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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 -
Cell Cycle (Georgetown, Tex.) Oct 2010The mechanisms that control E2F-1 activity are complex. We previously showed that Chk1 and Chk2 are required for E2F1 stabilization and p73 target gene induction...
The mechanisms that control E2F-1 activity are complex. We previously showed that Chk1 and Chk2 are required for E2F1 stabilization and p73 target gene induction following DNA damage. To gain further insight into the processes regulating E2F1 protein stability, we focused our investigation on the mechanisms responsible for regulating E2F1 turnover. Here we show that E2F1 is a substrate of the anaphase promoting complex or cyclosome (APC/C), a ubiquitin ligase that plays an important role in cell cycle progression. Ectopic expression of the APC/C activators Cdh1 and Cdc20 reduced the levels of co-expressed E2F-1 protein. Co-expression of DP1 with E2F1 blocked APC/C-induced E2F1 degradation, suggesting that the E2F1/DP1 heterodimer is protected from APC/C regulation. Following Cdc20 knockdown, E2F1 levels increased and remained stable in extracts over a time course, indicating that APC/C(Cdc20) is a primary regulator of E2F1 stability in vivo. Moreover, cell synchronization experiments showed that siRNA directed against Cdc20 induced an accumulation of E2F1 protein in prometaphase cells. These data suggest that APC/C(Cdc20) specifically targets E2F1 for degradation in early mitosis and reveal a novel mechanism for limiting free E2F1 levels in cells, failure of which may compromise cell survival and/or homeostasis.
Topics: Amino Acid Sequence; Anaphase-Promoting Complex-Cyclosome; Antigens, CD; Cadherins; Cdc20 Proteins; Cell Cycle Proteins; Checkpoint Kinase 1; Checkpoint Kinase 2; E2F1 Transcription Factor; HeLa Cells; Humans; Prometaphase; Protein Kinases; Protein Serine-Threonine Kinases; RNA, Small Interfering; Transcription Factor DP1; Ubiquitin-Protein Ligase Complexes
PubMed: 20948288
DOI: 10.4161/cc.9.19.13162 -
The EMBO Journal Jul 2009Regulation of BubR1 is central to the control of APC/C activity. We have found that BubR1 forms a complex with PCAF and is acetylated at lysine 250. Using mass...
Regulation of BubR1 is central to the control of APC/C activity. We have found that BubR1 forms a complex with PCAF and is acetylated at lysine 250. Using mass spectrometry and acetylated BubR1-specific antibodies, we have confirmed that BubR1 acetylation occurs at prometaphase. Importantly, BubR1 acetylation was required for checkpoint function, through the inhibition of ubiquitin-dependent BubR1 degradation. BubR1 degradation began before the onset of anaphase. It was noted that the pre-anaphase degradation was regulated by BubR1 acetylation. Degradation of an acetylation-mimetic form, BubR1-K250Q, was inhibited and chromosome segregation in cells expressing BubR1-K250Q was markedly delayed. By contrast, the acetylation-deficient mutant, BubR1-K250R, was unstable, and mitosis was accelerated in BubR1-K250R-expressing cells. Furthermore, we found that APC/C-Cdc20 was responsible for BubR1 degradation during mitosis. On the basis of our collective results, we propose that the acetylation status of BubR1 is a molecular switch that converts BubR1 from an inhibitor to a substrate of the APC/C complex, thus providing an efficient way to modulate APC/C activity and mitotic timing.
Topics: Acetylation; Amino Acid Sequence; Anaphase-Promoting Complex-Cyclosome; Animals; Cdc20 Proteins; Cell Cycle Proteins; Genes, cdc; HeLa Cells; Humans; Kinetochores; Mitosis; Molecular Sequence Data; Protein Serine-Threonine Kinases; Protein Stability; Sequence Alignment; Spindle Apparatus; Ubiquitin-Protein Ligase Complexes; Ubiquitination; p300-CBP Transcription Factors
PubMed: 19407811
DOI: 10.1038/emboj.2009.123 -
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 -
Molecular Biology of the Cell Feb 2019The nuclear envelope (NE) aids in organizing the interphase genome by tethering chromatin to the nuclear periphery. During mitotic entry, NE-chromatin contacts are...
The nuclear envelope (NE) aids in organizing the interphase genome by tethering chromatin to the nuclear periphery. During mitotic entry, NE-chromatin contacts are broken. Here, we report on the consequences of impaired NE removal from chromatin for cell division of human cells. Using a membrane-chromatin tether that cannot be dissociated when cells enter mitosis, we show that a failure in breaking membrane-chromatin interactions impairs mitotic chromatin organization, chromosome segregation and cytokinesis, and induces an aberrant NE morphology in postmitotic cells. In contrast, chromosome segregation and cell division proceed successfully when membrane attachment to chromatin is induced during metaphase, after chromosomes have been singularized and aligned at the metaphase plate. These results indicate that the separation of membranes and chromatin is critical during prometaphase to allow for proper chromosome compaction and segregation. We propose that one cause of these defects is the multivalency of membrane-chromatin interactions.
Topics: Cell Nucleus Shape; Chromatin; Chromosome Segregation; Endoplasmic Reticulum; HeLa Cells; Humans; Intracellular Membranes; M Phase Cell Cycle Checkpoints; Membrane Proteins; Metaphase; Mitosis; Nuclear Envelope; Protein Binding; Solubility
PubMed: 30586323
DOI: 10.1091/mbc.E18-10-0609 -
Journal of Cell Science Jan 2011The Aurora-A kinase has well-established roles in spindle assembly and function and is frequently overexpressed in tumours. Its abundance is cell cycle regulated, with a...
The Aurora-A kinase has well-established roles in spindle assembly and function and is frequently overexpressed in tumours. Its abundance is cell cycle regulated, with a peak in G2 and M phases, followed by regulated proteolysis at the end of mitosis. The microtubule-binding protein TPX2 plays a major role in regulating the activity and localisation of Aurora-A in mitotic cells. Here, we report a novel regulatory role of TPX2 and show that it protects Aurora-A from degradation both in interphase and in mitosis in human cells. Specifically, Aurora-A levels decrease in G2 and prometaphase cells silenced for TPX2, whereas degradation of Aurora-A is impaired in telophase cells overexpressing the Aurora-A-binding region of TPX2. The decrease in Aurora-A in TPX2-silenced prometaphases requires proteasome activity and the Cdh1 activator of the APC/C ubiquitin ligase. Reintroducing either full-length TPX2, or the Aurora-A-binding region of TPX2, but not a truncated TPX2 mutant lacking the Aurora-A-interaction domain, restores Aurora-A levels in TPX2-silenced prometaphases. The control by TPX2 of Aurora-A stability is independent of its ability to activate Aurora-A and to localise it to the spindle. These results highlight a novel regulatory level impinging on Aurora-A and provide further evidence for the central role of TPX2 in regulation of Aurora-A.
Topics: Aurora Kinases; Cell Cycle Proteins; Cell Line, Tumor; G2 Phase; Humans; Microtubule-Associated Proteins; Mitosis; Nuclear Proteins; Protein Binding; Protein Serine-Threonine Kinases; Protein Stability; Protein Structure, Tertiary
PubMed: 21147853
DOI: 10.1242/jcs.075457 -
Current Biology : CB Jun 2004To test current models for how unattached and untense kinetochores prevent Cdc20 activation of the anaphase-promoting complex/cyclosome (APC/C) throughout the spindle... (Comparative Study)
Comparative Study
BACKGROUND
To test current models for how unattached and untense kinetochores prevent Cdc20 activation of the anaphase-promoting complex/cyclosome (APC/C) throughout the spindle and the cytoplasm, we used GFP fusions and live-cell imaging to quantify the abundance and dynamics of spindle checkpoint proteins Mad1, Mad2, Bub1, BubR1, Mps1, and Cdc20 at kinetochores during mitosis in living PtK2 cells.
RESULTS
Unattached kinetochores in prometaphase bound on average only a small fraction (estimated at 500-5000 molecules) of the total cellular pool of each spindle checkpoint protein. Measurements of fluorescence recovery after photobleaching (FRAP) showed that GFP-Cdc20 and GFP-BubR1 exhibit biphasic exponential kinetics at unattached kinetochores, with approximately 50% displaying very fast kinetics (t1/2 of approximately 1-3 s) and approximately 50% displaying slower kinetics similar to the single exponential kinetics of GFP-Mad2 and GFP-Bub3 (t1/2 of 21-23 s). The slower phase of GFP-Cdc20 likely represents complex formation with Mad2 since it was tension insensitive and, unlike the fast phase, it was absent at metaphase kinetochores that lack Mad2 but retain Cdc20 and was absent at unattached prometaphase kinetochores for the Cdc20 derivative GFP-Cdc20delta1-167, which lacks the major Mad2 binding domain but retains kinetochore localization. GFP-Mps1 exhibited single exponential kinetics at unattached kinetochores with a t1/2 of approximately 10 s, whereas most GFP-Mad1 and GFP-Bub1 were much more stable components.
