Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 106.220
Filtrar
Mais filtros

Intervalo de ano de publicação
1.
Cell ; 187(7): 1701-1718.e28, 2024 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-38503283

RESUMO

Biomolecules incur damage during stress conditions, and damage partitioning represents a vital survival strategy for cells. Here, we identified a distinct stress granule (SG), marked by dsRNA helicase DHX9, which compartmentalizes ultraviolet (UV)-induced RNA, but not DNA, damage. Our FANCI technology revealed that DHX9 SGs are enriched in damaged intron RNA, in contrast to classical SGs that are composed of mature mRNA. UV exposure causes RNA crosslinking damage, impedes intron splicing and decay, and triggers DHX9 SGs within daughter cells. DHX9 SGs promote cell survival and induce dsRNA-related immune response and translation shutdown, differentiating them from classical SGs that assemble downstream of translation arrest. DHX9 modulates dsRNA abundance in the DHX9 SGs and promotes cell viability. Autophagy receptor p62 is activated and important for DHX9 SG disassembly. Our findings establish non-canonical DHX9 SGs as a dedicated non-membrane-bound cytoplasmic compartment that safeguards daughter cells from parental RNA damage.


Assuntos
RNA , Grânulos de Estresse , Citoplasma , RNA Mensageiro/genética , Estresse Fisiológico , Humanos , Células HeLa
2.
Cell ; 187(16): 4272-4288.e20, 2024 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-39013469

RESUMO

Vesicle trafficking is a fundamental process that allows for the sorting and transport of specific proteins (i.e., "cargoes") to different compartments of eukaryotic cells. Cargo recognition primarily occurs through coats and the associated proteins at the donor membrane. However, it remains unclear whether cargoes can also be selected at other stages of vesicle trafficking to further enhance the fidelity of the process. The WDR11-FAM91A1 complex functions downstream of the clathrin-associated AP-1 complex to facilitate protein transport from endosomes to the TGN. Here, we report the cryo-EM structure of human WDR11-FAM91A1 complex. WDR11 directly and specifically recognizes a subset of acidic clusters, which we term super acidic clusters (SACs). WDR11 complex assembly and its binding to SAC-containing proteins are indispensable for the trafficking of SAC-containing proteins and proper neuronal development in zebrafish. Our studies thus uncover that cargo proteins could be recognized in a sequence-specific manner downstream of a protein coat.


Assuntos
Microscopia Crioeletrônica , Transporte Proteico , Peixe-Zebra , Humanos , Animais , Endossomos/metabolismo , Células HEK293 , Células HeLa , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/química , Ligação Proteica
3.
Cell ; 187(14): 3619-3637.e27, 2024 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-38851188

RESUMO

Mitochondrial dynamics play a critical role in cell fate decisions and in controlling mtDNA levels and distribution. However, the molecular mechanisms linking mitochondrial membrane remodeling and quality control to mtDNA copy number (CN) regulation remain elusive. Here, we demonstrate that the inner mitochondrial membrane (IMM) protein mitochondrial fission process 1 (MTFP1) negatively regulates IMM fusion. Moreover, manipulation of mitochondrial fusion through the regulation of MTFP1 levels results in mtDNA CN modulation. Mechanistically, we found that MTFP1 inhibits mitochondrial fusion to isolate and exclude damaged IMM subdomains from the rest of the network. Subsequently, peripheral fission ensures their segregation into small MTFP1-enriched mitochondria (SMEM) that are targeted for degradation in an autophagic-dependent manner. Remarkably, MTFP1-dependent IMM quality control is essential for basal nucleoid recycling and therefore to maintain adequate mtDNA levels within the cell.


