RESUMO
The Chaperonin Containing Tailless polypeptide 1 (CCT) complex is an essential protein folding machine with a diverse clientele of substrates, including many proteins with ß-propeller domains. Here, we determine the structures of human CCT in complex with its accessory co-chaperone, phosducin-like protein 1 (PhLP1), in the process of folding Gß5, a component of Regulator of G protein Signaling (RGS) complexes. Cryoelectron microscopy (cryo-EM) and image processing reveal an ensemble of distinct snapshots that represent the folding trajectory of Gß5 from an unfolded molten globule to a fully folded ß-propeller. These structures reveal the mechanism by which CCT directs Gß5 folding through initiating specific intermolecular contacts that facilitate the sequential folding of individual ß sheets until the propeller closes into its native structure. This work directly visualizes chaperone-mediated protein folding and establishes that CCT orchestrates folding by stabilizing intermediates through interactions with surface residues that permit the hydrophobic core to coalesce into its folded state.
Assuntos
Proteínas de Ligação ao GTP , Chaperonas Moleculares , Humanos , Microscopia Crioeletrônica , Chaperonas Moleculares/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Dobramento de Proteína , Transdução de Sinais , ChaperoninasRESUMO
Optimum protein function and biochemical activity critically depends on water availability because solvent thermodynamics drive protein folding and macromolecular interactions1. Reciprocally, macromolecules restrict the movement of 'structured' water molecules within their hydration layers, reducing the available 'free' bulk solvent and therefore the total thermodynamic potential energy of water, or water potential. Here, within concentrated macromolecular solutions such as the cytosol, we found that modest changes in temperature greatly affect the water potential, and are counteracted by opposing changes in osmotic strength. This duality of temperature and osmotic strength enables simple manipulations of solvent thermodynamics to prevent cell death after extreme cold or heat shock. Physiologically, cells must sustain their activity against fluctuating temperature, pressure and osmotic strength, which impact water availability within seconds. Yet, established mechanisms of water homeostasis act over much slower timescales2,3; we therefore postulated the existence of a rapid compensatory response. We find that this function is performed by water potential-driven changes in macromolecular assembly, particularly biomolecular condensation of intrinsically disordered proteins. The formation and dissolution of biomolecular condensates liberates and captures free water, respectively, quickly counteracting thermal or osmotic perturbations of water potential, which is consequently robustly buffered in the cytoplasm. Our results indicate that biomolecular condensation constitutes an intrinsic biophysical feedback response that rapidly compensates for intracellular osmotic and thermal fluctuations. We suggest that preserving water availability within the concentrated cytosol is an overlooked evolutionary driver of protein (dis)order and function.
Assuntos
Substâncias Macromoleculares , Proteínas , Solventes , Termodinâmica , Água , Morte Celular , Citosol/química , Citosol/metabolismo , Homeostase , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Concentração Osmolar , Pressão , Proteínas/química , Proteínas/metabolismo , Solventes/química , Solventes/metabolismo , Temperatura , Fatores de Tempo , Água/química , Água/metabolismoRESUMO
A new interactive annotation interface supports a detailed molecular animation of the SARS-CoV-2 life cycle. With this tool, users can interactively explore the data used to create the animation and engage in scientific discourse through comments and questions.
Assuntos
COVID-19 , SARS-CoV-2 , HumanosRESUMO
Target of rapamycin (TOR) is a serine/threonine protein kinase conserved in most eukaryote organisms. TOR assembles into two multiprotein complexes (TORC1 and TORC2), which function as regulators of cellular growth and homeostasis by serving as direct transducers of extracellular biotic and abiotic signals, and, through their participation in intrinsic feedback loops, respectively. TORC1, the better-studied complex, is mainly involved in cell volume homeostasis through regulating accumulation of proteins and other macromolecules, while the functions of the lesser-studied TORC2 are only now starting to emerge. In this Cell Science at a Glance article and accompanying poster, we aim to highlight recent advances in our understanding of TORC2 signalling, particularly those derived from studies in yeast wherein TORC2 has emerged as a major regulator of cell surface homeostasis.
Assuntos
Sirolimo , Serina-Treonina Quinases TOR , Membrana Celular , Homeostase , Alvo Mecanístico do Complexo 2 de Rapamicina , Serina-Treonina Quinases TOR/genéticaRESUMO
This article describes four fluorescent membrane tension probes that have been designed, synthesized, evaluated, commercialized and applied to current biology challenges in the context of the NCCR Chemical Biology. Their names are Flipper-TR®, ER Flipper-TR®, Lyso Flipper-TR®, and Mito Flipper-TR®. They are available from Spirochrome.
