Spindle Microtubule Assembly Research Articles

Target Identification and Mechanistic Characterization of Indole Terpenoid Mimics: Proper Spindle Microtubule Assembly Is Essential for Cdh1-Mediated Proteolysis of CENP-A.

Centromere protein A (CENP-A), a centromere-specific histone H3 variant, is crucial for kinetochore positioning and chromosome segregation. However, its regulatory mechanism in human cells remains incompletely understood. A structure-activity relationship (SAR) study of the cell-cycle-arresting indole terpenoid mimic JP18 leads to the discovery of two more potent analogs, (+)-6-Br-JP18 and (+)-6-Cl-JP18. Tubulin is identified as a potential cellular target of these halogenated analogs by using the drug affinity responsive target stability (DARTS) based method. X-ray crystallography analysis reveals that both molecules bind to the colchicine-binding site of β-tubulin. Treatment of human cells with microtubule-targeting agents (MTAs), including these two compounds, results in CENP-A accumulation by destabilizing Cdh1, a co-activator of the anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase. This study establishes a link between microtubule dynamics and CENP-A accumulation using small-molecule tools and highlights the role of Cdh1 in CENP-A proteolysis.

Antifungal Activity and Action Mechanisms of 2,4-Di-tert-butylphenol against Ustilaginoidea virens.

Ustilaginoidea virens is a destructive phytopathogenic fungus that causes false smut disease in rice. In this study, the natural product 2,4-di-tert-butylphenol (2,4-DTBP) was found to be an environmentally friendly and effective agent for the first time, which exhibited strong antifungal activity against U. virens, with an EC50 value of 0.087 mmol/L. The scanning electron microscopy, fluorescence staining, and biochemical assays indicated that 2,4-DTBP could destroy the cell wall, cell membrane, and cellular redox homeostasis of U. virens, ultimately resulting in fungal cell death. Through the transcriptomic analysis, a total of 353 genes were significantly upregulated and 367 genes were significantly downregulated, focusing on the spindle microtubule assembly, cell wall and membrane, redox homeostasis, mycotoxin biosynthesis, and intracellular metabolism. These results enhanced the understanding of the antifungal activity and action mechanisms of 2,4-DTBP against U. virens, supporting it to be a potential antifungal agent for the control of false smut disease.

TACC3-ch-TOG interaction regulates spindle microtubule assembly by controlling centrosomal recruitment of γ-TuRC.

γ-Tubulin ring complex (γ-TuRC), composed of γ-tubulin and multiple γ-tubulin complex proteins (GCPs), serves as the major microtubule nucleating complex in animal cells. However, several γ-TuRC-associated proteins have been shown to control its function. Centrosomal adaptor protein, TACC3, is one such γ-TuRC-interacting factor that is essential for proper mitotic spindle assembly across organisms. ch-TOG is another microtubule assembly promoting protein, which interacts with TACC3 and cooperates in mitotic spindle assembly. However, the mechanism how TACC3-ch-TOG interaction regulates microtubule assembly and the γ-TuRC functions at the centrosomes remain unclear. Here, we show that deletion of the ch-TOG-binding region in TACC3 enhances recruitment of the γ-TuRC proteins to centrosomes and aggravates spindle microtubule assembly in human cells. Loss of TACC3-ch-TOG binding imparts stabilization on TACC3 interaction with the γ-TuRC proteins and it does so by stimulating TACC3 phosphorylation and thereby enhancing phospho-TACC3 recruitment to the centrosomes. We also show that localization of ch-TOG at the centrosomes is substantially reduced and the same on the spindle microtubules is increased in its TACC3-unbound condition. Additional results reveal that ch-TOG depletion stimulates γ-tubulin localization on the spindles without significantly affecting the centrosomal γ-tubulin level. The results indicate that ch-TOG binding to TACC3 controls TACC3 phosphorylation and TACC3-mediated stabilization of the γ-TuRCs at the centrosomes. They also implicate that the spatio-temporal control of TACC3 phosphorylation via ch-TOG-binding ensures mitotic spindle assembly to the optimal level.

