Cell Division最新文献

Background: Ral family is a member of Ras-like GTPase superfamily, which includes RalA and RalB. RalA/B play important roles in many cell biological functions, including cytoskeleton dynamics, cell division, membrane transport, gene expression and signal transduction. However, whether RalA/B involve into the mammalian oocyte meiosis is still unclear. This study aimed to explore the roles of RalA/B during mouse oocyte maturation.

Results: Our results showed that RalA/B expressed at all stages of oocyte maturation, and they were enriched at the spindle periphery area after meiosis resumption. The injection of RalA/B siRNAs into the oocytes significantly disturbed the polar body extrusion, indicating the essential roles of RalA/B for oocyte maturation. We observed that in the RalA/B knockdown oocytes the actin filament fluorescence intensity was significantly increased at the both cortex and cytoplasm, and the chromosomes were failed to locate near the cortex, indicating that RalA/B regulate actin dynamics for spindle migration in mouse oocytes. Moreover, we also found that the Golgi apparatus distribution at the spindle periphery was disturbed after RalA/B depletion.

Conclusions: In summary, our results indicated that RalA/B affect actin dynamics for chromosome positioning and Golgi apparatus distribution in mouse oocytes.

Background: Cancer cell aggregation is a key process involved in the formation of tumor cell clusters. It has recently been shown that clusters of circulating tumor cells (CTCs) have an increased metastatic potential compared to isolated circulating tumor cells. Several widely used chemotherapeutic agents that target the cytoskeleton microtubules and cause cell cycle arrest at mitosis have been reported to modulate CTC number or the size of CTC clusters.

Results: In this study, we investigated in vitro the impact of mitotic arrest on the ability of breast tumor cells to form clusters. By using live imaging and quantitative image analysis, we found that MCF-7 cancer cell aggregation is compromised upon incubation with paclitaxel or vinorelbine, two chemotherapeutic drugs that target microtubules. In line with these results, we observed that MCF-7 breast cancer cells experimentally synchronized and blocked in metaphase aggregated poorly and formed loose clusters. To monitor clustering at the single-cell scale, we next developed and validated an in vitro assay based on live video-microscopy and custom-designed micro-devices. The study of cluster formation from MCF-7 cells that express the fluorescent marker LifeAct-mCherry using this new assay allowed showing that substrate anchorage-independent clustering of MCF-7 cells was associated with the formation of actin-dependent highly dynamic cell protrusions. Metaphase-synchronized and blocked cells did not display such protrusions, and formed very loose clusters that failed to compact.

Conclusions: Altogether, our results suggest that mitotic arrest induced by microtubule-targeting anticancer drugs prevents cancer cell clustering and therefore, could reduce the metastatic potential of circulating tumor cells.

Background: Infantile hemangioma (IH) is the most common benign tumor in children. Long noncoding RNAs (lncRNAs) play a critical role in tumorigenesis. However, the expression levels and biological functions of lncRNAs in IH have not been well-studied. This study aimed to analyze the expression profile of lncRNAs and mRNAs in proliferating and involuting IHs.

Methods: The expression profiles of lncRNAs and mRNAs in proliferating and involuting IHs were identified by microarray analysis. Subsequently, detailed bioinformatics analyses were performed. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) analyses were conducted to validate the microarray results.

Results: In total, 146 differentially expressed (DE) lncRNAs and 374 DE mRNAs were identified. The DE mRNAs were enriched mostly in angiogenesis-related biological processes (BPs) and pathways by bioinformatics analysis. In addition, metabolism-related BPs (e.g., "glycogen biosynthetic process" and "metabolic process") and pathways (e.g., "oxidative phosphorylation") were identified. A lncRNA-mRNA co-expression network was constructed from 42 DE lncRNAs and 217 DE mRNAs. Twelve lncRNAs were predicted to have cis-regulated target genes. The microarray analysis results were validated by qRT-PCR using 5 randomly selected lncRNAs and 13 mRNAs. The IHC results revealed that both LOXL2 and FPK-1 exhibited higher protein expression levels in proliferating IH than in involuting IH. Moreover, inhibition of PFK-1 could suppress hemangioma-derived endothelial cell proliferation and migration, induce cell arrest, and reduce glucose uptake and lactate and ATP production.

Conclusions: The findings suggest that the identified DE lncRNAs and mRNAs may be associated with the pathogenesis of IH. The data presented herein can improve our understanding of IH development and provide direction for further studies investigating the mechanism underlying IH.

Background: Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) has opened new therapeutic possibilities. However, karyotypic abnormalities detected in iPSCs compromised their utility, especially chromosomal aberrations found at early passages raised serious safety concerns. The mechanism underlying the chromosomal abnormality in early-passage iPSCs is not known.

