV3 also inhibit the CLIC/GEEC pathway upon over-expression. Expression of caveolar protein leads to reduction in formation of early CLIC/GEEC carriers, as detected by quantitative electron microscopy analysis. Furthermore, the CLIC/GEEC pathway is upregulated in cells lacking CAV1/Cavin-1 or with reduced expression of Cavin-1 and Cavin-3. Inhibition by caveolins can be mimicked by the isolated caveolin scaffolding domain and is associated with perturbed diffusion of lipid microdomain components, as revealed by fluorescence recovery after photobleaching (FRAP) studies. In the absence of cavins (and caveolae) CAV1 is itself endocytosed preferentially through the CLIC/GEEC pathway, but the pathway loses polarization and sorting attributes with consequences for membrane dynamics and endocytic polarization in migrating cells and adult muscle tissue. We also found that noncaveolar Cavin-1 can act as a modulator for the activity of the key regulator of the CLIC/GEEC pathway, Cdc42. This work provides new insights into the regulation of noncaveolar clathrin-independent endocytosis by specific caveolar proteins, illustrating multiple levels of crosstalk between these pathways. We show for the first time a role for specific cavins in regulating the CLIC/GEEC pathway, provide a new tool to study this pathway, identify caveola-independent functions of the cavins and propose a novel mechanism for inhibition of the CLIC/GEEC pathway by caveolin.
Asunto(s)
Caveolas/metabolismo , Caveolina 1/metabolismo , Endocitosis/fisiología , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Unión al ARN/metabolismo , Células 3T3 , Animales , Células COS , Movimiento Celular , Fenómenos Fisiológicos Celulares , Chlorocebus aethiops , Colesterol/metabolismo , Clatrina , Endocitosis/genética , Activación Enzimática , Proteínas Ligadas a GPI/metabolismo , Receptores de Hialuranos/metabolismo , Proteínas de la Membrana/genética , Ratones , Interferencia de ARN , ARN Interferente Pequeño , Proteínas de Unión al ARN/genética , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Sphingomyelin (SM) is a major sphingolipid in mammalian cells and is reported to form specific lipid domains together with cholesterol. However, methods to examine the membrane distribution of SM are limited. We demonstrated in model membranes that fluorescent protein conjugates of 2 specific SM-binding toxins, lysenin (Lys) and equinatoxin II (EqtII), recognize different membrane distributions of SM; Lys exclusively binds clustered SM, whereas EqtII preferentially binds dispersed SM. Freeze-fracture immunoelectron microscopy showed that clustered but not dispersed SM formed lipid domains on the cell surface. Glycolipids and the membrane concentration of SM affect the SM distribution pattern on the plasma membrane. Using derivatives of Lys and EqtII as SM distribution-sensitive probes, we revealed the exclusive accumulation of SM clusters in the midbody at the time of cytokinesis. Interestingly, apical membranes of differentiated epithelial cells exhibited dispersed SM distribution, whereas SM was clustered in basolateral membranes. Clustered but not dispersed SM was absent from the cell surface of acid sphingomyelinase-deficient Niemann-Pick type A cells. These data suggest that both the SM content and membrane distribution are crucial for pathophysiological events bringing therapeutic perspective in the role of SM membrane distribution.
Asunto(s)
Citocinesis/fisiología , Esfingomielinas/metabolismo , Animales , Células COS , Membrana Celular/metabolismo , Polaridad Celular , Supervivencia Celular , Chlorocebus aethiops , ADN Complementario/metabolismo , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/citología , Fibroblastos/metabolismo , Células HeLa , Humanos , Lactante , Liposomas/metabolismo , Masculino , Microscopía de Fuerza Atómica , Microscopía Confocal , Microscopía Inmunoelectrónica , Enfermedad de Niemann-Pick Tipo A/genética , Proteínas Recombinantes/metabolismoRESUMEN
In recent years, Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) has emerged as a flexible method that enables semi-automated volume ultrastructural imaging. We present a toolset for adherent cells that enables tracking and finding cells, previously identified in light microscopy (LM), in the FIB-SEM, along with the automatic acquisition of high-resolution volume datasets. We detect the underlying grid pattern in both modalities (LM and EM), to identify common reference points. A combination of computer vision techniques enables complete automation of the workflow. This includes setting the coincidence point of both ion and electron beams, automated evaluation of the image quality and constantly tracking the sample position with the microscope's field of view reducing or even eliminating operator supervision. We show the ability to target the regions of interest in EM within 5 µm accuracy while iterating between different targets and implementing unattended data acquisition. Our results demonstrate that executing volume acquisition in multiple locations autonomously is possible in EM.
