RESUMO
Biomolecules contain various heterogeneities in their structures and local chemical properties, and their functions emerge through the dynamics encoded by these heterogeneities. Molecular dynamics model-based studies will greatly contribute to the elucidation of such chemical/mechanical structure-dynamics-function relationships and the mechanisms that generate them. Coarse-grained molecular dynamics models with appropriately designed nonuniform local interactions play an important role in considering the various phenomena caused by large molecular complexes consisting of various proteins and DNA such as nuclear chromosomes. Therefore, in this chapter, we will introduce a method for constructing a coarse-grained molecular dynamics model that simulates the global behavior of each chromosome in the nucleus of a mammalian cell containing many giant chromosomes.
Assuntos
Núcleo Celular , Simulação de Dinâmica Molecular , Núcleo Celular/metabolismo , Núcleo Celular/química , Animais , Humanos , Cromossomos/química , DNA/química , DNA/metabolismo , MamíferosRESUMO
The generation of quality data from a single-nucleus profiling experiment requires nuclei to be isolated from tissues in a gentle and efficient manner. Nuclei isolation must be carefully optimized across tissue types to preserve nuclear architecture, prevent nucleic acid degradation, and remove unwanted contaminants. Here, we present an optimized workflow for generating a single-nucleus suspension from ocular tissues of the embryonic chicken that is compatible with various downstream workflows. The described protocol enables the rapid isolation of a high yield of aggregate-free nuclei from the embryonic chicken eye without compromising nucleic acid integrity, and the nuclei suspension is compatible with single-nucleus RNA and ATAC sequencing. We detail several stopping points, either via cryopreservation or fixation, to enhance workflow adaptability. Further, we provide a guide through multiple QC points and demonstrate proof-of-principle using two commercially available kits. Finally, we demonstrate that existing in silico genotyping methods can be adopted to computationally derive biological replicates from a single pool of chicken nuclei, greatly reducing the cost of biological replication and allowing researchers to consider sex as a variable during analysis. Together, this tutorial represents a cost-effective, simple, and effective approach to single-nucleus profiling of embryonic chicken eye tissues and is likely to be easily modified to be compatible with similar tissue types.
Assuntos
Núcleo Celular , Galinhas , Análise de Célula Única , Animais , Núcleo Celular/metabolismo , Núcleo Celular/genética , Embrião de Galinha , Análise de Célula Única/métodos , Olho/embriologia , Olho/metabolismo , Criopreservação/métodos , Sequenciamento de Cromatina por Imunoprecipitação/métodosRESUMO
BACKGROUND: Radiation-induced liver fibrosis (RILF) is a common manifestation of radiation-induced liver injury (RILI) and is caused primarily by activated hepatic stellate cells (HSCs). Circular RNAs (circRNAs) play critical roles in various diseases, but little is known about the function and mechanism of circRNAs in RILF. METHODS: RNA pull-down and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to screen binding proteins of hsa_circ_0096498 (circ96498). RNA-binding protein immunoprecipitation, RNA pull-down and nuclear and cytoplasmic protein extraction were conducted to confirm the interaction between circ96498 and eukaryotic initiation factor 4A3 (EIF4A3). RNA sequencing was performed to screen target genes regulated by EIF4A3. HSCs with altered circ96498 and cell division cycle 42 (CDC42) expression were used to assess irradiated HSC activation. Circ96498 inhibition and CDC42 blockade were evaluated in RILF mouse models. RESULTS: In this study, we identified a radiation-sensitive circ96498, which was highly expressed in the irradiated HSCs of paracancerous tissues from RILI patients. Circ96498 inhibited the proliferation but promoted the apoptosis of irradiated HSCs, suppressed the secretion of proinflammatory cytokines IL-1ß, IL-6 and TNF-α, and decreased the expression of profibrotic markers (α-SMA and collagen 1) in irradiated HSCs. Mechanistically, irradiation induced the transport of EIF4A3 into the nucleus, and nuclear EIF4A3 increased the stability of CDC42 mRNA and increased CDC42 expression, thereby promoting HSC activation through the NF-κB and JNK/Smad2 pathways. However, the binding of circ96498 to EIF4A3 impeded the translocation of EIF4A3 into the nucleus, resulting in the inhibition of CDC42 expression and subsequent HSC activation. Furthermore, circ96498 knockdown promoted the development of the early and late stages of RILF in a mouse model, which was mitigated by CDC42 blockade. CONCLUSIONS: Collectively, our findings elucidate the involvement of the circ96498/EIF4A3/CDC42 axis in inhibiting irradiated HSC activation, which offers a novel approach for RILF prevention and treatment.
