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1.
Nat Commun ; 12(1): 4170, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234130

RESUMO

Genome organization is driven by forces affecting transcriptional state, but the relationship between transcription and genome architecture remains unclear. Here, we identified the Drosophila transcription factor Motif 1 Binding Protein (M1BP) in physical association with the gypsy chromatin insulator core complex, including the universal insulator protein CP190. M1BP is required for enhancer-blocking and barrier activities of the gypsy insulator as well as its proper nuclear localization. Genome-wide, M1BP specifically colocalizes with CP190 at Motif 1-containing promoters, which are enriched at topologically associating domain (TAD) borders. M1BP facilitates CP190 chromatin binding at many shared sites and vice versa. Both factors promote Motif 1-dependent gene expression and transcription near TAD borders genome-wide. Finally, loss of M1BP reduces chromatin accessibility and increases both inter- and intra-TAD local genome compaction. Our results reveal physical and functional interaction between CP190 and M1BP to activate transcription at TAD borders and mediate chromatin insulator-dependent genome organization.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Animais , Animais Geneticamente Modificados , Linhagem Celular , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Proteínas de Drosophila/genética , Técnicas de Silenciamento de Genes , Genoma de Inseto , Elementos Isolantes/genética , Masculino , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , RNA-Seq , Proteínas Repressoras/genética , Fatores de Transcrição/genética
2.
Nat Commun ; 12(1): 4194, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234144

RESUMO

Photomorphogenesis, light-mediated development, is an essential feature of all terrestrial plants. While chloroplast development and brassinosteroid (BR) signaling are known players in photomorphogenesis, proteins that regulate both pathways have yet to be identified. Here we report that DE-ETIOLATION IN THE DARK AND YELLOWING IN THE LIGHT (DAY), a membrane protein containing DnaJ-like domain, plays a dual-role in photomorphogenesis by stabilizing the BR receptor, BRI1, as well as a key enzyme in chlorophyll biosynthesis, POR. DAY localizes to both the endomembrane and chloroplasts via its first transmembrane domain and chloroplast transit peptide, respectively, and interacts with BRI1 and POR in their respective subcellular compartments. Using genetic analysis, we show that DAY acts independently on BR signaling and chlorophyll biogenesis. Collectively, this work uncovers DAY as a factor that simultaneously regulates BR signaling and chloroplast development, revealing a key regulator of photomorphogenesis that acts across cell compartments.


Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Membrana/metabolismo , Morfogênese/fisiologia , Proteínas Quinases/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Brassinosteroides/metabolismo , Clorofila/biossíntese , Cloroplastos/metabolismo , Estiolamento/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/isolamento & purificação , Luz , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Morfogênese/efeitos da radiação , Mutação , Plantas Geneticamente Modificadas , Proteínas Quinases/genética , RNA-Seq , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Plântula/crescimento & desenvolvimento , Transdução de Sinais/fisiologia
3.
Eur J Med Res ; 26(1): 75, 2021 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-34256840

