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Asian Journal of Andrology 2020Many studies have shown that microRNAs (miRNAs) play vital roles during the spermatogenesis. However, little is known about the altered miRNA profiles of testicular...
Many studies have shown that microRNAs (miRNAs) play vital roles during the spermatogenesis. However, little is known about the altered miRNA profiles of testicular tissues in nonobstructive azoospermia (NOA). Using microarray technology, the miRNA expression profiles of testicular biopsies from patients with NOA and of normal testicular tissues were determined. Bioinformatics analyses were conducted to predict the enriched biological processes and functions of identified miRNAs. The microarray data were validated by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), the results of which were then validated with a larger sample size. Correlations between the miRNA expression levels and clinical characteristics were analyzed. Receiver operating characteristic (ROC) curve analysis was used to evaluate the diagnostic ability of miRNAs for azoospermia. Hierarchical clustering showed that 129 miRNAs were significantly differentially expressed between the NOA and control groups. Bioinformatics analysis indicated that the differentially expressed miRNAs were involved in spermatogenesis, cell cycle, and mitotic prometaphase. In the subsequent qRT-PCR assays, the selected miRNA expression levels were consistent with the microarray results, and similar validated results were obtained with a larger sample size. Some clinical characteristics were significantly associated with the expression of certain miRNAs. In particular, we identified a combination of two miRNAs (miR-10b-3p and miR-34b-5p) that could serve as a predictive biomarker of azoospermia. This study provides altered miRNA profiles of testicular biopsies from NOA patients and examines the roles of miRNAs in spermatogenesis. These profiles may be useful for predicting and diagnosing the presence of testicular sperm in individuals with azoospermia.
Topics: Adult; Azoospermia; Biopsy; Cluster Analysis; Computational Biology; Follicle Stimulating Hormone; Gene Expression Profiling; Humans; Luteinizing Hormone; Male; MicroRNAs; Reverse Transcriptase Polymerase Chain Reaction; Spermatogenesis; Testis; Testosterone; Tissue Array Analysis
PubMed: 31134916
DOI: 10.4103/aja.aja_35_19 -
PloS One 2019During mitosis, the structure of the Endoplasmic Reticulum (ER) displays a dramatic reorganization and remodeling, however, the mechanism driving these changes is poorly...
During mitosis, the structure of the Endoplasmic Reticulum (ER) displays a dramatic reorganization and remodeling, however, the mechanism driving these changes is poorly understood. Hairpin-containing ER transmembrane proteins that stabilize ER tubules have been identified as possible factors to promote these drastic changes in ER morphology. Recently, the Reticulon and REEP family of ER shaping proteins have been shown to heavily influence ER morphology by driving the formation of ER tubules, which are known for their close proximity with microtubules. Here, we examine the role of microtubules and other cytoskeletal factors in the dynamics of a Drosophila Reticulon, Reticulon-like 1 (Rtnl1), localization to spindle poles during mitosis in the early embryo. At prometaphase, Rtnl1 is enriched to spindle poles just prior to the ER retention motif KDEL, suggesting a possible recruitment role for Rtnl1 in the bulk localization of ER to spindle poles. Using image analysis-based methods and precise temporal injections of cytoskeletal inhibitors in the early syncytial Drosophila embryo, we show that microtubules are necessary for proper Rtnl1 localization to spindles during mitosis. Lastly, we show that astral microtubules, not microfilaments, are necessary for proper Rtnl1 localization to spindle poles, and is largely independent of the minus-end directed motor protein dynein. This work highlights the role of the microtubule cytoskeleton in Rtnl1 localization to spindles during mitosis and sheds light on a pathway towards inheritance of this major organelle.
Topics: Animals; Drosophila Proteins; Drosophila melanogaster; Dyneins; Endoplasmic Reticulum; Kinesins; Microtubules; Mitosis; Spindle Poles
PubMed: 31877164
DOI: 10.1371/journal.pone.0226327 -
The Journal of Biological Chemistry Jun 2023Mitotic kinetochores are initially captured by dynamic microtubules via a "search-and-capture" mechanism. The microtubule motor, dynein, is critical for kinetochore...