Assuntos
DNA Mitocondrial , Mitocôndrias , Dinâmica Mitocondrial , Membranas Mitocondriais , Proteínas Mitocondriais , DNA Mitocondrial/metabolismo , DNA Mitocondrial/genética , Proteínas Mitocondriais/metabolismo , Humanos , Membranas Mitocondriais/metabolismo , Mitocôndrias/metabolismo , Animais , Células HeLa , Camundongos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Autofagia
4.
Cell ; 186(16): 3460-3475.e23, 2023 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-37478862

RESUMO

All eukaryotes require intricate protein networks to translate developmental signals into accurate cell fate decisions. Mutations that disturb interactions between network components often result in disease, but how the composition and dynamics of complex networks are established remains poorly understood. Here, we identify the E3 ligase UBR5 as a signaling hub that helps degrade unpaired subunits of multiple transcriptional regulators that act within a network centered on the c-Myc oncoprotein. Biochemical and structural analyses show that UBR5 binds motifs that only become available upon complex dissociation. By rapidly turning over unpaired transcription factor subunits, UBR5 establishes dynamic interactions between transcriptional regulators that allow cells to effectively execute gene expression while remaining receptive to environmental signals. We conclude that orphan quality control plays an essential role in establishing dynamic protein networks, which may explain the conserved need for protein degradation during transcription and offers opportunities to modulate gene expression in disease.


Assuntos
Fatores de Transcrição , Ubiquitina-Proteína Ligases , Humanos , Expressão Gênica , Células HEK293 , Células HeLa , Mutação , Transdução de Sinais , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
5.
Cell ; 186(2): 346-362.e17, 2023 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-36638793

RESUMO

Ribosomes frequently stall during mRNA translation, resulting in the context-dependent activation of quality control pathways to maintain proteostasis. However, surveillance mechanisms that specifically respond to stalled ribosomes with an occluded A site have not been identified. We discovered that the elongation factor-1α (eEF1A) inhibitor, ternatin-4, triggers the ubiquitination and degradation of eEF1A on stalled ribosomes. Using a chemical genetic approach, we unveiled a signaling network comprising two E3 ligases, RNF14 and RNF25, which are required for eEF1A degradation. Quantitative proteomics revealed the RNF14 and RNF25-dependent ubiquitination of eEF1A and a discrete set of ribosomal proteins. The ribosome collision sensor GCN1 plays an essential role by engaging RNF14, which directly ubiquitinates eEF1A. The site-specific, RNF25-dependent ubiquitination of the ribosomal protein RPS27A/eS31 provides a second essential signaling input. Our findings illuminate a ubiquitin signaling network that monitors the ribosomal A site and promotes the degradation of stalled translation factors, including eEF1A and the termination factor eRF1.


Assuntos
Proteínas de Ligação a RNA , Transativadores , Proteínas de Transporte/metabolismo , Fatores de Alongamento de Peptídeos/genética , Biossíntese de Proteínas , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Humanos , Células HeLa , Células HEK293 , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo , Fator 1 de Elongação de Peptídeos/metabolismo
6.
Cell ; 185(24): 4634-4653.e22, 2022 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-36347254

RESUMO

Understanding the basis for cellular growth, proliferation, and function requires determining the roles of essential genes in diverse cellular processes, including visualizing their contributions to cellular organization and morphology. Here, we combined pooled CRISPR-Cas9-based functional screening of 5,072 fitness-conferring genes in human HeLa cells with microscopy-based imaging of DNA, the DNA damage response, actin, and microtubules. Analysis of >31 million individual cells identified measurable phenotypes for >90% of gene knockouts, implicating gene targets in specific cellular processes. Clustering of phenotypic similarities based on hundreds of quantitative parameters further revealed co-functional genes across diverse cellular activities, providing predictions for gene functions and associations. By conducting pooled live-cell screening of ∼450,000 cell division events for 239 genes, we additionally identified diverse genes with functional contributions to chromosome segregation. Our work establishes a resource detailing the consequences of disrupting core cellular processes that represents the functional landscape of essential human genes.


