Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 759.055
Filtrar
1.
J Vis Exp ; (191)2023 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-36715426

RESUMO

The differentiation of natural killer (NK) cells from human pluripotent stem cells allows for research on and the manufacture of clinical-grade cellular products for immunotherapy. Described here is a two-phase protocol that uses a serum-free commercial medium and a cocktail of cytokines (interleukin [IL]-3, IL-7, IL-15, stem cell factor [SCF], and FMS-like tyrosine kinase 3 ligand [Ftl3L]) to differentiate human expanded potential stem cells (hEPSCs) into cells that possess NK cell properties in vitro with both 3-dimensional (3D) and 2-dimensional (2D) culture technology. Following this protocol, CD3-CD56+ or CD45+CD56+ NK cells are consistently generated. When cocultured with tumor targets for 3 h, the differentiated products display mild cytotoxicity as compared to an IL-2-independent permanent cell line, NK92mi cells. The protocol preserves the complexity of the differentiation microenvironment by the generation of 3D structures, thus facilitating the study of the spatial relationships between immune cells and their niches. Meanwhile, the 2D culture system enables the routine phenotypical validation of cell differentiation without harming the delicate differentiation niche.


Assuntos
Técnicas de Cocultura , Células Matadoras Naturais , Células-Tronco , Humanos , Diferenciação Celular , Linhagem Celular , Citocinas/metabolismo , Citotoxicidade Imunológica , Células Matadoras Naturais/citologia , Células-Tronco/citologia
2.
Cell ; 186(1): 80-97.e26, 2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36608661

RESUMO

Glucose is a universal bioenergy source; however, its role in controlling protein interactions is unappreciated, as are its actions during differentiation-associated intracellular glucose elevation. Azido-glucose click chemistry identified glucose binding to a variety of RNA binding proteins (RBPs), including the DDX21 RNA helicase, which was found to be essential for epidermal differentiation. Glucose bound the ATP-binding domain of DDX21, altering protein conformation, inhibiting helicase activity, and dissociating DDX21 dimers. Glucose elevation during differentiation was associated with DDX21 re-localization from the nucleolus to the nucleoplasm where DDX21 assembled into larger protein complexes containing RNA splicing factors. DDX21 localized to specific SCUGSDGC motif in mRNA introns in a glucose-dependent manner and promoted the splicing of key pro-differentiation genes, including GRHL3, KLF4, OVOL1, and RBPJ. These findings uncover a biochemical mechanism of action for glucose in modulating the dimerization and function of an RNA helicase essential for tissue differentiation.


Assuntos
RNA Helicases DEAD-box , Glucose , Queratinócitos , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , RNA Helicases DEAD-box/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Glucose/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Humanos
3.
Int J Mol Sci ; 24(2)2023 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-36675161

RESUMO

Lysosomes are acidic Ca2+ storage organelles that actively generate local Ca2+ signaling events to regulate a plethora of cell functions. Here, we characterized lysosomal Ca2+ signals in mouse renal collecting duct (CD) cells and we assessed their putative role in aquaporin 2 (AQP2)-dependent water reabsorption. Bafilomycin A1 and ML-SA1 triggered similar Ca2+ oscillations, in the absence of extracellular Ca2+, by alkalizing the acidic lysosomal pH or activating the lysosomal cation channel mucolipin 1 (TRPML1), respectively. TRPML1-dependent Ca2+ signals were blocked either pharmacologically or by lysosomes' osmotic permeabilization, thus indicating these organelles as primary sources of Ca2+ release. Lysosome-induced Ca2+ oscillations were sustained by endoplasmic reticulum (ER) Ca2+ content, while bafilomycin A1 and ML-SA1 did not directly interfere with ER Ca2+ homeostasis per se. TRPML1 activation strongly increased AQP2 apical expression and depolymerized the actin cytoskeleton, thereby boosting water flux in response to an hypoosmotic stimulus. These effects were strictly dependent on the activation of the Ca2+/calcineurin pathway. Conversely, bafilomycin A1 led to perinuclear accumulation of AQP2 vesicles without affecting water permeability. Overall, lysosomal Ca2+ signaling events can be differently decoded to modulate Ca2+-dependent cellular functions related to the dock/fusion of AQP2-transporting vesicles in principal cells of the CD.


