ResearchPad - 21 https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Cell polarity–dependent centrosome separation in the <i>C. elegans</i> embryo]]> https://www.researchpad.co/article/elastic_article_15326 In animal cells, faithful chromosome segregation depends on the assembly of a bipolar spindle driven by the timely separation of the two centrosomes. Here we took advantage of the highly stereotypical cell divisions in Caenorhabditis elegans embryos to identify new regulators of centrosome separation. We find that at the two-cell stage, the somatic AB cell initiates centrosome separation later than the germline P1 cell. This difference is strongly exacerbated by the depletion of the kinesin-13 KLP-7/MCAK, resulting in incomplete centrosome separation at NEBD in AB but not P1. Our genetic and cell biology data indicate that this phenotype depends on cell polarity via the enrichment in AB of the mitotic kinase PLK-1, which itself limits the cortical localization of the dynein-binding NuMA orthologue LIN-5. We postulate that the timely separation of centrosomes is regulated in a cell type–dependent manner.

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<![CDATA[The postmitotic midbody: Regulating polarity, stemness, and proliferation]]> https://www.researchpad.co/article/elastic_article_15325 Abscission, the final stage of cell division, requires well-orchestrated changes in endocytic trafficking, microtubule severing, actin clearance, and the physical sealing of the daughter cell membranes. These processes are highly regulated, and any missteps in localized membrane and cytoskeleton dynamics often lead to a delay or a failure in cell division. The midbody, a microtubule-rich structure that forms during cytokinesis, is a key regulator of abscission and appears to function as a signaling platform coordinating cytoskeleton and endosomal dynamics during the terminal stages of cell division. It was long thought that immediately following abscission and the conclusion of cell division, the midbody is either released or rapidly degraded by one of the daughter cells. Recently, the midbody has gained prominence for exerting postmitotic functions. In this review, we detail the role of the midbody in orchestrating abscission, as well as discuss the relatively new field of postabscission midbody biology, particularly focusing on how it may act to regulate cell polarity and its potential to regulate cell tumorigenicity or stemness.

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<![CDATA[Regulation of ETAA1-mediated ATR activation couples DNA replication fidelity and genome stability]]> https://www.researchpad.co/article/elastic_article_15322 The ATR kinase is a master regulator of the cellular response to DNA replication stress. Activation of ATR relies on dual pathways involving the TopBP1 and ETAA1 proteins, both of which harbor ATR-activating domains (AADs). However, the exact contribution of the recently discovered ETAA1 pathway to ATR signaling in different contexts remains poorly understood. Here, using an unbiased CRISPR-Cas9–based genome-scale screen, we show that the ATR-stimulating function of ETAA1 becomes indispensable for cell fitness and chromosome stability when the fidelity of DNA replication is compromised. We demonstrate that the ATR-activating potential of ETAA1 is controlled by cell cycle– and replication stress–dependent phosphorylation of highly conserved residues within its AAD, and that the stimulatory impact of these modifications is required for the ability of ETAA1 to prevent mitotic chromosome abnormalities following replicative stress. Our findings suggest an important role of ETAA1 in protecting against genome instability arising from incompletely duplicated DNA via regulatory control of its ATR-stimulating potential.

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<![CDATA[Delineating the contribution of Spc105-bound PP1 to spindle checkpoint silencing and kinetochore microtubule attachment regulation]]> https://www.researchpad.co/article/elastic_article_15320 Accurate chromosome segregation during cell division requires the spindle assembly checkpoint (SAC), which detects unattached kinetochores, and an error correction mechanism that destabilizes incorrect kinetochore–microtubule attachments. While the SAC and error correction are both regulated by protein phosphatase 1 (PP1), which silences the SAC and stabilizes kinetochore–microtubule attachments, how these distinct PP1 functions are coordinated remains unclear. Here, we investigate the contribution of PP1, docked on its conserved kinetochore receptor Spc105/Knl1, to SAC silencing and attachment regulation. We find that Spc105-bound PP1 is critical for SAC silencing but dispensable for error correction; in fact, reduced PP1 docking on Spc105 improved chromosome segregation and viability of mutant/stressed states. We additionally show that artificially recruiting PP1 to Spc105/Knl1 before, but not after, chromosome biorientation interfered with error correction. These observations lead us to propose that recruitment of PP1 to Spc105/Knl1 is carefully regulated to ensure that chromosome biorientation precedes SAC silencing, thereby ensuring accurate chromosome segregation.