Assuntos
Sistemas CRISPR-Cas , Genes Essenciais , Humanos , Células HeLa , Técnicas de Inativação de Genes , Fenótipo
7.
Cell ; 184(9): 2412-2429.e16, 2021 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-33852913

RESUMO

Cellular versatility depends on accurate trafficking of diverse proteins to their organellar destinations. For the secretory pathway (followed by approximately 30% of all proteins), the physical nature of the vessel conducting the first portage (endoplasmic reticulum [ER] to Golgi apparatus) is unclear. We provide a dynamic 3D view of early secretory compartments in mammalian cells with isotropic resolution and precise protein localization using whole-cell, focused ion beam scanning electron microscopy with cryo-structured illumination microscopy and live-cell synchronized cargo release approaches. Rather than vesicles alone, the ER spawns an elaborate, interwoven tubular network of contiguous lipid bilayers (ER exit site) for protein export. This receptacle is capable of extending microns along microtubules while still connected to the ER by a thin neck. COPII localizes to this neck region and dynamically regulates cargo entry from the ER, while COPI acts more distally, escorting the detached, accelerating tubular entity on its way to joining the Golgi apparatus through microtubule-directed movement.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Microtúbulos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Transporte Biológico Ativo , Células HeLa , Humanos , Transporte Proteico
8.
Cell ; 184(14): 3612-3625.e17, 2021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34115980

RESUMO

Biomolecular condensation is a widespread mechanism of cellular compartmentalization. Because the "survival of motor neuron protein" (SMN) is implicated in the formation of three different membraneless organelles (MLOs), we hypothesized that SMN promotes condensation. Unexpectedly, we found that SMN's globular tudor domain was sufficient for dimerization-induced condensation in vivo, whereas its two intrinsically disordered regions (IDRs) were not. Binding to dimethylarginine (DMA) modified protein ligands was required for condensate formation by the tudor domains in SMN and at least seven other fly and human proteins. Remarkably, asymmetric versus symmetric DMA determined whether two distinct nuclear MLOs-gems and Cajal bodies-were separate or "docked" to one another. This substructure depended on the presence of either asymmetric or symmetric DMA as visualized with sub-diffraction microscopy. Thus, DMA-tudor interaction modules-combinations of tudor domains bound to their DMA ligand(s)-represent versatile yet specific regulators of MLO assembly, composition, and morphology.


Assuntos
Arginina/análogos & derivados , Condensados Biomoleculares/metabolismo , Proteínas do Complexo SMN/química , Proteínas do Complexo SMN/metabolismo , Animais , Arginina/metabolismo , Núcleo Celular/metabolismo , Corpos Enovelados/metabolismo , Drosophila melanogaster/metabolismo , Células HEK293 , Células HeLa , Humanos , Ligantes , Metilação , Camundongos , Modelos Biológicos , Células NIH 3T3 , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Ribonucleoproteínas Nucleares Pequenas/metabolismo
9.
Cell ; 184(18): 4680-4696.e22, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34380047

RESUMO

Mutations causing amyotrophic lateral sclerosis (ALS) often affect the condensation properties of RNA-binding proteins (RBPs). However, the role of RBP condensation in the specificity and function of protein-RNA complexes remains unclear. We created a series of TDP-43 C-terminal domain (CTD) variants that exhibited a gradient of low to high condensation propensity, as observed in vitro and by nuclear mobility and foci formation. Notably, a capacity for condensation was required for efficient TDP-43 assembly on subsets of RNA-binding regions, which contain unusually long clusters of motifs of characteristic types and density. These "binding-region condensates" are promoted by homomeric CTD-driven interactions and required for efficient regulation of a subset of bound transcripts, including autoregulation of TDP-43 mRNA. We establish that RBP condensation can occur in a binding-region-specific manner to selectively modulate transcriptome-wide RNA regulation, which has implications for remodeling RNA networks in the context of signaling, disease, and evolution.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Regiões 3' não Traduzidas/genética , Sequência de Bases , Núcleo Celular/metabolismo , Células HEK293 , Células HeLa , Homeostase , Humanos , Mutação/genética , Motivos de Nucleotídeos/genética , Transição de Fase , Mutação Puntual/genética , Poli A/metabolismo , Ligação Proteica , Multimerização Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Deleção de Sequência
10.
Cell ; 184(12): 3318-3332.e17, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34038702