Assuntos
Aquaporina 2 , Túbulos Renais Coletores , Lisossomos , Água , Animais , Camundongos , Aquaporina 2/genética , Aquaporina 2/metabolismo , Lisossomos/genética , Lisossomos/metabolismo , Macrolídeos/farmacologia , Macrolídeos/metabolismo , Água/metabolismo , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/metabolismo
4.
Int J Mol Sci ; 24(2)2023 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-36675167

RESUMO

Four Ras guanine nucleotide-releasing proteins (RasGRP1 through 4) belong to the family of guanine nucleotide exchange factors (GEFs). RasGRPs catalyze the release of GDP from small GTPases Ras and Rap and facilitate their transition from an inactive GDP-bound to an active GTP-bound state. Thus, they regulate critical cellular responses via many downstream GTPase effectors. Similar to other RasGRPs, the catalytic module of RasGRP1 is composed of the Ras exchange motif (REM) and Cdc25 domain, and the EF hands and C1 domain contribute to its cellular localization and regulation. RasGRP1 can be activated by a diacylglycerol (DAG)-mediated membrane recruitment and protein kinase C (PKC)-mediated phosphorylation. RasGRP1 acts downstream of the T cell receptor (TCR), B cell receptors (BCR), and pre-TCR, and plays an important role in the thymocyte maturation and function of peripheral T cells, B cells, NK cells, mast cells, and neutrophils. The dysregulation of RasGRP1 is known to contribute to numerous disorders that range from autoimmune and inflammatory diseases and schizophrenia to neoplasia. Given its position at the crossroad of cell development, inflammation, and cancer, RASGRP1 has garnered interest from numerous disciplines. In this review, we outline the structure, function, and regulation of RasGRP1 and focus on the existing knowledge of the role of RasGRP1 in leukemia and other cancers.


Assuntos
Fatores de Troca do Nucleotídeo Guanina , Sistema Imunitário , Neoplasias , Humanos , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/imunologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Nucleotídeos de Guanina , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/metabolismo , Receptores de Antígenos de Linfócitos T , Linfócitos T/imunologia , Sistema Imunitário/citologia , Sistema Imunitário/imunologia
6.
PLoS One ; 18(1): e0278607, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36696395

RESUMO

Here we report urine-derived cell (UDC) culture and subsequent use for cloning which resulted in the successful development of cloned canine pups, which have remained healthy into adulthood. Bovine UDCs were used in vitro to establish comparative differences between cell sources. UDCs were chosen as a readily available and noninvasive source for obtaining cells. We analyzed the viability of cells stored in urine over time and could consistently culture cells which had remained in urine for 48hrs. Cells were shown to be viable and capable of being transfected with plasmids. Although primarily of epithelial origin, cells were found from multiple lineages, indicating that they enter the urine from more than one source. Held in urine, at 4°C, the majority of cells maintained their membrane integrity for several days. When compared to in vitro fertilization (IVF) derived embryos or those from traditional SCNT, UDC derived embryos did not differ in total cell number or in the number of DNA breaks, measured by TUNEL stain. These results indicate that viable cells can be obtained from multiple species' urine, capable of being used to produce live offspring at a comparable rate to other cell sources, evidenced by a 25% pregnancy rate and 2 live births with no losses in the canine UDC cloning trial. This represents a noninvasive means to recover the breeding capacity of genetically important or infertile animals. Obtaining cells in this way may provide source material for human and animal studies where cells are utilized.


Assuntos
Clonagem de Organismos , Nascido Vivo , Animais , Cães , Feminino , Gravidez , Clonagem de Organismos/métodos , Clonagem de Organismos/veterinária , Nascido Vivo/veterinária , Taxa de Gravidez , Urina/citologia
7.
Science ; 379(6627): 84-88, 2023 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-36603070

RESUMO

The central nervous system is lined by meninges, classically known as dura, arachnoid, and pia mater. We show the existence of a fourth meningeal layer that compartmentalizes the subarachnoid space in the mouse and human brain, designated the subarachnoid lymphatic-like membrane (SLYM). SLYM is morpho- and immunophenotypically similar to the mesothelial membrane lining of peripheral organs and body cavities, and it encases blood vessels and harbors immune cells. Functionally, the close apposition of SLYM with the endothelial lining of the meningeal venous sinus permits direct exchange of small solutes between cerebrospinal fluid and venous blood, thus representing the mouse equivalent of the arachnoid granulations. The functional characterization of SLYM provides fundamental insights into brain immune barriers and fluid transport.