Assuntos
Núcleo Celular , Fator de Iniciação 4A em Eucariotos , Células Estreladas do Fígado , Cirrose Hepática , RNA Circular , Proteína cdc42 de Ligação ao GTP , Humanos , Animais , RNA Circular/genética , RNA Circular/metabolismo , Fator de Iniciação 4A em Eucariotos/metabolismo , Fator de Iniciação 4A em Eucariotos/genética , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Cirrose Hepática/genética , Núcleo Celular/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Masculino , Camundongos , Transporte Ativo do Núcleo Celular , Camundongos Endogâmicos C57BL , Proliferação de Células , Apoptose/efeitos da radiação , RNA Helicases DEAD-boxRESUMO
The members of the evolutionary conserved actin-binding Ezrin, Radixin and Moesin (ERM) protein family are involved in numerous key cellular processes in the cytoplasm. In the last decades, ERM proteins, like actin and other cytoskeletal components, have also been shown to be functional components of the nucleus; however, the molecular mechanism behind their nuclear activities remained unclear. Therefore, our primary aim was to identify the nuclear protein interactome of the single Drosophila ERM protein, Moesin. We demonstrate that Moesin directly interacts with the Mediator complex through direct binding to its Med15 subunit, and the presence of Moesin at the regulatory regions of the Hsp70Ab heat shock gene was found to be Med15-dependent. Both Moesin and Med15 bind to heat shock factor (Hsf), and they are required for proper Hsp gene expression under physiological conditions. Moreover, we confirmed that Moesin, Med15 and Hsf are able to bind the monomeric form of actin and together they form a complex in the nucleus. These results elucidate a mechanism by which ERMs function within the nucleus. Finally, we present the direct interaction of the human orthologues of Drosophila Moesin and Med15, which highlights the evolutionary significance of our finding.
Assuntos
Núcleo Celular , Proteínas de Drosophila , Resposta ao Choque Térmico , Proteínas dos Microfilamentos , Ligação Proteica , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Núcleo Celular/metabolismo , Humanos , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/genética , Complexo Mediador/metabolismo , Complexo Mediador/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Actinas/metabolismo , Regulação da Expressão Gênica , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de MembranaRESUMO
Malassezia is a lipophilic commensal yeast that resides mainly on the mammalian skin and is also found to associate with the internal organs. Dysbiosis of Malassezia is related to several diseases and often escapes detection as it is difficult to culture and maintain. Malassezia cell wall differs from other budding yeasts like S. cerevisiae due to the difference in the lipid content and is difficult to transform. In this study, we present a methodology to stain Malassezia's nucleus and perform cell cycle studies. However, staining presents a challenge due to its exceptionally thick cell wall with high lipid content, hindering conventional methods. Our novel methodology addresses this challenge and enables the staining of the Malassezia nucleus with a low background. This would allow researchers to visualize the overall nuclear health specifically nuclear morphology and analyze DNA content, crucial for cell cycle progression. By employing DNA-specific dyes like DAPI or Hoechst, we can observe the nuclear structure, and using PI we can differentiate cells in distinct cell cycle phases using techniques like flow cytometry. This novel staining methodology unlocks the door for in-depth cell cycle analysis in Malassezia which has challenged us through ages being refractory to genetic manipulations, paving the way for a deeper understanding of this commensal fungus and its potential role in human health.