RESUMO

Long-term subcellular intravital imaging in mammals is vital to study diverse intercellular behaviors and organelle functions during native physiological processes. However, optical heterogeneity, tissue opacity, and phototoxicity pose great challenges. Here, we propose a computational imaging framework, termed digital adaptive optics scanning light-field mutual iterative tomography (DAOSLIMIT), featuring high-speed, high-resolution 3D imaging, tiled wavefront correction, and low phototoxicity with a compact system. By tomographic imaging of the entire volume simultaneously, we obtained volumetric imaging across 225 × 225 × 16 µm3, with a resolution of up to 220 nm laterally and 400 nm axially, at the millisecond scale, over hundreds of thousands of time points. To establish the capabilities, we investigated large-scale cell migration and neural activities in different species and observed various subcellular dynamics in mammals during neutrophil migration and tumor cell circulation.


Assuntos
Algoritmos , Imageamento Tridimensional , Óptica e Fotônica , Tomografia , Animais , Cálcio/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Movimento Celular , Drosophila , Células HeLa , Humanos , Larva/fisiologia , Fígado/diagnóstico por imagem , Masculino , Camundongos Endogâmicos C57BL , Neoplasias/patologia , Ratos Sprague-Dawley , Razão Sinal-Ruído , Frações Subcelulares/fisiologia , Fatores de Tempo , Peixe-Zebra
11.
Cell ; 184(21): 5448-5464.e22, 2021 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-34624221

RESUMO

Structural maintenance of chromosomes (SMC) complexes organize genome topology in all kingdoms of life and have been proposed to perform this function by DNA loop extrusion. How this process works is unknown. Here, we have analyzed how loop extrusion is mediated by human cohesin-NIPBL complexes, which enable chromatin folding in interphase cells. We have identified DNA binding sites and large-scale conformational changes that are required for loop extrusion and have determined how these are coordinated. Our results suggest that DNA is translocated by a spontaneous 50 nm-swing of cohesin's hinge, which hands DNA over to the ATPase head of SMC3, where upon binding of ATP, DNA is clamped by NIPBL. During this process, NIPBL "jumps ship" from the hinge toward the SMC3 head and might thereby couple the spontaneous hinge swing to ATP-dependent DNA clamping. These results reveal mechanistic principles of how cohesin-NIPBL and possibly other SMC complexes mediate loop extrusion.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA/química , Conformação de Ácido Nucleico , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/química , DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Hidrólise , Cinética , Microscopia de Força Atômica , Modelos Moleculares , Proteínas Nucleares/metabolismo , Conformação Proteica , Coesinas
12.
Cell ; 184(21): 5419-5431.e16, 2021 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-34597582

RESUMO

Many enveloped viruses require the endosomal sorting complexes required for transport (ESCRT) pathway to exit infected cells. This highly conserved pathway mediates essential cellular membrane fission events, which restricts the acquisition of adaptive mutations to counteract viral co-option. Here, we describe duplicated and truncated copies of the ESCRT-III factor CHMP3 that block ESCRT-dependent virus budding and arose independently in New World monkeys and mice. When expressed in human cells, these retroCHMP3 proteins potently inhibit release of retroviruses, paramyxoviruses, and filoviruses. Remarkably, retroCHMP3 proteins have evolved to reduce interactions with other ESCRT-III factors and have little effect on cellular ESCRT processes, revealing routes for decoupling cellular ESCRT functions from viral exploitation. The repurposing of duplicated ESCRT-III proteins thus provides a mechanism to generate broad-spectrum viral budding inhibitors without blocking highly conserved essential cellular ESCRT functions.