Asunto(s)
Imagenología Tridimensional , Microscopía Electrónica de Volumen , Microscopía Electrónica de Rastreo , Imagenología Tridimensional/métodos , Programas InformáticosRESUMEN
Crossing the blood-brain barrier is a crucial, rate-limiting step of brain metastasis. Understanding of the mechanisms of cancer cell extravasation from brain microcapillaries is limited as the underlying cellular and molecular processes cannot be adequately investigated using in vitro models and endpoint in vivo experiments. Using ultrastructural and functional imaging, we demonstrate that dynamic changes of activated brain microcapillaries promote the mandatory first steps of brain colonization. Successful extravasation of arrested cancer cells occurred when adjacent capillary endothelial cells (EC) entered into a distinct remodeling process. After extravasation, capillary loops were formed, which was characteristic of aggressive metastatic growth. Upon cancer cell arrest in brain microcapillaries, matrix-metalloprotease 9 (MMP9) was expressed. Inhibition of MMP2/9 and genetic perturbation of MMP9 in cancer cells, but not the host, reduced EC projections, extravasation, and brain metastasis outgrowth. These findings establish an active role of ECs in the process of cancer cell extravasation, facilitated by cross-talk between the two cell types. This extends our understanding of how host cells can contribute to brain metastasis formation and how to prevent it. SIGNIFICANCE: Tracking single extravasating cancer cells using multimodal correlative microscopy uncovers a brain seeding mechanism involving endothelial remodeling driven by cancer cell-derived MMP9, which might enable the development of approaches to prevent brain metastasis. See related commentary by McCarty, p. 1167.
Asunto(s)
Neoplasias Encefálicas , Endotelio Vascular , Humanos , Endotelio Vascular/patología , Células Endoteliales/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Encéfalo/patología , Neoplasias Encefálicas/patología , Línea Celular TumoralRESUMEN
Life exists in three dimensions, but until the turn of the century most electron microscopy methods provided only 2D image data. Recently, electron microscopy techniques capable of delving deep into the structure of cells and tissues have emerged, collectively called volume electron microscopy (vEM). Developments in vEM have been dubbed a quiet revolution as the field evolved from established transmission and scanning electron microscopy techniques, so early publications largely focused on the bioscience applications rather than the underlying technological breakthroughs. However, with an explosion in the uptake of vEM across the biosciences and fast-paced advances in volume, resolution, throughput and ease of use, it is timely to introduce the field to new audiences. In this Primer, we introduce the different vEM imaging modalities, the specialized sample processing and image analysis pipelines that accompany each modality and the types of information revealed in the data. We showcase key applications in the biosciences where vEM has helped make breakthrough discoveries and consider limitations and future directions. We aim to show new users how vEM can support discovery science in their own research fields and inspire broader uptake of the technology, finally allowing its full adoption into mainstream biological imaging.
RESUMEN
Volume electron microscopy (EM) is a time-consuming process - often requiring weeks or months of continuous acquisition for large samples. In order to compare the ultrastructure of a number of individuals or conditions, acquisition times must therefore be reduced. For resin-embedded samples, one solution is to selectively target smaller regions of interest by trimming with an ultramicrotome. This is a difficult and labour-intensive process, requiring manual positioning of the diamond knife and sample, and much time and training to master. Here, we have developed a semi-automated workflow for targeting with a modified ultramicrotome. We adapted two recent commercial systems to add motors for each rotational axis (and also each translational axis for one system), allowing precise and automated movement. We also developed a user-friendly software to convert X-ray images of resin-embedded samples into angles and cutting depths for the ultramicrotome. This is provided as an open-source Fiji plugin called Crosshair. This workflow is demonstrated by targeting regions of interest in a series of Platynereis dumerilii samples.