Methods: Human dermal fibroblasts (HDFs) were stimulated with KMOS (KLF4, cMYC, OCT4 and SOX2) proteins to enhance their proliferative capacity and many vigorous clones were obtained. Clonal reprogramming was carried out by KMOS mRNAs transfection to confirm the 'chromosomal mutagenicity' of reprogramming process. Subculturing was performed to examine karyotypic stability of iPSCs after the re-establishment of stemness. And antioxidant N-acetyl-cysteine (NAC) was added to the culture medium for further confirmming the mutagenicity in the first few days of reprogramming.

Results: Chromosomal aberrations were found in a small percentage of newly induced iPS clones by reprogramming transcription factors. Clonal reprogramming ruled out the aberrant chromosomes inherited from rare karyotypically abnormal parental cell subpopulation. More importantly, the antioxidant NAC effectively reduced the occurrence of chromosomal aberrations at the early stage of reprogramming. Once iPS cell lines were established, they restored karyotypic stability in subsequent subculturing.

Conclusions: Our results provided the first line of evidence for the 'chromosomal mutagenicity' of reprogramming process.

Background: XMU-MP-1 is an inhibitor of the Hippo pathway kinases MST1/2 and has been shown to promote the downstream activation of the pro-proliferative, pro-regenerative and anti-apoptotic transcriptional regulator YAP1. We tested whether XMU-MP-1 can activate YAP1 in a model human mini-organ, namely the hair follicle, to determine whether it can be pharmacologically exploited to promote regeneration in the hair follicle as a novel strategy to treat pathological hair loss disorders.

Results: XMU-MP-1 treatment inhibited MOB1 phosphorylation but did not increase active YAP1 in the hair follicle. Rather than promote proliferation, XMU-MP-1 serendipitously decreased the number of Ki-67+, EdU+ and phospho histone H3+ hair matrix keratinocytes and antagonised the cytotoxic effects of paclitaxel.

Conclusions: XMU-MP-1 perturbs epithelial cell cycle progression in a model human mini-organ. This may arise as an off-target effect, especially when XMU-MP-1 has been described to strongly inhibit 21 additional kinases beyond MST1/2. Therefore, whilst these effects may be dependent on tissue context, researchers should exercise caution when interpreting the effects of XMU-MP-1, especially in tissues with actively proliferating cell populations.

The evolutionarily conserved Cdc25 phosphatase is an essential protein that removes inhibitory phosphorylation moieties on the mitotic regulator Cdc2. Together with the Wee1 kinase, a negative regulator of Cdc2 activity, Cdc25 is thus a central regulator of cell cycle progression in Schizosaccharomyces pombe. The expression and activity of Cdc25 is dependent on the activity of the Target of Rapamycin Complex 1 (TORC1). TORC1 inhibition leads to the activation of Cdc25 and repression of Wee1, leading to advanced entry into mitosis. Withdrawal of nitrogen leads to rapid Cdc25 degradation via the ubiquitin- dependent degradation pathway by the Pub1 E3- ligase. Caffeine is believed to mediate the override of DNA damage checkpoint signalling, by inhibiting the activity of the ataxia telangiectasia mutated (ATM)/Rad3 homologues. This model remains controversial, as TORC1 appears to be the preferred target of caffeine in vivo. Recent studies suggest that caffeine induces DNA damage checkpoint override by inducing the nuclear accumulation of Cdc25 in S. pombe. Caffeine may thus modulate Cdc25 activity and stability via inhibition of TORC1. A clearer understanding of the mechanisms by which caffeine stabilises Cdc25, may provide novel insights into how TORC1 and DNA damage signalling is integrated.

Background: The enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase and induces the trimethylation of histone H3 lysine 27 (H3K27me3) in the promoter of many key genes; EZH2 acts as a transcriptional repressor and is an epigenetic regulator for several cancers. However, the role of EZH2 in nonneoplastic diseases, such as kidney diseases, is unknown and has been investigated.

Materials and method: NRK-52E cells were treated with DZNep, a potent inhibitor of EZH2, with different concentrations and for different times to evaluate the apoptosis level of NRK-52E cells by Western blot and Flow cytometry analysis. The binding of EZH2 to the Deptor promoter was determined by ChIP assay.

Results: The inhibition of EZH2 with 3-deazaneplanocin A (DZNep), a specific inhibitor of EZH2, led to the apoptosis of NRK-52E cells and the inhibition of mTORC1 and mTORC2 activity. A ChIP assay demonstrated that EZH2 bound the promoter region of Deptor, an endogenous inhibitor of mTORC1 and mTORC2, and regulated the transcription of Deptor by modulating H3K27me3 in its promoter region. Further experiments were performed to examine the effects of EZH2 inhibition on cisplatin-induced injured cells. Cisplatin induced the activation of mTORC1 and mTORC2 and apoptosis in NRK-52E cells, and DZNep inhibited mTORC1 and mTORC2 activity and aggravated cell apoptosis.