RESUMO

BACKGROUND: The aim of this study was to evaluate the expression of four up/down-regulated inflammatory miRNAs and their mRNA targets in the serum samples of COVID-19 patients with different grades. Also, we investigated the relative expression of these miRNAs and mRNAs during hospitalization. METHODS: In this cross-sectional study, 5 mL of blood sample were taken from COVID-19 patients with different grades and during hospitalization from several health centers of Yazd, Tehran, and Zahedan province of Iran from December 20, 2020 to March 2, 2021. The relative expression of miRNAs and mRNAs was evaluated by q-PCR. RESULTS: We found that the relative expression of hsa-miR-31-3p, hsa-miR-29a-3p, and hsa-miR-126-3p was significantly decreased and the relative expression of their mRNA targets (ZMYM5, COL5A3, and CAMSAP1) was significantly increased with the increase of disease grade. Conversely, the relative expression of hsa-miR-17-3p was significantly increased and its mRNA target (DICER1) was significantly decreased with the increase of disease grade. This pattern was exactly seen during hospitalization of COVID-19 patients who did not respond to treatment. In COVID-19 patients who responded to treatment, the expression of selected miRNAs and their mRNA targets returned to the normal level. A negative significant correlation was seen between (1) the expression of hsa-miR-31-3p and ZMYM5, (2) hsa-miR-29a-3p and COL5A3, (3) hsa-miR-126-3p and CAMSAP1, and (4) hsa-miR-17-3p and DICER1 in COVID-19 patients with any grade (P < 0.05) and during hospitalization. CONCLUSIONS: In this study, we gained a more accurate understanding of the expression of up/down-regulated inflammatory miRNAs in the blood of COVID-19 patients. The obtained data may help us in the diagnosis and prognosis of COVID-19. TRIAL REGISTRATION: The ethics committee of Zahedan University of Medical Sciences, Zahedan, Iran. (Ethical Code: IR.ZAUMS.REC.1399.316) was registered for this project.


Assuntos
COVID-19/genética , Perfilação da Expressão Gênica , MicroRNAs/genética , RNA Mensageiro/genética , COVID-19/sangue , COVID-19/virologia , Proteínas de Transporte/genética , Colágeno/genética , Estudos Transversais , RNA Helicases DEAD-box/genética , Hospitalização/estatística & dados numéricos , Humanos , Irã (Geográfico) , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/genética , Ribonuclease III/genética , SARS-CoV-2/fisiologia , Índice de Gravidade de Doença
4.
Anticancer Res ; 41(7): 3349-3361, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34230131

RESUMO

BACKGROUND/AIM: The present study investigated the oncogenic functions of TACC3 in the progression of gastric cancer (GC). MATERIALS AND METHODS: We analysed TACC3 in relation to cell growth, invasion capability, expression of epithelial-mesenchymal transition (EMT)-related markers, and ERK/Akt/cyclin D1 signaling factors. The correlation between the immunohistochemically confirmed expression of TACC3 and clinical factors was also analyzed. RESULTS: The increased proliferation and invasion of TACC3-over-expressing GC cells was accompanied by altered regulation of EMT-associated markers and activation of ERK/Akt/cyclin D1 signaling. Immunohistochemical analysis of TACC3 in human GC tissues revealed that its expression is correlated with aggressive characteristics and poor prognosis of intestinal-type GC. CONCLUSION: TACC3 contributes to gastric tumorigenesis by promoting EMT via the ERK/Akt/cyclin D1 signaling pathway. The correlation between TACC3 expression and multiple clinicopathological variables implies that its effective therapeutic targeting in GC will depend on the tumor subtype.


Assuntos
Carcinogênese/genética , Ciclina D1/genética , Transição Epitelial-Mesenquimal/genética , Sistema de Sinalização das MAP Quinases/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Proto-Oncogênicas c-akt/genética , Neoplasias Gástricas/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Transdução de Sinais/genética , Estômago/patologia , Neoplasias Gástricas/patologia
5.
Adv Exp Med Biol ; 1208: 117-130, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34260025

RESUMO

Thanks to the advances in optical microscope technology and our knowledge of autophagic biomarkers, single-molecule events of autophagy are now accessible to human eyes. Different proteins are involved hierarchically in the biogenesis and maturation of autophagosomes. Detecting these autophagy-related proteins either by immunostaining or fluorescent protein labelling makes the dynamic autophagic process visible. However, low antibody specificity and weak endogenous expression of autophagy-related proteins in certain tissues limit the applicability of immunostaining in autophagy detection. To cope with this, live-cell imaging combined with various fluorescent probes has been developed and employed in monitoring autophagy. As the most widely used autophagic biomarker, LC3 can be used to visualize autophagosomes, and fluorescent probes targeting LC3, i.e., RFP/mCherry-GFP-LC3, and GFP-LC3-RFP-LC3ΔG, can examine autophagy flux dynamically and quantitatively. In addition, the application of novel fluorophores such as Keima helps to detect the temporal and spatial characteristics of autophagy. Furthermore, selective autophagy can be clarified by labelling corresponding substrates and autophagosomes or lysosomes simultaneously. With the help of two-photon microscopy, the process of autophagy in live animals has been uncovered. Here, we summarize the methods for observing autophagy by optical microscopy and the selection of fluorescent markers.