Assuntos
Corantes Fluorescentes , Potencial da Membrana Mitocondrial , Corantes , Microscopia de FluorescênciaRESUMO
As the scientific community accumulates diverse data describing how molecular mechanisms occur, creating and sharing visual models that integrate the richness of this information has become increasingly important to help us explore, refine, and communicate our hypotheses. Three-dimensional (3D) animation is a powerful tool to capture dynamic hypotheses that are otherwise difficult or impossible to visualize using traditional 2D illustration techniques. This perspective discusses the current and future roles that 3D animation can play in the research sphere.
Assuntos
Imageamento Tridimensional , Imageamento Tridimensional/métodosRESUMO
Microsporidia are divergent fungal pathogens that employ a harpoon-like apparatus called the polar tube (PT) to invade host cells. The PT architecture and its association with neighboring organelles remain poorly understood. Here, we use cryo-electron tomography to investigate the structural cell biology of the PT in dormant spores from the human-infecting microsporidian species, Encephalitozoon intestinalis . Segmentation and subtomogram averaging of the PT reveal at least four layers: two protein-based layers surrounded by a membrane, and filled with a dense core. Regularly spaced protein filaments form the structural skeleton of the PT. Combining cryo-electron tomography with cellular modeling, we propose a model for the 3-dimensional organization of the polaroplast, an organelle that is continuous with the membrane layer that envelops the PT. Our results reveal the ultrastructure of the microsporidian invasion apparatus in situ , laying the foundation for understanding infection mechanisms.
RESUMO
In collaboration with educators and researchers, we created an online resource called Phase Separation 101 to help undergraduate students understand the basics of liquid-liquid phase separation, an emerging and complex concept in cell biology for which visual resources are still scarce. This work presents the workflow and visual communication strategies that we followed to build scientifically accurate visualizations of dynamic processes.
Assuntos
Computadores , Estudantes , HumanosRESUMO
Microsporidia are an early-diverging group of fungal pathogens that infect a wide range of hosts. Several microsporidian species infect humans, and infections can lead to fatal disease in immunocompromised individuals. As obligate intracellular parasites with highly reduced genomes, microsporidia are dependent on metabolites from their hosts for successful replication and development. Our knowledge of how microsporidian parasites develop inside the host remains rudimentary, and our understanding of the intracellular niche occupied by microsporidia has thus far relied largely on 2D TEM images and light microscopy. Here, we use serial block face scanning electron microscopy (SBF-SEM) to capture 3D snapshots of the human-infecting microsporidian, Encephalitozoon intestinalis , within host cells. We track the development of E. intestinalis through its life cycle, which allows us to propose a model for how its infection organelle, the polar tube, is assembled de novo in each developing spore. 3D reconstructions of parasite-infected cells provide insights into the physical interactions between host cell organelles and parasitophorous vacuoles, which contain the developing parasites. The host cell mitochondrial network is substantially remodeled during E. intestinalis infection, leading to mitochondrial fragmentation. SBF-SEM analysis shows changes in mitochondrial morphology in infected cells, and live-cell imaging provides insights into mitochondrial dynamics during infection. Together, our data provide insights into parasite development, polar tube assembly, and microsporidia-induced mitochondrial remodeling in the host cell.
RESUMO
Microsporidia are an early-diverging group of fungal pathogens with a wide host range. Several microsporidian species cause opportunistic infections in humans that can be fatal. As obligate intracellular parasites with highly reduced genomes, microsporidia are dependent on host metabolites for successful replication and development. Our knowledge of microsporidian intracellular development remains rudimentary, and our understanding of the intracellular niche occupied by microsporidia has relied on 2D TEM images and light microscopy. Here, we use serial block-face scanning electron microscopy (SBF-SEM) to capture 3D snapshots of the human-infecting species, Encephalitozoon intestinalis, within host cells. We track E. intestinalis development through its life cycle, which allows us to propose a model for how its infection organelle, the polar tube, is assembled de novo in developing spores. 3D reconstructions of parasite-infected cells provide insights into the physical interactions between host cell organelles and parasitophorous vacuoles, which contain the developing parasites. The host cell mitochondrial network is substantially remodeled during E. intestinalis infection, leading to mitochondrial fragmentation. SBF-SEM analysis shows changes in mitochondrial morphology in infected cells, and live-cell imaging provides insights into mitochondrial dynamics during infection. Our data provide insights into parasite development, polar tube assembly, and microsporidia-induced host mitochondria remodeling.
Assuntos
Encephalitozoon , Microsporídios , Parasitos , Animais , Humanos , Imageamento TridimensionalRESUMO
Condensates have emerged as a new way to understand how cells are organized, and have been invoked to play crucial roles in essentially all cellular processes. In this view, the cell is occupied by numerous assemblies, each composed of member proteins and nucleic acids that preferentially interact with each other. However, available visual representations of condensates fail to communicate the growing body of knowledge about how condensates form and function. The resulting focus on only a subset of the potential implications of condensates can skew interpretations of results and hinder the generation of new hypotheses. Here we summarize the discussion from a workshop that brought together cell biologists, visualization and computation specialists, and other experts who specialize in thinking about space and ways to represent it. We place the recent advances in condensate research in a historical perspective that describes evolving views of the cell; highlight different attributes of condensates that are not well-served by current visual conventions; and survey potential approaches to overcome these challenges. An important theme of these discussions is that the new understanding on the roles of condensates exposes broader challenges in visual representations that apply to cell biological research more generally.