ASPM Is a Prognostic Biomarker and Correlates With Immune Infiltration in Kidney Renal Clear Cell Carcinoma and Liver Hepatocellular Carcinoma.

BackgroundAbnormal spindle microtubule assembly (ASPM) is a centrosomal protein and that is related to a poor clinical prognosis and recurrence. However, the relationship between ASPM expression, tumor immunity, and the prognosis of different cancers remains unclear.MethodsASPM expression and its influence on tumor prognosis were analyzed using the Tumor Immune Estimation Resource (TIMER), UALCAN, OncoLnc, and Gene Expression Profiling Interactive Analysis (GEPIA) databases. The relationship between ASPM expression and tumor immunity was analyzed using the TIMER and GEPIA databases, and the results were further verified using qPCR, western blot, and multiplex quantitative immuno fluorescence.ResultsThe results showed that ASPM expression was significantly higher in most cancer tissues than in corresponding normal tissues, including kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), pancreatic adenocarcinoma (PAAD), and breast invasive carcinoma (BRCA). ASPM expression was significantly higher in late-stage cancers than in early-stages cancers (e.g., KIRC, KIRP, LIHC, LUAD, and BRCA; p < 0.05), demonstrating a possible role of ASPM in cancer progression and invasion. Moreover, our data showed that high ASPM expression was associated with poor overall survival, and disease-specific survival in KIRC and LIHC (p < 0.05). Besides, Cox hazard regression analysis results showed that ASPM may be an independent prognostic factor for KIRC and LIHC. ASPM expression showed a strong correlation with tumor-infiltrating B cells, CD8+ T cells, and M2 macrophages in KIRC and LIHC.ConclusionsThese findings demonstrate that the high expression of ASPM indicates poor prognosis as well as increased levels of immune cell infiltration in KIRC and LIHC. ASPM expression may serve as a novel prognostic biomarker for both the clinical outcome and immune cell infiltration in KIRC and LIHC.

Functional midbody assembly in the absence of a central spindle.

Contractile ring constriction during cytokinesis is thought to compact central spindle microtubules to form the midbody, an antiparallel microtubule bundle at the intercellular bridge. In Caenorhabditis elegans, central spindle microtubule assembly requires targeting of the CLASP family protein CLS-2 to the kinetochores in metaphase and spindle midzone in anaphase. CLS-2 targeting is mediated by the CENP-F-like HCP-1/2, but their roles in cytokinesis and midbody assembly are not known. We found that although HCP-1 and HCP-2 mostly function cooperatively, HCP-1 plays a more primary role in promoting CLS-2-dependent central spindle microtubule assembly. HCP-1/2 codisrupted embryos did not form central spindles but completed cytokinesis and formed functional midbodies capable of supporting abscission. These central spindle-independent midbodies appeared to form via contractile ring constriction-driven bundling of astral microtubules at the furrow tip. This work suggests that, in the absence of a central spindle, astral microtubules can support midbody assembly and that midbody assembly is more predictive of successful cytokinesis than central spindle assembly.

Mutation screening of multiple Pakistani MCPH families revealed novel and recurrent protein-truncating mutations of ASPM.

Autosomal primary microcephaly (MCPH) is a heterogenetic disorder that affects brain's cerebral cortex size and leads to a reduction in the cranial vault. Along with the hallmark feature of reduced head circumference, microcephalic patients also exhibit a variable degree of intellectual disability as well. Genetic studies have reported 28 MCPH genes, most of which produce microtubule-associated proteins and are involved in cell division. Herein this study, 14 patients from seven Pashtun origin Pakistani families of primary microcephaly were analyzed. Mutation analysis was performed through targeted Sanger DNA sequencing on the basis of phenotype-linked genetic makeup. Genetic analysis in one family found a novel pathogenic DNA change in the abnormal spindle microtubule assembly (ASPM) gene (NM_018136.4:c.3871dupGA), while the rest of the families revealed recurrent nonsense mutation c.3978G>A (p.Trp1326*) in the same gene. The novel reported frameshift insertion presumably truncates the protein p.(Lys1291Glyfs*14) and deletes the N-terminus domains. Identification of novel ASPM-truncating mutation expands the mutational spectrum of the ASPM gene, while mapping of recurrent mutation c.3978G>A (p.Trp1326*) will aid in establishing its founder effect in the Khyber Pakhtunkhwa (KPK) inhabitant population of Pakistan and should be suggestively screened for premarital counseling of MCPH susceptible families. Most of the recruited families are related to first-degree consanguinity. Hence, all the family elders were counseled to avoid intrafamilial marriages.

HN1 interacts with γ-tubulin to regulate centrosomes in advanced prostate cancer cells

ABSTRACT Prostate cancer is one of the most common cancer for men worldwide with advanced forms showing supernumerary or clustered centrosomes. Hematological and neurological expressed 1 (HN1) also known as Jupiter Microtubule Associated Homolog 1 (JPT1) belongs to a small poorly understood family of genes that are evolutionarily conserved across vertebrate species. The co-expression network of HN1 from the TCGA PRAD dataset indicates the putative role of HN1 in centrosome-related processes in the context of prostate cancer. HN1 expression is low in normal RWPE-1 cells as compared to cancerous androgen-responsive LNCaP and androgen insensitive PC-3 cells. HN1 overexpression resulted in differential response for cell proliferation and cell cycle changes in RWPE-1, LNCaP, and PC-3 cells. Since HN1 overexpression increased the proliferation rate in PC-3 cells, these cells were used for functional characterization of HN1 in advanced prostate carcinogenesis. Furthermore, alterations in HN expression led to an increase in abnormal to normal nuclei ratio and increased chromosomal aberrations in PC-3 cells. We observed the co-localization of HN1 with γ-tubulin foci in prostate cancer cells, further validated by immunoprecipitation. HN1 was observed as physically associated with γ-tubulin and its depletion led to increased γ-tubulin foci and disruption in microtubule spindle assembly. Higher HN1 expression was correlated with prostate cancer as compared to normal tissues. The restoration of HN1 expression after silencing suggested that it has a role in centrosome clustering, implicating a potential role of HN1 in cell division as well as in prostate carcinogenesis warranting further studies.

The critical role of B4GALT4 in promoting microtubule spindle assembly in HCC through the regulation of PLK1 and RHAMM expression.

Beta 1,4-galactosyltransferase (B4GALT)-family glycosyltransferases are involved in multiple biological processes promoting cancer progression, regulating the dynamic network of cancer cell proliferation and apoptosis, and are associated with metastasis. However, their roles in the dysregulation of expressions and functions in hepatocellular carcinoma (HCC) remain unclear. Herein, bioinformatic approaches have been applied to investigate their expression profiles, and to obtain correlations between gene expressions and clinicopathological parameters as well as downstream target genes in HCC. Multiple databases were used to screen the expressions of B4GALT family members in tumor tissues, and to evaluate their prognostic value among HCC patients in different aspects. Results indicated an overall upregulation of B4GALTs' transcription levels in tumor tissues and a strong correlation with poor prognosis. Through Gene Ontology analysis, gene set enrichment analysis, and verification of single-cell RNA sequencing data, we established a connection between the B4GALT family and microtubule spindle assembly, which particularly highlighted the role of B4GALT4 in this phenomenon. B4GALT4 knockdown downregulated the production of lumican, and repressed the expressions of polo-like kinase 1 and RHAMM by regulating the transforming growth factor-beta pathway, thus suggesting that B4GALT4 is a critical promotor for HCC. We believe that these studies will provide valuable insight into the role of B4GALT family members in HCC and lead to the development of new strategies to improve the outcomes for patients with HCC.

Selective targeting of non-centrosomal AURKA functions through use of a targeted protein degradation tool

Targeted protein degradation tools are becoming a new therapeutic modality, allowing small molecule ligands to be reformulated as heterobifunctional molecules (PROteolysis Targeting Chimeras, PROTACs) that recruit ubiquitin ligases to targets of interest, leading to ubiquitination and destruction of the targets. Several PROTACs against targets of clinical interest have been described, but detailed descriptions of the cell biology modulated by PROTACs are missing from the literature. Here we describe the functional characterization of a PROTAC derived from AURKA inhibitor MLN8237 (alisertib). We demonstrate efficient and specific destruction of both endogenous and overexpressed AURKA by Cereblon-directed PROTACs. At the subcellular level, we find differential targeting of AURKA on the mitotic spindle compared to centrosomes. The phenotypic consequences of PROTAC treatment are therefore distinct from those mediated by alisertib, and in mitotic cells differentially regulate centrosome- and chromatin- based microtubule spindle assembly pathways. In interphase cells PROTAC-mediated clearance of non-centrosomal AURKA modulates the cytoplasmic role played by AURKA in mitochondrial dynamics, whilst the centrosomal pool is refractory to PROTAC-mediated clearance. Our results point to differential sensitivity of subcellular pools of substrate, governed by substrate conformation or localization-dependent accessibility to PROTAC action, a phenomenon not previously described for this new class of degrader compounds.

Spastin interacts with CRMP5 to promote spindle organization in mouse oocytes by severing microtubules.

Microtubule-severing protein (MTSP) is critical for the survival of both mitotic and postmitotic cells. However, the study of MTSP during meiosis of mammalian oocytes has not been reported. We found that spastin, a member of the MTSP family, was highly expressed in oocytes and aggregated in spindle microtubules. After knocking down spastin by specific siRNA, the spindle microtubule density of meiotic oocytes decreased significantly. When the oocytes were cultured in vitro, the oocytes lacking spastin showed an obvious maturation disorder. Considering the microtubule-severing activity of spastin, we speculate that spastin on spindles may increase the number of microtubule broken ends by severing the microtubules, therefore playing a nucleating role, promoting spindle assembly and ensuring normal meiosis. In addition, we found the colocalization and interaction of collapsin response mediator protein 5 (CRMP5) and spastin in oocytes. CRMP5 can provide structural support and promote microtubule aggregation, creating transportation routes, and can interact with spastin in the microtubule activity of nerve cells (30). Knocking down CRMP5 may lead to spindle abnormalities and developmental disorders in oocytes. Overexpression of spastin may reverse the abnormal phenotype caused by the deletion of CRMP5. In summary, our data support a model in which the interaction between spastin and CRMP5 promotes the assembly of spindle microtubules in oocytes by controlling microtubule dynamics, therefore ensuring normal meiosis.

Identification of Hub Genes Associated With Immune Infiltration and Predict Prognosis in Hepatocellular Carcinoma via Bioinformatics Approaches.

AimsIn the cancer-related research field, there is currently a major need for a greater number of valuable biomarkers to predict the prognosis of hepatocellular carcinoma (HCC). In this study, we aimed to screen hub genes related to immune cell infiltration and explore their prognostic value for HCC.MethodsWe analyzed five datasets (GSE46408, GSE57957, GSE74656, GSE76427, and GSE87630) from the Gene Expression Omnibus database to screen the differentially expressed genes (DEGs). A protein–protein interaction network of the DEGs was constructed using the Search Tool for the Retrieval of Interacting Genes; then, the hub genes were identified. Functional enrichment of the genes was performed on the Metascape website. Next, the expression of these hub genes was validated in several databases, including Oncomine, Gene Expression Profiling Interactive Analysis 2 (GEPIA2), and Human Protein Atlas. We explored the correlations between the hub genes and infiltrated immune cells in the TIMER2.0 database. The survival curves were generated in GEPIA2, and the univariate and multivariate Cox regression analyses were performed using TIMER2.0.ResultsThe top ten hub genes [DNA topoisomerase II alpha (TOP2A), cyclin B2 (CCNB2), protein regulator of cytokinesis 1 (PRC1), Rac GTPase-activating protein 1 (RACGAP1), aurora kinase A (AURKA), cyclin-dependent kinase inhibitor 3 (CDKN3), nucleolar and spindle-associated protein 1 (NUSAP1), cell division cycle-associated 5 (CDCA5), abnormal spindle microtubule assembly (ASPM), and non-SMC condensin I complex subunit G (NCAPG)] were identified in subsequent analysis. These genes are most markedly enriched in cell division, suggesting their close association with tumorigenesis. Multi-database analyses validated that the hub genes were upregulated in HCC tissues. All hub genes positively correlated with several types of immune infiltration, including B cells, CD4+ T cells, macrophages, and dendritic cells. Furthermore, these hub genes served as independent prognostic factors, and the expression of these hub genes combing with the macrophage levels could help predict an unfavorable prognosis of HCC.ConclusionIn sum, these hub genes (TOP2A, CCNB2, PRC1, RACGAP1, AURKA, CDKN3, NUSAP1, CDCA5, ASPM, and NCAPG) may be pivotal markers for prognostic prediction as well as potentially work as targets for immune-based intervention strategies in HCC.

Identification of Abnormal Spindle Microtubule Assembly as a Promising Therapeutic Target for Osteosarcoma.

ObjectiveTo demonstrate the expression of abnormal spindle microtubule assembly (ASPM) in clinical osteosarcoma tissue specimens collected in our hospital, and to explore the function of ASPM in osteosarcoma in vitro and in vivo.MethodsTissue specimens from 82 cases of osteosarcoma were collected and analyzed by immunohistochemistry assay. We also investigated the relationship between ASPM expression and clinicopathological characteristics in the patients. We transfected shASPM plasmid and the empty control plasmid, respectively, and then used quantitative polymerase chain reaction and western blot analysis to detect ASPM expression. Cell colony assay and MTT were used to observe the proliferation ability. In vivo study was undertaken to explore the ASPM function further.ResultsIn this study, ASPM showed high expression in osteosarcoma tissue samples compared with non‐tumor normal tissues. ASPM was positively correlated with clinical pathological characteristics, including tumor size (P = 0.024) and clinical stage (P = 0.045). Our results further showed that ASPM depletion dramatically inhibited the proliferation of osteosarcoma cells (with fewer cells in the sh‐RNA‐ASPM group compared with the control group(P < 0.05, respectively), and the in vivo assays further confirmed that ASPM ablation markedly blocked tumor growth compared with control (P < 0.05).ConclusionOur data provides strong evidence that the high expression of ASPM in osteosarcoma promotes proliferation in vitro and in vivo, indicating its potential role as an osteosarcoma therapeutic target.

Integrated Bioinformatic Analysis Identifies Networks and Promising Biomarkers for Hepatitis B Virus-Related Hepatocellular Carcinoma.

Chronic infection with hepatitis B virus (HBV) has long been recognized as a dominant hazard factor for hepatocellular carcinoma (HCC) and accounts for at least half of HCC instances globally. However, the underlying molecular mechanism of HBV-linked HCC has not been completely elucidated. Here, three microarray datasets, totally containing 170 tumoral samples and 181 adjacent normal tissues from the liver of patients suffering from HBV-related HCC assembled from the Gene Expression Omnibus (GEO) database, were subjected to integrated analysis of differentially expressed genes (DEGs). Subsequently, the analysis of function and pathway enrichment as well as the protein-protein interaction network (PPI) was performed. The ten hub genes screened out from the PPI network were further subjected to expression profile and survival analysis. Overall, 329 DEGs (67 upregulated and 262 downregulated) were identified. Ten DEGs with the highest degree of connectivity included cyclin-dependent kinase 1 (CDK1), cyclin B1 (CCNB1), cyclin B2 (CCNB2), PDZ-binding kinase (PBK), abnormal spindle microtubule assembly (ASPM), nuclear division cycle 80 (NDC80), aurora kinase A (AURKA), targeting protein for xenopus kinesin-like protein 2 (TPX2), kinesin family member 2C (KIF2C), and centromere protein F (CENPF). Kaplan-Meier analysis unveiled that overexpression levels of KIF2C and TPX2 were relevant to both the poor overall survival and relapse-free survival. In summary, the hub genes validated in the present study may provide promising targets for the diagnosis, prognosis, and therapy of HBV-associated HCC. Additionally, our work uncovers various crucial biological components (e.g., extracellular exosome) and signaling pathways that participate in the progression of HCC induced by HBV, serving comprehensive knowledge of the mechanisms regarding HBV-related HCC.

Screening and Identification of Therapeutic Targets for Pulmonary Arterial Hypertension Through Microarray Technology.

Pulmonary arterial hypertension (PAH) is a rare but fatal disease characterized by vascular cell proliferation; the pathogenesis of PAH has yet to be fully elucidated. Publicly available genetic data were downloaded from the Gene Expression Omnibus (GEO) database, and gene set enrichment analysis (GSEA) was used to determine significant differences in gene expression between tissues with PAH and healthy lung tissues. Differentially expressed genes (DEGs) were identified using the online tool, GEO2R, and functional annotation of DEGs was performed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Next, the construction and module analysis of the protein–protein interaction (PPI) network and verification of the expression level of hub genes was performed. Finally, prediction and enrichment analysis of microRNAs associated with the hub genes was carried out. A total of 110 DEGs were detected by screening PAH and healthy lung samples. The expression of nine genes [polo-like kinase 4 (PLK4), centromere protein U, kinesin family member 20B, structural maintenance of chromosome 2 (SMC2), abnormal spindle microtubule assembly, Fanconi Anemia complementation group I, kinesin family member 18A, spindle apparatus coiled-coil protein 1, and MIS18 binding protein 1] was elevated in PAH; this was statistically significant compared with their expression in healthy lung tissue, and they were identified as hub genes. GO and KEGG analysis showed that the variations in DEGs were abundant in DNA-templated transcription, sister chromatid cohesion, mitotic nuclear division, cell proliferation, and regulation of the actin cytoskeleton. In conclusion, this study has successfully identified hub genes and key pathways of PAH, with a total of 110 DEGs and nine hub genes related to PAH, especially the PLK4 and SMC2 genes, thus providing important clues for the in-depth understanding of the molecular mechanism of PAH and providing potential therapeutic targets.

Bioinformatics analysis reveals meaningful markers and outcome predictors in HBV-associated hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common type of malignant neoplasm of the liver with high morbidity and mortality. Extensive research into the pathology of HCC has been performed; however, the molecular mechanisms underlying the development of hepatitis B virus-associated HCC have remained elusive. Thus, the present study aimed to identify critical genes and pathways associated with the development and progression of HCC. The expression profiles of the GSE121248 dataset were downloaded from the Gene Expression Omnibus database and the differentially expressed genes (DEGs) were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genome (KEGG) analyses were performed by using the Database for Annotation, Visualization and Integrated Discovery. Subsequently, protein-protein interaction (PPI) networks were constructed for detecting hub genes. In the present study, 1,153 DEGs (777 upregulated and 376 downregulated genes) were identified and the PPI network yielded 15 hub genes. GO analysis revealed that the DEGs were primarily enriched in ‘protein binding’, ‘cytoplasm’ and ‘extracellular exosome’. KEGG analysis indicated that DEGs were accumulated in ‘metabolic pathways’, ‘chemical carcinogenesis’ and ‘fatty acid degradation’. After constructing the PPI network, cyclin-dependent kinase 1, cyclin B1, cyclin A2, mitotic arrest deficient 2 like 1, cyclin B2, DNA topoisomerase IIα, budding uninhibited by benzimidazoles (BUB)1, TTK protein kinase, non-SMC condensin I complex subunit G, NDC80 kinetochore complex component, aurora kinase A, kinesin family member 11, cell division cycle 20, BUB1B and abnormal spindle microtubule assembly were identified as hub genes based on the high degree of connectivity by using Cytoscape software. In addition, overall survival (OS) and disease-free survival (DFS) analyses were performed using the Gene Expression Profiling Interactive Analysis online database, which revealed that the increased expression of all hub genes were associated with poorer OS and DFS outcomes. Receiver operating characteristic curves were constructed using GraphPad prism 7.0 software. The results confirmed that 15 hub genes were able to distinguish HCC form normal tissues. Furthermore, the expression levels of three key genes were analyzed in tumor and normal samples of the Human Protein Atlas database. The present results may provide further insight into the underlying mechanisms of HCC and potential therapeutic targets for the treatment of this disease.

Aurora A inhibition disrupts chromosome condensation and spindle assembly during the first embryonic division in pigs.

As common overexpression of Aurora A in various tumours, much attention has focused on its function in inducing cancer, and its value in cancer therapeutics, considerably less is known regarding its role in the first cleavage division of mammalian embryos. Here, we highlight an indispensable role of Aurora A during the first mitotic division progression of pig embryos just after meiosis. The expression and spatiotemporal localization of Aurora A were initially assessed in pig embryos during the first mitotic division by Western blot analysis and indirect immunofluorescent staining. Then, the potential role of Aurora A was further evaluated using a highly selective Aurora A inhibitor, MLN8054, during this mitotic progression in pig embryos. Aurora A was found to express and exhibit a specific dynamic intracellular localization pattern during the first mitotic division in pig embryos. Aurora A was diffused in the cytoplasm at the prophase stage, and then exhibited a dynamic intracellular localization which was tightly associated with the chromosome and spindle dynamics throughout subsequent mitotic phases. Inhibition of Aurora A by MLN8054 treatment led to the failure of the first cleavage, with the majority of embryos being arrested in prophase of the mitotic division. Further subcellular structure examination showed that Aurora A inhibition not only led to the failure of spindle microtubule assembly, but also resulted in severe defects in chromosome condensation, accompanied by an obvious decrease in p-TACC3(S558) expression during the prophase of the first mitosis. Together, these results illustrated that Aurora A is crucial for both spindle assembly and chromosome condensation during the first mitotic division in pig embryos, and that the regulation of Aurora A may be associated with its effects on p-TACC3(S558) expression.

Screening and Identification of Key Biomarkers for Bladder Cancer: A Study Based on TCGA and GEO Data.

Bladder cancer (BLCA) is a common malignant cancer, and it is the most common genitourinary cancer in the world. The recurrence rate is the highest of all cancers, and the treatment of BLCA has only slightly improved over the past 30 years. Genetic and environmental factors play an important role in the development and progression of BLCA. However, the mechanism of cancer development remains to be proven. Therefore, the identification of potential oncogenes is urgent for developing new therapeutic directions and designing novel biomarkers for the diagnosis and prognosis of BLCA. Based on this need, we screened overlapping differentially expressed genes (DEG) from the GSE7476, GSE13507, and TCGA BLCA datasets. To identify the central genes from these DEGs, we performed a protein-protein interaction network analysis. To investigate the role of DEGs and the underlying mechanisms in BLCA, we performed Gene Ontology (GO) and Kyoto Gene and Genomic Encyclopedia (KEGG) analysis; we identified the hub genes via different evaluation methods in cytoHubba and then selected the target genes by performing survival analysis. Finally, the relationship between these target genes and tumour immunity was analysed to explore the roles of these genes. In summary, our current studies indicate that both cell division cycle 20 (CDC20) and abnormal spindle microtubule assembly (ASPM) genes are potential prognostic biomarkers for BLCA. It may also be a potential immunotherapeutic target with future clinical significance.

Screening and Identification of Key Biomarkers in Pancreatic Cancer: Evidence from Bioinformatic Analysis.