Mitotic kinetochores are initially captured by dynamic microtubules via a "search-and-capture" mechanism. The microtubule motor, dynein, is critical for kinetochore capture as it has been shown to transport microtubule-attached chromosomes toward the spindle pole during prometaphase. The microtubule-binding nuclear division cycle 80 (Ndc80) complex that is recruited to kinetochores in prophase is known to play a central role in forming kinetochore-microtubule (kMT) attachments in metaphase. It is not yet clear, however, how Ndc80 contributes to initial kMT capture during prometaphase. Here, by combining CRISPR/Cas9-mediated knockout and RNAi technology with assays specific to study kMT capture, we show that mitotic cells lacking Ndc80 exhibit substantial defects in this function during prometaphase. Rescue experiments show that Ndc80 mutants deficient in microtubule-binding are unable to execute proper kMT capture. While cells inhibited of dynein alone are predominantly able to make initial kMT attachments, cells co-depleted of Ndc80 and dynein show severe defects in kMT capture. Further, we use an in vitro total internal reflection fluorescence microscopy assay to reconstitute microtubule capture events, which suggest that Ndc80 and dynein coordinate with each other for microtubule plus-end capture and that the phosphorylation status of Ndc80 is critical for productive kMT capture. A novel interaction between Ndc80 and dynein that we identify in prometaphase extracts might be critical for efficient plus-end capture. Thus, our studies, for the first time, identify a distinct event in the formation of initial kMT attachments, which is directly mediated by Ndc80 and in coordination with dynein is required for efficient kMT capture and chromosome alignment.
Topics: Dyneins; Kinetochores; Nuclear Proteins; Microtubules; Mitosis; Spindle Apparatus; Microtubule-Associated Proteins; Cell Cycle Proteins
PubMed: 37060995
DOI: 10.1016/j.jbc.2023.104711 -
Current Biology : CB Jun 2024Faithful chromosome segregation requires that sister chromatids establish bi-oriented kinetochore-microtubule attachments. The spindle assembly checkpoint (SAC) prevents...
Faithful chromosome segregation requires that sister chromatids establish bi-oriented kinetochore-microtubule attachments. The spindle assembly checkpoint (SAC) prevents premature anaphase onset with incomplete attachments. However, how microtubule attachment and checkpoint signaling are coordinated remains unclear. The conserved kinase Mps1 initiates SAC signaling by localizing transiently to kinetochores in prometaphase and is released upon bi-orientation. Using biochemistry, structure predictions, and cellular assays, we shed light on this dynamic behavior in Saccharomyces cerevisiae. A conserved N-terminal segment of Mps1 binds the neck region of Ndc80:Nuf2, the main microtubule receptor of kinetochores. Mutational disruption of this interface, located at the backside of the paired CH domains and opposite the microtubule-binding site, prevents Mps1 localization, eliminates SAC signaling, and impairs growth. The same interface of Ndc80:Nuf2 binds the microtubule-associated Dam1 complex. We demonstrate that the error correction kinase Ipl1/Aurora B controls the competition between Dam1 and Mps1 for the same binding site. Thus, binding of the Dam1 complex to Ndc80:Nuf2 may release Mps1 from the kinetochore to promote anaphase onset.
Topics: Saccharomyces cerevisiae Proteins; Kinetochores; Saccharomyces cerevisiae; Protein Serine-Threonine Kinases; Microtubules; Cell Cycle Proteins; Microtubule-Associated Proteins; Nuclear Proteins
PubMed: 38776902
DOI: 10.1016/j.cub.2024.03.062 -
Current Biology : CB Feb 2021The precise regulation of microtubule dynamics over time and space in dividing cells is critical for several mitotic mechanisms that ultimately enable cell...
The precise regulation of microtubule dynamics over time and space in dividing cells is critical for several mitotic mechanisms that ultimately enable cell proliferation, tissue organization, and development. Astral microtubules, which extend from the centrosome toward the cell cortex, must be present for the mitotic spindle to properly orient, as well as for the faithful execution of anaphase and cytokinesis. However, little is understood about how the dynamic properties of astral microtubules are regulated spatiotemporally, or the contribution of astral microtubule dynamics to spindle positioning. The mitotic regulator Cdk1-CyclinB promotes destabilization of centrosomal microtubules and increased microtubule dynamics as cells enter mitosis, but how Cdk1 activity modulates astral microtubule stability, and whether it impacts spindle positioning, is unknown. Here, we uncover a mechanism revealing that Cdk1 destabilizes astral microtubules in prometaphase and thereby influences spindle reorientation. Phosphorylation of the EB1-dependent microtubule plus-end tracking protein GTSE1 by Cdk1 in early mitosis abolishes its interaction with EB1 and recruitment to microtubule plus ends. Loss of Cdk1 activity, or mutation of phosphorylation sites in GTSE1, induces recruitment of GTSE1 to growing microtubule plus ends in mitosis. This decreases the catastrophe frequency of astral microtubules and causes an increase in the number of long astral microtubules reaching the cell cortex, which restrains the ability of cells to reorient spindles along the long cellular axis in early mitosis. Astral microtubules thus must not only be present but also dynamic to allow the spindle to reorient, a state assisted by selective destabilization of long astral microtubules via Cdk1.