Assuntos
Citocinese , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , HIV-1/fisiologia , Proteínas do Envelope Viral/metabolismo , Liberação de Vírus , Animais , Morte Celular , Sobrevivência Celular , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Células HEK293 , Células HeLa , Humanos , Interferons/metabolismo , Mamíferos/genética , Camundongos Endogâmicos C57BL , RNA/metabolismo , Transdução de Sinais , Proteínas de Transporte Vesicular/metabolismo , Montagem de Vírus , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo
13.
Cell ; 184(12): 3125-3142.e25, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-33930289

RESUMO

The N6-methyladenosine (m6A) RNA modification is used widely to alter the fate of mRNAs. Here we demonstrate that the C. elegans writer METT-10 (the ortholog of mouse METTL16) deposits an m6A mark on the 3' splice site (AG) of the S-adenosylmethionine (SAM) synthetase pre-mRNA, which inhibits its proper splicing and protein production. The mechanism is triggered by a rich diet and acts as an m6A-mediated switch to stop SAM production and regulate its homeostasis. Although the mammalian SAM synthetase pre-mRNA is not regulated via this mechanism, we show that splicing inhibition by 3' splice site m6A is conserved in mammals. The modification functions by physically preventing the essential splicing factor U2AF35 from recognizing the 3' splice site. We propose that use of splice-site m6A is an ancient mechanism for splicing regulation.


Assuntos
Adenosina/análogos & derivados , Sítios de Splice de RNA/genética , Splicing de RNA/genética , Fator de Processamento U2AF/metabolismo , Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Caenorhabditis elegans/genética , Sequência Conservada/genética , Dieta , Células HeLa , Humanos , Íntrons/genética , Metionina Adenosiltransferase , Metilação , Metiltransferases/química , Camundongos , Mutação/genética , Conformação de Ácido Nucleico , Ligação Proteica , Precursores de RNA/química , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno , S-Adenosilmetionina , Transcriptoma/genética
14.
Cell ; 184(24): 5950-5969.e22, 2021 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-34741801

RESUMO

The biogenesis of mammalian autophagosomes remains to be fully defined. Here, we used cellular and in vitro membrane fusion analyses to show that autophagosomes are formed from a hitherto unappreciated hybrid membrane compartment. The autophagic precursors emerge through fusion of FIP200 vesicles, derived from the cis-Golgi, with endosomally derived ATG16L1 membranes to generate a hybrid pre-autophagosomal structure, HyPAS. A previously unrecognized apparatus defined here controls HyPAS biogenesis and mammalian autophagosomal precursor membranes. HyPAS can be modulated by pharmacological agents whereas its formation is inhibited upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or by expression of SARS-CoV-2 nsp6. These findings reveal the origin of mammalian autophagosomal membranes, which emerge via convergence of secretory and endosomal pathways, and show that this process is targeted by microbial factors such as coronaviral membrane-modulating proteins.


Assuntos
Autofagossomos/virologia , COVID-19/virologia , Autofagia , COVID-19/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Endossomos/fisiologia , Endossomos/virologia , Complexo de Golgi/fisiologia , Células HEK293 , Células HeLa , Humanos , Fusão de Membrana , Microscopia Confocal , Fagossomos/metabolismo , Fagossomos/virologia , Proteínas Qa-SNARE/biossíntese , Receptores sigma/biossíntese , SARS-CoV-2 , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/biossíntese , Sinaptotagminas/biossíntese , Receptor Sigma-1
15.
Cell ; 184(4): 899-911.e13, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33545089