Asunto(s)
Microtomía , Poliquetos , Animales , Humanos , Microscopía Electrónica de Rastreo , Microtomía/métodos , Programas Informáticos , FijiRESUMEN
Receptor degradation terminates signaling by activated receptor tyrosine kinases. Degradation of EGFR occurs in lysosomes and requires the switching of RAB5 for RAB7 on late endosomes to enable their fusion with the lysosome, but what controls this critical switching is poorly understood. We show that the tyrosine kinase FER alters PKCδ function by phosphorylating it on Y374, and that phospho-Y374-PKCδ prevents RAB5 release from nascent late endosomes, thereby inhibiting EGFR degradation and promoting the recycling of endosomal EGFR to the cell surface. The rapid association of phospho-Y374-PKCδ with EGFR-containing endosomes is diminished by PTPN14, which dephosphorylates phospho-Y374-PKCδ. In triple-negative breast cancer cells, the FER-dependent phosphorylation of PKCδ enhances EGFR signaling and promotes anchorage-independent cell growth. Importantly, increased Y374-PKCδ phosphorylation correlating with arrested late endosome maturation was identified in â¼25% of triple-negative breast cancer patients, suggesting that dysregulation of this pathway may contribute to their pathology.
Asunto(s)
Endocitosis , Proteína Quinasa C-delta/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteolisis , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Endocitosis/efectos de los fármacos , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Activación Enzimática/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/genética , Receptores ErbB/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Mitógenos/farmacología , Fosforilación/efectos de los fármacos , Fosfotirosina/metabolismo , Estabilidad Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Proteínas Tirosina Fosfatasas no Receptoras/deficiencia , Proteínas Tirosina Fosfatasas no Receptoras/metabolismo , Proteolisis/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Tiempo , Ubiquitinación/efectos de los fármacos , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Eukaryotic phytoplankton have a small global biomass but play major roles in primary production and climate. Despite improved understanding of phytoplankton diversity and evolution, we largely ignore the cellular bases of their environmental plasticity. By comparative 3D morphometric analysis across seven distant phytoplankton taxa, we observe constant volume occupancy by the main organelles and preserved volumetric ratios between plastids and mitochondria. We hypothesise that phytoplankton subcellular topology is modulated by energy-management constraints. Consistent with this, shifting the diatom Phaeodactylum from low to high light enhances photosynthesis and respiration, increases cell-volume occupancy by mitochondria and the plastid CO2-fixing pyrenoid, and boosts plastid-mitochondria contacts. Changes in organelle architectures and interactions also accompany Nannochloropsis acclimation to different trophic lifestyles, along with respiratory and photosynthetic responses. By revealing evolutionarily-conserved topologies of energy-managing organelles, and their role in phytoplankton acclimation, this work deciphers phytoplankton responses at subcellular scales.
Asunto(s)
Metabolismo Energético , Imagenología Tridimensional , Fitoplancton/citología , Fitoplancton/fisiología , Aclimatación/efectos de la radiación , Metabolismo Energético/efectos de la radiación , Luz , Microalgas/metabolismo , Microalgas/efectos de la radiación , Microalgas/ultraestructura , Mitocondrias/metabolismo , Mitocondrias/efectos de la radiación , Mitocondrias/ultraestructura , Fitoplancton/efectos de la radiación , Fitoplancton/ultraestructura , Plastidios/metabolismo , Fracciones Subcelulares/metabolismoRESUMEN
The evolutionary origin of metazoan cell types such as neurons and muscles is not known. Using whole-body single-cell RNA sequencing in a sponge, an animal without nervous system and musculature, we identified 18 distinct cell types. These include nitric oxidesensitive contractile pinacocytes, amoeboid phagocytes, and secretory neuroid cells that reside in close contact with digestive choanocytes that express scaffolding and receptor proteins. Visualizing neuroid cells by correlative x-ray and electron microscopy revealed secretory vesicles and cellular projections enwrapping choanocyte microvilli and cilia. Our data show a communication system that is organized around sponge digestive chambers, using conserved modules that became incorporated into the pre- and postsynapse in the nervous systems of other animals.
