79,004 results on '"Cell Nucleus"'
Search Results
2. Mechanisms of Chromosome Folding and Nuclear Organization: Their Interplay and Open Questions
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Job Dekker and Leonid A. Mirny
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Cell Nucleus ,Genome ,Chromosome ,Compartmentalization (psychology) ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Chromatin ,Chromosomes ,Folding (chemistry) ,medicine.anatomical_structure ,Evolutionary biology ,medicine ,Interphase ,Nucleus ,Mitosis ,Spatial organization - Abstract
Microscopy and genomic approaches provide detailed descriptions of the three-dimensional folding of chromosomes and nuclear organization. The fundamental question is how activity of molecules at the nanometer scale can lead to complex and orchestrated spatial organization at the scale of chromosomes and the whole nucleus. At least three key mechanisms can bridge across scales: (1) tethering of specific loci to nuclear landmarks leads to massive reorganization of the nucleus; (2) spatial compartmentalization of chromatin, which is driven by molecular affinities, results in spatial isolation of active and inactive chromatin; and (3) loop extrusion activity of SMC (structural maintenance of chromosome) complexes can explain many features of interphase chromatin folding and underlies key phenomena during mitosis. Interestingly, many features of chromosome organization ultimately result from collective action and the interplay between these mechanisms, and are further modulated by transcription and topological constraints. Finally, we highlight some outstanding questions that are critical for our understanding of nuclear organization and function. We believe many of these questions can be answered in the coming years.
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- 2024
3. Uncovering the Principles of Genome Folding by 3D Chromatin Modeling
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Frank Alber, Yuxiang Zhan, Asli Yildirim, and Lorenzo Boninsegna
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Folding (chemistry) ,Cell Nucleus ,Genome ,Genomics ,Computational biology ,Biology ,Chromatin Assembly and Disassembly ,General Biochemistry, Genetics and Molecular Biology ,Chromatin Assembly ,Chromatin ,Chromosomes - Abstract
Our understanding of how genomic DNA is tightly packed inside the nucleus, yet is still accessible for vital cellular processes, has grown dramatically over recent years with advances in microscopy and genomics technologies. Computational methods have played a pivotal role in the structural interpretation of experimental data, which helped unravel some organizational principles of genome folding. Here, we give an overview of current computational efforts in mechanistic and data-driven 3D chromatin structure modeling. We discuss strengths and limitations of different methods and evaluate the added value and benefits of computational approaches to infer the 3D structural and dynamic properties of the genome and its underlying mechanisms at different scales and resolution, ranging from the dynamic formation of chromatin loops and topological associated domains to nuclear compartmentalization of chromatin and nuclear bodies.
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- 2024
4. Essential Roles for RNA in Shaping Nuclear Organization
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Sofia A. Quinodoz and Mitchell Guttman
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Cell Nucleus ,RNA, Untranslated ,Protein molecules ,Mechanism (biology) ,Nuclear organization ,RNA ,Computational biology ,DNA ,Biology ,General Biochemistry, Genetics and Molecular Biology ,chemistry.chemical_compound ,chemistry ,RNA, Long Noncoding - Abstract
It has long been proposed that nuclear RNAs might play an important role in organizing the structure of the nucleus. Initial experiments performed more than 30 years ago found that global disruption of RNA led to visible rearrangements of nuclear organization. Yet, this idea remained controversial for many years, in large part because it was unclear what specific RNAs might be involved, and which specific nuclear structures might be dependent on RNA. Over the past few years, the contributions of RNA to organizing nuclear structures have become clearer with the discovery that many nuclear bodies are enriched for specific noncoding RNAs (ncRNAs); in specific cases, ncRNAs have been shown to be essential for establishment and maintenance of these nuclear structures. More recently, many different ncRNAs have been shown to play critical roles in initiating the three-dimensional (3D) spatial organization of DNA, RNA, and protein molecules in the nucleus. These examples, combined with global imaging and genomic experiments, have begun to paint a picture of a broader role for RNA in nuclear organization and to uncover a unifying mechanism that may explain why RNA is a uniquely suited molecule for this role. In this review, we provide an overview of the history of RNA and nuclear structure and discuss key examples of RNA-mediated bodies, the global roles of ncRNAs in shaping nuclear structure, and emerging insights into mechanisms of RNA-mediated nuclear organization.
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- 2024
5. The Nuclear Lamina
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Karen L. Reddy, Ashley J. Melendez-Perez, and Xianrong Wong
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Cell Nucleus ,Nuclear Lamina ,Nuclear Envelope ,DNA replication ,Biology ,Mechanotransduction, Cellular ,General Biochemistry, Genetics and Molecular Biology ,Chromatin ,Lamins ,Cell biology ,medicine.anatomical_structure ,RNA splicing ,medicine ,Inner membrane ,Nuclear lamina ,Nuclear membrane ,Transcription factor ,Lamin - Abstract
Lamins interact with a host of nuclear membrane proteins, transcription factors, chromatin regulators, signaling molecules, splicing factors, and even chromatin itself to form a nuclear subcompartment, the nuclear lamina, that is involved in a variety of cellular processes such as the governance of nuclear integrity, nuclear positioning, mitosis, DNA repair, DNA replication, splicing, signaling, mechanotransduction and -sensation, transcriptional regulation, and genome organization. Lamins are the primary scaffold for this nuclear subcompartment, but interactions with lamin-associated peptides in the inner nuclear membrane are self-reinforcing and mutually required. Lamins also interact, directly and indirectly, with peripheral heterochromatin domains called lamina-associated domains (LADs) and help to regulate dynamic 3D genome organization and expression of developmentally regulated genes.
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- 2024
6. Nuclear size and shape control
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Gautam Dey and Helena Cantwell
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Cell Nucleus ,Nuclear morphology ,Nuclear division ,Nuclear Envelope ,Evolutionary biology ,Cell Biology ,Biology ,Cholesterol homeostasis ,Developmental Biology ,Shape control - Abstract
The nucleus displays a wide range of sizes and shapes in different species and cell types, yet its size scaling and many of the key structural constituents that determine its shape are highly conserved. In this review, we discuss the cellular properties and processes that contribute to nuclear size and shape control, drawing examples from across eukaryotes and highlighting conserved themes and pathways. We then outline physiological roles that have been uncovered for specific nuclear morphologies and disease pathologies associated with aberrant nuclear morphology. We argue that a comparative approach, assessing and integrating observations from different systems, will be a powerful way to help us address the open questions surrounding functional roles of nuclear size and shape in cell physiology.
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- 2022
7. Data on Biology Described by a Researcher at Kanazawa University (An Efficient Method for Isolating and Purifying Nuclei from Mice Brain for Single-Molecule Imaging Using High-Speed Atomic Force Microscopy).
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ATOMIC force microscopy ,NUCLEAR pore complex ,BRAIN imaging ,BIOLOGY ,RESEARCH personnel - Abstract
Researchers at Kanazawa University in Japan have developed a new method for isolating and purifying nuclei from the mouse brain for single-molecule imaging using high-speed atomic force microscopy (HS-AFM). The study addresses the limitations of traditional optical imaging techniques, which are unable to achieve the required resolution for observing organelles and proteins within cells. The researchers utilized the rapid strainer microfiltration (RSM) protocol to prepare high-quality nuclei from the mouse brain and then used HS-AFM to study the spatiotemporal dynamics of nuclear pores. This research provides valuable insights into the dynamics of nuclear pores and their function in controlling the movement of molecules between the cell nucleus and cytoplasm. [Extracted from the article]
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- 2024
8. Circular RNAs as biomarkers and therapeutic targets in cancer
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Aamir Ahmad, Gjumrakch Aliev, Guang Yang, Valentin Pavlov, Ilgiz Gareev, Ozal Beylerli, and Aferin Beilerli
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Cell Nucleus ,0301 basic medicine ,Cancer Research ,biology ,RNA ,RNA polymerase II ,RNA, Circular ,Computational biology ,MicroRNAs ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Transcription (biology) ,Circular RNA ,Neoplasms ,030220 oncology & carcinogenesis ,microRNA ,RNA splicing ,Biomarkers, Tumor ,biology.protein ,Humans ,Cancer biomarkers ,Gene ,Biomarkers - Abstract
Circular RNAs (circRNAs) are a class of single-stranded closed non-coding RNA molecules (ncRNAs), which are formed as a result of reverse splicing of mRNAs. Despite their relative abundance, an interest in understanding their regulatory importance is rather recent. High stability, abundance and evolutionary conservation among species underline some of their important traits. CircRNAs perform a variety of cellular functions ranging from miRNA and proteins sponges to transcriptional modulation and splicing. Additionally, most circRNAs are expressed aberrantly in pathological conditions suggesting their possible exploitation as diagnostic biomarkers. Their covalent closed cyclic structure resulting in resistance to RNases further makes them suitable as cancer biomarkers. Studies involving human tumors have verified differences in the expression profiles of circRNAs, indicating a regulatory role in cancer pathogenesis and metastasis. As endogenous competitive RNA, circRNAs can regulate tumor proliferation and invasion. Further, some circRNAs located in the nucleus can regulate transcription of genes by binding to RNA polymerase II. In this review, we elaborate the characteristics, functions and mechanisms of action of circRNAs in cancer. We also discuss the possibility of using circRNAs as potential therapeutic targets and biomarkers for cancer.
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- 2022
9. Cancer cells employ an evolutionarily conserved polyploidization program to resist therapy
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Sarah R. Amend, Emma U. Hammarlund, Robert Axelrod, Kenneth J. Pienta, Joel S. Brown, and Robert H. Austin
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0301 basic medicine ,Cancer Research ,Biology ,Genome ,Cancer recurrence ,Polyploidy ,03 medical and health sciences ,0302 clinical medicine ,Neoplasms ,medicine ,Humans ,Lethal cancer ,Cell Nucleus ,Chromatin/genetics ,Cell Enlargement ,Cancer ,Therapeutic resistance ,medicine.disease ,Neoplasms/genetics ,Chromatin ,Cell biology ,Cell and molecular biology ,Whole genome doubling ,030104 developmental biology ,030220 oncology & carcinogenesis ,Cancer cell ,Polyploid giant cancer cells ,Convergent evolution - Abstract
Unusually large cancer cells with abnormal nuclei have been documented in the cancer literature since 1858. For more than 100 years, they have been generally disregarded as irreversibly senescent or dying cells, too morphologically misshapen and chromatin too disorganized to be functional. Cell enlargement, accompanied by whole genome doubling or more, is observed across organisms, often associated with mitigation strategies against environmental change, severe stress, or the lack of nutrients. Our comparison of the mechanisms for polyploidization in other organisms and non-transformed tissues suggest that cancer cells draw from a conserved program for their survival, utilizing whole genome doubling and pausing proliferation to survive stress. These polyaneuploid cancer cells (PACCs) are the source of therapeutic resistance, responsible for cancer recurrence and, ultimately, cancer lethality.
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- 2022
10. Organization of the Pluripotent Genome
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Patrick S.L. Lim and Eran Meshorer
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Cell Nucleus ,Pluripotent Stem Cells ,0303 health sciences ,Genome ,Mechanism (biology) ,Epigenome ,Computational biology ,Biology ,Embryonic stem cell ,General Biochemistry, Genetics and Molecular Biology ,Chromatin ,Epigenesis, Genetic ,03 medical and health sciences ,0302 clinical medicine ,PERSPECTIVES ,Animals ,Humans ,Gene Regulatory Networks ,Epigenetics ,Induced pluripotent stem cell ,030217 neurology & neurosurgery ,Function (biology) ,030304 developmental biology - Abstract
In the past several decades, the establishment of in vitro models of pluripotency has ushered in a golden era for developmental and stem cell biology. Research in this arena has led to profound insights into the regulatory features that shape early embryonic development. Nevertheless, an integrative theory of the epigenetic principles that govern the pluripotent nucleus remains elusive. Here, we summarize the epigenetic characteristics that define the pluripotent state. We cover what is currently known about the epigenome of pluripotent stem cells and reflect on the use of embryonic stem cells as an experimental system. In addition, we highlight insights from super-resolution microscopy, which have advanced our understanding of the form and function of chromatin, particularly its role in establishing the characteristically "open chromatin" of pluripotent nuclei. Further, we discuss the rapid improvements in 3C-based methods, which have given us a means to investigate the 3D spatial organization of the pluripotent genome. This has aided the adaptation of prior notions of a "pluripotent molecular circuitry" into a more holistic model, where hotspots of co-interacting domains correspond with the accumulation of pluripotency-associated factors. Finally, we relate these earlier hypotheses to an emerging model of phase separation, which posits that a biophysical mechanism may presuppose the formation of a pluripotent-state-defining transcriptional program.