RESUMO

Changes in appendage structure underlie key transitions in vertebrate evolution. Addition of skeletal elements along the proximal-distal axis facilitated critical transformations, including the fin-to-limb transition that permitted generation of diverse modes of locomotion. Here, we identify zebrafish mutants that form supernumerary long bones in their pectoral fins. These new bones integrate into musculature, form joints, and articulate with neighboring elements. This phenotype is caused by activating mutations in previously unrecognized regulators of appendage patterning, vav2 and waslb, that function in a common pathway. This pathway is required for appendage development across vertebrates, and loss of Wasl in mice causes defects similar to those seen in murine Hox mutants. Concordantly, formation of supernumerary bones requires Hox11 function, and mutations in the vav2/wasl pathway drive enhanced expression of hoxa11b, indicating developmental homology with the forearm. Our findings reveal a latent, limb-like pattern ability in fins that is activated by simple genetic perturbation.


Assuntos
Osso e Ossos/embriologia , Extremidades/embriologia , Peixe-Zebra/embriologia , Actinas/metabolismo , Nadadeiras de Animais/embriologia , Animais , Sequência de Bases , Padronização Corporal , Sistemas CRISPR-Cas/genética , Linhagem da Célula , Epistasia Genética , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Genes Reporter , Células HeLa , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Mutação/genética , Fenótipo , Filogenia , Transdução de Sinais/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
16.
Cell ; 182(5): 1140-1155.e18, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32814015

RESUMO

The endosomal sorting complex required for transport-III (ESCRT-III) catalyzes membrane fission from within membrane necks, a process that is essential for many cellular functions, from cell division to lysosome degradation and autophagy. How it breaks membranes, though, remains unknown. Here, we characterize a sequential polymerization of ESCRT-III subunits that, driven by a recruitment cascade and by continuous subunit-turnover powered by the ATPase Vps4, induces membrane deformation and fission. During this process, the exchange of Vps24 for Did2 induces a tilt in the polymer-membrane interface, which triggers transition from flat spiral polymers to helical filament to drive the formation of membrane protrusions, and ends with the formation of a highly constricted Did2-Ist1 co-polymer that we show is competent to promote fission when bound on the inside of membrane necks. Overall, our results suggest a mechanism of stepwise changes in ESCRT-III filament structure and mechanical properties via exchange of the filament subunits to catalyze ESCRT-III activity.


Assuntos
Membrana Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Fusão de Membrana/fisiologia , Adenosina Trifosfatases/metabolismo , Linhagem Celular Tumoral , Endossomos/metabolismo , Células HeLa , Humanos , Polimerização , Transporte Proteico/fisiologia
17.
Cell ; 182(5): 1125-1139.e18, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32822574

RESUMO

Maternal decidual NK (dNK) cells promote placentation, but how they protect against placental infection while maintaining fetal tolerance is unclear. Here we show that human dNK cells highly express the antimicrobial peptide granulysin (GNLY) and selectively transfer it via nanotubes to extravillous trophoblasts to kill intracellular Listeria monocytogenes (Lm) without killing the trophoblast. Transfer of GNLY, but not other cell death-inducing cytotoxic granule proteins, strongly inhibits Lm in human placental cultures and in mouse and human trophoblast cell lines. Placental and fetal Lm loads are lower and pregnancy success is greatly improved in pregnant Lm-infected GNLY-transgenic mice than in wild-type mice that lack GNLY. This immune defense is not restricted to pregnancy; peripheral NK (pNK) cells also transfer GNLY to kill bacteria in macrophages and dendritic cells without killing the host cell. Nanotube transfer of GNLY allows dNK to protect against infection while leaving the maternal-fetal barrier intact.