RESUMO
The cytosolic Chaperonin Containing Tailless polypeptide 1 (CCT) complex is an essential protein folding machine with a diverse clientele of substrates, including many proteins with ß-propeller domains. Here, we determined structures of CCT in complex with its accessory co-chaperone, phosducin-like protein 1 (PhLP1), in the process of folding Gß5, a component of Regulator of G protein Signaling (RGS) complexes. Cryo-EM and image processing revealed an ensemble of distinct snapshots that represent the folding trajectory of Gß5 from an unfolded molten globule to a fully folded ß-propeller. These structures reveal the mechanism by which CCT directs Gß5 folding through initiating specific intermolecular contacts that facilitate the sequential folding of individual ß-sheets until the propeller closes into its native structure. This work directly visualizes chaperone-mediated protein folding and establishes that CCT directs folding by stabilizing intermediates through interactions with surface residues that permit the hydrophobic core to coalesce into its folded state.
RESUMO
Target of rapamycin complex 2 (TORC2) is a conserved protein kinase that regulates multiple plasma membrane (PM)-related processes, including endocytosis. Direct, chemical inhibition of TORC2 arrests endocytosis but with kinetics that is relatively slow and therefore inconsistent with signaling being mediated solely through simple phosphorylation cascades. Here, we show that in addition to and independently from regulation of the phosphorylation of endocytic proteins, TORC2 also controls endocytosis by modulating PM tension. Elevated PM tension, upon TORC2 inhibition, impinges on endocytosis at two different levels by (1) severing the bonds between the PM adaptor proteins Sla2 and Ent1 and the actin cytoskeleton and (2) hindering recruitment of Rvs167, an N-BAR-containing protein important for vesicle fission to endocytosis sites. These results underline the importance of biophysical cues in the regulation of cellular and molecular processes.
Assuntos
Proteínas do Citoesqueleto/genética , Endocitose/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Proteínas dos Microfilamentos/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genética , Citoesqueleto de Actina/genética , Membrana Celular/genética , Citoplasma/genética , Fosforilação , Saccharomyces cerevisiae/genética , Transdução de Sinais/genéticaRESUMO
The target of rapamycin complex 2 (TORC2) plays a key role in maintaining the homeostasis of plasma membrane (PM) tension. TORC2 activation following increased PM tension involves redistribution of the Slm1 and 2 paralogues from PM invaginations known as eisosomes into membrane compartments containing TORC2. How Slm1/2 relocalization is triggered, and if/how this plays a role in TORC2 inactivation with decreased PM tension, is unknown. Using osmotic shocks and palmitoylcarnitine as orthogonal tools to manipulate PM tension, we demonstrate that decreased PM tension triggers spontaneous, energy-independent reorganization of pre-existing phosphatidylinositol-4,5-bisphosphate into discrete invaginated membrane domains, which cluster and inactivate TORC2. These results demonstrate that increased and decreased membrane tension are sensed through different mechanisms, highlighting a role for membrane lipid phase separation in mechanotransduction.
Assuntos
Membrana Celular/metabolismo , Proteínas Fúngicas/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Mecanotransdução Celular , Fosfatidilinositol 4,5-Difosfato/metabolismo , Saccharomyces cerevisiae/metabolismo , Sistemas do Segundo Mensageiro , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Membrana Celular/efeitos dos fármacos , Proteínas do Citoesqueleto , Ativação Enzimática , Proteínas Fúngicas/genética , Cinética , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Mecanotransdução Celular/efeitos dos fármacos , Pressão Osmótica , Palmitoilcarnitina/farmacologia , Transporte Proteico , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sistemas do Segundo Mensageiro/efeitos dos fármacosRESUMO
To systematically explore complex genetic interactions, we constructed ~200,000 yeast triple mutants and scored negative trigenic interactions. We selected double-mutant query genes across a broad spectrum of biological processes, spanning a range of quantitative features of the global digenic interaction network and tested for a genetic interaction with a third mutation. Trigenic interactions often occurred among functionally related genes, and essential genes were hubs on the trigenic network. Despite their functional enrichment, trigenic interactions tended to link genes in distant bioprocesses and displayed a weaker magnitude than digenic interactions. We estimate that the global trigenic interaction network is ~100 times as large as the global digenic network, highlighting the potential for complex genetic interactions to affect the biology of inheritance, including the genotype-to-phenotype relationship.