Assuntos
Encéfalo , Espaço Subaracnóideo , Animais , Humanos , Camundongos , Dura-Máter/citologia , Dura-Máter/fisiologia , Endotélio/citologia , Endotélio/fisiologia , Espaço Subaracnóideo/citologia , Espaço Subaracnóideo/fisiologia , Epitélio/fisiologia , Encéfalo/anatomia & histologia , Encéfalo/imunologia , Líquido Cefalorraquidiano/fisiologia
8.
Sci Adv ; 9(3): eade5348, 2023 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-36652509

RESUMO

A critical stage of T cell development is ß-selection; at this stage, the T cell receptor ß (TCRß) chain is generated, and the developing T cell starts to acquire antigenic specificity. Progression through ß-selection is assisted by low-affinity interactions between the nascent TCRß chain and peptide presented on stromal major histocompatibility complex and cues provided by the niche. In this study, we identify a cue within the developing T cell niche that is critical for T cell development. E-cadherin mediates cell-cell interactions and influences cell fate in many developmental systems. In developing T cells, E-cadherin contributed to the formation of an immunological synapse and the alignment of the mitotic spindle with the polarity axis during division, which facilitated subsequent T cell development. Collectively, these data suggest that E-cadherin facilitates interactions with the thymic niche to coordinate the ß-selection stage of T cell development.


Assuntos
Caderinas , Linfócitos T , Animais , Camundongos , Caderinas/metabolismo , Comunicação Celular , Receptores de Antígenos de Linfócitos T alfa-beta , Linfócitos T/citologia , Linfócitos T/metabolismo , Timo , Fuso Acromático/metabolismo
9.
J Neurosci ; 43(2): 211-220, 2023 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-36639906

RESUMO

In the developing cortex, excitatory neurons migrate along the radial fibers to their final destinations and build up synaptic connection with each other to form functional circuitry. The shaping of neuronal morphologies by actin cytoskeleton dynamics is crucial for neuronal migration. However, it is largely unknown how the distribution and assembly of the F-actin cytoskeleton are coordinated. In the present study, we found that an actin regulatory protein, coronin 2B, is indispensable for the transition from a multipolar to bipolar morphology during neuronal migration in ICR mice of either sex. Loss of coronin 2B led to heterotopic accumulation of migrating neurons in the intermediate zone along with reduced dendritic complexity and aberrant neuronal activity in the cortical plate. This was accompanied by increased seizure susceptibility, suggesting the malfunction of cortical development in coronin 2B-deficient brains. Coronin 2B knockdown disrupted the distribution of the F-actin cytoskeleton at the leading processes, while the migration defect in coronin 2B-deficient neurons was partially rescued by overexpression of Rac1 and its downstream actin-severing protein, cofilin. Our results collectively reveal the physiological function of coronin 2B during neuronal migration whereby it maintains the proper distribution of activated Rac1 and the F-actin cytoskeleton.SIGNIFICANCE STATEMENT Deficits in neuronal migration during cortical development result in various neurodevelopmental disorders (e.g., focal cortical dysplasia, periventricular heterotopia, epilepsy, etc.). Most signaling pathways that control neuronal migration process converge to regulate actin cytoskeleton dynamics. Therefore, it is important to understand how actin dynamics is coordinated in the critical processes of neuronal migration. Herein, we report that coronin 2B is a key protein that regulates neuronal migration through its ability to control the distribution of the actin cytoskeleton and its regulatory signaling protein Rac1 during the multipolar-bipolar transition in the intermediate zone, providing insights into the molecular machinery that drives the migration process of newborn neurons.