Assuntos
Antígenos de Diferenciação de Linfócitos T/imunologia , Bactérias/imunologia , Movimento Celular/imunologia , Células Matadoras Naturais/imunologia , Trofoblastos/imunologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Feminino , Células HeLa , Humanos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Placenta/imunologia , Placenta/microbiologia , Gravidez , Ratos , Células THP-1 , Trofoblastos/microbiologia
18.
Cell ; 183(7): 1801-1812.e13, 2020 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-33308477

RESUMO

Cellular stress leads to reprogramming of mRNA translation and formation of stress granules (SGs), membraneless organelles consisting of mRNA and RNA-binding proteins. Although the function of SGs remains largely unknown, it is widely assumed they contain exclusively non-translating mRNA. Here, we re-examine this hypothesis using single-molecule imaging of mRNA translation in living cells. Although we observe non-translating mRNAs are preferentially recruited to SGs, we find unequivocal evidence that mRNAs localized to SGs can undergo translation. Our data indicate that SG-associated translation is not rare, and the entire translation cycle (initiation, elongation, and termination) can occur on SG-localized transcripts. Furthermore, translating mRNAs can be observed transitioning between the cytosol and SGs without changing their translational status. Together, these results demonstrate that mRNA localization to SGs is compatible with translation and argue against a direct role for SGs in inhibition of protein synthesis.


Assuntos
Grânulos Citoplasmáticos/metabolismo , Biossíntese de Proteínas/genética , Transporte de RNA/genética , Imagem Individual de Molécula , Estresse Fisiológico , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Citosol/metabolismo , Células HeLa , Humanos , Fases de Leitura Aberta/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
19.
Cell ; 183(2): 474-489.e17, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33035451

RESUMO

Mg2+ is the most abundant divalent cation in metazoans and an essential cofactor for ATP, nucleic acids, and countless metabolic enzymes. To understand how the spatio-temporal dynamics of intracellular Mg2+ (iMg2+) are integrated into cellular signaling, we implemented a comprehensive screen to discover regulators of iMg2+ dynamics. Lactate emerged as an activator of rapid release of Mg2+ from endoplasmic reticulum (ER) stores, which facilitates mitochondrial Mg2+ (mMg2+) uptake in multiple cell types. We demonstrate that this process is remarkably temperature sensitive and mediated through intracellular but not extracellular signals. The ER-mitochondrial Mg2+ dynamics is selectively stimulated by L-lactate. Further, we show that lactate-mediated mMg2+ entry is facilitated by Mrs2, and point mutations in the intermembrane space loop limits mMg2+ uptake. Intriguingly, suppression of mMg2+ surge alleviates inflammation-induced multi-organ failure. Together, these findings reveal that lactate mobilizes iMg2+ and links the mMg2+ transport machinery with major metabolic feedback circuits and mitochondrial bioenergetics.


Assuntos
Retículo Endoplasmático/metabolismo , Ácido Láctico/metabolismo , Magnésio/metabolismo , Animais , Células COS , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Chlorocebus aethiops , Retículo Endoplasmático/fisiologia , Feminino , Células HeLa , Células Hep G2 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo
20.
Cell ; 181(2): 346-361.e17, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32302572

RESUMO

Stressed cells shut down translation, release mRNA molecules from polysomes, and form stress granules (SGs) via a network of interactions that involve G3BP. Here we focus on the mechanistic underpinnings of SG assembly. We show that, under non-stress conditions, G3BP adopts a compact auto-inhibited state stabilized by electrostatic intramolecular interactions between the intrinsically disordered acidic tracts and the positively charged arginine-rich region. Upon release from polysomes, unfolded mRNAs outcompete G3BP auto-inhibitory interactions, engendering a conformational transition that facilitates clustering of G3BP through protein-RNA interactions. Subsequent physical crosslinking of G3BP clusters drives RNA molecules into networked RNA/protein condensates. We show that G3BP condensates impede RNA entanglement and recruit additional client proteins that promote SG maturation or induce a liquid-to-solid transition that may underlie disease. We propose that condensation coupled to conformational rearrangements and heterotypic multivalent interactions may be a general principle underlying RNP granule assembly.


Assuntos
Grânulos Citoplasmáticos/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Células HeLa , Humanos , Conformação de Ácido Nucleico , Organelas/metabolismo , Fosforilação , RNA Mensageiro/metabolismo , Estresse Fisiológico/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA