RESUMO
Cytotoxic T lymphocytes (CTLs) and natural killer cells kill virus-infected and tumor cells through the polarized release of perforin and granzymes. Perforin is a pore-forming toxin that creates a lesion in the plasma membrane of the target cell through which granzymes enter the cytosol and initiate apoptosis. Endosomal sorting complexes required for transport (ESCRT) proteins are involved in the repair of small membrane wounds. We found that ESCRT proteins were precisely recruited in target cells to sites of CTL engagement immediately after perforin release. Inhibition of ESCRT machinery in cancer-derived cells enhanced their susceptibility to CTL-mediated killing. Thus, repair of perforin pores by ESCRT machinery limits granzyme entry into the cytosol, potentially enabling target cells to resist cytolytic attack.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte , Glicoproteínas de Membrana , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Granzimas/metabolismo , Glicoproteínas de Membrana/metabolismo , Perforina/genética , Perforina/metabolismo , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Linfócitos T Citotóxicos/metabolismoRESUMO
Understanding cellular architecture is essential for understanding biology. Electron microscopy (EM) uniquely visualizes cellular structures with nanometre resolution. However, traditional methods, such as thin-section EM or EM tomography, have limitations in that they visualize only a single slice or a relatively small volume of the cell, respectively. Focused ion beam-scanning electron microscopy (FIB-SEM) has demonstrated the ability to image small volumes of cellular samples with 4-nm isotropic voxels1. Owing to advances in the precision and stability of FIB milling, together with enhanced signal detection and faster SEM scanning, we have increased the volume that can be imaged with 4-nm voxels by two orders of magnitude. Here we present a volume EM atlas at such resolution comprising ten three-dimensional datasets for whole cells and tissues, including cancer cells, immune cells, mouse pancreatic islets and Drosophila neural tissues. These open access data (via OpenOrganelle2) represent the foundation of a field of high-resolution whole-cell volume EM and subsequent analyses, and we invite researchers to explore this atlas and pose questions.
Assuntos
Conjuntos de Dados como Assunto , Disseminação de Informação , Microscopia Eletrônica de Varredura , Organelas/ultraestrutura , Animais , Linhagem Celular , Células Cultivadas , Drosophila melanogaster/citologia , Drosophila melanogaster/ultraestrutura , Feminino , Complexo de Golgi/ultraestrutura , Humanos , Interfase , Ilhotas Pancreáticas/citologia , Masculino , Camundongos , Microscopia Eletrônica de Varredura/métodos , Microscopia Eletrônica de Varredura/normas , Microtúbulos/ultraestrutura , Neuroglia/ultraestrutura , Neurônios/ultraestrutura , Publicação de Acesso Aberto , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/ultraestrutura , Ribossomos/ultraestrutura , Vesículas Sinápticas/ultraestrutura , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/ultraestruturaRESUMO
Within cells, the spatial compartmentalization of thousands of distinct proteins serves a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) can elucidate protein spatial relationships to global ultrastructure, but has suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional cryogenic SR and focused ion beam-milled block-face EM across entire vitreously frozen cells. The approach preserves ultrastructure while enabling independent SR and EM workflow optimization. We discovered unexpected protein-ultrastructure relationships in mammalian cells including intranuclear vesicles containing endoplasmic reticulum-associated proteins, web-like adhesions between cultured neurons, and chromatin domains subclassified on the basis of transcriptional activity. Our findings illustrate the value of a comprehensive multimodal view of ultrastructural variability across whole cells.
Assuntos
Células/ultraestrutura , Microscopia Crioeletrônica/métodos , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Animais , Células COS , Adesão Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Congelamento , Células HeLa , Humanos , CamundongosRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
We demonstrate gas cluster ion beam scanning electron microscopy (SEM), in which wide-area ion milling is performed on a series of thick tissue sections. This three-dimensional electron microscopy technique acquires datasets with <10 nm isotropic resolution of each section, and these can then be stitched together to span the sectioned volume. Incorporating gas cluster ion beam SEM into existing single-beam and multibeam SEM workflows should be straightforward, increasing reliability while improving z resolution by a factor of three or more.