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<![CDATA[DNA replication and mitotic entry: A brake model for cell cycle progression]]> https://www.researchpad.co/article/elastic_article_15318 The core function of the cell cycle is to duplicate the genome and divide the duplicated DNA into two daughter cells. These processes need to be carefully coordinated, as cell division before DNA replication is complete leads to genome instability and cell death. Recent observations show that DNA replication, far from being only a consequence of cell cycle progression, plays a key role in coordinating cell cycle activities. DNA replication, through checkpoint kinase signaling, restricts the activity of cyclin-dependent kinases (CDKs) that promote cell division. The S/G2 transition is therefore emerging as a crucial regulatory step to determine the timing of mitosis. Here we discuss recent observations that redefine the coupling between DNA replication and cell division and incorporate these insights into an updated cell cycle model for human cells. We propose a cell cycle model based on a single trigger and sequential releases of three molecular brakes that determine the kinetics of CDK activation.

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<![CDATA[CDK1-mediated CENP-C phosphorylation modulates CENP-A binding and mitotic kinetochore localization]]> https://www.researchpad.co/article/elastic_article_15314 The kinetochore is essential for faithful chromosome segregation during mitosis. To form a functional kinetochore, constitutive centromere-associated network (CCAN) proteins are assembled on the centromere chromatin that contains the centromere-specific histone CENP-A. CENP-C, a CCAN protein, directly interacts with the CENP-A nucleosome to nucleate the kinetochore structure. As CENP-C is a hub protein for kinetochore assembly, it is critical to address how the CENP-A–CENP-C interaction is regulated during cell cycle progression. To address this question, we investigated the CENP-C C-terminal region, including a conserved CENP-A–binding motif, in both chicken and human cells and found that CDK1-mediated phosphorylation of CENP-C facilitates its binding to CENP-A in vitro and in vivo. We observed that CENP-A binding is involved in CENP-C kinetochore localization during mitosis. We also demonstrate that the CENP-A–CENP-C interaction is critical for long-term viability in human RPE-1 cells. These results provide deeper insights into protein-interaction network plasticity in centromere proteins during cell cycle progression.

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<![CDATA[Two <i>S. pombe</i> septation phases differ in ingression rate, septum structure, and response to F-actin loss]]> https://www.researchpad.co/article/elastic_article_15305 In fission yeast, cytokinesis requires a contractile actomyosin ring (CR) coupled to membrane and septum ingression. Septation proceeds in two phases. In anaphase B, the septum ingresses slowly. During telophase, the ingression rate increases, and the CR becomes dispensable. Here, we explore the relationship between the CR and septation by analyzing septum ultrastructure, ingression, and septation proteins in cells lacking F-actin. We show that the two phases of septation correlate with septum maturation and the response of cells to F-actin removal. During the first phase, the septum is immature and, following F-actin removal, rapidly loses the Bgs1 glucan synthase from the membrane edge and fails to ingress. During the second phase, the rapidly ingressing mature septum can maintain a Bgs1 ring and septum ingression without F-actin, but ingression becomes Cdc42 and exocyst dependent. Our results provide new insights into fungal cytokinesis and reveal the dual function of CR as an essential landmark for the concentration of Bgs1 and a contractile structure that maintains septum shape and synthesis.

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<![CDATA[A user-friendly, high-throughput tool for the precise fluorescent quantification of deoxyribonucleoside triphosphates from biological samples]]> https://www.researchpad.co/article/Nb4c51b4b-2039-4b16-b950-76549442337e Cells maintain a fine-tuned, dynamic concentration balance in the pool of deoxyribonucleoside 5′-triphosphates (dNTPs). This balance is essential for physiological processes including cell cycle control or antiviral defense. Its perturbation results in increased mutation frequencies, replication arrest and may promote cancer development. An easily accessible and relatively high-throughput method would greatly accelerate the exploration of the diversified consequences of dNTP imbalances. The dNTP incorporation based, fluorescent TaqMan-like assay published by Wilson et al. has the aforementioned advantages over mass spectrometry, radioactive or chromatography based dNTP quantification methods. Nevertheless, the assay failed to produce reliable data in several biological samples. Therefore, we applied enzyme kinetics analysis on the fluorescent dNTP incorporation curves and found that the Taq polymerase exhibits a dNTP independent exonuclease activity that decouples signal generation from dNTP incorporation. Furthermore, we found that both polymerization and exonuclease activities are unpredictably inhibited by the sample matrix. To resolve these issues, we established a kinetics based data analysis method which identifies the signal generated by dNTP incorporation. We automated the analysis process in the nucleoTIDY software which enables even the inexperienced user to calculate the final and accurate dNTP amounts in a 96-well-plate setup within minutes.

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<![CDATA[Mylk3 null C57BL/6N mice develop cardiomyopathy, whereas Nnt null C57BL/6J mice do not]]> https://www.researchpad.co/article/N10807206-d34a-4cb8-8f17-90e30d8c5708

Genetic differences between C57BL/6 substrains lead to different cardiovascular traits; a null mutation in Mylk3 likely causes cardiomyopathy in C57BL/6N mice, whereas C57BL/6J Nnt-null mice do not develop cardiomyopathy.

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<![CDATA[CLASP2 binding to curved microtubule tips promotes flux and stabilizes kinetochore attachments]]> https://www.researchpad.co/article/N6bf105bd-3502-4ac3-942f-3873dd79be05

Girão et al. use structure-guided functional mutants of CLASP2 to show that recognition of growing microtubule plus-ends through EB–protein interaction and the ability to associate with curved microtubule protofilaments through TOG2 and TOG3 domains promote growth and stabilization of kinetochore–microtubules required for poleward flux.

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<![CDATA[Rating scales for rare neurological diseases]]> https://www.researchpad.co/article/N2866cbdf-eac2-48bd-94a5-fe95e6cf2602 ]]> <![CDATA[SET binding to Sgo1 inhibits Sgo1–cohesin interactions and promotes chromosome segregation]]> https://www.researchpad.co/article/N81632380-ba05-4c76-92a2-f9d5bc255519

Sgo1 is essential for centromeric cohesion protection; however, how it is disabled at anaphase onset is not quite understood. Qu et al. report that SET inhibits Sgo1–cohesin interactions by binding Sgo1 during mitosis, thus promoting centromeric cohesion resolution and timely chromosome segregation.

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<![CDATA[High-resolution imaging reveals how the spindle midzone impacts chromosome movement]]> https://www.researchpad.co/article/N4f28886a-0344-4e88-90c5-4dda28a829c5

Microtubule bundles in the spindle midzone have been reported to either promote or hinder chromosome movement. Pamula et al. examine the assembly dynamics of midzone microtubule bundles during anaphase and how chromosome segregation is impacted by aberrant bundle assembly.

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<![CDATA[Knockdown of Rab7a suppresses the proliferation, migration, and xenograft tumor growth of breast cancer cells]]> https://www.researchpad.co/article/5c800fe4d5eed0c484a9688e

Breast cancer is a common invasive cancer in women. Ras-related protein Rab-7a (Rab7a) is involved in late endocytic trafficking, while its role in breast cancer is largely unclear. In the present study, we investigated the role of Rab7a in breast cancer. Comparing with adjacent breast tissues, Rab7a expression was increased in breast cancer tissues. Using lentivirus-mediated knockdown strategy, we found that Rab7a silencing inhibited the proliferation and colony formation of MDA-MB-231 cells. Apoptosis and G2 cell cycle arrest were induced in Rab7a knockdown. By contrast, Rab7a suppressed the apoptosis and promoted proliferation and colony formation of MCF-7 cells. The migration of MDA-MB-231 cells was suppressed by Rab7a knockdown. In vivo, depletion of Rab7a inhibited the xenograft tumor development of MDA-MB-231 cells. Altogether, our results highlight the novel function of Rab7a in the proliferation, invasion, and xenograft tumor development of breast cancer cells.

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<![CDATA[p16INK4a inhibits the proliferation of osteosarcoma cells through regulating the miR-146b-5p/TRAF6 pathway]]> https://www.researchpad.co/article/5c6863efd5eed0c484023c78

Down-regulation of p16INK4a and miR-146b-5p contributes to tumorigenesis in osteosarcoma (OS). However, the correlation between p16INK4a and miR-146b-5p in OS proliferation remains largely unknown. In the present study, we demonstrated that miR-146b-5p expression was positively correlated with p16INK4a in OS, but inversely correlated with TNF receptor associated factor 6 (TRAF6) expression. Overexpression of miR-146b-5p dramatically suppressed OS cell proliferation. Mechanistically, we validated TRAF6 as a direct functional target of miR-146b-5p and found that miR-146b-5p overexpression significantly decreased the level of phosphorylated PI3k and Akt, which are the pivotal downstream effectors of TRAF6. Moreover, TRAF6 expression was positively correlated with Ki-67 but inversely correlated with miR-146b-5p expression. In OS cells, silencing of TRAF6 mimicked the anti-tumor effects of miR-146b-5p. p16INK4a is an important tumor suppressor gene frequently down-regulated in OS. We found that this inhibitory effect is associated with the suppression of the miR-146b-5p, and is mediated via up-regulating TRAF6 expression. Our findings identified p16INK4a and miR-146b-5p as tumor suppressors, and suggested p16INK4a, miR-146b-5p and TRAF6 as potential therapeutic candidates for malignant OS.

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<![CDATA[Inhibited corneal neovascularization in rabbits following corneal alkali burn by double-target interference for VEGF and HIF-1α]]> https://www.researchpad.co/article/5c6863c4d5eed0c484023b20

Expression of hypoxia-inducible factor (HIF) 1α has been observed in corneal neovascularization (CNV). Vascular endothelial growth factor (VEGF), one of the most well-known angiogenic factors in CNV, is under the regulation of HIF-1. The present study aims to investigate the synergistic effects of VEGF and HIF-1α gene silencing on alkali burn-induced CNV in rabbits. The models of rabbits in corneal alkali burn were established. SiRNA recombinant adenovirus was used to explore the synergistic effects of VEGF and HIF-1α gene silencing on alkali burn-induced CNV. CNV area and ultrastructure of cornea were observed. The expression of VEGF and HIF-1α was detected. CNV was observed in rabbits following alkali burn. In addition, overexpressed VEGF and HIF-1α was also observed in rabbits following alkali burn. Then, silencing HIF-1α or silencing VEGF decreased area of CNV, inhibited neovascularization and improved pathological changes, while double-target interference for VEGF and HIF-1α decreased area of CNV inhibited neovascularization, and improved pathological changes to a greater extent. Our study provides evidences emphasizing the distinct notion that VEGF and HIF-1α play the contributory role in alkali burn-induced CNV as a result of double-target interference for VEGF and HIF-1α inhibiting CNV in rabbits following corneal alkali burn.

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<![CDATA[LncRNA GAS8-AS1 suppresses papillary thyroid carcinoma cell growth through the miR-135b-5p/CCND2 axis]]> https://www.researchpad.co/article/5c4b9416d5eed0c48487dd0b

The aim of the present study was to investigate the potential role of GAS8 antisense RNA 1 (GAS8-AS1) in papillary thyroid carcinoma (PTC). PcDNA3.1-GAS8-AS1 and si-GAS8-AS1, miR-135b-5p mimic and si-CCND2 were transfected into PTC cells. Cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8). QRT-PCR was used to determine expressions of GAS8-AS1, miR-135b-5p, and CCND2, and Western blot were detected protein level of CCND2. The miRNA target gene prediction site TargetScan was used to predict potential targets of GAS8-AS1 and miR-135b-5p. Cell cycle progression was analyzed by flow cytometry. We found that GAS8-AS1 was down-regulated in PTC cell lines and inhibited proliferation and cycle of PTC cell. GAS8-AS1 directly targets miR-135b-5p, and GAS8-AS1 could regulate a downstream target of miR-135b-5p, Cyclin G2 (CCNG2), in an miR-135b-5p-mediated manner. In addition, we also proved that overexpressed GAS8-AS1 inhibited tumor formation in vivo. GAS8-AS1 suppresses PTC cell growth through the miR-135b-5p/CCND2 axis.

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<![CDATA[MiRNA-575 suppresses angiogenesis by targeting Rab5-MEK-ERK pathway in endothelial cells]]> https://www.researchpad.co/article/5c4b941cd5eed0c48487de14

Hypertension is a major risk factor for the development of atherosclerosis. Increased carotid intima-media thickness (CIMT) is generally considered as an early marker of atherosclerosis. Recently, circulating miRNAs have been implicated both as sensitive biomarkers and key regulators in the development of atherosclerosis. However, the biological functions and molecular regulatory mechanisms for miR-575 on angiogenesis remain unknown. In our study, we first identified up-regulation of circulating miR-575 in plasma of essential hypertensive patients with increased CIMT (iCIMT) compared with those patients with normal CIMT (nCIMT). Furthermore, the overexpression of miR-575 in human umbilical vein endothelial cells (HUVECs) by its mimics significantly inhibited migration and proliferation as well as induction of apoptosis of HUVECs. Inhibition of miR-575 performed the reverse effects of HUVECs. We further suggested Rab5B was the downstream target of miR-575 and knockdown of Rab5B significantly inhibited migration and proliferation of HUVECs. Overexpression of Rab5B largely rescued the miR-575-mediated impairment of angiogenesis processes including: cell proliferation, migration, and apoptosis as well as activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK-ERK) signaling. Therefore, our results uncover a novel role of miR-575 in endothelial cells, implying a potential biomarker and clinical target for atherosclerosis in hypertensive patients.