Pancreatic cancer (PC) whose mortality is comparable to morbidity is a highly fatal disease. Early approaches of diagnosis and treatment for PC are quite limited, so it is of great urgency to figure out the exact tumorigenesis and development mechanism of PC. To identify the related molecular markers of pancreatic oncogenesis, we downloaded three microarray datasets (GSE63111, GSE101448, and GSE107610) from Gene Expression Omnibus (GEO) database. The common differentially expressed genes (DEGs) among them were identified, and the corresponding function enrichment analyses were accomplished. The protein-protein interaction network was conducted by Search Tool for the Retrieval of Interacting Genes (STRING), and the corresponding module analysis was accomplished by Cytoscape. There were 55 DEGs found in total. The molecular function and biological processes (BP) of these DEGs mainly include cytokinesis, mitotic nuclear division, cell division, cell proliferation, microtubule-based movement, and mineral absorption. Among the 55 DEGs, 14 hub genes were further confirmed and it was concluded that they mainly function in mitotic cytokinesis, microtubule-based movement, mitotic chromosome condensation, and mitotic spindle assembly from the BP analysis. The survival analysis showed that all the 14 hub genes, especially nucleolar and spindle associated protein 1 and abnormal spindle microtubule assembly, may involve in the tumorigenesis and development of PC. And they might be used as new biomarkers for auxiliary diagnosis and potential targets for immunotherapy of PC.

α-Fodrin is required for the organization of functional microtubules during mitosis

ABSTRACTThe cytoskeleton protein α-fodrin plays a major role in maintaining structural stability of membranes. It was also identified as part of the brain γ-tubulin ring complex, the major microtubule nucleator. Here, we investigated the requirement of α-fodrin for microtubule spindle assembly during mitotic progression. We found that α-fodrin depletion results in abnormal mitosis with uncongressed chromosomes, leading to prolonged activation of the spindle assembly checkpoint and a severe mitotic delay. Further, α-fodrin repression led to the formation of shortened spindles with unstable kinetochore-microtubule attachments. We also found that the mitotic kinesin CENP-E had reduced levels at kinetochores to likely account for the chromosome misalignment defects in α-fodrin-depleted cells. Importantly, we showed these cells to exhibit reduced levels of detyrosinated α-tubulin, which primarily drives CENP-E localization. Since proper microtubule dynamics and chromosome alignment are required for completion of normal mitosis, this study reveals an unforeseen role of α-fodrin in regulating mitotic progression. Future studies on these lines of observations should reveal important mechanistic insight for fodrin’s involvement in cancer.

Evolution of ASPM coding variation in apes and associations with brain structure in chimpanzees.

Studying genetic mechanisms underlying primate brain morphology can provide insight into the evolution of human brain structure and cognition. In humans, loss-of-function mutations in the gene coding for ASPM (Abnormal Spindle Microtubule Assembly) have been associated with primary microcephaly, which is defined by a significantly reduced brain volume, intellectual disability and delayed development. However, less is known about the effects of common ASPM variation in humans and other primates. In this study, we characterized the degree of coding variation at ASPM in a large sample of chimpanzees (N = 241), and examined potential associations between genotype and various measures of brain morphology. We identified and genotyped five non-synonymous polymorphisms in exons 3 (V588G), 18 (Q2772K, K2796E, C2811Y) and 27 (I3427V). Using T1-weighted magnetic resonance imaging of brains, we measured total brain volume, cerebral gray and white matter volume, cerebral ventricular volume, and cortical surface area in the same chimpanzees. We found a potential association between ASPM V588G genotype and cerebral ventricular volume but not with the other measures. Additionally, we found that chimpanzee, bonobo, and human lineages each independently show a signature of accelerated ASPM protein evolution. Overall, our results suggest the potential effects of ASPM variation on cerebral cortical development, and emphasize the need for further functional studies. These results are the first evidence suggesting ASPM variation might play a role in shaping natural variation in brain structure in nonhuman primates.