Assuntos
Encéfalo/ultraestrutura , Córtex Cerebral/ultraestrutura , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Microscopia Eletrônica de Varredura/métodos , Animais , Drosophila melanogaster , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fixação de TecidosRESUMO
Lipid droplets (LDs) are neutral lipid storage organelles that transfer lipids to various organelles including peroxisomes. Here, we show that the hereditary spastic paraplegia protein M1 Spastin, a membrane-bound AAA ATPase found on LDs, coordinates fatty acid (FA) trafficking from LDs to peroxisomes through two interrelated mechanisms. First, M1 Spastin forms a tethering complex with peroxisomal ABCD1 to promote LD-peroxisome contact formation. Second, M1 Spastin recruits the membrane-shaping ESCRT-III proteins IST1 and CHMP1B to LDs via its MIT domain to facilitate LD-to-peroxisome FA trafficking, possibly through IST1- and CHMP1B-dependent modifications in LD membrane morphology. Furthermore, LD-to-peroxisome FA trafficking mediated by M1 Spastin is required to relieve LDs of lipid peroxidation. M1 Spastin's dual roles in tethering LDs to peroxisomes and in recruiting ESCRT-III components to LD-peroxisome contact sites for FA trafficking may underlie the pathogenesis of diseases associated with defective FA metabolism in LDs and peroxisomes.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Ácidos Graxos/metabolismo , Gotículas Lipídicas/metabolismo , Peroxissomos/metabolismo , Espastina/metabolismo , Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Motivos de Aminoácidos , Transporte Biológico , Células HeLa , Humanos , Hidrólise , Ácidos Láuricos/metabolismo , Modelos Biológicos , Proteínas Mutantes/metabolismo , Proteínas Oncogênicas/metabolismo , Espastina/químicaRESUMO
Focal adhesions (FAs) link the extracellular matrix to the actin cytoskeleton to mediate cell adhesion, migration, mechanosensing and signalling. FAs have conserved nanoscale protein organization, suggesting that the position of proteins within FAs regulates their activity and function. Vinculin binds different FA proteins to mediate distinct cellular functions, but how vinculin's interactions are spatiotemporally organized within FAs is unknown. Using interferometric photoactivation localization super-resolution microscopy to assay vinculin nanoscale localization and a FRET biosensor to assay vinculin conformation, we found that upward repositioning within the FA during FA maturation facilitates vinculin activation and mechanical reinforcement of FAs. Inactive vinculin localizes to the lower integrin signalling layer in FAs by binding to phospho-paxillin. Talin binding activates vinculin and targets active vinculin higher in FAs where vinculin can engage retrograde actin flow. Thus, specific protein interactions are spatially segregated within FAs at the nanoscale to regulate vinculin activation and function.
Assuntos
Adesões Focais/metabolismo , Nanoestruturas , Nanotecnologia/métodos , Vinculina/metabolismo , Actinas/química , Actinas/metabolismo , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Transferência Ressonante de Energia de Fluorescência , Adesões Focais/genética , Humanos , Integrinas/química , Integrinas/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência/métodos , Modelos Moleculares , Mutação , Paxilina/química , Paxilina/genética , Paxilina/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , Talina/química , Talina/genética , Talina/metabolismo , Vinculina/química , Vinculina/genéticaRESUMO
The molecular mechanism responsible for capturing, sorting and retrieving vesicle membrane proteins following triggered exocytosis is not understood. Here we image the post-fusion release and then capture of a vesicle membrane protein, the vesicular acetylcholine transporter, from single vesicles in living neuroendocrine cells. We combine these measurements with super-resolution interferometric photo-activation localization microscopy and electron microscopy, and modelling to map the nanometer-scale topography and architecture of the structures responsible for the transporter's capture following exocytosis. We show that after exocytosis, the transporter rapidly diffuses into the plasma membrane, but most travels only a short distance before it is locally captured over a dense network of membrane-resident clathrin-coated structures. We propose that the extreme density of these structures acts as a short-range diffusion trap. They quickly sequester diffusing vesicle material and limit its spread across the membrane. This system could provide a means for clathrin-mediated endocytosis to quickly recycle vesicle proteins in highly excitable cells.
Assuntos
Fusão de Membrana/fisiologia , Proteínas de Membrana/fisiologia , Proteínas Vesiculares de Transporte de Acetilcolina/fisiologia , Animais , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Clatrina/fisiologia , Clatrina/ultraestrutura , Endocitose/fisiologia , Exocitose/fisiologia , Proteínas de Membrana/ultraestrutura , Microscopia Eletrônica , Microscopia de Interferência/métodos , Células PC12/fisiologia , Ratos , Vesículas Sinápticas/fisiologia , Vesículas Sinápticas/ultraestrutura , Proteínas Vesiculares de Transporte de Acetilcolina/ultraestruturaRESUMO
Cell adhesions to the extracellular matrix (ECM) are necessary for morphogenesis, immunity and wound healing. Focal adhesions are multifunctional organelles that mediate cell-ECM adhesion, force transmission, cytoskeletal regulation and signalling. Focal adhesions consist of a complex network of trans-plasma-membrane integrins and cytoplasmic proteins that form a <200-nm plaque linking the ECM to the actin cytoskeleton. The complexity of focal adhesion composition and dynamics implicate an intricate molecular machine. However, focal adhesion molecular architecture remains unknown. Here we used three-dimensional super-resolution fluorescence microscopy (interferometric photoactivated localization microscopy) to map nanoscale protein organization in focal adhesions. Our results reveal that integrins and actin are vertically separated by a â¼40-nm focal adhesion core region consisting of multiple protein-specific strata: a membrane-apposed integrin signalling layer containing integrin cytoplasmic tails, focal adhesion kinase and paxillin; an intermediate force-transduction layer containing talin and vinculin; and an uppermost actin-regulatory layer containing zyxin, vasodilator-stimulated phosphoprotein and α-actinin. By localizing amino- and carboxy-terminally tagged talins, we reveal talin's polarized orientation, indicative of a role in organizing the focal adhesion strata. The composite multilaminar protein architecture provides a molecular blueprint for understanding focal adhesion functions.
Assuntos
Matriz Extracelular/metabolismo , Integrinas/metabolismo , Actinas/metabolismo , Animais , Adesão Celular , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Matriz Extracelular/ultraestrutura , Humanos , Camundongos , Modelos BiológicosRESUMO
We introduce a method for optically imaging intracellular proteins at nanometer spatial resolution. Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated, localized (to approximately 2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a superresolution image. We used this method--termed photoactivated localization microscopy--to image specific target proteins in thin sections of lysosomes and mitochondria; in fixed whole cells, we imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane.