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1.
Cancer Res ; 83(8): 1299-1314, 2023 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-36652557

RESUMO

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.


Assuntos
Neoplasias Encefálicas , Endotélio Vascular , Humanos , Endotélio Vascular/patologia , Células Endoteliais/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral
2.
J Cell Biol ; 222(3)2023 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-36562752

RESUMO

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.


Assuntos
Imageamento Tridimensional , Microscopia Eletrônica de Volume , Microscopia Eletrônica de Varredura , Imageamento Tridimensional/métodos , Software
3.
Elife ; 112022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36378502

RESUMO

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.


Assuntos
Microtomia , Poliquetos , Animais , Humanos , Microscopia Eletrônica de Varredura , Microtomia/métodos , Software , Fiji
4.
ISME J ; 16(10): 2348-2359, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35804051

RESUMO

Parasites are widespread and diverse in oceanic plankton and many of them infect single-celled algae for survival. How these parasites develop and scavenge energy within the host and how the cellular organization and metabolism of the host is altered remain open questions. Combining quantitative structural and chemical imaging with time-resolved transcriptomics, we unveil dramatic morphological and metabolic changes of the marine parasite Amoebophrya (Syndiniales) during intracellular infection, particularly following engulfment and digestion of nutrient-rich host chromosomes. Changes include a sequential acristate and cristate mitochondrion with a 200-fold increase in volume, a 13-fold increase in nucleus volume, development of Golgi apparatus and a metabolic switch from glycolysis (within the host) to TCA (free-living dinospore). Similar changes are seen in apicomplexan parasites, thus underlining convergent traits driven by metabolic constraints and the infection cycle. In the algal host, energy-producing organelles (plastid, mitochondria) remain relatively intact during most of the infection. We also observed that sugar reserves diminish while lipid droplets increase. Rapid infection of the host nucleus could be a "zombifying" strategy, allowing the parasite to digest nutrient-rich chromosomes and escape cytoplasmic defense, whilst benefiting from maintained carbon-energy production of the host cell.


Assuntos
Dinoflagellida , Microalgas , Parasitos , Animais , Carbono , Açúcares
5.
Nat Rev Methods Primers ; 2: 51, 2022 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-37409324

RESUMO

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.

6.
Science ; 374(6568): 717-723, 2021 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-34735222

RESUMO

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.


Assuntos
Evolução Biológica , Poríferos/citologia , Animais , Comunicação Celular , Extensões da Superfície Celular/ultraestrutura , Cílios/fisiologia , Cílios/ultraestrutura , Sistema Digestório/citologia , Mesoderma/citologia , Sistema Nervoso/citologia , Fenômenos Fisiológicos do Sistema Nervoso , Óxido Nítrico/metabolismo , Poríferos/genética , Poríferos/metabolismo , RNA-Seq , Vesículas Secretórias/ultraestrutura , Transdução de Sinais , Análise de Célula Única , Transcriptoma
7.
Environ Microbiol ; 23(11): 6569-6586, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34499794

RESUMO

Photosymbiosis is widespread and ecologically important in the oceanic plankton but remains poorly studied. Here, we used multimodal subcellular imaging to investigate the photosymbiosis between colonial Collodaria and their microalga dinoflagellate (Brandtodinium). We showed that this symbiosis is very dynamic whereby symbionts interact with different host cells via extracellular vesicles within the colony. 3D electron microscopy revealed that the photosynthetic apparatus of the microalgae was more voluminous in symbiosis compared to free-living while the mitochondria volume was similar. Stable isotope probing coupled with NanoSIMS showed that carbon and nitrogen were stored in the symbiotic microalga in starch granules and purine crystals respectively. Nitrogen was also allocated to the algal nucleolus. In the host, low 13 C transfer was detected in the Golgi. Metal mapping revealed that intracellular iron concentration was similar in free-living and symbiotic microalgae (c. 40 ppm) and twofold higher in the host, whereas copper concentration increased in symbionts and was detected in the host cell and extracellular vesicles. Sulfur concentration was around two times higher in symbionts (chromatin and pyrenoid) than their host. This study improves our understanding on the functioning of this oceanic photosymbiosis and paves the way for more studies to further assess its biogeochemical significance.


Assuntos
Dinoflagellida , Microalgas , Fotossíntese , Plâncton , Simbiose
8.
Proc Natl Acad Sci U S A ; 118(27)2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34215695

RESUMO

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.


Assuntos
Metabolismo Energético , Haptófitas/metabolismo , Plâncton/citologia , Simbiose , Ciclo do Carbono , Divisão Celular , Núcleo Celular/metabolismo , Microalgas/citologia , Mitocôndrias/metabolismo , Fotossíntese , Plastídeos/metabolismo
9.
Nat Commun ; 12(1): 1049, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33594064

RESUMO

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.


Assuntos
Metabolismo Energético , Imageamento Tridimensional , Fitoplâncton/citologia , Fitoplâncton/fisiologia , Aclimatação/efeitos da radiação , Metabolismo Energético/efeitos da radiação , Luz , Microalgas/metabolismo , Microalgas/efeitos da radiação , Microalgas/ultraestrutura , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Mitocôndrias/ultraestrutura , Fitoplâncton/efeitos da radiação , Fitoplâncton/ultraestrutura , Plastídeos/metabolismo , Frações Subcelulares/metabolismo
10.
J Cell Biol ; 220(2)2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33411917

RESUMO

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 ∼25% of triple-negative breast cancer patients, suggesting that dysregulation of this pathway may contribute to their pathology.


Assuntos
Endocitose , Proteína Quinase C-delta/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteólise , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Ativação Enzimática/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Mitógenos/farmacologia , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Tirosina Fosfatases não Receptoras/deficiência , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Proteólise/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Ubiquitinação/efeitos dos fármacos , Proteínas rab de Ligação ao GTP/metabolismo
11.
Cell Host Microbe ; 28(6): 853-866.e5, 2020 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-33245857

RESUMO

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.


Assuntos
COVID-19/genética , Retículo Endoplasmático/ultraestrutura , SARS-CoV-2/ultraestrutura , Compartimentos de Replicação Viral/ultraestrutura , COVID-19/diagnóstico por imagem , COVID-19/patologia , COVID-19/virologia , Morte Celular/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/virologia , Humanos , Microscopia Eletrônica , Pandemias , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , Compartimentos de Replicação Viral/metabolismo , Replicação Viral/genética
12.
Sci Rep ; 10(1): 2004, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32029771

RESUMO

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.

13.
Nat Commun ; 10(1): 3312, 2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31346174

RESUMO

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.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Lisossomos/metabolismo , Animais , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/fisiopatologia , Humanos , Insulina/química , Secreção de Insulina , Células Secretoras de Insulina/citologia , Macroautofagia , Masculino , Camundongos Endogâmicos C57BL , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
14.
Cell Rep ; 27(12): 3602-3617.e5, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31216478

RESUMO

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.


Assuntos
Hepacivirus/fisiologia , Hepatite C/virologia , Membranas Intracelulares/virologia , Gotículas Lipídicas/fisiologia , Proteínas não Estruturais Virais/metabolismo , Montagem de Vírus , Replicação Viral , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/virologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Hepatite C/genética , Hepatite C/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Gotículas Lipídicas/virologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , RNA Viral/análise , RNA Viral/genética , Análise Espaço-Temporal , Células Tumorais Cultivadas
15.
Curr Biol ; 29(6): 968-978.e4, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30827917

RESUMO

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.


Assuntos
Haptófitas/fisiologia , Rhizaria/fisiologia , Simbiose/fisiologia , Divisão Celular , Tamanho Celular , Haptófitas/citologia , Haptófitas/metabolismo , Fotossíntese
16.
Dev Cell ; 49(1): 77-88.e7, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30880002

RESUMO

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.


Assuntos
Antiporters/genética , Macrófagos/metabolismo , Proteínas de Membrana Transportadoras/genética , Microglia/metabolismo , Fagossomos/ultraestrutura , Animais , Apoptose/genética , Compartimento Celular/genética , Células HeLa , Humanos , Macrófagos/ultraestrutura , Camundongos , Microglia/ultraestrutura , Neurônios/metabolismo , Neurônios/ultraestrutura , Fagócitos/ultraestrutura , Fagocitose/genética , Fagossomos/genética , Células RAW 264.7 , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
17.
J Vis Exp ; (139)2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30247481

RESUMO

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.


Assuntos
Células Imobilizadas/metabolismo , Técnicas Histológicas/métodos , Microscopia Eletrônica/métodos , Humanos
18.
Nat Commun ; 9(1): 1228, 2018 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-29581545

RESUMO

Microglia are highly motile glial cells that are proposed to mediate synaptic pruning during neuronal circuit formation. Disruption of signaling between microglia and neurons leads to an excess of immature synaptic connections, thought to be the result of impaired phagocytosis of synapses by microglia. However, until now the direct phagocytosis of synapses by microglia has not been reported and fundamental questions remain about the precise synaptic structures and phagocytic mechanisms involved. Here we used light sheet fluorescence microscopy to follow microglia-synapse interactions in developing organotypic hippocampal cultures, complemented by a 3D ultrastructural characterization using correlative light and electron microscopy (CLEM). Our findings define a set of dynamic microglia-synapse interactions, including the selective partial phagocytosis, or trogocytosis (trogo-: nibble), of presynaptic structures and the induction of postsynaptic spine head filopodia by microglia. These findings allow us to propose a mechanism for the facilitatory role of microglia in synaptic circuit remodeling and maturation.


Assuntos
Microglia/fisiologia , Modelos Biológicos , Pseudópodes/fisiologia , Sinapses/fisiologia , Animais , Hipocampo/fisiologia , Antígeno de Macrófago 1/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Plasticidade Neuronal , Fagocitose , Terminações Pré-Sinápticas/fisiologia , Transdução de Sinais
19.
Traffic ; 19(5): 354-369, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29451726

RESUMO

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.


Assuntos
Lisossomos/ultraestrutura , Biogênese de Organelas , Tomografia com Microscopia Eletrônica/métodos , Células HeLa , Humanos , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/metabolismo , Imagem Óptica/métodos
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