Asunto(s)
Evolución Biológica , Poríferos/citología , Animales , Comunicación Celular , Extensiones de la Superficie Celular/ultraestructura , Cilios/fisiología , Cilios/ultraestructura , Sistema Digestivo/citología , Mesodermo/citología , Sistema Nervioso/citología , Fenómenos Fisiológicos del Sistema Nervioso , Óxido Nítrico/metabolismo , Poríferos/genética , Poríferos/metabolismo , RNA-Seq , Vesículas Secretoras/ultraestructura , Transducción de Señal , Análisis de la Célula Individual , TranscriptomaRESUMEN
Alignment of stacks of serial images generated by Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) is generally performed using translations only, either through slice-by-slice alignments with SIFT or alignment by template matching. However, limitations of these methods are two-fold: the introduction of a bias along the dataset in the z-direction which seriously alters the morphology of observed organelles and a missing compensation for pixel size variations inherent to the image acquisition itself. These pixel size variations result in local misalignments and jumps of a few nanometers in the image data that can compromise downstream image analysis. We introduce a novel approach which enables affine transformations to overcome local misalignments while avoiding the danger of introducing a scaling, rotation or shearing trend along the dataset. Our method first computes a template dataset with an alignment method restricted to translations only. This pre-aligned dataset is then smoothed selectively along the z-axis with a median filter, creating a template to which the raw data is aligned using affine transformations. Our method was applied to FIB-SEM datasets and showed clear improvement of the alignment along the z-axis resulting in a significantly more accurate automatic boundary segmentation using a convolutional neural network.
RESUMEN
Pathogenesis induced by SARS-CoV-2 is thought to result from both an inflammation-dominated cytokine response and virus-induced cell perturbation causing cell death. Here, we employ an integrative imaging analysis to determine morphological organelle alterations induced in SARS-CoV-2-infected human lung epithelial cells. We report 3D electron microscopy reconstructions of whole cells and subcellular compartments, revealing extensive fragmentation of the Golgi apparatus, alteration of the mitochondrial network and recruitment of peroxisomes to viral replication organelles formed by clusters of double-membrane vesicles (DMVs). These are tethered to the endoplasmic reticulum, providing insights into DMV biogenesis and spatial coordination of SARS-CoV-2 replication. Live cell imaging combined with an infection sensor reveals profound remodeling of cytoskeleton elements. Pharmacological inhibition of their dynamics suppresses SARS-CoV-2 replication. We thus report insights into virus-induced cytopathic effects and provide alongside a comprehensive publicly available repository of 3D datasets of SARS-CoV-2-infected cells for download and smooth online visualization.
Asunto(s)
COVID-19/genética , Retículo Endoplásmico/ultraestructura , SARS-CoV-2/ultraestructura , Compartimentos de Replicación Viral/ultraestructura , COVID-19/diagnóstico por imagen , COVID-19/patología , COVID-19/virología , Muerte Celular/genética , Retículo Endoplásmico/genética , Retículo Endoplásmico/virología , Humanos , Microscopía Electrónica , Pandemias , SARS-CoV-2/genética , SARS-CoV-2/patogenicidad , Compartimentos de Replicación Viral/metabolismo , Replicación Viral/genéticaRESUMEN
Phagocytic immune cells such as microglia can engulf and process pathogens and dying cells with high efficiency while still maintaining their dynamic behavior and morphology. Effective intracellular processing of ingested cells is likely to be crucial for microglial function, but the underlying cellular mechanisms are poorly understood. Using both living fish embryos and mammalian macrophages, we show that processing depends on the shrinkage and packaging of phagosomes into a unique cellular compartment, the gastrosome, with distinct molecular and ultra-structural characteristics. Loss of the transporter Slc37a2 blocks phagosomal shrinkage, resulting in the expansion of the gastrosome and the dramatic bloating of the cell. This, in turn, affects the ability of microglia to phagocytose and migrate toward brain injuries. Thus, this work identifies a conserved crucial step in the phagocytic pathway of immune cells and provides a potential entry point for manipulating their behavior in development and disease.