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- 2023
11. Two novel STAT1 mutations cause Mendelian susceptibility to mycobacterial disease
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Jiarui Wang, Xianqin Zhang, Tingting Zou, Dazhi Zhang, Wenqiang Liu, Zhenxing Liu, Xuejie Peng, Zhengyi Ni, Yang Tan, Meiqi Hou, Mi Zhou, Chao Yuan, and Xiaopei Zhou
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Male ,Mutant ,Biophysics ,Virulence ,Chromosomal translocation ,medicine.disease_cause ,Biochemistry ,chemistry.chemical_compound ,Protein Domains ,medicine ,Humans ,Genetic Predisposition to Disease ,Amino Acid Sequence ,STAT1 ,Molecular Biology ,Gene ,Cell Nucleus ,Genetics ,Mycobacterium Infections ,Mutation ,Base Sequence ,biology ,Promoter ,DNA ,Cell Biology ,Pedigree ,Protein Transport ,HEK293 Cells ,STAT1 Transcription Factor ,chemistry ,biology.protein ,Female ,Mutant Proteins ,HeLa Cells ,Protein Binding ,Subcellular Fractions - Abstract
Mendelian susceptibility to mycobacterial disease (MSMD) is a rare monogenetic disease, which is characterized by susceptibility to some weakly virulent mycobacteria. Here, we explored the pathogenic genes and molecular mechanisms of MSMD patients. We recruited three patients diagnosed with MSMD from two families. Two novel mutations (c.1228A > G, p.K410E and c.2071A > G, p.M691V) in STAT1 gene were identified from two families. The translocation of K410E mutant STAT1 protein into nucleus was not affected. The binding ability between gamma-activating sequence (GAS) and K410E mutant STAT1 protein was significantly reduced, which will reduce the interaction between STAT1 protein with the promoters of target genes. The M691V mutant STAT1 protein cannot translocate into the nucleus after IFN-γ stimulation, which will affect the STAT1 protein form gamma-activating factors (GAF) and bind the GAS in the promoter region of downstream target genes. Taken together, our results showed that the mutation of K410E led to impaired binding of STAT1 to target DNA, and the mutation of M691V prevented the transport of STAT1 into the nucleus, which led to MSMD. Together, we identified two novel mutations (c.1228A > G, p.K410E and c.2071A > G, p.M691V) in STAT1 gene in MSMD patients, and deciphered the molecular mechanism of MSMD caused by STAT1 mutations.
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- 2022
12. Yolk platelets impede nuclear expansion in Xenopus embryos
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Yasuhiro Iwao, Sora Shimogama, and Yuki Hara
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Blastomeres ,food.ingredient ,Embryo, Nonmammalian ,Xenopus ,Embryonic Development ,Cleavage (embryo) ,Endoplasmic Reticulum ,Xenopus laevis ,food ,Yolk ,medicine ,Animals ,Interphase ,Molecular Biology ,Cell Size ,Cell Nucleus ,biology ,Chemistry ,Endoplasmic reticulum ,Cell Membrane ,Cell Biology ,Cell cycle ,biology.organism_classification ,Cell biology ,medicine.anatomical_structure ,Cytoplasm ,Nucleus ,Developmental Biology - Abstract
During metazoan early embryogenesis, the intracellular properties of proteins and organelles change dynamically through rapid cleavage. In particular, a change in the nucleus size is known to contribute to embryonic development-dependent cell cycle and gene expression regulation. Here, we compared the nuclear sizes of various blastomeres from developing Xenopus embryos and analyzed the mechanisms that control the nuclear expansion dynamics by manipulating the amount of intracellular components in a cell-free system. There was slower nuclear expansion during longer interphase durations in blastomeres from vegetal hemispheres than those from animal hemispheres. Furthermore, upon recapitulating interphase events by manipulating the concentration of yolk platelets, which are originally rich in the vegetal blastomeres, in cell-free cytoplasmic extracts, there was slower nuclear expansion and DNA replication as compared to normal yolk-free conditions. Under these conditions, the supplemented yolk platelets accumulated around the nucleus in a microtubule-dependent manner and impeded organization of the endoplasmic reticulum network. Overall, we propose that yolk platelets around the nucleus reduce membrane supply from the endoplasmic reticulum to the nucleus, resulting in slower nuclear expansion in the yolk-rich vegetal blastomeres.
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- 2022
13. The role of SAF-A/hnRNP U in regulating chromatin structure
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Nick Gilbert, Mattia Marenda, and Elena Lazarova
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Cell Nucleus ,fungi ,Scaffold attachment factor A ,RNA-Binding Proteins ,RNA ,Heterogeneous-Nuclear Ribonucleoprotein U ,Biology ,Cell function ,Chromatin ,Cell biology ,Transcription (biology) ,Genetics ,Interphase ,Mitosis ,Developmental Biology ,Chromatin organisation - Abstract
Scaffold attachment factor A (SAF-A) or hnRNP U is a nuclear RNA-binding protein with a well-documented role in processing newly transcribed RNA. Recent studies also indicate that SAF-A can oligomerise in an ATP-dependent manner and interact with RNA to form a dynamic nuclear mesh. This mesh is thought to regulate nuclear and chromatin architecture, yet a mechanistic understanding is lacking. Here, we review developments in the field to understand how the SAF-A/RNA mesh affects chromatin organisation in interphase and mitosis. As SAF-A has an intrinsically disordered domain we discuss how the chromatin mesh is related to nuclear phase-separated condensates, which in other situations have been shown to regulate transcription and cell functions. Finally, we infer possible links between diseases emerging from SAF-A mutations and its role in chromatin organisation and regulation.
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- 2022
14. Epigenetic evidence for distinct contributions of resident and acquired myonuclei during long-term exercise adaptation using timed in vivo myonuclear labeling
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Yuan Wen, Kevin A. Murach, Ferdinand von Walden, and Cory M. Dungan
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Male ,Time Factors ,Satellite Cells, Skeletal Muscle ,Physiology ,Green Fluorescent Proteins ,Muscle Fibers, Skeletal ,Mice, Transgenic ,Biology ,Epigenesis, Genetic ,Mice ,Physical Conditioning, Animal ,Animals ,Epigenetics ,Transcription factor ,Cell Nucleus ,Staining and Labeling ,Rapid Report ,Protein turnover ,Promoter ,Cell Biology ,Methylation ,biology.organism_classification ,Adaptation, Physiological ,Cell biology ,DNA methylation ,Satellite (biology) ,Stem cell - Abstract
Muscle fibers are syncytial postmitotic cells that can acquire exogenous nuclei from resident muscle stem cells, called satellite cells. Myonuclei are added to muscle fibers by satellite cells during conditions such as load-induced hypertrophy. It is difficult to dissect the molecular contributions of resident versus satellite cell-derived myonuclei during adaptation due to the complexity of labeling distinct nuclear populations in multinuclear cells without label transference between nuclei. To sidestep this barrier, we used a genetic mouse model where myonuclear DNA can be specifically and stably labeled via nonconstitutive H2B-GFP at any point in the lifespan. Resident myonuclei (Mn) were GFP-tagged in vivo before 8 wk of progressive weighted wheel running (PoWeR) in adult mice (>4-mo-old). Resident + satellite cell-derived myonuclei (Mn+SC Mn) were labeled at the end of PoWeR in a separate cohort. Following myonuclear isolation, promoter DNA methylation profiles acquired with low-input reduced representation bisulfite sequencing (RRBS) were compared to deduce epigenetic contributions of satellite cell-derived myonuclei during adaptation. Resident myonuclear DNA has hypomethylated promoters in genes related to protein turnover, whereas the addition of satellite cell-derived myonuclei shifts myonuclear methylation profiles to favor transcription factor regulation and cell-cell signaling. By comparing myonucleus-specific methylation profiling to previously published single-nucleus transcriptional analysis in the absence (Mn) versus the presence of satellite cells (Mn+SC Mn) with PoWeR, we provide evidence that satellite cell-derived myonuclei may preferentially supply specific ribosomal proteins to growing myofibers and retain an epigenetic “memory” of prior stem cell identity. These data offer insights on distinct epigenetic myonuclear characteristics and contributions during adult muscle growth.
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- 2022
15. Swine acute diarrhea syndrome coronavirus replication is reduced by inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway
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Yuru Han, Shufeng Feng, Jiyu Zhang, Li Feng, Jianfei Chen, Zhaoyang Jing, Hongyan Shi, Jianbo Liu, Liaoyuan Zhang, Da Shi, Dakai Liu, Tingshuai Feng, Xin Zhang, Yiming Wang, and Zhaoyang Ji
- Subjects
MAPK/ERK pathway ,MAP Kinase Signaling System ,Swine ,Viral protein ,viruses ,Apoptosis ,Biology ,Virus Replication ,medicine.disease_cause ,behavioral disciplines and activities ,Article ,Virus ,Cell Line ,chemistry.chemical_compound ,Biosynthesis ,Virology ,Chlorocebus aethiops ,mental disorders ,Viral replication ,medicine ,Animals ,Protein Kinase Inhibitors ,Vero Cells ,Coronavirus ,Cell Nucleus ,Mitogen-Activated Protein Kinase 1 ,Gene knockdown ,Mitogen-Activated Protein Kinase 3 ,Alphacoronavirus ,virus diseases ,respiratory tract diseases ,Cell biology ,Swine acute diarrhea syndrome coronavirus (SADS-CoV) ,chemistry ,Gene Knockdown Techniques ,Host-Pathogen Interactions ,ERK pathway - Abstract
Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly discovered enteric coronavirus. We have previously shown that the caspase-dependent FASL-mediated and mitochondrion-mediated apoptotic pathways play a central role in SADS-CoV-induced apoptosis, which facilitates viral replication. However, the roles of intracellular signaling pathways in SADS-CoV-mediated cell apoptosis and the relative advantages that such pathways confer on the host or virus remain largely unknown. In this study, we show that SADS-CoV induces the activation of ERK during infection, irrespective of viral biosynthesis. The knockdown or chemical inhibition of ERK1/2 significantly suppressed viral protein expression and viral progeny production. The inhibition of ERK activation also circumvented SADS-CoV-induced apoptosis. Taken together, these data suggest that ERK activation is important for SADS-CoV replication, and contributes to the virus-mediated changes in host cells. Our findings demonstrate the takeover of a particular host signaling mechanism by SADS-CoV and identify a potential approach to inhibiting viral spread.
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- 2022
16. Developmental trajectory of monopronucleated zygotes after in vitro fertilization when they include both male and female genomes
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Xingqiang Wei, Noritoshi Enatsu, Kohyu Furuhashi, Shoji Kokeguchi, Junko Otsuki, Masahide Shiotani, and Toshiroh Iwasaki
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Adult ,Male ,Cytoplasm ,Zygote ,medicine.medical_treatment ,Embryonic Development ,Fertilization in Vitro ,Polar Bodies ,Biology ,Time-Lapse Imaging ,Cohort Studies ,Andrology ,Human fertilization ,medicine ,Humans ,Sperm Injections, Intracytoplasmic ,Retrospective Studies ,Cell Nucleus ,Sex Chromosomes ,In vitro fertilisation ,Genome, Human ,Obstetrics and Gynecology ,Second polar body ,Embryo ,Oocyte ,Sperm ,medicine.anatomical_structure ,Developmental trajectory ,Reproductive Medicine ,Female - Abstract
Objective To examine the cause of monopronucleated zygote (1PN) formation that includes both maternal and paternal genomes. Design Retrospective cohort study. Setting Private fertility clinic. Patient(s) A total of 44 1PN and 726 2-pronuclear zygotes from 702 patients were observed using 2 different time-lapse observation systems. Intervention(s) Previously recorded time lapse data were reviewed to examine the mechanism of 1PN formation. Main Outcome Measure(s) The distance between the position of the second polar body extrusion and the fertilization cone or epicenter/starting position of the cytoplasmic wave was measured, and the consequent data were analyzed. Cytoplasmic waves were confirmed using vector analysis software. Result(s) The cut-off value for the difference in the distance between the position of the second polar body extrusion and the fertilization cone or the epicenter/starting position of the cytoplasmic wave was 17 μm (AUC: 0.987, 95% CI: 0.976–0.999) for the Embryo Scope and 18 μm (AUC: 0.972, 95% CI: 0.955–0.988) for the iBIS time-lapse observation systems. Conclusion(s) In this study, it was found with a high degree of accuracy that a monopronucleus is formed when the fusion of the sperm takes place within 18 μm from the point of the second polar body extrusion. The theoretical chance of 1PN occurrence after in vitro fertilization is 2.7% when the sperm is considered to be fused anywhere in the plasma membrane of an oocyte.
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- 2022
17. Protective effect of dexmedetomidine in cecal ligation perforation-induced acute lung injury through HMGB1/RAGE pathway regulation and pyroptosis activation
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Huaqin Sun, Mingsun Fang, Hongyi Hu, Xiaoping Xu, Zhehao Liang, and Tao Tao
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Male ,animal diseases ,Acute Lung Injury ,Receptor for Advanced Glycation End Products ,Perforation (oil well) ,Bioengineering ,Lung injury ,Pharmacology ,Protective Agents ,HMGB1 ,Applied Microbiology and Biotechnology ,NF-κB ,Cell Line ,RAGE (receptor) ,Intensive care ,polycyclic compounds ,Pyroptosis ,Animals ,Medicine ,RNA, Messenger ,HMGB1 Protein ,Cecum ,Ligation ,Lung ,Cell Nucleus ,biology ,business.industry ,Lentivirus ,RAGE ,General Medicine ,respiratory system ,respiratory tract diseases ,Mice, Inbred C57BL ,Disease Models, Animal ,Protein Transport ,Myeloperoxidase ,biology.protein ,Cytokines ,Tumor necrosis factor alpha ,Inflammation Mediators ,business ,TP248.13-248.65 ,hormones, hormone substitutes, and hormone antagonists ,Dexmedetomidine ,Research Article ,Research Paper ,Signal Transduction ,Biotechnology - Abstract
Dexmedetomidine (DEX) has been reported to attenuate cecal ligation perforation (CLP)-stimulated acute lung injury (ALI) by downregulating HMGB1 and RAGE. This study aimed to further investigate the specific mechanisms of RAGE and its potential-related mechanisms of DEX on ALI models in vitro and in vivo. The in vitro and in vivo ALI models were established by lipopolysaccharide treatment in MLE-12 cells and CLP in mice, respectively. The effect of DEX on pathological alteration was investigated by HE staining. Thereafter, the myeloperoxidase (MPO) activity and inflammatory cytokine levels were respectively detected to assess the lung injury of mice using commercial kits. The expression levels of HMGB1, RAGE, NF-κB, and pyroptosis-related molecules were detected by RT-qPCR and Western blot. HE staining showed that lung injury, increased inflammatory cell infiltration, and lung permeability was found in the ALI mice, and DEX treatment significantly attenuated lung tissue damage induced by CLP. The MPO activity and inflammatory cytokines (TNF-α, IL-1β, and NLRP3) levels were also significantly reduced after DEX treatment compared with those in the ALI mice. Moreover, DEX activated the HMGB1/RAGE/NF-κB pathway and upregulated the pyroptosis-related proteins. However, the protective DEX effect was impaired by RAGE overexpression in ALI mice and MLE-12 cells. Additionally, DEX treatment significantly suppressed HMGB1 translocation from the nucleus region to the cytoplasm, and this effect was reversed by RAGE overexpression. These findings suggested that DEX may be a useful ALI treatment, and the protective effects on ALI mice may be through the inhibition of HMGB1/RAGE/NF-κB pathway and cell pyroptosis., Graphical abstractDexmedetomidine (DEX) has protective effects on acute lung injury (ALI) in vitro and in vivo. The possible mechanisms may be closely associated with inflammatory response, caspase-1-mediated cell pyroptosis, and high-mobility group protein 1 (HMGB1)/receptor for advanced glycation end products (RAGE)/nuclear factor-κB pathway. Moreover, DEX could promote the HMGB1 translocation from the cytoplasm to the nucleus in lipopolysaccharide-activated MLE-12 cells, whereas the action of RAGE overexpression was opposite.
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- 2021
18. Pyruvate kinase M2 (PKM2) interacts with activating transcription factor 2 (ATF2) to bridge glycolysis and pyroptosis in microglia
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Yi Zhang, Xin Ge, Mengmeng Li, Dongmei Zhang, Xue-qin Wang, Feng Ji, Hongjian Lu, Chengwei Duan, Xiangyang Zhu, Jian-bin Su, and Xueyan Wang
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Lipopolysaccharides ,Male ,Pyruvate Kinase ,Immunology ,PKM2 ,Cell Line ,Pyroptosis ,Animals ,Glycolysis ,Gene Silencing ,Phosphorylation ,Molecular Biology ,Neuroinflammation ,Cell Nucleus ,Inflammation ,Activating Transcription Factor 2 ,biology ,Chemistry ,Brain ,Warburg effect ,Aerobiosis ,Activating transcription factor 2 ,Up-Regulation ,Cell biology ,Mice, Inbred C57BL ,Protein Transport ,biology.protein ,Microglia ,Pyruvate kinase ,Protein Binding - Abstract
Pyruvate kinase M2 (PKM2), a glycolytic rate-limiting enzyme, reportedly plays an important role in tumorigenesis and the inflammatory response by regulating the metabolic reprogramming. However, its contribution to microglial activation during neuroinflammation is still unknown. In this study, we observed an enhanced glycolysis level in the lipopolysaccharide (LPS)-activated microglia. Utilizing the glycolysis inhibitor 2-DG, we proved that LPS requires glycolysis to induce microglial pyroptosis. Moreover, the protein expression, dimer/monomer formation, phosphorylation and nuclear translocation of PKM2 were all increased by LPS. Silencing PKM2 or preventing its nuclear translocation by TEPP-46 significantly alleviated the LPS-induced inflammatory response and pyroptosis in microglia. Employing biological mass spectrometry combined with immunoprecipitation technology, we identified for the first time that PKM2 interacts with activating transcription factor 2 (ATF2) in microglia. Inhibition of glycolysis or preventing PKM2 nuclear aggregation significantly reduced the phosphorylation and activation of ATF2. Furthermore, knocking down ATF2 reduced the LPS-induced pyroptosis of microglia. In vivo, we showed the LPS-induced pyroptosis in the cerebral cortex tissues of mice, and first found that an increased PKM2 expression was co-localized with ATF2 in the inflamed mice brain. Collectively, our data suggested for the first time that PKM2, a key rate-limiting enzyme of the Warburg effect, directly interacts with the pro-inflammatory transcription factor ATF2 to bridge glycolysis and pyroptosis in microglia, which might be a pivotal crosstalk between metabolic reprogramming and neuroinflammation in the CNS.
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- 2021
19. IFI16 Isoforms with Cytoplasmic and Nuclear Locations Play Differential Roles in Recognizing Invaded DNA Viruses
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Jingfei Zhu, Dapei Li, Yanghua Qin, Zhengrong Chen, Lifen Xie, Zigang Qiao, Yongdong Yan, Haiping Yao, and Feng Ma
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Cell Nucleus ,Gene isoform ,viruses ,Immunology ,DNA Viruses ,Nuclear Proteins ,Transfection ,Biology ,Phosphoproteins ,Virus ,Cell biology ,Nuclear DNA ,chemistry.chemical_compound ,chemistry ,Transcription (biology) ,Humans ,Protein Isoforms ,Immunology and Allergy ,Gene ,Cells, Cultured ,Nuclear localization sequence ,DNA - Abstract
IFN-γ–inducible protein 16 (IFI16) recognizes viral DNAs from both nucleus-replicating viruses and cytoplasm-replicating viruses. Isoform 2 of IFI16 (IFI16-iso2) with nuclear localization sequence (NLS) has been studied extensively as a well-known DNA sensor. However, the characteristics and functions of other IFI16 isoforms are almost unknown. Here, we find that IFI16-iso1, with exactly the same length as IFI16-iso2, lacks the NLS and locates in the cytoplasm. To distinguish the functions of IFI16-iso1 and IFI16-iso2, we have developed novel nuclear viral DNA mimics that can be recognized by the nuclear DNA sensors, including IFI16-iso2 and hnRNPA2B1. The hexanucleotide motif 5′-AGTGTT-3′ DNA form of the nuclear localization sequence (DNLS) effectively drives cytoplasmic viral DNA nuclear translocation. These nuclear viral DNA mimics potently induce IFN-β and antiviral IFN-stimulated genes in human A549 cells, HEK293T cells, and mouse macrophages. The subcellular location difference of IFI16 isoforms determines their differential functions in recognizing viral DNA and activating type I IFN–dependent antiviral immunity. IFI16-iso1 preferentially colocalizes with cytoplasmic HSV60mer and cytoplasm-replicating vaccinia virus (VACV), whereas IFI16-iso2 mainly colocalizes with nuclear HSV60-DNLS and nucleus-replicating HSV-1. Compared with IFI16-iso2, IFI16-iso1 induces more transcription of IFN-β and IFN-stimulated genes, as well as stronger antiviral immunity upon HSV60mer transfection or VACV infection. IFI16-iso2, with the ability of nuclear-cytoplasmic shuttling, clears both invaded HSV type 1 and VACV significantly. However, IFI16-iso2 induces more type I IFN–dependent antiviral immunity than IFI16-iso1 upon HSV60-DNLS transfection or HSV type 1 infection. Our study has developed potent agonists for nuclear DNA sensors and also has demonstrated that IFI16 isoforms with cytoplasmic and nuclear locations play differential roles in innate immunity against DNA viruses.
- Published
- 2021
20. 3D genome organization in the central nervous system, implications for neuropsychological disorders
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Yan Jiang, Jie Weng, Yuhao Dong, and Daijing Sun
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Cell Nucleus ,Central Nervous System ,Genome ,Mechanism (biology) ,Central nervous system ,Neuropsychology ,Computational biology ,Biology ,medicine.disease ,Chromatin ,Chromosomes ,Eukaryotic Cells ,22q11 Deletion Syndrome ,medicine.anatomical_structure ,Schizophrenia ,Genetics ,medicine ,Trinucleotide repeat expansion ,Molecular Biology ,Genomic organization - Abstract
Chromosomes in eukaryotic cell nuclei are highly compacted and finely organized into hierarchical three-dimensional (3D) configuration. In recent years, scientists have gained deeper understandings of 3D genome structures and revealed novel evidence linking 3D genome organization to various important cell events on the molecular level. Most importantly, alteration of 3D genome architecture has emerged as an intriguing higher order mechanism that connects disease-related genetic variants in multiple heterogenous and polygenic neuropsychological disorders, delivering novel insights into the etiology. In this review, we provide a brief overview of the hierarchical structures of 3D genome and two proposed regulatory models, loop extrusion and phase separation. We then focus on recent Hi-C data in the central nervous system and discuss 3D genome alterations during normal brain development and in mature neurons. Most importantly, we make a comprehensive review on current knowledge and discuss the role of 3D genome in multiple neuropsychological disorders, including schizophrenia, repeat expansion disorders, 22q11 deletion syndrome, and others.
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- 2021
21. Nuclear positioning during development: Pushing, pulling and flowing
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Ojas Deshpande and Ivo A. Telley
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Cell Nucleus ,Cell division ,Context (language use) ,Cell Biology ,Cell cycle ,Biology ,Embryonic stem cell ,Cell biology ,medicine.anatomical_structure ,medicine ,Asymmetric cell division ,Animals ,Drosophila ,Cytoskeleton ,Nucleus ,Cytokinesis ,Developmental Biology - Abstract
The positioning of the nucleus, the central organelle of the cell, is an active and regulated process crucially linked to cell cycle, differentiation, migration, and polarity. Alterations in positioning have been correlated with cell and tissue function deficiency and genetic or chemical manipulation of nuclear position is embryonic lethal. Nuclear positioning is a precursor for symmetric or asymmetric cell division which is accompanied by fate determination of the daughter cells. Nuclear positioning also plays a key role during early embryonic developmental stages in insects, such as Drosophila, where hundreds of nuclei divide without cytokinesis and are distributed within the large syncytial embryo at roughly regular spacing. While the cytoskeletal elements and the linker proteins to the nucleus are fairly well characterised, including some of the force generating elements driving nuclear movement, there is considerable uncertainty about the biophysical mechanism of nuclear positioning, while the field is debating different force models. In this review, we highlight the current body of knowledge, discuss cell context dependent models of nuclear positioning, and outline open questions.
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- 2021
22. Decreased Lamin B1 Levels Affect Gene Positioning and Expression in Postmitotic Neurons
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Toshikazu Kakizaki, Hideki Uosaki, Azumi Noguchi, Kinichi Nakashima, Yuchio Yanagawa, Hirokazu Arakawa, Katsuhide Igarashi, Yuichi Uosaki, Takumi Takizawa, Ruri Kaneda, Kenji Ito, Hideyuki Nakashima, and Maky Ideta-Otsuka
- Subjects
0301 basic medicine ,Neurogenesis ,Locus (genetics) ,Biology ,gene positioning ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Transcription (biology) ,Gene expression ,medicine ,Transcriptional regulation ,Animals ,Humans ,Gene ,Cell Nucleus ,Neurons ,Genetics ,Lamin Type B ,maturation ,General Neuroscience ,General Medicine ,neuron ,Chromatin ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,gene expression ,chromatin ,Nucleus ,030217 neurology & neurosurgery ,Lamin ,nuclear lamina - Abstract
Gene expression programs and concomitant chromatin regulation change dramatically during the maturation of postmitotic neurons. Subnuclear positioning of gene loci is relevant to transcriptional regulation. However, little is known about subnuclear genome positioning in neuronal maturation. Using cultured murine hippocampal neurons, we found genomic locus 14qD2 to be enriched with genes that are upregulated during neuronal maturation. Reportedly, the locus is homologous to human 8p21.3, which has been extensively studied in neuropsychiatry and neurodegenerative diseases. Mapping of the 14qD2 locus in the nucleus revealed that it was relocated from the nuclear periphery to the interior. Moreover, we found a concomitant decrease in lamin B1 expression. Overexpression of lamin B1 in neurons using a lentiviral vector prevented the relocation of the 14qD2 locus and repressed the transcription of the Egr3 gene on this locus. Taken together, our results suggest that reduced lamin B1 expression during the maturation of neurons is important for appropriate subnuclear positioning of the genome and transcriptional programs.
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- 2021
23. A customised target capture sequencing tool for molecular identification of Aloe vera and relatives
- Author
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Martin Fritzsche, Nina Rønsted, Caroline Howard, Juan Viruel, Ryan Mate, Olwen M. Grace, Yannick Woudstra, and Thomas Bleazard
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0106 biological sciences ,Plant genetics ,Science ,Computational biology ,010603 evolutionary biology ,01 natural sciences ,Article ,Aloe vera ,03 medical and health sciences ,Target capture ,Aloe ,Phylogeny ,Plant Proteins ,030304 developmental biology ,Molecular identification ,Cell Nucleus ,2. Zero hunger ,0303 health sciences ,Multidisciplinary ,biology ,High-Throughput Nucleotide Sequencing ,Phylogenomics ,biology.organism_classification ,Biological Evolution ,Next-generation sequencing ,Genetic markers ,Medicine ,Transcriptome ,Genome, Plant - Abstract
Plant molecular identification studies have, until recently, been limited to the use of highly conserved markers from plastid and other organellar genomes, compromising resolution in highly diverse plant clades. Due to their higher evolutionary rates and reduced paralogy, low-copy nuclear genes overcome this limitation but are difficult to sequence with conventional methods and require high-quality input DNA. Aloe vera and its relatives (Asphodelaceae, subfamily Alooideae) are of economic interest for food and health products and have horticultural value. However, pressing conservation issues are increasing the need for a molecular identification tool to regulate the trade. With >600 species and an origin of ±15 million years ago, this predominantly African succulent plant clade is a diverse and taxonomically complex group for which low-copy nuclear genes would be desirable for accurate species discrimination. Unfortunately, with an average genome size of 16.76 pg, obtaining high coverage sequencing data for these genes would be prohibitively costly and computationally demanding. We used newly generated transcriptome data to design a customised RNA-bait panel targeting 189 low-copy nuclear genes in Alooideae. We demonstrate its efficacy in obtaining high-coverage sequence data for the target loci on Illumina sequencing platforms, including degraded DNA samples from museum specimens, with considerably improved phylogenetic resolution. This customised target capture sequencing protocol has the potential to confidently indicate phylogenetic relationships of Aloe vera and related species, as well as aid molecular identification applications.
- Published
- 2021
24. Curcumin protection against ultraviolet-induced photo-damage in Hacat cells by regulating nuclear factor erythroid 2-related factor 2
- Author
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Runxiang Li, Huilan Zhu, Miaojian Wan, Huiyan Deng, Huaping Li, Quan Chen, and Bihua Liang
- Subjects
Keratinocytes ,skin ,Cell Survival ,NF-E2-Related Factor 2 ,Ultraviolet Rays ,Apoptosis ,Bioengineering ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,Antioxidants ,Superoxide dismutase ,chemistry.chemical_compound ,ultraviolet ,medicine ,HaCaT Cells ,Humans ,oxidative stress ,curcumin ,Cell Proliferation ,Cell Nucleus ,biology ,integumentary system ,Cell growth ,Chemistry ,photo-damage ,General Medicine ,respiratory system ,Molecular biology ,Heme oxygenase ,HaCaT ,nuclear factor erythroid 2-related factor 2 ,Cytoprotection ,Catalase ,biology.protein ,Curcumin ,Oxidative stress ,TP248.13-248.65 ,Research Article ,Research Paper ,Biotechnology - Abstract
Curcumin suppressed ultraviolet (UV) induced skin carcinogenesis and activated the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. However, whether curcumin protects skin injury caused by UV is still unknown. A vitro model was established and curcumin effects on Hacat cells were detected. Nrf2 was knocked down in Hacat cells to verify the Nrf2 role in the protective effect of curcumin. Results indicated that ultraviolet A (UVA) (or ultraviolet B (UVB)) irradiation would lead to decreased cell proliferation, increased cell apoptosis, decreased catalase, heme oxygenase 1, and superoxide dismutase expression, and increased levels of protein carbonylation and malondialdehyde (p
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- 2021
25. Cell-cycle dependent GATA2 subcellular localization in mouse 2-cell embryos
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Manabu Kawahara, Masaya Komatsu, Hanako Bai, Hayato Tsukahara, Masashi Takahashi, and Takuya Wakai
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Blastomeres ,Time Factors ,Green Fluorescent Proteins ,Biophysics ,Biology ,Biochemistry ,Gene expression ,Animals ,Interphase ,Molecular Biology ,Mitosis ,Transcription factor ,Cell Nucleus ,Mice, Inbred ICR ,Reverse Transcriptase Polymerase Chain Reaction ,Cell Cycle ,GATA2 ,Gene Expression Regulation, Developmental ,Cell Biology ,Blastomere ,Cell cycle ,Protein subcellular localization prediction ,Cell biology ,GATA2 Transcription Factor ,Blastocyst ,Microscopy, Fluorescence ,Female - Abstract
GATA factors are essential transcription factors for embryonic development that broadly control the transcription of other genes. This study aimed to examine GATA2 protein localization in mouse embryos at the 2-cell stage, when drastic transformation in gene expression occurs for subsequent development in early embryos. We first analyzed GATA2 localization in 2-cell embryos at the interphase and mitotic phases by immunofluorescence analysis. In the interphase, GATA2 protein was localized in the nucleus, as a common transcription factor. In the mitotic phase, GATA2 protein was observed as a focally-aggregated spot around the nucleus of each blastomere. To explore the relationship between GATA2 protein localization and cell cycle progression in mouse 2-cell stage embryos, GFP-labeled GATA2 protein was overexpressed in the blastomere of 2-cell embryos. Overexpression of GFP-labeled GATA2 protein arrested cellular mitosis, focally aggregated GATA2 protein expression was not observed. This mitotic arrest by GATA2 overexpression was not accompanied with the upregulation of a 2-cell stage specific gene, murine endogenous retrovirus-L. These results suggest that GATA2 protein localization changes dynamically depending on cell cycle progression in mouse 2-cell embryos; in particular, focally aggregated localization of GATA2 in the mitotic phase requires appropriate cell cycle progression.
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- 2021
26. The economics of organellar gene loss and endosymbiotic gene transfer
- Author
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Steven L. Kelly
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Proteomics ,0106 biological sciences ,Chloroplasts ,Nuclear gene ,Gene Transfer, Horizontal ,QH301-705.5 ,Arabidopsis ,Computational biology ,Mitochondrion ,Biology ,QH426-470 ,ENCODE ,01 natural sciences ,Genome ,Chloroplast ,03 medical and health sciences ,0302 clinical medicine ,Organelle ,Genetics ,Biology (General) ,Genome, Chloroplast ,Symbiosis ,Gene loss endosymbiotic gene transfer ,Gene ,030304 developmental biology ,Cell Nucleus ,0303 health sciences ,Bacteria ,Host Microbial Interactions ,Endosymbiosis ,Research ,food and beverages ,Mitochondria ,Organellar genome ,Genome, Mitochondrial ,Genome, Plant ,030217 neurology & neurosurgery ,010606 plant biology & botany - Abstract
The endosymbiosis of the bacterial progenitors of mitochondrion and the chloroplast are landmark events in the evolution of life on earth. While both organelles have retained substantial proteomic and biochemical complexity, this complexity is not reflected in the content of their genomes. Instead, the organellar genomes encode fewer than 5% of genes found in living relatives of their ancestors. While many of the 95% of missing organellar genes have been discarded, others have been transferred to the host nuclear genome through a process known as endosymbiotic gene transfer. Here we demonstrate that the difference in the per-cell copy number of the organellar and nuclear genomes presents an energetic incentive to the cell to either delete genes or transfer them to the nuclear genome. We show that, for the majority transferred genes, the energy saved by nuclear-transfer exceeds the costs incurred from importing the encoded protein into the organelle where it can provide its function. Finally, we show that the net energy saved by endosymbiotic gene transfer can constitute an appreciable proportion of total cellular energy budgets, and is therefore sufficient to impart a selectable advantage to the cell. Thus, reduced cellular cost and improved energy efficiency likely played a role in the reductive evolution of mitochondrial and chloroplast genomes and the transfer of organellar genes to the nuclear genome.Significance statementThe endosymbioses of the mitochondrion and the chloroplast were each followed by substantial gene loss and transfer of organellar genes to the nuclear genome. Here we show that the high per-cell copy number of these organellar genomes creates an energetic incentive for the cell to discard genes or transfer them to the nuclear genome. Thus, organellar gene loss and endosymbiotic gene transfer can be intrinsically advantageous to the cell.
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- 2021
27. Reversible protein aggregation as cytoprotective mechanism against heat stress
- Author
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Silvia Salas-Pino, Rafael R. Daga, Paola Gallardo, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), and Junta de Andalucía
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Amyloid ,Cytoplasm ,NUCLEAR ,Protein aggregation ,Biology ,Proteomics ,Fungal Proteins ,Protein Aggregates ,QUALITY-CONTROL ,Stress, Physiological ,Transcription (biology) ,Yeasts ,Gene expression ,Genetics ,SHOCK RESPONSE ,Gene ,Cell Nucleus ,Mechanism (biology) ,GRANULES ,General Medicine ,Mini-Review ,Compartmentalization (psychology) ,INTRINSICALLY DISORDERED PROTEINS ,Stress Granules ,Solubility ,MOLECULAR CHAPERONES ,Cytoprotection ,AMYLOID FORMATION ,Biophysics ,PHASE-TRANSITION ,RNA ,Heat-Shock Response ,Intracellular ,Protein Binding ,MISFOLDED PROTEINS - Abstract
© The Author(s) 2021., Temperature fluctuation is one of the most frequent threats to which organisms are exposed in nature. The activation of gene expression programs that trigger the transcription of heat stress-protective genes is the main cellular response to resist high temperatures. In addition, reversible accumulation and compartmentalization of thermosensitive proteins in high-order molecular assemblies are emerging as critical mechanisms to ensure cellular protection upon heat stress. Here, we summarize representative examples of membrane-less intracellular bodies formed upon heat stress in yeasts and human cells and highlight how protein aggregation can be turned into a cytoprotective mechanism., This work was supported by the Ministerio de Ciencia, Innovación y Universidades (Grant: PGC2018-099849-B-I00 to R.R. Daga) and Junta de Andalucía-FEDER-UPO (grant: UPO-1264663 to S.S.P.).
- Published
- 2021
28. Nuage condensates: accelerators or circuit breakers for sRNA silencing pathways?
- Author
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Geraldine Seydoux and John Paul T. Ouyang
- Subjects
Biomolecular Condensates ,Cell Nucleus ,Small RNA ,Embryo, Nonmammalian ,Biology ,Compartmentalization (psychology) ,Argonaute ,Germ Cell Ribonucleoprotein Granules ,Cell Compartmentation ,Cell biology ,Germ Cells ,Argonaute Proteins ,Perspective ,Transfer RNA ,Animals ,Gene silencing ,RNA Interference ,RNA, Helminth ,RNA, Small Interfering ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Molecular Biology ,Biogenesis ,Function (biology) - Abstract
Nuage are RNA-rich condensates that assemble around the nuclei of developing germ cells. Many proteins required for the biogenesis and function of silencing small RNAs (sRNAs) enrich in nuage, and it is often assumed that nuage is the cellular site where sRNAs are synthesized and encounter target transcripts for silencing. Using C. elegans as a model, we examine the complex multicondensate architecture of nuage and review evidence for compartmentalization of silencing pathways. We consider the possibility that nuage condensates balance the activity of competing sRNA pathways and serve to limit, rather than enhance, sRNA amplification to protect transcripts from dangerous runaway silencing.
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- 2021
29. Genome-wide quantification of transcription factor binding at single-DNA-molecule resolution using methyl-transferase footprinting
- Author
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Judith B. Zaugg, Guido Barzaghi, Mike L. Smith, Arnaud R Krebs, and Rozemarijn Kleinendorst
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Bisulfite sequencing ,DNA Footprinting ,DNA footprinting ,RNA polymerase II ,Computational biology ,Article ,General Biochemistry, Genetics and Molecular Biology ,Mice ,chemistry.chemical_compound ,Transcription (biology) ,Animals ,Humans ,DNA Modification Methylases ,Transcription factor ,Gene Library ,Cell Nucleus ,Genome ,biology ,Chemistry ,High-Throughput Nucleotide Sequencing ,Mouse Embryonic Stem Cells ,DNA ,Sequence Analysis, DNA ,Single Molecule Imaging ,Footprinting ,Nucleosomes ,Bisulfite ,Gene Expression Regulation ,biology.protein ,RNA Polymerase II ,Software ,Transcription Factors - Abstract
Precise control of gene expression requires the coordinated action of multiple factors at cis-regulatory elements. We recently developed single-molecule footprinting to simultaneously resolve the occupancy of multiple proteins including transcription factors, RNA polymerase II and nucleosomes on single DNA molecules genome-wide. The technique combines the use of cytosine methyltransferases to footprint the genome with bisulfite sequencing to resolve transcription factor binding patterns at cis-regulatory elements. DNA footprinting is performed by incubating permeabilized nuclei with recombinant methyltransferases. Upon DNA extraction, whole-genome or targeted bisulfite libraries are prepared and loaded on Illumina sequencers. The protocol can be completed in 4-5 d in any laboratory with access to high-throughput sequencing. Analysis can be performed in 2 d using a dedicated R package and requires access to a high-performance computing system. Our method can be used to analyze how transcription factors cooperate and antagonize to regulate transcription.
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- 2021
30. Nucleo-cytoplasmic RNA distribution responsible for maintaining neuroinflammatory microenvironment
- Author
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Jianhua Peng, Xiancheng Qiu, Yuyan Liao, Chenghao Kuang, Long Gu, Lifang Zhang, Shigang Yin, Xi Kong, Yong Jiang, Ghosh Dipritu, Qianke Tao, Zheng Bao, and Yijing He
- Subjects
Cytoplasm ,Hemorrhage ,Biology ,RNA Transport ,Cell Line ,Mice ,medicine ,Animals ,Molecular Biology ,Gene ,Neuroinflammation ,Cell Nucleus ,Regulation of gene expression ,Microglia ,Gene Expression Profiling ,Computational Biology ,RNA ,Cell Biology ,Subcellular localization ,Antisense RNA ,Cell biology ,Disease Models, Animal ,Gene Ontology ,medicine.anatomical_structure ,Cellular Microenvironment ,Neuroinflammatory Diseases ,RNA, Long Noncoding ,Disease Susceptibility ,Research Paper - Abstract
Subcellular localization of transcripts is highly associated with regulation of gene expression, synthesis of protein, and also the development of the human brain cortex. Although many mechanisms are prevalent in the occurrence of neuroinflammation, the mechanisms based on differences in subcellular localization of transcripts have not been explored. To characterize the dynamic profile of nuclear and cytoplasmic transcripts during the progress of haemorrhage-induced neuroinflammation, we isolated nucleo-cytoplasmic RNA fractions of oxyhaemoglobin (oxy-Hb) treated microglia cells and sequenced both fractions. We discovered that cytoplasmic retained genes were the major forces to maintain the neuroinflammatory microenvironment with 10 hub genes and 40 conserved genes were identified. Moreover, antisense RNA Gm44096 and lincRNA Gm47270, which co-expressed with a crowd of inflammatory genes in the cytoplasm, were discovered as regulatory strategies for sustaining the neuroinflammatory microenvironment. Thus, our study provides a new perspective on understanding haemorrhage-induced neuroinflammation and also reveals a mechanism of lncRNA responsible for maintaining the neuroinflammatory microenvironment.
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- 2021
31. Pellino‐2 in nonimmune cells: novel interaction partners and intracellular localization
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Ove Bruland, Ingvild Aukrust, Ileana M. Cristea, Cecilie Bredrup, and Eyvind Rødahl
- Subjects
Nigericin ,Ubiquitin-Protein Ligases ,Active Transport, Cell Nucleus ,Biophysics ,Biochemistry ,chemistry.chemical_compound ,Structural Biology ,Two-Hybrid System Techniques ,Genetics ,medicine ,Humans ,Protein Interaction Maps ,Molecular Biology ,Cells, Cultured ,Cell Nucleus ,Innate immune system ,biology ,Chemistry ,Kinase ,Nuclear Proteins ,Potassium channel blocker ,Cell Biology ,Fibroblasts ,Cell biology ,Ubiquitin ligase ,HEK293 Cells ,biology.protein ,Tumor necrosis factor alpha ,Nuclear localization sequence ,Intracellular ,Protein Binding ,medicine.drug - Abstract
Pellino-2 is an E3 ubiquitin ligase that mediates intracellular signaling in innate immune pathways. Most studies of endogenous Pellino-2 have been performed in macrophages, but none in nonimmune cells. Using yeast two-hybrid screening and co-immunoprecipitation, we identified six novel interaction partners of Pellino-2, with various localizations: insulin receptor substrate 1, NIMA-related kinase 9, tumor necrosis factor receptor-associated factor 7, cyclin-F, roundabout homolog 1, and disheveled homolog 2. Pellino-2 showed cytoplasmic localization in a wide range of nonimmune cells under physiological potassium concentrations. Treatment with the potassium ionophore nigericin resulted in nuclear localization of Pellino-2, which was reversed by the potassium channel blocker tetraethylammonium. Live-cell imaging revealed intracellular migration of GFP-tagged Pellino-2. In summary, Pellino-2 interacts with proteins at different cellular locations, taking part in dynamic processes that change its intracellular localization influenced by potassium efflux. publishedVersion
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- 2021
32. Salicylic acid inducible nucleocytoplasmic shuttling of NPR1 fusion proteins in human cells
- Author
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Fatemeh Sadeghi, Irfan N Bandey, Xiaoyang Li, Badrinath Roysam, Navin Varadarajan, and Monish Kumar
- Subjects
Cytoplasm ,Recombinant Fusion Proteins ,Gene Expression ,Bioengineering ,Chromosomal translocation ,Applied Microbiology and Biotechnology ,Article ,Transactivation ,Humans ,Arabidopsis thaliana ,Cell Nucleus ,biology ,Arabidopsis Proteins ,Chemistry ,fungi ,HEK 293 cells ,biology.organism_classification ,Fusion protein ,Cell biology ,Protein Transport ,Crosstalk (biology) ,HEK293 Cells ,Synthetic Biology ,Salicylic Acid ,mCherry ,Nuclear localization sequence ,Biotechnology - Abstract
Ligand inducible proteins that enable precise and reversible control of nuclear translocation of passenger proteins have broad applications ranging from genetic studies in mammals to therapeutics that target diseases such as cancer and diabetes. One of the drawbacks of the current translocation systems is that the ligands used to control nuclear localization are either toxic or prone to crosstalk with endogenous protein cascades within live animals. We sought to take advantage of salicylic acid (SA), a small molecule that has been extensively used in humans. In plants, SA functions as a hormone that can mediate immunity and is sensed by the non-expressor of pathogenesis-related (NPR) proteins. Although it is well recognized that nuclear translocation of NPR1 is essential to promoting immunity in plants, the exact subdomain of Arabidopsis thaliana NPR1 (AtNPR1) essential for SA mediated nuclear translocation is controversial. Here, we utilized the fluorescent protein mCherry as the reporter to investigate the ability of SA to induce nuclear translocation of the full-length NPR1 protein or its C-terminal transactivation (TAD) domain using HEK293 cells as a heterologous system. HEK293 cells lack accessory plant proteins including NPR3/NPR4 and are thus ideally suited for studying the impact of SA-induced changes in NPR1. Our results obtained using a stable expression system show that the TAD of AtNPR1 is sufficient to enable the reversible SA mediated nuclear translocation of mCherry. Our studies advance a basic understanding of nuclear translocation mediated by the TAD of AtNPR1 and uncover a biotechnological tool for SA mediated nuclear localization. This article is protected by copyright. All rights reserved.
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- 2021
33. Nuclear S-nitrosylation impacts tissue regeneration in zebrafish
- Author
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Hon-Chiu Eastwood Leung, Gianfranco Matrone, Antrix Jain, Martin A. Denvir, John P. Cooke, Julie Rodor, Cristian Coarfa, Sung Yun Jung, Jong Min Choi, Kimal Rajapakshe, and Andy Baker
- Subjects
Male ,Tail ,Science ,General Physics and Astronomy ,Nerve Tissue Proteins ,Nitric Oxide ,Article ,General Biochemistry, Genetics and Molecular Biology ,Nitric oxide ,chemistry.chemical_compound ,Demethylase activity ,Animals ,Regeneration ,Zebrafish ,Cell Nucleus ,Histone Demethylases ,Multidisciplinary ,Mass spectrometry ,biology ,Regeneration (biology) ,Nitrosylation ,KDM1A ,General Chemistry ,S-Nitrosylation ,Zebrafish Proteins ,biology.organism_classification ,Cell biology ,Nitric oxide synthase ,chemistry ,Regenerative medicine ,Proteome ,Animal Fins ,biology.protein ,Self-renewal ,Female ,Co-Repressor Proteins ,Signal Transduction - Abstract
Despite the importance of nitric oxide signaling in multiple biological processes, its role in tissue regeneration remains largely unexplored. Here, we provide evidence that inducible nitric oxide synthase (iNos) translocates to the nucleus during zebrafish tailfin regeneration and is associated with alterations in the nuclear S-nitrosylated proteome. iNos inhibitors or nitric oxide scavengers reduce protein S-nitrosylation and impair tailfin regeneration. Liquid chromatography/tandem mass spectrometry reveals an increase of up to 11-fold in the number of S-nitrosylated proteins during regeneration. Among these, Kdm1a, a well-known epigenetic modifier, is S-nitrosylated on Cys334. This alters Kdm1a binding to the CoRest complex, thus impairing its H3K4 demethylase activity, which is a response specific to the endothelial compartment. Rescue experiments show S-nitrosylation is essential for tailfin regeneration, and we identify downstream endothelial targets of Kdm1a S-nitrosylation. In this work, we define S-nitrosylation as an essential post-translational modification in tissue regeneration., The role of the post-translational modifications in tissue regeneration is still not clearly understood. Here, the authors show that many nuclear proteins change S-nitrosylation state in the regenerating zebrafish tailfin, highlighting the importance of Kdm1a S-nitrosylation in the repair process.
- Published
- 2021
34. Lamin C is required to establish genome organization after mitosis
- Author
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Karen L. Reddy, Xianrong Wong, Jennifer C. Harr, Ashley J. Melendez-Perez, Victoria E. Hoskins, and Molly Gordon
- Subjects
congenital, hereditary, and neonatal diseases and abnormalities ,animal structures ,Euchromatin ,Nuclear Envelope ,QH301-705.5 ,Mitosis ,Biology ,QH426-470 ,Genome ,Chromosomes ,Mice ,Genetics ,Animals ,Humans ,Telophase ,Biology (General) ,Genomic organization ,Cell Nucleus ,Regulation of gene expression ,Nuclear Lamina ,Lamin Type B ,integumentary system ,Research ,Lamin Type A ,Chromatin ,Lamins ,Cell biology ,embryonic structures ,cardiovascular system ,Nuclear lamina ,Lamin - Abstract
Background The dynamic 3D organization of the genome is central to gene regulation and development. The nuclear lamina influences genome organization through the tethering of lamina-associated domains (LADs) to the nuclear periphery. Evidence suggests that lamins A and C are the predominant lamins involved in the peripheral association of LADs, potentially serving different roles. Results Here, we examine chromosome architecture in mouse cells in which lamin A or lamin C are downregulated. We find that lamin C, and not lamin A, is required for the 3D organization of LADs and overall chromosome organization. Striking differences in localization are present as cells exit mitosis and persist through early G1 and are linked to differential phosphorylation. Whereas lamin A associates with the nascent nuclear envelope (NE) during telophase, lamin C remains in the interior, surrounding globular LAD aggregates enriched on euchromatic regions. Lamin C association with the NE is delayed until several hours into G1 and correlates temporally and spatially with the post-mitotic NE association of LADs. Post-mitotic LAD association with the NE, and global 3D genome organization, is perturbed only in cells depleted of lamin C, and not lamin A. Conclusions Lamin C regulates LAD dynamics during exit from mitosis and is a key regulator of genome organization in mammalian cells. This reveals an unexpectedly central role for lamin C in genome organization, including inter-chromosomal LAD-LAD segregation and LAD scaffolding at the NE, raising intriguing questions about the individual and overlapping roles of lamin A/C in cellular function and disease.
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- 2021
35. STEAP3 promotes cancer cell proliferation by facilitating nuclear trafficking of EGFR to enhance RAC1-ERK-STAT3 signaling in hepatocellular carcinoma
- Author
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Haigang Li, Zhang-Hai He, Mu-Yan Cai, Dan Xie, Jie Luo, Ye-Qing Liu, and Li-li Wang
- Subjects
MAPK/ERK pathway ,Male ,rac1 GTP-Binding Protein ,Cancer Research ,Cell Cycle Proteins ,Tumour biomarkers ,Phosphorylation ,STAT3 ,Extracellular Signal-Regulated MAP Kinases ,Mice, Inbred BALB C ,biology ,Cell Cycle ,Liver Neoplasms ,Middle Aged ,Prognosis ,Phenotype ,ErbB Receptors ,Gene Expression Regulation, Neoplastic ,Protein Transport ,Treatment Outcome ,Hepatocellular carcinoma ,Disease Progression ,Neoplastic Stem Cells ,Female ,Oxidoreductases ,Signal Transduction ,STAT3 Transcription Factor ,Carcinoma, Hepatocellular ,MAP Kinase Signaling System ,Immunology ,Mice, Nude ,RAC1 ,Article ,Cellular and Molecular Neuroscience ,Antigen ,Cell Line, Tumor ,Spheroids, Cellular ,medicine ,Animals ,Humans ,RNA, Messenger ,Cell Proliferation ,Cell Nucleus ,QH573-671 ,business.industry ,Cell Biology ,Oncogenes ,medicine.disease ,digestive system diseases ,Cell culture ,Tumor progression ,biology.protein ,Cancer research ,business ,Cytology - Abstract
STEAP3 (Six-transmembrane epithelial antigen of the prostate 3, TSAP6, dudulin-2) has been reported to be involved in tumor progression in human malignancies. Nevertheless, how it participates in the progression of human cancers, especially HCC, is still unknown. In the present study, we found that STEAP3 was aberrantly overexpressed in the nuclei of HCC cells. In a large cohort of clinical HCC tissues, high expression level of nuclear STEAP3 was positively associated with tumor differentiation and poor prognosis (p
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- 2021
36. A new automated tool to quantify nucleoid distribution within mitochondrial networks
- Author
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Gerald Pfeffer, Matthew A Lines, Rasha Sabouny, Marc Germain, Mathieu Ouellet, Timothy E. Shutt, Hema Saranya Ilamathi, and Justine Desrochers-Goyette
- Subjects
DNA Replication ,Dynamins ,Mitochondrial DNA ,animal structures ,Fission ,Science ,Mutant ,Cellular imaging ,Biology ,Mitochondrion ,DNA, Mitochondrial ,Mitochondrial Dynamics ,Article ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Homeostasis ,Humans ,Distribution (pharmacology) ,Nucleoid ,030304 developmental biology ,Cell Nucleus ,Myosin Type II ,0303 health sciences ,Multidisciplinary ,Myosin Heavy Chains ,fungi ,Mitochondria ,Cell biology ,medicine.anatomical_structure ,embryonic structures ,bacteria ,Medicine ,Mitochondrial fission ,Nucleus ,030217 neurology & neurosurgery - Abstract
Mitochondrial DNA (mtDNA) maintenance is essential to sustain a functionally healthy population of mitochondria within cells. Proper mtDNA replication and distribution within mitochondrial networks are essential to maintain mitochondrial homeostasis. However, the fundamental basis of mtDNA segregation and distribution within mitochondrial networks is still unclear. To address these questions, we developed an algorithm, Mitomate tracker to unravel the global distribution of nucleoids within mitochondria. Using this tool, we decipher the semi-regular spacing of nucleoids across mitochondrial networks. Furthermore, we show that mitochondrial fission actively regulates mtDNA distribution by controlling the distribution of nucleoids within mitochondrial networks. Specifically, we found that primary cells bearing disease-associated mutations in the fission proteins DRP1 and MYH14 show altered nucleoid distribution, and acute enrichment of enlarged nucleoids near the nucleus. Further analysis suggests that the altered nucleoid distribution observed in the fission mutants is the result of both changes in network structure and nucleoid density. Thus, our study provides novel insights into the role of mitochondria fission in nucleoid distribution and the understanding of diseases caused by fission defects.
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- 2021
37. Starvation-induced proteasome assemblies in the nucleus link amino acid supply to apoptosis
- Author
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Benjamin H. Kwok, Nazar Mashtalir, Jean-Yves Masson, Karine Boulay, Oumaima Ahmed, Frédérick A. Mallette, El Bachir Affar, Maxime Uriarte, Claire Viallard, Anaïs Darracq, Przemyslaw Sapieha, Salima Daou, Bruno Larrivée, Djaileb Abdelhadi, Daryl A. Ronato, Marion Voide, Nadine Sen Nkwe, Laura Hulea, Eric Milot, Hugo Wurtele, Nicolas Desjardins-Lecavalier, Mikhail Sergeev, Haithem Barbour, Louis Masclef, Roch Tremblay, Mohammadjavad Paydar, and Clémence Messmer
- Subjects
Proteasome Endopeptidase Complex ,Science ,General Physics and Astronomy ,Apoptosis ,Protein degradation ,Autoantigens ,Article ,General Biochemistry, Genetics and Molecular Biology ,Deubiquitinating enzyme ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Ubiquitin ,Stress, Physiological ,Cell Line, Tumor ,Organelle ,Protein biosynthesis ,Animals ,Humans ,Amino Acids ,Exercise ,030304 developmental biology ,Cell Nucleus ,chemistry.chemical_classification ,0303 health sciences ,Multidisciplinary ,biology ,Catabolism ,Nutrients ,General Chemistry ,Fibroblasts ,Nutrient signalling ,Cell biology ,Amino acid ,DNA-Binding Proteins ,DNA Repair Enzymes ,Eukaryotic Cells ,chemistry ,Proteasome ,Starvation ,Protein Biosynthesis ,Proteolysis ,biology.protein ,030217 neurology & neurosurgery - Abstract
Eukaryotic cells have evolved highly orchestrated protein catabolic machineries responsible for the timely and selective disposal of proteins and organelles, thereby ensuring amino acid recycling. However, how protein degradation is coordinated with amino acid supply and protein synthesis has remained largely elusive. Here we show that the mammalian proteasome undergoes liquid-liquid phase separation in the nucleus upon amino acid deprivation. We termed these proteasome condensates SIPAN (Starvation-Induced Proteasome Assemblies in the Nucleus) and show that these are a common response of mammalian cells to amino acid deprivation. SIPAN undergo fusion events, rapidly exchange proteasome particles with the surrounding milieu and quickly dissolve following amino acid replenishment. We further show that: (i) SIPAN contain K48-conjugated ubiquitin, (ii) proteasome inhibition accelerates SIPAN formation, (iii) deubiquitinase inhibition prevents SIPAN resolution and (iv) RAD23B proteasome shuttling factor is required for SIPAN formation. Finally, SIPAN formation is associated with decreased cell survival and p53-mediated apoptosis, which might contribute to tissue fitness in diverse pathophysiological conditions., Upon starvation, cells coordinate protein disposal to recycle amino acids, although the role of the proteasome has been unclear. Here, the authors show that in the mammalian nucleus, proteasomes form condensates that dissolve following nutrient replenishment.
- Published
- 2021
38. Formation of nuclear condensates by the Mediator complex subunit Med15 in mammalian cells
- Author
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Wenxue Zhao, Miao Li, Jie Yao, Wei-jie Zeng, Xingding Zhang, Jian Chen, and Yuanyuan Shi
- Subjects
Cell imaging ,Physiology ,QH301-705.5 ,Protein subunit ,Mediator ,Plant Science ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Structural Biology ,Transcription (biology) ,Animals ,Biology (General) ,Promoter Regions, Genetic ,Ecology, Evolution, Behavior and Systematics ,Biomolecular Condensates ,Cell Nucleus ,Regulation of gene expression ,Mediator Complex ,Cell growth ,Proteins ,Cell Biology ,Nuclear condensates ,Cell biology ,General Agricultural and Biological Sciences ,Med15 ,Transcription ,Research Article ,Developmental Biology ,Biotechnology - Abstract
Background The Mediator complex is an evolutionarily conserved multi-subunit protein complex that plays major roles in transcriptional activation and is essential for cell growth, proliferation, and differentiation. Recent studies revealed that some Mediator subunits formed nuclear condensates that may facilitate enhancer-promoter interactions and gene activation. The assembly, regulation, and functions of these nuclear condensates remain to be further understood. Results We found that Med15, a subunit in the tail module of the Mediator complex, formed nuclear condensates through a novel mechanism. Nuclear foci of Med15 were detected by both immunostaining of endogenous proteins and live cell imaging. Like Med1 foci and many other biomolecular condensates, Med15 foci were sensitive to 1, 6-Hexanediol and showed rapid recovery during fluorescence recovery after photobleaching. Interestingly, overexpressing DYRK3, a dual-specificity kinase that controls the phase transition of membraneless organelles, appeared to disrupt Med1 foci and Med15 foci. We identified two regions that are required to form Med15 nuclear condensates: the glutamine-rich intrinsically disordered region (IDR) and a short downstream hydrophobic motif. The optodroplet assay revealed that both the IDR and the C-terminal region of Med15 contributed to intracellular phase separation. Conclusions We identified that the Mediator complex subunit Med15 formed nuclear condensates and characterized their features in living cells. Our work suggests that Med15 plays a role in the assembly of transcription coactivator condensates in the nucleus and identifies Med15 regions that contribute to phase separation.