Assuntos
Actinas , Proteínas dos Microfilamentos , Neurônios , Proteínas rac1 de Ligação ao GTP , Animais , Camundongos , Actinas/fisiologia , Movimento Celular/fisiologia , Camundongos Endogâmicos ICR , Proteínas dos Microfilamentos/fisiologia , Proteínas rac1 de Ligação ao GTP/fisiologia , Neurônios/citologia
10.
Nature ; 613(7944): 534-542, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36599984

RESUMO

To survive, animals must convert sensory information into appropriate behaviours1,2. Vision is a common sense for locating ethologically relevant stimuli and guiding motor responses3-5. How circuitry converts object location in retinal coordinates to movement direction in body coordinates remains largely unknown. Here we show through behaviour, physiology, anatomy and connectomics in Drosophila that visuomotor transformation occurs by conversion of topographic maps formed by the dendrites of feature-detecting visual projection neurons (VPNs)6,7 into synaptic weight gradients of VPN outputs onto central brain neurons. We demonstrate how this gradient motif transforms the anteroposterior location of a visual looming stimulus into the fly's directional escape. Specifically, we discover that two neurons postsynaptic to a looming-responsive VPN type promote opposite takeoff directions. Opposite synaptic weight gradients onto these neurons from looming VPNs in different visual field regions convert localized looming threats into correctly oriented escapes. For a second looming-responsive VPN type, we demonstrate graded responses along the dorsoventral axis. We show that this synaptic gradient motif generalizes across all 20 primary VPN cell types and most often arises without VPN axon topography. Synaptic gradients may thus be a general mechanism for conveying spatial features of sensory information into directed motor outputs.


Assuntos
Comportamento Animal , Drosophila , Neurônios , Desempenho Psicomotor , Sinapses , Animais , Encéfalo/citologia , Encéfalo/fisiologia , Drosophila/anatomia & histologia , Drosophila/citologia , Drosophila/fisiologia , Neurônios/fisiologia , Campos Visuais/fisiologia , Sinapses/metabolismo , Axônios , Dendritos , Reação de Fuga
11.
Nature ; 613(7944): 550-557, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36599986

RESUMO

Animals display substantial inter-species variation in the rate of embryonic development despite a broad conservation of the overall sequence of developmental events. Differences in biochemical reaction rates, including the rates of protein production and degradation, are thought to be responsible for species-specific rates of development1-3. However, the cause of differential biochemical reaction rates between species remains unknown. Here, using pluripotent stem cells, we have established an in vitro system that recapitulates the twofold difference in developmental rate between mouse and human embryos. This system provides a quantitative measure of developmental speed as revealed by the period of the segmentation clock, a molecular oscillator associated with the rhythmic production of vertebral precursors. Using this system, we show that mass-specific metabolic rates scale with the developmental rate and are therefore higher in mouse cells than in human cells. Reducing these metabolic rates by inhibiting the electron transport chain slowed down the segmentation clock by impairing the cellular NAD+/NADH redox balance and, further downstream, lowering the global rate of protein synthesis. Conversely, increasing the NAD+/NADH ratio in human cells by overexpression of the Lactobacillus brevis NADH oxidase LbNOX increased the translation rate and accelerated the segmentation clock. These findings represent a starting point for the manipulation of developmental rate, with multiple translational applications including accelerating the differentiation of human pluripotent stem cells for disease modelling and cell-based therapies.


Assuntos
Embrião de Mamíferos , Desenvolvimento Embrionário , Animais , Humanos , Camundongos , Diferenciação Celular , Desenvolvimento Embrionário/fisiologia , NAD/metabolismo , Oxirredução , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Especificidade da Espécie , Técnicas In Vitro , Transporte de Elétrons , Relógios Biológicos , Fatores de Tempo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo
12.
Cell Mol Biol Lett ; 28(1): 7, 2023 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-36694134