Conclusions: These data suggested that EZH2 inhibition increased the transcription of Deptor by modifying H3K27me3 in its promoter region, subsequently inhibited mTORC1 and mTORC2 activities, downregulated the expression of apoptosis suppressor genes, and finally led to apoptosis in renal tubular cells. The inhibition of EZH2 aggravated the cisplatin-induced injury in renal tubular cells by inactivating the mTOR complexes. The present study provides new insight into renal protection and suggests that EZH2 might be a target.

Cell division is orchestrated by the phosphorylation and dephosphorylation of thousands of proteins. These post-translational modifications underlie the molecular cascades converging to the activation of the universal mitotic kinase, Cdk1, and entry into cell division. They also govern the structural events that sustain the mechanics of cell division. While the role of protein kinases in mitosis has been well documented by decades of investigations, little was known regarding the control of protein phosphatases until the recent years. However, the regulation of phosphatase activities is as essential as kinases in controlling the activation of Cdk1 to enter M-phase. The regulation and the function of phosphatases result from post-translational modifications but also from the combinatorial association between conserved catalytic subunits and regulatory subunits that drive their substrate specificity, their cellular localization and their activity. It now appears that sequential dephosphorylations orchestrated by a network of phosphatase activities trigger Cdk1 activation and then order the structural events necessary for the timely execution of cell division. This review discusses a series of recent works describing the important roles played by protein phosphatases for the proper regulation of meiotic division. Many breakthroughs in the field of cell cycle research came from studies on oocyte meiotic divisions. Indeed, the meiotic division shares most of the molecular regulators with mitosis. The natural arrests of oocytes in G2 and in M-phase, the giant size of these cells, the variety of model species allowing either biochemical or imaging as well as genetics approaches explain why the process of meiosis has served as an historical model to decipher signalling pathways involved in the G2-to-M transition. The review especially highlights how the phosphatase PP2A-B55δ critically orchestrates the timing of meiosis resumption in amphibian oocytes. By opposing the kinase PKA, PP2A-B55δ controls the release of the G2 arrest through the dephosphorylation of their substrate, Arpp19. Few hours later, the inhibition of PP2A-B55δ by Arpp19 releases its opposing kinase, Cdk1, and triggers M-phase. In coordination with a variety of phosphatases and kinases, the PP2A-B55δ/Arpp19 duo therefore emerges as the key effector of the G2-to-M transition.

Background: Apoptosis plays pivotal roles in organ development and tissue homeostasis, with its major function to remove unhealthy cells that may compromise the fitness of the organism. Toll signaling, with the ancient evolutionary origin, regulates embryonic dorsal-ventral patterning, axon targeting and degeneration, and innate immunity. Using Drosophila as a genetic model, we characterized the role of Toll signaling in apoptotic cell death.

Results: We found that gain of Toll signaling is able to trigger caspase-dependent cell death in development. In addition, JNK activity is required for Toll-induced cell death. Furthermore, ectopic Toll expression induces the activation of JNK pathway. Moreover, physiological activation of Toll signaling is sufficient to produce JNK-dependent cell death. Finally, Toll signaling activates JNK-mediated cell death through promoting ROS production.

Conclusions: As Toll pathway has been evolutionarily conserved from Drosophila to human, this study may shed light on the mechanism of mammalian Toll-like receptors (TLRs) signaling in apoptotic cell death.

Background: Microtubule organization is essential for bipolar spindle assembly and chromosome segregation, which contribute to genome stability. Kinesin-5 Eg5 is known to be a crucial regulator in centrosome separation and spindle assembly in mammalian somatic cells, however, the functions and mechanisms of Eg5 in male meiotic cell division remain largely unknown.

Results: In this study, we have found that Eg5 proteins are expressed in mouse spermatogonia, spermatocytes and spermatids. After Eg5 inhibition by specific inhibitors Monastrol, STLC and Dimethylenastron, the meiotic spindles of dividing spermatocytes show spindle collapse and the defects in bipolar spindle formation. We demonstrate that Eg5 regulates spindle bipolarity and the maintenance of meiotic spindles in meiosis. Eg5 inhibition leads to monopolar spindles, spindle abnormalities and chromosome misalignment in cultured GC-2 spd cells. Furthermore, Eg5 inhibition results in the decrease of the spermatids and the abnormalities in mature sperms.

Conclusions: Our results have revealed an important role of kinesin-5 Eg5 in male meiosis and the maintenance of male fertility. We demonstrate that Eg5 is crucial for bipolar spindle assembly and chromosome alignment in dividing spermatocytes. Our data provide insights into the functions of Eg5 in meiotic spindle assembly of dividing spermatocytes.