- Published
- 2021
39. Single cell imaging-based chromatin biomarkers for tumor progression
- Author
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Saradha Venkatachalapathy, G. V. Shivashankar, Doorgesh Sharma Jokhun, and Madhavi Andhari
- Subjects
Biopsy ,Science ,Cell ,Biophysics ,Breast Neoplasms ,In Vitro Techniques ,Biology ,Article ,Machine Learning ,chemistry.chemical_compound ,Neoplasms ,Biomarkers, Tumor ,Image Processing, Computer-Assisted ,Tumor Microenvironment ,medicine ,Extracellular ,Humans ,Neoplasm Metastasis ,Cancer ,Probability ,Cell Nucleus ,Tumor microenvironment ,Multidisciplinary ,Computational Biology ,DNA ,Genomics ,Fibroblasts ,medicine.disease ,Phenotype ,Chromatin ,Computational biology and bioinformatics ,medicine.anatomical_structure ,chemistry ,Tumor progression ,Disease Progression ,Cancer research ,Medicine ,Collagen ,Biomarkers ,Protein Binding - Abstract
Tumour progression within the tissue microenvironment is accompanied by complex biomechanical alterations of the extracellular environment. While histopathology images provide robust biochemical markers for tumor progression in clinical settings, a quantitative single cell score using nuclear morphology and chromatin organization integrated with the long range mechanical coupling within the tumor microenvironment is missing. We propose that the spatial chromatin organization in individual nuclei characterises the cell state and their alterations during tumor progression. In this paper, we first built an image analysis pipeline and implemented it to classify nuclei from patient derived breast tissue biopsies of various cancer stages based on their nuclear and chromatin features. Replacing H&E with DNA binding dyes such as Hoescht stained tissue biopsies, we improved the classification accuracy. Using the nuclear morphology and chromatin organization features, we constructed a pseudo-time model to identify the chromatin state changes that occur during tumour progression. This enabled us to build a single-cell mechano-genomic score that characterises the cell state during tumor progression from a normal to a metastatic state. To gain further insights into the alterations in the local tissue microenvironments, we also used the nuclear orientations to identify spatial neighbourhoods that have been posited to drive tumor progression. Collectively, we demonstrate that image-based single cell chromatin and nuclear features are important single cell biomarkers for phenotypic mapping of tumor progression., Scientific Reports, 11 (1), ISSN:2045-2322
- Published
- 2021
40. AKIRIN2 controls the nuclear import of proteasomes in vertebrates
- Author
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Julian Jude, Michael Schutzbier, Robert Kalis, Matthias Hinterndorfer, Tobias Neumann, Elisabeth Roitinger, Gijs A. Versteeg, Irina Grishkovskaya, Melanie de Almeida, Hanna Brunner, Susanne Kandolf, Richard Imre, Kashish Singh, Sumit Deswal, Karl Mechtler, David Haselbach, Johannes Zuber, Thomas Lendl, Alexander Schleiffer, and Milica Vunjak
- Subjects
Male ,Proteasome Endopeptidase Complex ,Active Transport, Cell Nucleus ,Genes, myc ,Mitosis ,Biology ,03 medical and health sciences ,Cell Line, Tumor ,Humans ,Nuclear protein ,Transcription factor ,030304 developmental biology ,Cell Nucleus ,0303 health sciences ,Multidisciplinary ,030302 biochemistry & molecular biology ,Nuclear Proteins ,Cell sorting ,Chromatin ,Transport protein ,Cell biology ,DNA-Binding Proteins ,Proteasome ,Proteolysis ,Female ,CRISPR-Cas Systems ,Nuclear transport ,Protein Binding ,Transcription Factors - Abstract
Protein expression and turnover are controlled through a complex interplay of transcriptional, post-transcriptional and post-translational mechanisms to enable spatial and temporal regulation of cellular processes. To systematically elucidate such gene regulatory networks, we developed a CRISPR screening assay based on time-controlled Cas9 mutagenesis, intracellular immunostaining and fluorescence-activated cell sorting that enables the identification of regulatory factors independent of their effects on cellular fitness. We pioneered this approach by systematically probing the regulation of the transcription factor MYC, a master regulator of cell growth1–3. Our screens uncover a highly conserved protein, AKIRIN2, that is essentially required for nuclear protein degradation. We found that AKIRIN2 forms homodimers that directly bind to fully assembled 20S proteasomes to mediate their nuclear import. During mitosis, proteasomes are excluded from condensing chromatin and re-imported into newly formed daughter nuclei in a highly dynamic, AKIRIN2-dependent process. Cells undergoing mitosis in the absence of AKIRIN2 become devoid of nuclear proteasomes, rapidly causing accumulation of MYC and other nuclear proteins. Collectively, our study reveals a dedicated pathway controlling the nuclear import of proteasomes in vertebrates and establishes a scalable approach to decipher regulators in essential cellular processes. Using time-controlled CRISPR screens, the authors identify AKIRIN2 as a factor involved in the nuclear import of the proteasome.
- Published
- 2021
41. Generating a new mouse model for nuclear PTEN deficiency by a single K13R mutation
- Author
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Miho Iijima, Takashi Kato, Atsushi Igarashi, and Hiromi Sesaki
- Subjects
Cell Nucleus ,Neurons ,Mutation ,biology ,Phosphatase ,PTEN Phosphohydrolase ,Cell Biology ,medicine.disease_cause ,Article ,Cell biology ,Disease Models, Animal ,Mice ,medicine.anatomical_structure ,Genetics ,biology.protein ,medicine ,Animals ,Tensin ,Phosphorylation ,PTEN ,Nucleus ,Intracellular ,Nuclear localization sequence - Abstract
Many human diseases, including cancer and neurological abnormalities, are linked to deficiencies of phosphatase and tensin homolog deleted on chromosome ten (PTEN), a dual phosphatase that dephosphorylates both lipids and proteins. PTEN functions in multiple intracellular locations, including the plasma membrane and nucleus. Therefore, a critical challenge to understand the pathogenesis of PTEN-associated diseases is to determine the specific role of PTEN at different locations. Toward this goal, the current study generated a mouse line in which lysine 13, which is critical for the nuclear localization of PTEN, is changed to arginine in the lipid-binding domain using the CRISPR-Ca9 gene-editing system. We found that PTENK13R mice show a strong decrease in the localization of PTEN in the nucleus without affecting the protein stability, phosphatase activity, and phosphorylation in the C-terminal tail region. PTENK13R mice are viable but produce smaller neurons and develop microcephaly. These data demonstrate that PTENK13R mice provide a useful animal model to study the role of PTEN in the nucleus in vivo.
- Published
- 2021
42. U1 small nuclear ribonucleoprotein is essential for early larval development in silkworm, Bombyx mori
- Author
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Zulian Liu, Shengxiang Zhang, Dehong Yang, Yong Zhang, Yongping Huang, Xinran Zhang, Xiaoqian Zhang, and Xu Yang
- Subjects
genetic processes ,fungi ,Wild type ,Biology ,Bombyx ,biology.organism_classification ,environment and public health ,General Biochemistry, Genetics and Molecular Biology ,Ribonucleoprotein, U1 Small Nuclear ,Cell biology ,chemistry.chemical_compound ,Cell nucleus ,medicine.anatomical_structure ,chemistry ,Cytoplasm ,Bombyx mori ,Insect Science ,medicine ,Animals ,snRNP ,Agronomy and Crop Science ,Ecology, Evolution, Behavior and Systematics ,Ecdysone ,Small nuclear ribonucleoprotein ,Ribonucleoprotein - Abstract
U1 small nuclear ribonucleoproteins (U1 snRNP) associates with 5' splice sites in the form of ribonucleoprotein particles, is highly conserved among species. The physiological functions of U1 snRNP in a lepidopteran model insect Bombyx mori is mostly unknown. Here we showed that U1 snRNP plays an important role in the development of silkworm. Knockout of U1 snRNP in silkworm showed either delayed or stationary 1st-instar larva development compared with the wild type group. U1 snRNP deletion mutants exhibited abnormal cellular phenotypes with enlarged cell nucleus, scanty cytoplasm, and enlarged nuclei. RNA-seq analysis revealed that genes involved in metabolic pathway, biosynthesis of secondary metabolites and steroid hormone biosynthesis were significantly affected by U1 snRNP depletion. Taken together, our study suggests that U1 snRNP homeostasis plays an important role in silkworm development. This article is protected by copyright. All rights reserved.
- Published
- 2021
43. Multiplexed Single-Cell Plasmonic Immunoassay of Intracellular Signaling Proteins Enables Non-Destructive Monitoring of Cell Fate
- Author
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Hui He, Yanrong Wen, Quan Zhao, Jialing Zhao, and Zhen Liu
- Subjects
Cell Nucleus ,Immunoassay ,Cytoplasm ,Cell signaling ,biology ,Chemistry ,Cell ,Intracellular Signaling Peptides and Proteins ,Cell fate determination ,Analytical Chemistry ,Cell biology ,medicine.anatomical_structure ,Apoptosis ,medicine ,biology.protein ,Epidermal growth factor receptor ,Signal transduction ,Nucleus ,Signal Transduction - Abstract
It is of significant importance in cancer biology to identify signaling pathways that play key roles in cell fate determination. Dissecting cellular signaling pathways requires the measurement of a large number of signaling proteins. However, tools for simultaneously monitoring multiple signaling pathway components in single living cells remain limited at present. Herein, we describe an approach, termed multiplexed single-cell plasmonic immunosandwich assay (mxscPISA), for simultaneous detection of multiple signaling proteins in individual living cells. This approach enabled simultaneous non-destructive monitoring of multiple (up to five, currently the highest multiplexing capacity in living cells) cytoplasmic and nucleus signaling proteins in individual cells with ultrahigh detection sensitivity. As a proof of principle, the epidermal growth factor receptor (EGFR) pathway, which plays a central role in cell fate determination, was investigated using this approach in this study. We found that there were differential attenuation rate of pro-survival and accumulation rate of pro-death signaling protein of the EGFR pathway in response to EGFR inactivation. These findings implicate that, after EGFR inactivation, a transient imbalance between survival and apoptotic signaling outputs contributed to the final cell fate of death. The mxscPISA approach can be a promising tool to reveal a signaling dynamic pattern at the single-cell level and to identify key components of signaling pathways that contribute to the final cell fate using only a limited number of cells.