Assuntos
Autofagia , Proteínas Associadas aos Microtúbulos , Animais , Autofagossomos , Proteínas de Fluorescência Verde , Humanos , Lisossomos , Proteínas Associadas aos Microtúbulos/genética
6.
Nutrients ; 13(6)2021 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-34207686

RESUMO

Here, we performed a genome-wide search for methylation sites that contribute to the risk of obesity. We integrated methylation quantitative trait locus (mQTL) data with BMI GWAS information through a SNP-based multiomics approach to identify genomic regions where mQTLs for a methylation site co-localize with obesity risk SNPs. We then tested whether the identified site contributed to BMI through Mendelian randomization. We identified multiple methylation sites causally contributing to the risk of obesity. We validated these findings through a replication stage. By integrating expression quantitative trait locus (eQTL) data, we noted that lower methylation at cg21178254 site upstream of CCNL1 contributes to obesity by increasing the expression of this gene. Higher methylation at cg02814054 increases the risk of obesity by lowering the expression of MAST3, whereas lower methylation at cg06028605 contributes to obesity by decreasing the expression of SLC5A11. Finally, we noted that rare variants within 2p23.3 impact obesity by making the cg01884057 site more susceptible to methylation, which consequently lowers the expression of POMC, ADCY3 and DNAJC27. In this study, we identify methylation sites associated with the risk of obesity and reveal the mechanism whereby a number of these sites exert their effects. This study provides a framework to perform an omics-wide association study for a phenotype and to understand the mechanism whereby a rare variant causes a disease.


Assuntos
Metilação de DNA/genética , Epigenoma/genética , Predisposição Genética para Doença/genética , Obesidade/genética , Adenilil Ciclases/genética , Índice de Massa Corporal , Ciclinas/genética , Epigenômica/métodos , Estudo de Associação Genômica Ampla , Proteínas de Choque Térmico HSP40/genética , Humanos , Análise da Randomização Mendeliana , Proteínas Associadas aos Microtúbulos/genética , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Pró-Opiomelanocortina/genética , Proteínas Serina-Treonina Quinases/genética , Locos de Características Quantitativas , Fatores de Risco , Proteínas de Transporte de Sódio-Glucose/genética , Proteínas rab de Ligação ao GTP/genética
7.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34072837

RESUMO

The chromatin reader protein Spindlin1 plays an important role in epigenetic regulation, through which it has been linked to several types of malignant tumors. In the current work, we report on the development of novel analogs of the previously published lead inhibitor A366. In an effort to improve the activity and explore the structure-activity relationship (SAR), a series of 21 derivatives was synthesized, tested in vitro, and investigated by means of molecular modeling tools. Docking studies and molecular dynamics (MD) simulations were performed to analyze and rationalize the structural differences responsible for the Spindlin1 activity. The analysis of MD simulations shed light on the important interactions. Our study highlighted the main structural features that are required for Spindlin1 inhibitory activity, which include a positively charged pyrrolidine moiety embedded into the aromatic cage connected via a propyloxy linker to the 2-aminoindole core. Of the latter, the amidine group anchor the compounds into the pocket through salt bridge interactions with Asp184. Different protocols were tested to identify a fast in silico method that could help to discriminate between active and inactive compounds within the A366 series. Rescoring the docking poses with MM-GBSA calculations was successful in this regard. Because A366 is known to be a G9a inhibitor, the most active developed Spindlin1 inhibitors were also tested over G9a and GLP to verify the selectivity profile of the A366 analogs. This resulted in the discovery of diverse selective compounds, among which 1s and 1t showed Spindlin1 activity in the nanomolar range and selectivity over G9a and GLP. Finally, future design hypotheses were suggested based on our findings.