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<![CDATA[The effect of Euphorbia szovitsii Fisch. & C.A.Mey extract on the viability and the proliferation of MDA-MB-231 cell line]]> https://www.researchpad.co/article/5c4b9412d5eed0c48487dc77

Some medicinal herbs and compounds are known to target cancer cells, but the success of them as anticancer compounds depends to a large extent on their ability to activate pathways that kill cancer cells by arresting cell cycle and inducing apoptosis. The aim of the present study was to determine the anticancer effects of Euphorbia szovitsii Fisch. & C.A.Mey. on the breast cancer cells to reveal the underlying mechanism of its anti-breast cancer properties. In this experimental study, triple negative breast cancer cell line (MDA-MB-231) was cultivated in RPMI-1640 medium. Hydroalcoholic extract (70:30) of aerial parts of the plant was prepared. The cultured cells were treated with different concentrations (0–1000 μg/ml) of E. szovitsii extract for 24 and 48 h. Toxicity of the extract on MDA-MB-231 cells was examined using MTT (3-[4,5-dimethyl-2-thiazolyl]-2, 5 diphenyl tetrazolium bromide) test. The Annexin V–FITC Apoptosis Detection Kit was used to evaluate apoptosis and necrosis. Flow cytometry technique was employed to differentiate different phases of the cell cycle in the cells. Data were analyzed by GraphPad Prism and SPSS software. After 24 and 48 h, the IC50 values were respectively 76.78 (95% CI = 60.75–97.05; R = 0.8588) and 59.71 (95% CI = 46.25–77.09; R = 0.8543) μg/ml for E. szovitsii. The extract exhibited antiproliferative effects against MDA-MB-231 cells in a dose-dependent manner. Annexin V-FITC/PI assay confirmed that the extract was able to induce apoptosis in MDA-MB-231 cells. Moreover, treatment with the extract resulted in cell cycle arrest at G1 phase. Therefore, E. szovitsii could induce apoptosis and cycle arrest in the MDA-MB-231 cell line. It might be a good resource of natural products for producing anti-breast cancer drugs.

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<![CDATA[Huayu Tongmai Granules protects against vascular endothelial dysfunction via up-regulating miR-185 and down-regulating RAGE]]> https://www.researchpad.co/article/5c1c2c4bd5eed0c48446091e

Objective: Receptor of advanced glycation end products (RAGE) is a membrane protein that contributes to the initiation and progression of diabetic vascular complications, which is reported as a target of miR-185. Huayu Tongmai Granules is a Chinese herbal compound that is capable of treating diabetic angiopathy. The present study was designed to explore the molecular biological mechanism by which Huayu Tongmai Granules protects against diabetic angiopathy.

Methods: The rat model of diabetes and hyperglucose cell model were established. The blood glucose was detected to verify whether the model was successfully established. Besides, serum nitric oxide (NO) and reactive oxygen species (ROS) concentrations of the rats in each group were determined. The quantitative real-time PCR analysis was performed to examine the mRNA expression levels of miR-185 and other miRNAs in femoral artery of rats or human umbilical vein endothelial cell line. Additionally, the protein levels of RAGE or Bax were determined using Western blotting. Cell apoptosis was determined by terminal dUTP nick-end labeling assay or flow cytometry.

Results: In the present study, we found that Huayu Tongmai Granules significantly decreased blood glucose and serum ROS and up-regulated serum NO concentration. MiR-185 was low-expressed in diabetic rats; however, Huayu Tongmai Granules intervention up-regulated miR-185. Stable overexpression of miR-185 directly suppressed the expression of RAGE and further suppressed endothelial cell apoptosis.

Conclusion: Huayu Tongmai Granules appears to have a therapeutic effect on diabetic angiopathy that is most probably mediated by miR-185/RAGE axis.

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