RESUMO

BACKGROUND: Mechanotransduction mechanisms whereby periodontal ligament stem cells (PDLSCs) translate mechanical stress into biochemical signals and thereby trigger osteogenic programs necessary for alveolar bone remodeling are being deciphered. Low-density lipoprotein receptor-related protein 6 (LRP6), a Wnt transmembrane receptor, has been qualified as a key monitor for mechanical cues. However, the role of LRP6 in the mechanotransduction of mechanically induced PDLSCs remains obscure. METHODS: The Tension System and tooth movement model were established to determine the expression profile of LRP6. The loss-of-function assay was used to investigate the role of LRP6 on force-regulated osteogenic commitment in PDLSCs. The ability of osteogenic differentiation and proliferation was estimated by alkaline phosphatase (ALP) staining, ALP activity assay, western blotting, quantitative real-time PCR (qRT-PCR), and immunofluorescence. Crystalline violet staining was used to visualize cell morphological change. Western blotting, qRT-PCR, and phalloidin staining were adopted to affirm filamentous actin (F-actin) alteration. YAP nucleoplasmic localization was assessed by immunofluorescence and western blotting. YAP transcriptional response was evaluated by qRT-PCR. Cytochalasin D was used to determine the effects of F-actin on osteogenic commitment and YAP switch behavior in mechanically induced PDLSCs. RESULTS: LRP6 was robustly activated in mechanically induced PDLSCs and PDL tissues. LRP6 deficiency impeded force-dependent osteogenic differentiation and proliferation in PDLSCs. Intriguingly, LRP6 loss caused cell morphological aberration, F-actin dynamics disruption, YAP nucleoplasmic relocation, and subsequent YAP inactivation. Moreover, disrupted F-actin dynamics inhibited osteogenic differentiation, proliferation, YAP nuclear translocation, and YAP activation in mechanically induced PDLSCs. CONCLUSIONS: We identified that LRP6 in PDLSCs acted as the mechanosensor regulating mechanical stress-inducible osteogenic commitment via the F-actin/YAP cascade. Targeting LRP6 for controlling alveolar bone remodeling may be a prospective therapy to attenuate relapse of orthodontic treatment.


Assuntos
Actinas , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Osteogênese , Ligamento Periodontal , Células-Tronco , Actinas/genética , Actinas/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células , Células Cultivadas , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Mecanotransdução Celular/genética , Mecanotransdução Celular/fisiologia , Osteogênese/genética , Osteogênese/fisiologia , Ligamento Periodontal/citologia , Ligamento Periodontal/metabolismo , Células-Tronco/metabolismo
13.
Immun Inflamm Dis ; 11(1): e743, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36705422

RESUMO

INTRODUCTION: Chronic periodontitis (CP) is an inflammatory periodontal disease with high incidence and complex pathology. This research is aimed to investigate the function of exosomal miR-205-5p (Exo-miR-205-5p) in CP and the underlying molecular mechanisms. METHOD: Exo-miR-205-5p was isolated from miR-205-5p mimics-transfected periodontal ligament stem cells (PDLSCs), and subsequently cocultured with lipopolysaccharide (LPS)-induced cells or injected into LPS-treated rats. The mRNA expression of inflammatory factors and Th17/Treg-related factors were measured by quantitative real-time PCR. The contents of inflammatory factors and the percentages of Th17/Treg cells were measured by enzyme-linked immunosorbent assay and flow cytometry, respectively. Besides, the target relation between miR-205-5p and X-box binding protein 1 (XBP1) was explored. RESULTS: MiR-205-5p was downregulated in LPS-induced PDLSCs and corresponding exosomes. Exo-miR-205-5p inhibited inflammatory cell infiltration, decreased the production of TNF-α, IL-1ß, and IL-6, and decreased the percentage of Th17 cells in LPS-treated rats. In addition, XBP1 was a target of miR-205-5p. Overexpression of XBP1 weakened the effects of Exo-miR-205-5p on inhibiting inflammation and regulating Treg/Th17 balance in LPS-induced cells. CONCLUSIONS: Exo-miR-205-5p derived from PDLSCs relieves the inflammation and balances the Th17/Treg cells in CP through targeting XBP1.