Assuntos
Fenômenos Biofísicos , Proteínas de Ciclo Celular/química , Epigênese Genética , Proteínas Associadas aos Microtúbulos/química , Fosfoproteínas/química , Conformação Proteica , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/ultraestrutura , Entropia , Humanos , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/ultraestrutura , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Fosfoproteínas/ultraestrutura , Ligação Proteica , Relação Estrutura-Atividade
8.
J Cell Sci ; 134(11)2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34096607

RESUMO

Axons and dendrites are distinguished by microtubule polarity. In Drosophila, dendrites are dominated by minus-end-out microtubules, whereas axons contain plus-end-out microtubules. Local nucleation in dendrites generates microtubules in both orientations. To understand why dendritic nucleation does not disrupt polarity, we used live imaging to analyze the fate of microtubules generated at branch points. We found that they had different rates of success exiting the branch based on orientation: correctly oriented minus-end-out microtubules succeeded in leaving about twice as often as incorrectly oriented microtubules. Increased success relied on other microtubules in a parallel orientation. From a candidate screen, we identified Trim9 and kinesin-5 (Klp61F) as machinery that promoted growth of new microtubules. In S2 cells, Eb1 recruited Trim9 to microtubules. Klp61F promoted microtubule growth in vitro and in vivo, and could recruit Trim9 in S2 cells. In summary, the data argue that Trim9 and kinesin-5 act together at microtubule plus ends to help polymerizing microtubules parallel to pre-existing ones resist catastrophe.


Assuntos
Polaridade Celular , Dendritos , Cinesina/genética , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos , Polimerização
9.
Nat Commun ; 12(1): 3687, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140499

RESUMO

Microtubules are severed by katanin at distinct cellular locations to facilitate reorientation or amplification of dynamic microtubule arrays, but katanin targeting mechanisms are poorly understood. Here we show that a centrosomal microtubule-anchoring complex is used to recruit katanin in acentrosomal plant cells. The conserved protein complex of Msd1 (also known as SSX2IP) and Wdr8 is localized at microtubule nucleation sites along the microtubule lattice in interphase Arabidopsis cells. Katanin is recruited to these sites for efficient release of newly formed daughter microtubules. Our cell biological and genetic studies demonstrate that Msd1-Wdr8 acts as a specific katanin recruitment factor to cortical nucleation sites (but not to microtubule crossover sites) and stabilizes the association of daughter microtubule minus ends to their nucleation sites until they become severed by katanin. Molecular coupling of sequential anchoring and severing events by the evolutionarily conserved complex renders microtubule release under tight control of katanin activity.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Katanina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Células Vegetais/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Processamento de Imagem Assistida por Computador , Interfase , Katanina/genética , Espectrometria de Massas , Proteínas Associadas aos Microtúbulos/genética , Mutação , Fenótipo , Plantas Geneticamente Modificadas , Imagem com Lapso de Tempo , Tubulina (Proteína)/metabolismo
10.
Int J Mol Sci ; 22(9)2021 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-34063622