Assuntos
Ciclo Celular , Núcleo Celular , Malassezia , Coloração e Rotulagem , Núcleo Celular/metabolismo , Humanos , Coloração e Rotulagem/métodos , Citometria de Fluxo/métodos , Parede Celular/metabolismoRESUMO
N6-methyladenosine (m6A) is the most abundant post-transcriptional dynamic RNA modification process in eukaryotes, extensively implicated in cellular growth, embryonic development and immune homeostasis. One of the most profound biological functions of m6A is to regulate RNA metabolism, thereby determining the fate of RNA. Notably, the regulation of m6A-mediated organized RNA metabolism critically relies on the assembly of membraneless organelles (MLOs) in both the nucleus and cytoplasm, such as nuclear speckles, stress granules and processing bodies. In addition, m6A-associated MLOs exert a pivotal role in governing diverse RNA metabolic processes encompassing transcription, splicing, transport, decay and translation. However, emerging evidence suggests that dysregulated m6A levels contribute to the formation of pathological condensates in a range of human diseases, including tumorigenesis, reproductive diseases, neurological diseases and respiratory diseases. To date, the molecular mechanism by which m6A regulates the aggregation of biomolecular condensates associated with RNA metabolism is unclear. In this review, we comprehensively summarize the updated biochemical processes of m6A-associated MLOs, particularly focusing on their impact on RNA metabolism and their pivotal role in disease development and related biological mechanisms. Furthermore, we propose that m6A-associated MLOs could serve as predictive markers for disease progression and potential drug targets in the future.
Assuntos
Adenosina , RNA , Humanos , Adenosina/metabolismo , Adenosina/análogos & derivados , RNA/metabolismo , Organelas/metabolismo , Animais , Processamento Pós-Transcricional do RNA , Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patologia , Núcleo Celular/metabolismo , Citoplasma/metabolismoRESUMO
Automated diagnostic systems can enhance the accuracy and efficiency of pathological diagnoses, nuclear segmentation plays a crucial role in computer-aided diagnosis systems for histopathology. However, achieving accurate nuclear segmentation is challenging due to the complex background tissue structures and significant variations in cell morphology and size in pathological images. In this study, we have proposed a U-Net based deep learning model, called MA-Net(Multifunctional Aggregation Network), to accurately segmenting nuclei from H&E stained images. In contrast to previous studies that focused on improving a single module of the network, we applied feature fusion modules, attention gate units, and atrous spatial pyramid pooling to the encoder and decoder, skip connections, and bottleneck of U-Net, respectively, to enhance the network's performance in nuclear segmentation. The dice coefficient loss was used during model training to enhance the network's ability to segment small objects. We applied the proposed MA-Net to multiple public datasets, and comprehensive results showed that this method outperforms the original U-Net method and other state-of-the-art methods in nuclei segmentation tasks. The source code of our work can be found in https://github.com/LinaZhaoAIGroup/MA-Net.
Assuntos
Núcleo Celular , Aprendizado Profundo , Humanos , Processamento de Imagem Assistida por Computador/métodos , Redes Neurais de Computação , Diagnóstico por Computador/métodos , AlgoritmosRESUMO
Despite the importance of cellular senescence in human health, how damaged cells undergo senescence remains elusive. We have previously shown that promyelocytic leukemia nuclear body (PML-NBs) translocation of the ciliary FBF1 is essential for senescence induction in stressed cells. Here we discover that an early cellular event occurring in stressed cells is the transient assembly of stress-induced nucleus-to-cilium microtubule arrays (sinc-MTs). The sinc-MTs are distinguished by unusual polyglutamylation and unique polarity, with minus-ends nucleating near the nuclear envelope and plus-ends near the ciliary base. KIFC3, a minus-end-directed kinesin, is recruited to plus-ends of sinc-MTs and interacts with the centrosomal protein CENEXIN1. In damaged cells, CENEXIN1 co-translocates with FBF1 to PML-NBs. Deficiency of KIFC3 abolishes PML-NB translocation of FBF1 and CENEXIN1, as well as senescence initiation in damaged cells. Our study reveals that KIFC3-mediated nuclear transport of FBF1 along polyglutamylated sinc-MTs is a prerequisite for senescence induction in mammalian cells.
Assuntos
Núcleo Celular , Senescência Celular , Cílios , Cinesinas , Microtúbulos , Humanos , Cinesinas/metabolismo , Cinesinas/genética , Núcleo Celular/metabolismo , Microtúbulos/metabolismo , Cílios/metabolismo , Animais , Transporte Ativo do Núcleo Celular , CamundongosRESUMO
Importin ß-superfamily nuclear import receptors (NIRs) mitigate mislocalization and aggregation of RNA-binding proteins (RBPs), like FUS and TDP-43, which are implicated in neurodegenerative diseases. NIRs potently disaggregate RBPs by recognizing their nuclear localization signal (NLS). However, disease-causing mutations in NLS compromise NIR binding and activity. Here, we define features that characterize the anti-aggregation activity of NIR and NLS. We find that high binding affinity between NIR and NLS, and optimal NLS location relative to the aggregating domain plays a role in determining NIR disaggregation activity. A designed FUS chimera (FUSIBB), carrying the importin ß binding (IBB) domain, is solubilized by importin ß in vitro, translocated to the nucleus in cultured cells, and downregulates the expression of endogenous FUS. In this study, we posit that guiding the mutual recognition of NLSs and NIRs will aid the development of therapeutics, illustrated by the highly soluble FUSIBB replacing the aggregation-prone endogenous FUS.