Assuntos
Periodontite Crônica , MicroRNAs , Células-Tronco , Proteína 1 de Ligação a X-Box , Animais , Ratos , Periodontite Crônica/metabolismo , Periodontite Crônica/patologia , Inflamação/metabolismo , Lipopolissacarídeos/toxicidade , MicroRNAs/genética , Ligamento Periodontal/citologia , Ligamento Periodontal/patologia , Células-Tronco/metabolismo , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismo
14.
Nature ; 613(7943): 345-354, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36599983

RESUMO

Understanding how a subset of expressed genes dictates cellular phenotype is a considerable challenge owing to the large numbers of molecules involved, their combinatorics and the plethora of cellular behaviours that they determine1,2. Here we reduced this complexity by focusing on cellular organization-a key readout and driver of cell behaviour3,4-at the level of major cellular structures that represent distinct organelles and functional machines, and generated the WTC-11 hiPSC Single-Cell Image Dataset v1, which contains more than 200,000 live cells in 3D, spanning 25 key cellular structures. The scale and quality of this dataset permitted the creation of a generalizable analysis framework to convert raw image data of cells and their structures into dimensionally reduced, quantitative measurements that can be interpreted by humans, and to facilitate data exploration. This framework embraces the vast cell-to-cell variability that is observed within a normal population, facilitates the integration of cell-by-cell structural data and allows quantitative analyses of distinct, separable aspects of organization within and across different cell populations. We found that the integrated intracellular organization of interphase cells was robust to the wide range of variation in cell shape in the population; that the average locations of some structures became polarized in cells at the edges of colonies while maintaining the 'wiring' of their interactions with other structures; and that, by contrast, changes in the location of structures during early mitotic reorganization were accompanied by changes in their wiring.


Assuntos
Células-Tronco Pluripotentes Induzidas , Espaço Intracelular , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Análise de Célula Única , Conjuntos de Dados como Assunto , Interfase , Forma Celular , Mitose , Polaridade Celular , Sobrevivência Celular
15.
Nat Commun ; 14(1): 180, 2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36635295

RESUMO

The potential of pluripotent cells to respond to developmental cues and trigger cell differentiation is enhanced during the G1 phase of the cell cycle, but the molecular mechanisms involved are poorly understood. Variations in polycomb activity during interphase progression have been hypothesized to regulate the cell-cycle-phase-dependent transcriptional activation of differentiation genes during lineage transition in pluripotent cells. Here, we show that recruitment of Polycomb Repressive Complex 1 (PRC1) and associated molecular functions, ubiquitination of H2AK119 and three-dimensional chromatin interactions, are enhanced during S and G2 phases compared to the G1 phase. In agreement with the accumulation of PRC1 at target promoters upon G1 phase exit, cells in S and G2 phases show firmer transcriptional repression of developmental regulator genes that is drastically perturbed upon genetic ablation of the PRC1 catalytic subunit RING1B. Importantly, depletion of RING1B during retinoic acid stimulation interferes with the preference of mouse embryonic stem cells (mESCs) to induce the transcriptional activation of differentiation genes in G1 phase. We propose that incremental enrolment of polycomb repressive activity during interphase progression reduces the tendency of cells to respond to developmental cues during S and G2 phases, facilitating activation of cell differentiation in the G1 phase of the pluripotent cell cycle.


Assuntos
Histonas , Células-Tronco Pluripotentes , Complexo Repressor Polycomb 1 , Animais , Camundongos , Diferenciação Celular/genética , Cromatina/genética , Histonas/metabolismo , Interfase , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Células-Tronco Pluripotentes/citologia
16.
J Cell Biol ; 222(2)2023 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-36459066

RESUMO

Progressive accrual of senescent cells in aging and chronic diseases is associated with detrimental effects in tissue homeostasis. We found that senescent fibroblasts and epithelia were not only refractory to macrophage-mediated engulfment and removal, but they also paralyzed the ability of macrophages to remove bystander apoptotic corpses. Senescent cell-mediated efferocytosis suppression (SCES) was independent of the senescence-associated secretory phenotype (SASP) but instead required direct contact between macrophages and senescent cells. SCES involved augmented senescent cell expression of CD47 coinciding with increased CD47-modifying enzymes QPCT/L. SCES was reversible by interfering with the SIRPα-CD47-SHP-1 axis or QPCT/L activity. While CD47 expression increased in human and mouse senescent cells in vitro and in vivo, another ITIM-containing protein, CD24, contributed to SCES specifically in human epithelial senescent cells where it compensated for genetic deficiency in CD47. Thus, CD47 and CD24 link the pathogenic effects of senescent cells to homeostatic macrophage functions, such as efferocytosis, which we hypothesize must occur efficiently to maintain tissue homeostasis.