RESUMO

Ribonucleic acid export 1 (Rae1) is an important nucleoporin that participates in mRNA export during the interphase of higher eukaryotes and regulates the mitotic cell cycle. In this study, small RNA interference technology was used to knockdown Rae1, and immunofluorescence, immunoblotting, and chromosome spreading were used to study the role of Rae1 in mouse oocyte meiotic maturation. We found that Rae1 is a crucial regulator of meiotic maturation of mouse oocytes. After the resumption of meiosis (GVBD), Rae1 was concentrated on the kinetochore structure. The knockdown of Rae1 by a specific siRNA inhibited GVBD progression at 2 h, finally leading to a decreased 14 h polar body extrusion (PBE) rate. However, a comparable 14 h PBE rate was found in the control, and the Rae1 knockdown groups that had already undergone GVBD. Furthermore, we found elevated PBE after 9.5 h in the Rae1 knockdown oocytes. Further analysis revealed that Rae1 depletion significantly decreased the protein level of securin. In addition, we detected weakened kinetochore-microtubule (K-MT) attachments, misaligned chromosomes, and an increased incidence of aneuploidy in the Rae1 knockdown oocytes. Collectively, we propose that Rae1 modulates securin protein levels, which contribute to chromosome alignment, K-MT attachments, and aneuploidy in meiosis.


Assuntos
Meiose/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas à Matriz Nuclear/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Oócitos/metabolismo , Animais , Técnicas de Silenciamento de Genes , Técnicas de Maturação in Vitro de Oócitos , Cinetocoros/metabolismo , Camundongos , Oócitos/crescimento & desenvolvimento , Corpos Polares/metabolismo , RNA Mensageiro/genética , RNA Interferente Pequeno/genética
11.
Nat Commun ; 12(1): 3292, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078910

RESUMO

Autophagy regulates primary cilia formation, but the underlying mechanism is not fully understood. In this study, we identify NIMA-related kinase 9 (NEK9) as a GABARAPs-interacting protein and find that NEK9 and its LC3-interacting region (LIR) are required for primary cilia formation. Mutation in the LIR of NEK9 in mice also impairs in vivo cilia formation in the kidneys. Mechanistically, NEK9 interacts with MYH9 (also known as myosin IIA), which has been implicated in inhibiting ciliogenesis through stabilization of the actin network. MYH9 accumulates in NEK9 LIR mutant cells and mice, and depletion of MYH9 restores ciliogenesis in NEK9 LIR mutant cells. These results suggest that NEK9 regulates ciliogenesis by acting as an autophagy adaptor for MYH9. Given that the LIR in NEK9 is conserved only in land vertebrates, the acquisition of the autophagic regulation of the NEK9-MYH9 axis in ciliogenesis may have possible adaptive implications for terrestrial life.


Assuntos
Autofagia/genética , Cílios/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Cadeias Pesadas de Miosina/genética , Quinases Relacionadas a NIMA/genética , Sequência de Aminoácidos , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Linhagem Celular , Cílios/genética , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Rim/citologia , Rim/metabolismo , Fígado/citologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Quinases Relacionadas a NIMA/deficiência , Ligação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais
13.
J Cell Sci ; 134(10)2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-34042161

RESUMO

During anaphase, a microtubule-containing structure called the midzone forms between the segregating chromosomes. The midzone is composed of an antiparallel array of microtubules and numerous microtubule-associated proteins that contribute to midzone formation and function. In many cells, the midzone is an important source of signals that specify the location of contractile ring assembly and constriction. The midzone also contributes to the events of anaphase by generating forces that impact chromosome segregation and spindle elongation; some midzone components contribute to both processes. The results of recent experiments have increased our understanding of the importance of the midzone, a microtubule array that has often been overlooked. This Journal of Cell Science at a Glance article will review, and illustrate on the accompanying poster, the organization, formation and dynamics of the midzone, and discuss open questions for future research.