Asunto(s)
Antiportadores/genética , Macrófagos/metabolismo , Proteínas de Transporte de Membrana/genética , Microglía/metabolismo , Fagosomas/ultraestructura , Animales , Apoptosis/genética , Compartimento Celular/genética , Células HeLa , Humanos , Macrófagos/ultraestructura , Ratones , Microglía/ultraestructura , Neuronas/metabolismo , Neuronas/ultraestructura , Fagocitos/ultraestructura , Fagocitosis/genética , Fagosomas/genética , Células RAW 264.7 , Pez Cebra/genética , Pez Cebra/crecimiento & desarrolloRESUMEN
Photosymbiosis between single-celled hosts and microalgae is common in oceanic plankton, especially in oligotrophic surface waters. However, the functioning of this ecologically important cell-cell interaction and the subcellular mechanisms allowing the host to accommodate and benefit from its microalgae remain enigmatic. Here, using a combination of quantitative single-cell structural and chemical imaging techniques (FIB-SEM, nanoSIMS, Synchrotron X-ray fluorescence), we show that the structural organization, physiology, and trophic status of the algal symbionts (the haptophyte Phaeocystis) significantly change within their acantharian hosts compared to their free-living phase in culture. In symbiosis, algal cell division is blocked, photosynthesis is enhanced, and cell volume is increased by up to 10-fold with a higher number of plastids (from 2 to up to 30) and thylakoid membranes. The multiplication of plastids can lead to a 38-fold increase of the total plastid volume in a cell. Subcellular mapping of nutrients (nitrogen and phosphorous) and their stoichiometric ratios shows that symbiotic algae are impoverished in phosphorous and suggests a higher investment in energy-acquisition machinery rather than in growth. Nanoscale imaging also showed that the host supplies a substantial amount of trace metals (e.g., iron and cobalt), which are stored in algal vacuoles at high concentrations (up to 660 ppm). Sulfur mapping reveals a high concentration in algal vacuoles that may be a source of antioxidant molecules. Overall, this study unveils an unprecedented morphological and metabolic transformation of microalgae following their integration into a host, and it suggests that this widespread symbiosis is a farming strategy wherein the host engulfs and exploits microalgae.
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Haptophyta/fisiología , Rhizaria/fisiología , Simbiosis/fisiología , División Celular , Tamaño de la Célula , Haptophyta/citología , Haptophyta/metabolismo , FotosíntesisRESUMEN
The hepatitis C virus (HCV) is a major cause of chronic liver disease, affecting around 71 million people worldwide. Viral RNA replication occurs in a membranous compartment composed of double-membrane vesicles (DMVs), whereas virus particles are thought to form by budding into the endoplasmic reticulum (ER). It is unknown how these steps are orchestrated in space and time. Here, we established an imaging system to visualize HCV structural and replicase proteins in live cells and with high resolution. We determined the conditions for the recruitment of viral proteins to putative assembly sites and studied the dynamics of this event and the underlying ultrastructure. Most notable was the selective recruitment of ER membranes around lipid droplets where structural proteins and the viral replicase colocalize. Moreover, ER membranes wrapping lipid droplets were decorated with double membrane vesicles, providing a topological map of how HCV might coordinate the steps of viral replication and virion assembly.
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Hepacivirus/fisiología , Hepatitis C/virología , Membranas Intracelulares/virología , Gotas Lipídicas/fisiología , Proteínas no Estructurales Virales/metabolismo , Ensamble de Virus , Replicación Viral , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/virología , Hepatitis C/genética , Hepatitis C/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Gotas Lipídicas/virología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , ARN Viral/análisis , ARN Viral/genética , Análisis Espacio-Temporal , Células Tumorales CultivadasRESUMEN
Compromised function of insulin-secreting pancreatic ß cells is central to the development and progression of Type 2 Diabetes (T2D). However, the mechanisms underlying ß cell failure remain incompletely understood. Here, we report that metabolic stress markedly enhances macroautophagy-independent lysosomal degradation of nascent insulin granules. In different model systems of diabetes including of human origin, stress-induced nascent granule degradation (SINGD) contributes to loss of insulin along with mammalian/mechanistic Target of Rapamycin (mTOR)-dependent suppression of macroautophagy. Expression of Protein Kinase D (PKD), a negative regulator of SINGD, is reduced in diabetic ß cells. Pharmacological activation of PKD counters SINGD and delays the onset of T2D. Conversely, inhibition of PKD exacerbates SINGD, mitigates insulin secretion and accelerates diabetes. Finally, reduced levels of lysosomal tetraspanin CD63 prevent SINGD, leading to increased insulin secretion. Overall, our findings implicate aberrant SINGD in the pathogenesis of diabetes and suggest new therapeutic strategies to prevent ß cell failure.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Lisosomas/metabolismo , Animales , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/fisiopatología , Humanos , Insulina/química , Secreción de Insulina , Células Secretoras de Insulina/citología , Macroautofagia , Masculino , Ratones Endogámicos C57BL , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.