Assuntos
Regulação para Baixo , Sinais de Localização Nuclear , Proteína FUS de Ligação a RNA , beta Carioferinas , Proteína FUS de Ligação a RNA/metabolismo , Proteína FUS de Ligação a RNA/genética , Humanos , beta Carioferinas/metabolismo , beta Carioferinas/genética , Núcleo Celular/metabolismo , Ligação Proteica , Células HEK293 , Agregados Proteicos , Células HeLa , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/genética , Transporte Ativo do Núcleo CelularRESUMO
Adenoviral pVII proteins are multifunctional, highly basic, histone-like proteins that can bind to and transport the viral genome into the host cell nucleus. Despite the identification of several nuclear localization signals (NLSs) in the pVII protein of human adenovirus (HAdV)2, the mechanistic details of nuclear transport are largely unknown. Here we provide a full characterization of the nuclear import of precursor (Pre-) pVII protein from an ancient siadenovirus, frog siadenovirus 1 (FrAdV1), using a combination of structural, functional, and biochemical approaches. Two strong NLSs (termed NLSa and NLSd) interact with importin (IMP)ß1 and IMPα, respectively, and are the main drivers of nuclear import. A weaker NLS (termed NLSb) also contributes, together with an additional signal (NLSc) which we found to be important for nucleolar targeting and intranuclear binding. Expression of wild-type and NLS defective derivatives Pre-pVII in the presence of selective inhibitors of different nuclear import pathways revealed that, unlike its human counterpart, FrAdV1 Pre-pVII nuclear import is dependent on IMPα/ß1 and IMPß1, but not on transportin-1 (IMPß2). Clearly, AdVs evolved to maximize the nuclear import pathways for the pVII proteins, whose subcellular localization is the result of a complex process. Therefore, our results pave the way for an evolutionary comparison of the interaction of different AdVs with the host cell nuclear transport machinery.
Assuntos
Transporte Ativo do Núcleo Celular , Sinais de Localização Nuclear , Sinais de Localização Nuclear/metabolismo , Humanos , Núcleo Celular/metabolismo , beta Carioferinas/metabolismo , Animais , alfa Carioferinas/metabolismo , alfa Carioferinas/genética , Proteínas Virais/metabolismo , Proteínas Virais/genética , Adenoviridae/metabolismo , Adenoviridae/genética , Sequência de AminoácidosRESUMO
Nuclear pore complexes are massive protein gateways that control molecular exchange between the nucleus and cytoplasm. In this issue of Cell, Singh et al. provide the first high-resolution views of the elusive nuclear basket, which extends deep into the nucleus to coordinate functions from genome organization to mRNP export.
Assuntos
Núcleo Celular , Poro Nuclear , Núcleo Celular/metabolismo , Poro Nuclear/metabolismo , Humanos , Transporte Ativo do Núcleo Celular , Animais , Ribonucleoproteínas/metabolismoRESUMO
Reactive astrocytes play critical roles in the occurrence of various neurological diseases such as multiple sclerosis. Activation of astrocytes is often accompanied by a glycolysis-dominant metabolic switch. However, the role and molecular mechanism of metabolic reprogramming in activation of astrocytes have not been clarified. Here, we found that PKM2, a rate-limiting enzyme of glycolysis, displayed nuclear translocation in astrocytes of EAE (experimental autoimmune encephalomyelitis) mice, an animal model of multiple sclerosis. Prevention of PKM2 nuclear import by DASA-58 significantly reduced the activation of mice primary astrocytes, which was observed by decreased proliferation, glycolysis and secretion of inflammatory cytokines. Most importantly, we identified the ubiquitination-mediated regulation of PKM2 nuclear import by ubiquitin ligase TRIM21. TRIM21 interacted with PKM2, promoted its nuclear translocation and stimulated its nuclear activity to phosphorylate STAT3, NF-κB and interact with c-myc. Further single-cell RNA sequencing and immunofluorescence staining demonstrated that TRIM21 expression was upregulated in astrocytes of EAE. TRIM21 overexpressing in mice primary astrocytes enhanced PKM2-dependent glycolysis and proliferation, which could be reversed by DASA-58. Moreover, intracerebroventricular injection of a lentiviral vector to knockdown TRIM21 in astrocytes or intraperitoneal injection of TEPP-46, which inhibit the nuclear translocation of PKM2, effectively decreased disease severity, CNS inflammation and demyelination in EAE. Collectively, our study provides novel insights into the pathological function of nuclear glycolytic enzyme PKM2 and ubiquitination-mediated regulatory mechanism that are involved in astrocyte activation. Targeting this axis may be a potential therapeutic strategy for the treatment of astrocyte-involved neurological disease.