Assuntos
Anáfase , Fuso Acromático , Animais , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos , Vertebrados
14.
J Cell Sci ; 134(10)2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-34014309

RESUMO

In animal cells, a single cytoplasmic dynein motor mediates microtubule minus-end-directed transport, counterbalancing dozens of plus-end-directed kinesins. The remarkable ability of dynein to interact with a diverse cargo spectrum stems from its tightly regulated recruitment of cargo-specific adaptor proteins, which engage the dynactin complex to make a tripartite processive motor. Adaptor binding is governed by the homologous dynein light intermediate chain subunits LIC1 (DYNC1LI1) and LIC2 (DYNC1LI2), which exist in mutually exclusive dynein complexes that can perform both unique and overlapping functions. The intrinsically disordered and variable C-terminal domains of the LICs are indispensable for engaging a variety of structurally divergent adaptors. Here, we hypothesize that numerous spatiotemporally regulated permutations of posttranslational modifications of the LICs, as well as of the adaptors and cargoes, exponentially expand the spectrum of dynein-adaptor-cargo complexes. We thematically illustrate the possibilities that could generate a vast set of biochemical variations required to support the wide range of dynein functions.


Assuntos
Dineínas do Citoplasma , Dineínas , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Dineínas do Citoplasma/genética , Dineínas do Citoplasma/metabolismo , Complexo Dinactina/genética , Complexo Dinactina/metabolismo , Dineínas/genética , Dineínas/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo
15.
Nat Commun ; 12(1): 3176, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34039995

RESUMO

Chromosomes pair and synapse with their homologous partners to segregate correctly at the first meiotic division. Association of telomeres with the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex composed of SUN1 and KASH5 enables telomere-led chromosome movements and telomere bouquet formation, facilitating precise pairwise alignment of homologs. Here, we identify a direct interaction between SUN1 and Speedy A (SPDYA) and determine the crystal structure of human SUN1-SPDYA-CDK2 ternary complex. Analysis of meiosis prophase I process in SPDYA-binding-deficient SUN1 mutant mice reveals that the SUN1-SPDYA interaction is required for the telomere-LINC complex connection and the assembly of a ring-shaped telomere supramolecular architecture at the nuclear envelope, which is critical for efficient homologous pairing and synapsis. Overall, our results provide structural insights into meiotic telomere structure that is essential for meiotic prophase I progression.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Prófase Meiótica I , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Telômero/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/isolamento & purificação , Proteínas de Ciclo Celular/ultraestrutura , Linhagem Celular Tumoral , Cristalografia por Raios X , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/isolamento & purificação , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 2 Dependente de Ciclina/ultraestrutura , Feminino , Células HEK293 , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/ultraestrutura , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/isolamento & purificação , Proteínas Associadas aos Microtúbulos/ultraestrutura , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/ultraestrutura , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
16.
Int J Mol Sci ; 22(9)2021 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-33946513

RESUMO

Cells form a bipolar spindle during mitosis to ensure accurate chromosome segregation. Proper spindle architecture is established by a set of kinesin motors and microtubule-associated proteins. In most eukaryotes, kinesin-5 motors are essential for this process, and genetic or chemical inhibition of their activity leads to the emergence of monopolar spindles and cell death. However, these deficiencies can be rescued by simultaneous inactivation of kinesin-14 motors, as they counteract kinesin-5. We conducted detailed genetic analyses in fission yeast to understand the mechanisms driving spindle assembly in the absence of kinesin-5. Here, we show that deletion of the dri1 gene, which encodes a putative RNA-binding protein, can rescue temperature sensitivity caused by cut7-22, a fission yeast kinesin-5 mutant. Interestingly, kinesin-14/Klp2 levels on the spindles in the cut7 mutants were significantly reduced by the dri1 deletion, although the total levels of Klp2 and the stability of spindle microtubules remained unaffected. Moreover, RNA-binding motifs of Dri1 are essential for its cytoplasmic localization and function. We have also found that a portion of Dri1 is spatially and functionally sequestered by chaperone-based protein aggregates upon mild heat stress and limits cell division at high temperatures. We propose that Dri1 might be involved in post-transcriptional regulation through its RNA-binding ability to promote the loading of Klp2 on the spindle microtubules.


Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Fuso Acromático/metabolismo , Deleção de Genes , Temperatura Alta , Cinesina/genética , Cinesina/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Mutação , Agregados Proteicos , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Fuso Acromático/genética
17.
Mol Biol Rep ; 48(4): 3549-3559, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33866496

RESUMO

This study investigated the differentiation of transplanted transplanted mesenchymal stem cells MSCs into neuron-like cells, repair of erectile dysfunction (ED), and synergy of MSCs seeded to nanofibrous scaffolds with after transplantation around the injured cavernous nerve (CN) of rats. The synthesized polymer was electrospun in a rotating drum to prepare nanofiber meshes (NMs). Human MSCs were prepared and confirmed. Eight-week-old male Sprague-Dawley rats were divided into five groups of six each: group 1-sham operation; group 2-CN injury; group 3-MSCs treatment after CN injury; group 4-nanofibrous scaffold treatment after CN injury; and group 5-post-CN injury treatment combining a nanofibrous scaffold and MSCs (nano-MSCs). In the latter group, the damaged CN was instantly surrounded by an MSC-containing a nanofibrous scaffold in the aftermath of injury. Morphological analysis and immuno-histochemical staining in relation to nerves (Tuj1, NF, MAP2, MBP and peripherin), endothelium (vWF), smooth muscle (SMA), neurofilament (NF), and apoptosis (TUNEL) were performed. We evaluated the mean proportion expressed as a percentage of the ratio of muscle to collagen of penile cavernous smooth-muscle cells as well as the expression of cavernous SMA, NF, vWF, and TUNEL makers. Compared to the group free of CN injury, erectile function was markedly reduced in the group with CN injury at 2 and 4 weeks (p < 0.05). By contrast, compared to the sham operation group, erectile function was better in the group with MSC transplantation (p < 0.05). Similarly, by comparison to the group solely with hMSCs, erectile function was better in the group with nano-MSC transplantation (p < 0.05). Transplantation of MSCs demonstrated the neuronal differentiation. By contrast to MSCs on their own, neuronal differentiation was more significantly expressed in nano-MSCs. The mean proportion expressed as a percentage of the ratio of muscle to collagen of penile cavernous smooth-muscle cells, the expression of cavernous SMA, NF, vWF, and apoptosis improved in the cavernosum after transplantation. NMs showed synergy with MSCs for the repair of erectile dysfunction. Transplanted MSCs differentiated into neuron-like cells and repaired erectile dysfunction in the rats with CN injury. Transplanted MSCs increased the mean percentage of the collagen area of the caversnosum as well as the expression levels of cavernous neuronal, endothelial, smooth-muscle markers, and apoptosis.


Assuntos
Diferenciação Celular , Disfunção Erétil/terapia , Transplante de Células-Tronco Mesenquimais/métodos , Regeneração Nervosa , Traumatismos dos Nervos Periféricos/terapia , Actinas/genética , Actinas/metabolismo , Animais , Apoptose , Células Cultivadas , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/metabolismo , Pênis/inervação , Poliésteres/química , Ratos , Ratos Sprague-Dawley , Tecidos Suporte/química , Tubulina (Proteína)/metabolismo
18.
Int J Mol Sci ; 22(8)2021 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-33921398