- Published
- 2021
44. The necessity of nucleophagic modality
- Author
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Dalibor Mijaljica and Daniel J. Klionsky
- Subjects
Cell Nucleus ,Nucleophagy ,Nuclear Envelope ,Microautophagy ,Cellular homeostasis ,Saccharomyces cerevisiae ,Cell Biology ,Biology ,DNA, Ribosomal ,Cell biology ,medicine.anatomical_structure ,Autophagy ,medicine ,Late nucleophagy ,Inner membrane ,Nuclear lamina ,Nuclear pore ,Nuclear membrane ,Commentary and Views ,Molecular Biology - Abstract
Nucleophagy, the selective subtype of autophagy that predominantly targets only a selected and (nonessential) portion of the nucleus, and rarely the nucleus in its entirety, for degradation, reinforces the paradigm that nucleophagy recycling is a meticulous and highly delicate process guarded by fail-safe mechanisms. Our goal in this commentary is to encourage autophagy researchers and other scientists to explore nucleophagy blind spots and gain advanced insights into the diverse roles of this process and its selective modality as they pertain to intranuclear quality control and cellular homeostasis. Identifying and deciphering nucleophagic signaling, regulation, molecular mechanism(s) and its mediators, cargo composition and nuclear membrane dynamics under numerous physiological and/or pathological settings will provide important advances in our understanding of this critical type of organelle-selective autophagy. Abbreviations: INM, inner nuclear membrane; LN, late nucleophagy; mRNA, messenger RNA; NE, nuclear envelope; NL, nuclear lamina; NPC(s), nuclear pore complex(es); NVJ(s), nucleus-vacuole junction(s); ONM, outer nuclear membrane; PMN, piecemeal microautophagy of the nucleus; PND, programmed nuclear death; PNuD, programmed nuclear destruction; rDNA/rRNA, ribosomal DNA/RNA
- Published
- 2021
45. Components and Mechanisms of Nuclear Mechanotransduction
- Author
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Philipp Niethammer
- Subjects
Cell Nucleus ,Laminopathy ,Cell Biology ,Biology ,medicine.disease ,Mechanotransduction, Cellular ,Genome ,Article ,Chromatin ,Cell nucleus ,medicine.anatomical_structure ,Gene expression ,medicine ,Biophysics ,Mechanotransduction ,Nuclear membrane ,Nucleus ,Developmental Biology - Abstract
The cell nucleus is best known as the container of the genome. Its envelope provides a barrier for passive macromolecule diffusion, which enhances the control of gene expression. As its largest and stiffest organelle, the nucleus also defines the minimal space requirements of a cell. Internal or external pressures that deform a cell to its physical limits cause a corresponding nuclear deformation. Evidence is consolidating that the nucleus, in addition to its genetic functions, serves as a physical sensing device for critical cell body deformation. Nuclear mechanotransduction allows cells to adapt their acute behaviors, mechanical stability, paracrine signaling, and fate to their physical surroundings. This review summarizes the basic chemical and mechanical properties of nuclear components, and how these properties are thought to be utilized for mechanosensing.
- Published
- 2021
46. Arabidopsis thaliana PrimPol is a primase and lesion bypass DNA polymerase with the biochemical characteristics to cope with DNA damage in the nucleus, mitochondria, and chloroplast
- Author
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Alfredo Cruz-Ramírez, Paola L. García-Medel, Alma Fuentes-Pascacio, Luis G. Brieba, Antolín Peralta-Castro, Noe Baruch-Torres, and José A. Pedroza-García
- Subjects
DNA Replication ,DNA Repair ,DNA polymerase ,DNA damage ,Science ,Arabidopsis ,DNA, Single-Stranded ,DNA Primase ,DNA-Directed DNA Polymerase ,Article ,DNA sequencing ,chemistry.chemical_compound ,Polymerase ,Cell Nucleus ,Multidisciplinary ,biology ,DNA synthesis ,Arabidopsis Proteins ,food and beverages ,DNA ,Multifunctional Enzymes ,Enzymes ,Mitochondria ,Cell biology ,chemistry ,biology.protein ,Medicine ,Primase ,DNA Damage ,Mitochondrial DNA replication - Abstract
PrimPol is a novel Primase–Polymerase that synthesizes RNA and DNA primers de novo and extents from these primers as a DNA polymerase. Animal PrimPol is involved in nuclear and mitochondrial DNA replication by virtue of its translesion DNA synthesis (TLS) and repriming activities. Here we report that the plant model Arabidopsis thaliana encodes a functional PrimPol (AtPrimPol). AtPrimPol is a low fidelity and a TLS polymerase capable to bypass DNA lesions, like thymine glycol and abasic sites, by incorporating directly across these lesions or by skipping them. AtPrimPol is also an efficient primase that preferentially recognizes the single-stranded 3′-GTCG-5′ DNA sequence, where the 3′-G is cryptic. AtPrimPol is the first DNA polymerase that localizes in three cellular compartments: nucleus, mitochondria, and chloroplast. In vitro, AtPrimPol synthesizes primers that are extended by the plant organellar DNA polymerases and this reaction is regulated by organellar single-stranded binding proteins. Given the constant exposure of plants to endogenous and exogenous DNA-damaging agents and the enzymatic capabilities of lesion bypass and re-priming of AtPrimPol, we postulate a predominant role of this enzyme in avoiding replication fork collapse in all three plant genomes, both as a primase and as a TLS polymerase.
- Published
- 2021
47. RIPK1 regulates starvation resistance by modulating aspartate catabolism
- Author
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Yedan Zhong, Xiaofen Wu, Xinyu Mei, Ying Li, Daichao Xu, Zhangdan Xie, Yuan Guo, Nan Liu, and Zheng-Jiang Zhu
- Subjects
Programmed cell death ,Aspartate homeostasis ,endocrine system diseases ,Cell Survival ,Sp1 Transcription Factor ,Science ,Citric Acid Cycle ,General Physics and Astronomy ,Biology ,AMP-Activated Protein Kinases ,General Biochemistry, Genetics and Molecular Biology ,Article ,Cell Line ,RIPK1 ,Mice ,Adenosine Triphosphate ,medicine ,Autophagy ,Animals ,Autophagy-Related Protein-1 Homolog ,Humans ,Metabolomics ,Starvation ,Cell Nucleus ,Aspartic Acid ,Multidisciplinary ,Catabolism ,Autophagosomes ,AMPK ,nutritional and metabolic diseases ,General Chemistry ,ULK1 ,Cell biology ,Animals, Newborn ,Receptor-Interacting Protein Serine-Threonine Kinases ,medicine.symptom ,hormones, hormone substitutes, and hormone antagonists ,Signal Transduction - Abstract
RIPK1 is a crucial regulator of cell death and survival. Ripk1 deficiency promotes mouse survival in the prenatal period while inhibits survival in the early postnatal period without a clear mechanism. Metabolism regulation and autophagy are critical to neonatal survival from severe starvation at birth. However, the mechanism by which RIPK1 regulates starvation resistance and survival remains unclear. Here, we address this question by discovering the metabolic regulatory role of RIPK1. First, metabolomics analysis reveals that Ripk1 deficiency specifically increases aspartate levels in both mouse neonates and mammalian cells under starvation conditions. Increased aspartate in Ripk1−/− cells enhances the TCA flux and ATP production. The energy imbalance causes defective autophagy induction by inhibiting the AMPK/ULK1 pathway. Transcriptional analyses demonstrate that Ripk1−/− deficiency downregulates gene expression in aspartate catabolism by inactivating SP1. To summarize, this study reveals that RIPK1 serves as a metabolic regulator responsible for starvation resistance., RIPK1 is critical for normal development and cell death. Here, the authors identify a metabolic role for RIPK1 in aspartate homeostasis, as increased aspartate levels in RIPK1-deficient cells inhibits starvation-induced autophagy by ULK1.
- Published
- 2021
48. Corticosteroid receptors as a model for the Hsp90•immunophilin-based transport machinery
- Author
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Gisela Ileana Mazaira, Graciela Piwien Pilipuk, and Mario D. Galigniana
- Subjects
Cell Nucleus ,Receptors, Steroid ,biology ,Chemistry ,Endocrinology, Diabetes and Metabolism ,medicine.medical_treatment ,Dynein ,Hsp90 ,Steroid ,Cell biology ,Receptors, Glucocorticoid ,Endocrinology ,medicine.anatomical_structure ,Cytoplasm ,Heat shock protein ,medicine ,biology.protein ,Humans ,HSP90 Heat-Shock Proteins ,Immunophilins ,Receptor ,Nucleus ,Cellular compartment ,Molecular Chaperones - Abstract
Steroid receptors form soluble heterocomplexes with the 90-kDa heat-shock protein (Hsp90) and other chaperones and co-chaperones. The assembly and composition of the oligomer is influenced by the presence and nature of the bound steroid. Although these receptors shuttle dynamically in and out of the nucleus, their primary localization in the absence of steroid can be mainly cytoplasmic, mainly nuclear, or partitioned into both cellular compartments. Upon steroid binding, receptors become localized to the nucleus via the transportosome, a retrotransport molecular machinery that comprises Hsp90, a high-molecular-weight immunophilin, and dynein motors. This molecular machinery, first evidenced in steroid receptors, can also be used by other soluble proteins. In this review, we dissect the complete model of this transport machinery system.
- Published
- 2021
49. Rett syndrome linked to defects in forming the MeCP2/Rbfox/LASR complex in mouse models
- Author
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Yuhan Zhang, Hong Zhu, Wei-Kang Wang, Jingyi Hui, Ji-Song Guan, Yan Jiang, Jinsong Li, Xing Fu, Ping Wu, Catherine C. L. Wong, Lin Zhang, Ying Huang, Shen-Fei Wang, and Jinbiao Ma
- Subjects
congenital, hereditary, and neonatal diseases and abnormalities ,Methyl-CpG-Binding Protein 2 ,Protein subunit ,Science ,General Physics and Astronomy ,RNA-binding protein ,Rett syndrome ,Nerve Tissue Proteins ,RNA-binding proteins ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Heterogeneous-Nuclear Ribonucleoproteins ,Article ,MECP2 ,Mice ,Protein Domains ,mental disorders ,medicine ,Rett Syndrome ,Animals ,Humans ,Gene ,Cell Nucleus ,Multidisciplinary ,Alternative splicing ,General Chemistry ,Exons ,medicine.disease ,Cell biology ,nervous system diseases ,Alternative Splicing ,Disease Models, Animal ,Protein Subunits ,HEK293 Cells ,Multiprotein Complexes ,RNA splicing ,Synaptic plasticity ,Mutation ,Female ,RNA Splicing Factors - Abstract
Rett syndrome (RTT) is a severe neurological disorder and a leading cause of intellectual disability in young females. RTT is mainly caused by mutations found in the X-linked gene encoding methyl-CpG binding protein 2 (MeCP2). Despite extensive studies, the molecular mechanism underlying RTT pathogenesis is still poorly understood. Here, we report MeCP2 as a key subunit of a higher-order multiunit protein complex Rbfox/LASR. Defective MeCP2 in RTT mouse models disrupts the assembly of the MeCP2/Rbfox/LASR complex, leading to reduced binding of Rbfox proteins to target pre-mRNAs and aberrant splicing of Nrxns and Nlgn1 critical for synaptic plasticity. We further show that MeCP2 disease mutants display defective condensate properties and fail to promote phase-separated condensates with Rbfox proteins in vitro and in cultured cells. These data link an impaired function of MeCP2 with disease mutation in splicing control to its defective properties in mediating the higher-order assembly of the MeCP2/Rbfox/LASR complex., MeCP2 mutations can cause Rett syndrome, a severe childhood neurological disorder. Here the authors show that MeCP2 mediates the higher-order assembly of a large splicing complex Rbfox/LASR, which is disrupted in the mouse models of Rett syndrome.
- Published
- 2021
50. Lamin post-translational modifications: emerging toggles of nuclear organization and function
- Author
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Laura A. Murray-Nerger and Ileana M. Cristea
- Subjects
congenital, hereditary, and neonatal diseases and abnormalities ,animal structures ,Biology ,Biochemistry ,Article ,03 medical and health sciences ,0302 clinical medicine ,Ubiquitin ,medicine ,Humans ,Muscular dystrophy ,Intermediate filament ,Molecular Biology ,030304 developmental biology ,Cell Nucleus ,0303 health sciences ,Progeria ,integumentary system ,medicine.disease ,Chromatin ,Lamins ,Cell biology ,embryonic structures ,biology.protein ,Nuclear lamina ,Protein Processing, Post-Translational ,030217 neurology & neurosurgery ,Lamin ,Function (biology) - Abstract
Nuclear lamins are ancient type V intermediate filaments with diverse functions that include maintaining nuclear shape, mechanosignaling, tethering and stabilizing chromatin, regulating gene expression, and contributing to cell cycle progression. Despite these numerous roles, an outstanding question has been how lamins are regulated. Accumulating work indicates that a range of lamin post-translational modifications (PTMs) control their functions both in homeostatic cells and in disease states such as progeria, muscular dystrophy, and viral infection. Here, we review the current knowledge of the diverse types of PTMs that regulate lamins in a site-specific manner. We highlight methods that can be used to characterize lamina PTMs whose functions are currently unknown and provide a perspective on the future of the lamin PTM field.
- Published
- 2021
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