Topics: Anaphase; Animals; CDC2 Protein Kinase; Humans; Mice; Microtubule-Associated Proteins; Microtubules; Prometaphase; Protein Stability; Spindle Apparatus
PubMed: 33333009
DOI: 10.1016/j.cub.2020.11.040 -
Cell Research Aug 2019Oriented cell divisions are controlled by a conserved molecular cascade involving Gαi, LGN, and NuMA. Here, we show that NDP52 regulates spindle orientation via...
Oriented cell divisions are controlled by a conserved molecular cascade involving Gαi, LGN, and NuMA. Here, we show that NDP52 regulates spindle orientation via remodeling the polar cortical actin cytoskeleton. siRNA-mediated NDP52 suppression surprisingly revealed a ring-like compact subcortical F-actin architecture surrounding the spindle in prophase/prometaphase cells, which resulted in severe defects of astral microtubule growth and an aberrant spindle orientation. Remarkably, NDP52 recruited the actin assembly factor N-WASP and regulated the dynamics of the subcortical F-actin ring in mitotic cells. Mechanistically, NDP52 was found to bind to phosphatidic acid-containing vesicles, which absorbed cytoplasmic N-WASP to regulate local filamentous actin growth at the polar cortex. Our TIRFM analyses revealed that NDP52-containing vesicles anchored N-WASP and shortened the length of actin filaments in vitro. Based on these results we propose that NDP52-containing vesicles regulate cortical actin dynamics through N-WASP to accomplish a spatiotemporal regulation between astral microtubules and the actin network for proper spindle orientation and precise chromosome segregation. In this way, intracellular vesicles cooperate with microtubules and actin filaments to regulate proper mitotic progression. Since NDP52 is absent from yeast, we reason that metazoans have evolved an elaborate spindle positioning machinery to ensure accurate chromosome segregation in mitosis.
Topics: Actin Cytoskeleton; Actins; Chromosome Segregation; Cytoplasmic Vesicles; HEK293 Cells; HeLa Cells; Humans; Microtubules; Mitosis; Nuclear Proteins; RNA Interference; Spindle Apparatus; Wiskott-Aldrich Syndrome Protein, Neuronal
PubMed: 31201383
DOI: 10.1038/s41422-019-0189-9 -
Asian Pacific Journal of Cancer... Sep 2021We aim to enhance the effectiveness of curcumin analog PGV-1 through co-treatment with diosmin, a citrus flavonoid, on 4T1 cells and evaluate the molecular targets...
OBJECTIVE
We aim to enhance the effectiveness of curcumin analog PGV-1 through co-treatment with diosmin, a citrus flavonoid, on 4T1 cells and evaluate the molecular targets underlying its effect on the cell cycle.
METHODS
Cytotoxic effects were performed by MTT assay against 4T1 cells. The May Grünwald-Giemsa staining was used to observe cell cycle arrest. The senescence was assayed with SA-ß-gal staining. Bioinformatic studies were utilized to discover protein targets of PGV-1 and diosmin on triple-negative breast cancer (TNBC) using SwissTargetPrediction, then exploration of protein targets was performed using the TCGA dataset via the UALCAN website. Kaplan-Meier was performed using GraphPad with data from the TCGA dataset via Oncoln. Using MOE 2010, we conducted the binding affinity between PGV-1 and diosmin to protein targets.
RESULTS
PGV-1 and diosmin showed cytotoxic effect with IC50 values of 9 µM and 389 µM, respectively, and the combined cytotoxic assay exhibited a synergistic effect with a combination index (CI) of <1. PGV-arrested 4T1 cells in pro-metaphase and induced mitotic catastrophe, while the combination of diosmin with PGV-1 increased the number of mitotic catastrophes. The SA-ß-gal assay revealed that both compounds were capable of inducing senescence in 4T1 cells. Study bioinformatics and molecular docking showed that PGV-1 and diosmin target cell cycle regulatory proteins in TNBC, namely CDK1, KIF11, and AURKA. Thus, the combination of diosmin and PGV-1 modulating the cell cycle that causes senescence and catastrophic death of 4T1 cancer cells is related to the inhibition of these cell cycle proteins.
CONCLUSION
Diosmin enhances the cytotoxic effect of PGV-1 synergistically on 4T1 cancer cells, which correlates to the increasing senescence and mitotic catastrophe. The synergistic effect of the co-treatment is likely to target AURKA, CDK1, and KIF11. The combination of PGV-1 and diosmin performs a potential as a combinatorial anticancer drug for TNBC.
Topics: Antineoplastic Agents; Apoptosis Regulatory Proteins; Curcumin; Diosmin; Drug Therapy, Combination; Female; Humans; Mitosis; Triple Negative Breast Neoplasms
PubMed: 34582664
DOI: 10.31557/APJCP.2021.22.9.2929 -
Development (Cambridge, England) Sep 2022Mutations that occur in RNA-splicing machinery may contribute to hematopoiesis-related diseases. How splicing factor mutations perturb hematopoiesis, especially in the...