Assuntos
Astrócitos , Encefalomielite Autoimune Experimental , Ribonucleoproteínas , Regulação para Cima , Animais , Astrócitos/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/genética , Camundongos , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Hormônios Tireóideos/metabolismo , Hormônios Tireóideos/genética , Proteínas de Ligação a Hormônio da Tireoide , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Camundongos Endogâmicos C57BL , Piruvato Quinase/metabolismo , Piruvato Quinase/genética , Transporte Ativo do Núcleo Celular , Feminino , Glicólise , Ubiquitinação , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Núcleo Celular/metabolismoRESUMO
Alterations in nuclear structure and function are hallmarks of cancer cells. Little is known about these changes in Cancer-Associated Fibroblasts (CAFs), crucial components of the tumor microenvironment. Loss of the androgen receptor (AR) in human dermal fibroblasts (HDFs), which triggers early steps of CAF activation, leads to nuclear membrane changes and micronuclei formation, independent of cellular senescence. Similar changes occur in established CAFs and are reversed by restoring AR activity. AR associates with nuclear lamin A/C, and its loss causes lamin A/C nucleoplasmic redistribution. AR serves as a bridge between lamin A/C and the protein phosphatase PPP1. Loss of AR decreases lamin-PPP1 association and increases lamin A/C phosphorylation at Ser 301, a characteristic of CAFs. Phosphorylated lamin A/C at Ser 301 binds to the regulatory region of CAF effector genes of the myofibroblast subtype. Expression of a lamin A/C Ser301 phosphomimetic mutant alone can transform normal fibroblasts into tumor-promoting CAFs.
Assuntos
Fibroblastos Associados a Câncer , Núcleo Celular , Lamina Tipo A , Receptores Androgênicos , Humanos , Receptores Androgênicos/metabolismo , Receptores Androgênicos/genética , Lamina Tipo A/metabolismo , Lamina Tipo A/genética , Fosforilação , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Núcleo Celular/metabolismo , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 1/genética , Fibroblastos/metabolismo , Membrana Nuclear/metabolismo , Masculino , Microambiente TumoralRESUMO
More than a decade after the discovery of the classical cytoplasmic IκB proteins, IκBζ was identified as an additional member of the IκB family. Unlike cytoplasmic IκB proteins, IκBζ has distinct features, including its nuclear localization, preferential binding to NF-κB subunits, unique expression properties, and specialized role in NF-κB regulation. While the activation of NF-κB is primarily controlled by cytoplasmic IκB members at the level of nuclear entry, IκBζ provides an additional layer of NF-κB regulation in the nucleus, enabling selective gene activation. Human genome-wide association studies (GWAS) and gene knockout experiments in mice have elucidated the physiological and pathological roles of IκBζ. Despite the initial focus to its role in activated macrophages, IκBζ has since been recognized as a key player in the IL-17-triggered production of immune molecules in epithelial cells, which has garnered significant clinical interest. Recent research has also unveiled a novel molecular function of IκBζ, linking NF-κB and the POU transcription factors through its N-terminal region, whose role had remained elusive for many years.