Assuntos
Apoptose , Antígeno CD47 , Macrófagos , Fenótipo Secretor Associado à Senescência , Animais , Humanos , Camundongos , Antígeno CD47/genética , Macrófagos/citologia , Regulação para Cima , Aminoaciltransferases/metabolismo , Antígeno CD24/metabolismo
17.
Appl Immunohistochem Mol Morphol ; 31(1): 57-63, 2023 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-36121292

RESUMO

Osteoarthritis (OA) is the main joint disease associated with aging. Previous studies have confirmed that both osteopontin (OPN) and αvß3 integrin are involved in the progression of knee OA. The purpose of this study was to determine the expression of OPN and αvß3 integrin and chondrocyte senescence levels in OA. Forty-six cartilage tissues from normal and knee OA patients were divided into 4 groups of normal, minor, moderate, and severe lesions based on the Mankin score. Immunohistochemistry and western blotting were used to determine the expression of αvß3, OPN, and senescent-associated-ß-galactosidase (SAß-gal) in articular cartilage. Then, Spearman's correlation was used to analyze the correlations between the Mankin scores and αvß3, OPN and SAß-gal. Pearson correlation analysis was used to analyze the correlations among αvß3, OPN, and SAß-gal. The expression of OPN, αvß3, and SAß-gal in articular cartilage was explored. αvß3, OPN, and SAß-gal proteins were all elevated in OA cartilage, and the correlation coefficient between the Mankin score and the average optical density value of αvß3, OPN, SAß-gal were r =0.60, r =0.75, and r =0.87, respectively, all P <0.001; the correlation between the average optical density value of αvß3 and OPN was r =0.3191, P <0.05; the correlation between αvß3 and SAß-gal was r =0.4955, P <0.001; and the correlation between OPN and SAß-gal was r =0.7821, P <0.001. The correlations among αvß3, OPN, and SAß-gal expression in articular cartilage might be important in OA progression and pathogenesis. Nonetheless, more research is needed to elucidate the exact contribution of αvß3, OPN, and SAß-gal to the degenerative process of OA.


Assuntos
Cartilagem , Condrócitos , Integrina alfaVbeta3 , Osteopontina , Humanos , Gravidade do Paciente , Integrina alfaVbeta3/metabolismo , Condrócitos/citologia , Senescência Celular
18.
Diabetologia ; 66(1): 223-240, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36260124

RESUMO

AIMS/HYPOTHESIS: Senescent renal tubular cells may be linked to diabetic kidney disease (DKD)-related tubulopathy. We studied mice with or without diabetes in which hedgehog interacting protein (HHIP) was present or specifically knocked out in renal tubules (HhipRT-KO), hypothesising that local deficiency of HHIP in the renal tubules would attenuate tubular cell senescence, thereby preventing DKD tubulopathy. METHODS: Low-dose streptozotocin was employed to induce diabetes in both HhipRT-KO and control (Hhipfl/fl) mice. Transgenic mice overexpressing Hhip in renal proximal tubular cells (RPTC) (HhipRPTC-Tg) were used for validation, and primary RPTCs and human RPTCs (HK2) were used for in vitro studies. Kidney morphology/function, tubular senescence and the relevant molecular measurements were assessed. RESULTS: Compared with Hhipfl/fl mice with diabetes, HhipRT-KO mice with diabetes displayed lower blood glucose levels, normalised GFR, ameliorated urinary albumin/creatinine ratio and less severe DKD, including tubulopathy. Sodium-glucose cotransporter 2 (SGLT2) expression was attenuated in RPTCs of HhipRT-KO mice with diabetes compared with Hhipfl/fl mice with diabetes. In parallel, an increased tubular senescence-associated secretory phenotype involving release of inflammatory cytokines (IL-1ß, IL-6 and monocyte chemoattractant protein-1) and activation of senescence markers (p16, p21, p53) in Hhipfl/fl mice with diabetes was attenuated in HhipRT-KO mice with diabetes. In contrast, HhipRPTC-Tg mice had increased tubular senescence, which was inhibited by canagliflozin in primary RPTCs. In HK2 cells, HHIP overexpression or recombinant HHIP increased SGLT2 protein expression and promoted cellular senescence by targeting both ataxia-telangiectasia mutated and ataxia-telangiectasia and Rad3-related-mediated cell arrest. CONCLUSIONS/INTERPRETATION: Tubular HHIP deficiency prevented DKD-related tubulopathy, possibly via the inhibition of SGLT2 expression and cellular senescence.