Mutations that occur in RNA-splicing machinery may contribute to hematopoiesis-related diseases. How splicing factor mutations perturb hematopoiesis, especially in the differentiation of erythro-myeloid progenitors (EMPs), remains elusive. Dhx38 is a pre-mRNA splicing-related DEAH box RNA helicase, for which the physiological functions and splicing mechanisms during hematopoiesis currently remain unclear. Here, we report that Dhx38 exerts a broad effect on definitive EMPs as well as the differentiation and maintenance of hematopoietic stem and progenitor cells (HSPCs). In dhx38 knockout zebrafish, EMPs and HSPCs were found to be arrested in mitotic prometaphase, accompanied by a 'grape' karyotype, owing to the defects in chromosome alignment. Abnormal alternatively spliced genes related to chromosome segregation, the microtubule cytoskeleton, cell cycle kinases and DNA damage were present in the dhx38 mutants. Subsequently, EMPs and HSPCs in dhx38 mutants underwent P53-dependent apoptosis. This study provides novel insights into alternative splicing regulated by Dhx38, a process that plays a crucial role in the proliferation and differentiation of fetal EMPs and HSPCs.
Topics: Alternative Splicing; Animals; Hematopoiesis; Hematopoietic Stem Cells; Myeloid Progenitor Cells; Zebrafish
PubMed: 35929537
DOI: 10.1242/dev.200450 -
Pharmaceuticals (Basel, Switzerland) Jan 2023Melanoma is considered the most aggressive form of skin cancer, showing high metastatic potential and persistent high mortality rates despite the introduction of...
Melanoma is considered the most aggressive form of skin cancer, showing high metastatic potential and persistent high mortality rates despite the introduction of immunotherapy and targeted therapies. Thus, it is important to identify new drug candidates for melanoma. The design of hybrid molecules, with different pharmacophore fragments combined in the same scaffold, is an interesting strategy for obtaining new multi-target and more effective anticancer drugs. We designed nine hybrid compounds bearing piperine and chlorogenic acid pharmacophoric groups and evaluated their antitumoral potential on melanoma cells with distinct mutational profiles SK-MEL-147, CHL-1 and WM1366. We identified the compound named PQM-277 () to be the most cytotoxic one, inhibiting mitosis progression and promoting an accumulation of cells in pro-metaphase and metaphase by altering the expression of genes that govern G2/M transition and mitosis onset. Compound downregulated , , , , , and , and upregulated . Molecular docking showed that could interact with the CUL1-RBX1 complex, which activity is necessary to trigger molecular events essential for FOXM1 transactivation and, in turn, G2/M gene expression. In addition, compound effectively induced apoptosis by increasing ratio. Our findings demonstrate that is an important antitumor candidate prototype and support further investigations to evaluate its potential for melanoma treatment, especially for refractory cases to BRAF/MEK inhibitors.
PubMed: 37259298
DOI: 10.3390/ph16020145 -
The International Journal of... Nov 2020The nuclear pore protein NUP62 localizes to spindle poles in mitosis and plays a role in maintaining centrosome homeostasis. In this study, we found that NUP62-depleted...
The nuclear pore protein NUP62 localizes to spindle poles in mitosis and plays a role in maintaining centrosome homeostasis. In this study, we found that NUP62-depleted cells exhibited a defective spindle assembly checkpoint (SAC) and that depletion of NUP62 caused a slight decrease in MAD2 protein levels after nocodazole treatment. However, depletion of NUP62 did not cause a failure in kinetochore localization of the SAC proteins BUBR1, MAD1, and MAD2 in prometaphase. NUP62 depletion slightly prolonged mitotic timing but did not affect cell doubling time. In addition, NUP62 depletion caused a SAC defect and induced aneuploidy in human neural stem cells. Furthermore, overexpression of NUP62Q391P, a mutant protein causing autosomal recessive infantile bilateral striatal necrosis, resulted in a defect in the SAC, indicating that the amino acid residue Q391 in NUP62 is crucial for its effect on the SAC. Overall, we conclude that NUP62 maintains the SAC downstream of kinetochores and thereby ensures maintenance of chromosomal stability.
Topics: Amino Acid Substitution; Cell Cycle Checkpoints; Chromosomal Instability; Chromosomes, Human; HCT116 Cells; HeLa Cells; Humans; Membrane Glycoproteins; Mutation, Missense; Nuclear Pore Complex Proteins; Spindle Apparatus
PubMed: 32905854
DOI: 10.1016/j.biocel.2020.105843