Assuntos
Núcleo Celular , NF-kappa B , Humanos , NF-kappa B/metabolismo , Animais , Núcleo Celular/metabolismo , Proteínas I-kappa B/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de SinalRESUMO
GRK2 and arrestin3, key players in the functional regulation of G protein-coupled receptors (GPCRs), are ubiquitinated by Mdm2, a nuclear protein. The agonist-induced increase in arrestin3 ubiquitination occurs in the nucleus, underscoring the crucial role of its nuclear translocation in this process. The ubiquitination of arrestin3 occurs in the nucleus, highlighting the pivotal role of its nuclear translocation in this process. In contrast, GRK2 cannot translocate into the nucleus; thus, facilitation of the cytosolic translocation of nuclear Mdm2 is required to ubiquitinate GRK2 in the cytosol. Among the explored cellular components and processes, arrestin, Gßγ, clathrin, and receptor phosphorylation were found to be required for the nuclear import of arrestin3, the ubiquitination of arrestin3 in the nucleus, nuclear export of Mdm2, and the ubiquitination of GRK2 in the cytosol. In conclusion, our findings demonstrate that agonist-induced ubiquitination of arrestin3 in the nucleus is interconnected with cytosolic GRK2 ubiquitination.
Assuntos
Transporte Ativo do Núcleo Celular , Núcleo Celular , Citosol , Quinase 2 de Receptor Acoplado a Proteína G , Proteínas Proto-Oncogênicas c-mdm2 , Ubiquitinação , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Humanos , Citosol/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Núcleo Celular/metabolismo , Fosforilação , Arrestinas/metabolismo , Células HEK293 , AnimaisRESUMO
Thus far, multiple techniques for single cell analysis have been developed, yet we lack a relatively simple tool to assess DNA and RNA from the same cell at whole-transcriptome and whole-genome depths. Here we present an updated method for physical separation of cytoplasmic RNA from the nuclei, which allows for simultaneous studies of DNA and RNA from the same single cell. The method consists of three steps-(1) immobilization of a single cell on solid substrate, (2) hypotonic lysis of immobilized single cell, and (3) separation of cytosol containing aqueous phase and immobilized nucleus. We found that DNA and RNA extracted from single cell using our approach is suitable for downstream sequencing-based applications. We demonstrated that the coverage of transcriptome and genome sequencing data obtained after DNA/RNA separation is similar to that observed without separation. We also showed that the separation procedure does not create any noticeable bias in observed mutational load or mutation spectra. Thus, our method can serve as a tool for simultaneous complex analysis of the genome and transcriptome, providing necessary information on the relationship between somatic mutations and the regulation of gene expression.
Assuntos
Análise de Célula Única , Transcriptoma , Análise de Célula Única/métodos , Transcriptoma/genética , Humanos , RNA/genética , DNA/genética , Mutação , Genoma Humano , Núcleo Celular/genética , Núcleo Celular/metabolismo , Perfilação da Expressão Gênica/métodosRESUMO
The Notch receptor is a pleiotropic signaling protein that translates intercellular ligand interactions into changes in gene expression via the nuclear localization of the Notch intracellular Domain (NICD). Using a combination of immunohistochemistry, RNA in situ, Optogenetics and super-resolution live imaging of transcription in human cells, we show that the N1ICD can form condensates that positively facilitate Notch target gene expression. We determined that N1ICD undergoes Phase Separation Coupled Percolation (PSCP) into transcriptional condensates, which recruit, enrich, and encapsulate a broad set of core transcriptional proteins. We show that the capacity for condensation is due to the intrinsically disordered transcriptional activation domain of the N1ICD. In addition, the formation of such transcriptional condensates acts to promote Notch-mediated super enhancer-looping and concomitant activation of the MYC protooncogene expression. Overall, we introduce a novel mechanism of Notch1 activity in which discrete changes in nuclear N1ICD abundance are translated into the assembly of transcriptional condensates that facilitate gene expression by enriching essential transcriptional machineries at target genomic loci.
Assuntos
Elementos Facilitadores Genéticos , Receptor Notch1 , Receptor Notch1/metabolismo , Receptor Notch1/genética , Humanos , Regulação da Expressão Gênica , Núcleo Celular/metabolismo , Separação de FasesRESUMO
Cells maintain homeostasis via dynamic regulation of stress response pathways. Stress pathways transiently induce response regulons via negative feedback loops, but the extent to which individual genes provide feedback has not been comprehensively measured for any pathway. Here, we disrupted the induction of each gene in the Saccharomyces cerevisiae heat shock response (HSR) and quantified cell growth and HSR dynamics following heat shock. The screen revealed a core feedback loop governing the expression of the chaperone Hsp70 reinforced by an auxiliary feedback loop controlling Hsp70 subcellular localization. Mathematical modeling and live imaging demonstrated that multiple HSR targets converge to promote Hsp70 nuclear localization via its release from cytosolic condensates. Following ethanol stress, a distinct set of factors similarly converged on Hsp70, suggesting that nonredundant subsets of the HSR regulon confer feedback under different conditions. Flexible spatiotemporal feedback loops may broadly organize stress response regulons and expand their adaptive capacity.