Assuntos
Proteínas de Transporte , Diabetes Mellitus Tipo 1 , Glicoproteínas de Membrana , Transportador 2 de Glucose-Sódio , Animais , Humanos , Camundongos , Diabetes Mellitus Tipo 1/genética , Células Epiteliais , Proteínas Hedgehog , Transportador 2 de Glucose-Sódio/genética , Proteínas de Transporte/genética , Glicoproteínas de Membrana/genética , Camundongos Transgênicos , Diabetes Mellitus Experimental/genética , Túbulos Renais/citologia , Senescência Celular
19.
Nucleic Acids Res ; 51(2): 919-934, 2023 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-36583339

RESUMO

Protein synthesis by the ribosome requires large-scale rearrangements of the 'small' subunit (SSU; ∼1 MDa), including inter- and intra-subunit rotational motions. However, with nearly 2000 structures of ribosomes and ribosomal subunits now publicly available, it is exceedingly difficult to design experiments based on analysis of all known rotation states. To overcome this, we developed an approach where the orientation of each SSU head and body is described in terms of three angular coordinates (rotation, tilt and tilt direction) and a single translation. By considering the entire RCSB PDB database, we describe 1208 fully-assembled ribosome complexes and 334 isolated small subunits, which span >50 species. This reveals aspects of subunit rearrangements that are universal, and others that are organism/domain-specific. For example, we show that tilt-like rearrangements of the SSU body (i.e. 'rolling') are pervasive in both prokaryotic and eukaryotic (cytosolic and mitochondrial) ribosomes. As another example, domain orientations associated with frameshifting in bacteria are similar to those found in eukaryotic ribosomes. Together, this study establishes a common foundation with which structural, simulation, single-molecule and biochemical efforts can more precisely interrogate the dynamics of this prototypical molecular machine.


Assuntos
Subunidades Ribossômicas , Ribossomos , Eucariotos/citologia , Biossíntese de Proteínas , Subunidades Ribossômicas/genética , Ribossomos/metabolismo , Rotação , Células Procarióticas , Fenômenos Biomecânicos
20.
Cell ; 186(1): 112-130.e20, 2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36580912

RESUMO

How SARS-CoV-2 penetrates the airway barrier of mucus and periciliary mucins to infect nasal epithelium remains unclear. Using primary nasal epithelial organoid cultures, we found that the virus attaches to motile cilia via the ACE2 receptor. SARS-CoV-2 traverses the mucus layer, using motile cilia as tracks to access the cell body. Depleting cilia blocks infection for SARS-CoV-2 and other respiratory viruses. SARS-CoV-2 progeny attach to airway microvilli 24 h post-infection and trigger formation of apically extended and highly branched microvilli that organize viral egress from the microvilli back into the mucus layer, supporting a model of virus dispersion throughout airway tissue via mucociliary transport. Phosphoproteomics and kinase inhibition reveal that microvillar remodeling is regulated by p21-activated kinases (PAK). Importantly, Omicron variants bind with higher affinity to motile cilia and show accelerated viral entry. Our work suggests that motile cilia, microvilli, and mucociliary-dependent mucus flow are critical for efficient virus replication in nasal epithelia.


Assuntos
COVID-19 , Sistema Respiratório , SARS-CoV-2 , Humanos , Cílios/fisiologia , Cílios/virologia , COVID-19/virologia , Sistema Respiratório/citologia , Sistema Respiratório/virologia , SARS-CoV-2/fisiologia , Microvilosidades/fisiologia , Microvilosidades/virologia , Internalização do Vírus , Células Epiteliais/fisiologia , Células Epiteliais/virologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...