CONCLUSIONS
Our data support catalytic models of checkpoint activation where Mad1 and Bub1 are mainly resident, Mad2 free of Mad1, BubR1 and Bub3 free of Bub1, Cdc20, and Mps1 dynamically exchange as part of the diffuse wait-anaphase signal; and Mad2 interacts with Cdc20 at unattached kinetochores.
Topics: Anaphase-Promoting Complex-Cyclosome; Blotting, Western; Calcium-Binding Proteins; Cdc20 Proteins; Cell Cycle Proteins; Diagnostic Imaging; Fluorescence Recovery After Photobleaching; Green Fluorescent Proteins; HeLa Cells; Humans; Kinetochores; Luminescent Proteins; Mad2 Proteins; Metalloproteins; Mitosis; Models, Biological; Nuclear Proteins; Phosphoproteins; Precipitin Tests; Protein Kinases; Protein Serine-Threonine Kinases; RNA-Binding Proteins; Repressor Proteins; Ribosomal Proteins; Signal Transduction; Transfection; Ubiquitin-Protein Ligase Complexes
PubMed: 15182668
DOI: 10.1016/j.cub.2004.05.053 -
Nature Cell Biology Oct 2007The first female meiotic division (meiosis I, MI) is uniquely prone to chromosome segregation errors through non-disjunction, resulting in trisomies and early pregnancy...
The first female meiotic division (meiosis I, MI) is uniquely prone to chromosome segregation errors through non-disjunction, resulting in trisomies and early pregnancy loss. Here, we show a fundamental difference in the control of mammalian meiosis that may underlie such susceptibility. It involves a reversal in the well-established timing of activation of the anaphase-promoting complex (APC) by its co-activators cdc20 and cdh1. APC(cdh1) was active first, during prometaphase I, and was needed in order to allow homologue congression, as loss of cdh1 speeded up MI, leading to premature chromosome segregation and a non-disjunction phenotype. APC(cdh1) targeted cdc20 for degradation, but did not target securin or cyclin B1. These were degraded later in MI through APC(cdc20), making cdc20 re-synthesis essential for successful meiotic progression. The switch from APC(cdh1) to APC(cdc20) activity was controlled by increasing CDK1 and cdh1 loss. These findings demonstrate a fundamentally different mechanism of control for the first meiotic division in mammalian oocytes that is not observed in meioses of other species.
Topics: Anaphase-Promoting Complex-Cyclosome; Animals; Blotting, Western; Carrier Proteins; Cdc20 Proteins; Cell Cycle Proteins; Cyclin B; Cyclin B1; Female; Meiosis; Mice; Microscopy, Fluorescence; Oocytes; Prometaphase; Securin; Time Factors; Ubiquitin-Protein Ligase Complexes
PubMed: 17891138
DOI: 10.1038/ncb1640 -
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 -
Journal of Visualized Experiments : JoVE Dec 2017In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk of conceiving...
In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk of conceiving an aneuploid fetus increases dramatically and this phenomenon is known as the maternal age effect. One requirement for accurate chromosome segregation during the meiotic divisions is maintenance of sister chromatid cohesion during the extended prophase period that oocytes experience. Cytological evidence in both humans and model organisms suggests that meiotic cohesion deteriorates during the aging process. In addition, segregation errors in human oocytes are most prevalent during meiosis I, consistent with premature loss of arm cohesion. The use of model organisms is critical for unraveling the mechanisms that underlie age-dependent loss of cohesion. Drosophila melanogaster offers several advantages for studying the regulation of meiotic cohesion in oocytes. However, until recently, only genetic tests were available to assay for loss of arm cohesion in oocytes of different genotypes or under different experimental conditions. Here, a detailed protocol is provided for using fluorescence in situ hybridization (FISH) to directly visualize defects in arm cohesion in prometaphase I and metaphase I arrested Drosophila oocytes. By generating a FISH probe that hybridizes to the distal arm of the X chromosome and collecting confocal Z stacks, a researcher can visualize the number of individual FISH signals in three dimensions and determine whether sister chromatid arms are separated. The procedure outlined makes it possible to quantify arm cohesion defects in hundreds of Drosophila oocytes. As such, this method provides an important tool for investigating the mechanisms that contribute to cohesion maintenance as well as the factors that lead to its demise during the aging process.
Topics: Animals; Drosophila; Female; Humans; In Situ Hybridization, Fluorescence; Metaphase; Oocytes; Prometaphase
PubMed: 29286418
DOI: 10.3791/56802