Assuntos
Retroalimentação Fisiológica , Regulação Fúngica da Expressão Gênica , Proteínas de Choque Térmico HSP70 , Resposta ao Choque Térmico , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Núcleo Celular/metabolismo , Regulon/genéticaRESUMO
HIV-1 infection requires passage of the viral core through the nuclear pore of the cell, a process that depends on functions of the viral capsid. Recent studies have shown that HIV-1 cores enter the nucleus prior to capsid disassembly. Interactions of the viral capsid with the nuclear pore complex are necessary but not sufficient for nuclear entry, and the mechanism by which the viral core traverses the comparably sized nuclear pore is unknown. Here we show that the HIV-1 core is highly elastic and that this property is linked to nuclear entry and infectivity. Using atomic force microscopy-based approaches, we found that purified wild type cores rapidly returned to their normal conical morphology following a severe compression. Results from independently performed molecular dynamic simulations of the mature HIV-1 capsid also revealed its elastic property. Analysis of four HIV-1 capsid mutants that exhibit impaired nuclear entry revealed that the mutant viral cores are brittle. Adaptation of two of the mutant viruses in cell culture resulted in additional substitutions that restored elasticity and rescued infectivity and nuclear entry. We also show that capsid-targeting compound PF74 and the antiviral drug Lenacapavir reduce core elasticity and block HIV-1 nuclear entry at concentrations that preserve interactions between the viral core and the nuclear envelope. Our results indicate that elasticity is a fundamental property of the HIV-1 core that enables nuclear entry, thereby facilitating infection. These results provide new insights into the role of the capsid in HIV-1 nuclear entry and the antiviral mechanisms of HIV-1 capsid inhibitors.
Assuntos
Elasticidade , Infecções por HIV , HIV-1 , HIV-1/fisiologia , Humanos , Infecções por HIV/virologia , Infecções por HIV/metabolismo , Internalização do Vírus , Capsídeo/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Simulação de Dinâmica Molecular , Microscopia de Força Atômica , Poro Nuclear/metabolismo , Indóis , Fenilalanina/análogos & derivadosRESUMO
BACKGROUND: Eukaryotic cells are highly compartmentalized by a variety of organelles that carry out specific cellular processes. The position of these organelles within the cell is elaborately regulated and vital for their function. For instance, the position of lysosomes relative to the nucleus controls their degradative capacity and is altered in pathophysiological conditions. The molecular components orchestrating the precise localization of organelles remain incompletely understood. A confounding factor in these studies is the fact that organelle positioning is surprisingly non-trivial to address e.g., perturbations that affect the localization of organelles often lead to secondary phenotypes such as changes in cell or organelle size. These phenotypes could potentially mask effects or lead to the identification of false positive hits. To uncover and test potential molecular components at scale, accurate and easy-to-use analysis tools are required that allow robust measurements of organelle positioning. RESULTS: Here, we present an analysis workflow for the faithful, robust, and quantitative analysis of organelle positioning phenotypes. Our workflow consists of an easy-to-use Fiji plugin and an R Shiny App. These tools enable users without background in image or data analysis to (1) segment single cells and nuclei and to detect organelles, (2) to measure cell size and the distance between detected organelles and the nucleus, (3) to measure intensities in the organelle channel plus one additional channel, (4) to measure radial intensity profiles of organellar markers, and (5) to plot the results in informative graphs. Using simulated data and immunofluorescent images of cells in which the function of known factors for lysosome positioning has been perturbed, we show that the workflow is robust against common problems for the accurate assessment of organelle positioning such as changes of cell shape and size, organelle size and background. CONCLUSIONS: OrgaMapper is a versatile, robust, and easy-to-use automated image analysis workflow that can be utilized in microscopy-based hypothesis testing and screens. It effectively allows for the mapping of the intracellular space and enables the discovery of novel regulators of organelle positioning.