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Nat Commun ; 11(1): 3709, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709843


Cryo-electron tomography combined with subtomogram averaging (StA) has yielded high-resolution structures of macromolecules in their native context. However, high-resolution StA is not commonplace due to beam-induced sample drift, images with poor signal-to-noise ratios (SNR), challenges in CTF correction, and limited particle number. Here we address these issues by collecting tilt series with a higher electron dose at the zero-degree tilt. Particles of interest are then located within reconstructed tomograms, processed by conventional StA, and then re-extracted from the high-dose images in 2D. Single particle analysis tools are then applied to refine the 2D particle alignment and generate a reconstruction. Use of our hybrid StA (hStA) workflow improved the resolution for tobacco mosaic virus from 7.2 to 4.4 Å and for the ion channel RyR1 in crowded native membranes from 12.9 to 9.1 Å. These resolution gains make hStA a promising approach for other StA projects aimed at achieving subnanometer resolution.

Canais de Cálcio/química , Microscopia Crioeletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Tomografia com Microscopia Eletrônica/métodos , Substâncias Macromoleculares/química , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Razão Sinal-Ruído , Imagem Individual de Molécula , Fluxo de Trabalho
Sci Adv ; 5(7): eaav9732, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31328159


LRH-1 (liver receptor homolog-1/NR5a2) is an orphan nuclear receptor, which regulates glucose and lipid metabolism, as well as intestinal inflammation via the transcriptional control of intestinal glucocorticoid synthesis. Predominantly expressed in epithelial cells, its expression and role in immune cells are presently enigmatic. LRH-1 was found to be induced in immature and mature T lymphocytes upon stimulation. T cell-specific deletion of LRH-1 causes a drastic loss of mature peripheral T cells. LRH-1-depleted CD4+ T cells exert strongly reduced activation-induced proliferation in vitro and in vivo and fail to mount immune responses against model antigens and to induce experimental intestinal inflammation. Similarly, LRH-1-deficient cytotoxic CD8+ T cells fail to control viral infections. This study describes a novel and critical role of LRH-1 in T cell maturation, functions, and immopathologies and proposes LRH-1 as an emerging pharmacological target in the treatment of T cell-mediated inflammatory diseases.

Imunomodulação , Receptores Citoplasmáticos e Nucleares/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Apoptose/genética , Biomarcadores , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Colite/etiologia , Colite/metabolismo , Colite/patologia , Citocinas/metabolismo , Citotoxicidade Imunológica , Suscetibilidade a Doenças , Feminino , Deleção de Genes , Humanos , Isotipos de Imunoglobulinas/imunologia , Masculino , Camundongos , Receptores Citoplasmáticos e Nucleares/metabolismo
Nature ; 571(7765): 429-433, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31292547


Balanced fusion and fission are key for the proper function and physiology of mitochondria1,2. Remodelling of the mitochondrial inner membrane is mediated by the dynamin-like protein mitochondrial genome maintenance 1 (Mgm1) in fungi or the related protein optic atrophy 1 (OPA1) in animals3-5. Mgm1 is required for the preservation of mitochondrial DNA in yeast6, whereas mutations in the OPA1 gene in humans are a common cause of autosomal dominant optic atrophy-a genetic disorder that affects the optic nerve7,8. Mgm1 and OPA1 are present in mitochondria as a membrane-integral long form and a short form that is soluble in the intermembrane space. Yeast strains that express temperature-sensitive mutants of Mgm19,10 or mammalian cells that lack OPA1 display fragmented mitochondria11,12, which suggests that Mgm1 and OPA1 have an important role in inner-membrane fusion. Consistently, only the mitochondrial outer membrane-not the inner membrane-fuses in the absence of functional Mgm113. Mgm1 and OPA1 have also been shown to maintain proper cristae architecture10,14; for example, OPA1 prevents the release of pro-apoptotic factors by tightening crista junctions15. Finally, the short form of OPA1 localizes to mitochondrial constriction sites, where it presumably promotes mitochondrial fission16. How Mgm1 and OPA1 perform their diverse functions in membrane fusion, scission and cristae organization is at present unknown. Here we present crystal and electron cryo-tomography structures of Mgm1 from Chaetomium thermophilum. Mgm1 consists of a GTPase (G) domain, a bundle signalling element domain, a stalk, and a paddle domain that contains a membrane-binding site. Biochemical and cell-based experiments demonstrate that the Mgm1 stalk mediates the assembly of bent tetramers into helical filaments. Electron cryo-tomography studies of Mgm1-decorated lipid tubes and fluorescence microscopy experiments on reconstituted membrane tubes indicate how the tetramers assemble on positively or negatively curved membranes. Our findings convey how Mgm1 and OPA1 filaments dynamically remodel the mitochondrial inner membrane.

Chaetomium/química , Microscopia Crioeletrônica , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas de Ligação ao GTP/química , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/química , Cristalografia por Raios X , Proteínas Fúngicas/ultraestrutura , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/ultraestrutura , Galactosilceramidas/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/ultraestrutura , Modelos Moleculares , Domínios Proteicos , Multimerização Proteica