RESUMO

The intervertebral disc is the largest avascular organ. Autophagy is an important cell survival mechanism by self-digestion and recycling damaged components under stress, primarily nutrient deprivation. Resident cells would utilize autophagy to cope with the harsh disc environment. Our objective was to elucidate the roles of human disc cellular autophagy. In human disc cells, serum deprivation and pro-inflammatory interleukin-1ß (IL-1ß) stimulation increased autophagy marker microtubule-associated protein 1 light chain 3 (LC3)-II and decreased autophagy substrate p62/sequestosome 1 (p62/SQSTM1), indicating enhanced autophagy. Then, RNA interference (RNAi) of autophagy-related gene 5 (ATG5), essential for autophagy, showed decreases in ATG5 protein (26.8%-27.4%, p < 0.0001), which suppressed early-stage autophagy with decreased LC3-II and increased p62/SQSTM1. Cell viability was maintained by ATG5 RNAi in serum-supplemented media (95.5%, p = 0.28) but reduced in serum-free media (80.4%, p = 0.0013) with IL-1ß (69.9%, p = 0.0008). Moreover, ATG5 RNAi accelerated IL-1ß-induced changes in apoptosis and senescence. Meanwhile, ATG5 RNAi unaffected IL-1ß-induced catabolic matrix metalloproteinase release, down-regulated anabolic gene expression, and mitogen-activated protein kinase pathway activation. Lysosomotropic chloroquine supplementation presented late-stage autophagy inhibition with apoptosis and senescence induction, while catabolic enzyme production was modest. Disc-tissue analysis detected age-related changes in ATG5, LC3-II, and p62/SQSTM1. In summary, autophagy protects against human disc cellular apoptosis and senescence rather than extracellular matrix catabolism.


Assuntos
Proteína 5 Relacionada à Autofagia/genética , Disco Intervertebral/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteína Sequestossoma-1/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/efeitos dos fármacos , Autofagia/genética , Linhagem Celular , Sobrevivência Celular/genética , Senescência Celular/efeitos dos fármacos , Cloroquina/farmacologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-1beta/genética , Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/patologia , Masculino , Metabolismo/efeitos dos fármacos , Pessoa de Meia-Idade
19.
Nat Commun ; 12(1): 2409, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33893302

RESUMO

During cytokinesis, the actin cytoskeleton is partitioned into two spatially distinct actin isoform specific networks: a ß-actin network that generates the equatorial contractile ring, and a γ-actin network that localizes to the cell cortex. Here we demonstrate that the opposing regulation of the ß- and γ-actin networks is required for successful cytokinesis. While activation of the formin DIAPH3 at the cytokinetic furrow underlies ß-actin filament production, we show that the γ-actin network is specifically depleted at the cell poles through the localized deactivation of the formin DIAPH1. During anaphase, CLIP170 is delivered by astral microtubules and displaces IQGAP1 from DIAPH1, leading to formin autoinhibition, a decrease in cortical stiffness and localized membrane blebbing. The contemporaneous production of a ß-actin contractile ring at the cell equator and loss of γ-actin from the poles is required to generate a stable cytokinetic furrow and for the completion of cell division.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Citocinese , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Centrossomo/metabolismo , Forminas/genética , Forminas/metabolismo , Células HeLa , Humanos , Microscopia de Força Atômica , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
20.
Mol Cell ; 81(9): 2031-2040.e8, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33909989

RESUMO

Autophagy is a fundamental catabolic process that uses a unique post-translational modification, the conjugation of ATG8 protein to phosphatidylethanolamine (PE). ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway involving conjugation of ATG8 to single membranes (CASM) at endolysosomal compartments, with key functions in immunity, vision, and neurobiology. It is widely assumed that CASM involves the same conjugation of ATG8 to PE, but this has not been formally tested. Here, we discover that all ATG8s can also undergo alternative lipidation to phosphatidylserine (PS) during CASM, induced pharmacologically, by LC3-associated phagocytosis or influenza A virus infection, in mammalian cells. Importantly, ATG8-PS and ATG8-PE adducts are differentially delipidated by the ATG4 family and bear different cellular dynamics, indicating significant molecular distinctions. These results provide important insights into autophagy signaling, revealing an alternative form of the hallmark ATG8 lipidation event. Furthermore, ATG8-PS provides a specific "molecular signature" for the non-canonical autophagy pathway.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagossomos/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Autofagia , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfatidilserinas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/genética , Autofagossomos/patologia , Família da Proteína 8 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Feminino , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Vírus da Influenza A/patogenicidade , Macrolídeos/farmacologia , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Monensin/farmacologia , Fagocitose , Fosfatidiletanolaminas/metabolismo , Células RAW 264.7 , Transdução de Sinais
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