The functional significance of ATM phosphorylation of Bub3 on Serine 135 in sensitivity to DNA damaging agents.

e15048 Background: Identification of biomarkers to assess and modify the sensitivity of cancer cells to radiotherapy and chemotherapy is critical to improve cancer treatment outcome. Budding uninhibited by benzimidazoles 3 (Bub3) is a mitotic checkpoint protein, and it is frequently overexpressed in many cancers and associated with low survival rates. Bub3 is involved in the repair of DNA damage induced by radiotherapy and chemotherapy. We recently identified that the ATM kinase phosphorylated Bub3 on Serine 135 (S135) via the stable isotope labeled amino acid in cell culture -mass spectrometry analysis. This study aims to explore the mechanism of ATM-phosphorylated Bub3 in the DNA damage response (DDR) and its effect on tumor sensitivity to DNA damaging agents. Methods: The radiosensitivity of the cells was detected by clonal formation assay, the proliferation ability of the cells was detected by the MTS assay. The expression of DDR protein γ-H2AX in the nucleus was detected by immunofluorescence assay. Genomic instability was observed by multinuclear formation. Co-immunoprecipitation and Western Blot were used to explore the internal mechanism of ATM phosphorylation of Bub3 in DDR. Results: We showed that ionizing radiation (IR) could induce Bub3 S/TQ (the specific ATM consensus motif) phosphorylation in an ATM dependent manner. Mutation of Bub3 Serine 135 to alanine (S135A) led to a phosphorylation defect. Phenotypic experiments showed hypersensitivity to IR in cells expressing Bub3 S135A. Bub3 S135A prolonged existence of γ-H2AX foci and increased the proportion of cells containing micronuclei. Further, we found that Ku70 and Ku80 showed a significant increase after IR in their interactions with Bub3, while Bub3 S135A mutation significantly reduced the interaction, leading to impaired DNA repair. Conclusions: We demonstrate that Bub3 S135 phosphorylation mediated by ATM is essential for an optimal DDR and disruption of this pathway increases tumor sensitivity to DNA damaging agents.

Mitotic checkpoint protein Mad1 is required for early Nup153 recruitment to chromatin and nuclear envelope integrity

ABSTRACTNucleoporin Nup153 is a multifunctional protein and a known binding partner of mitotic checkpoint protein Mad1 (also known as MAD1L1). The functional relevance of their interaction has remained elusive. Here, we have further dissected the interface and functional interplay of Nup153 and Mad1. Using in situ proximity ligation assays, we found that the presence of a nuclear envelope (NE) is a prerequisite for the Nup153–Mad1 association. Time-lapse microscopy revealed that depletion of Mad1 delayed recruitment of Nup153 to anaphase chromatin, which was often accompanied by a prolongation of anaphase. Furthermore, as seen by electron microscopic and three-dimensional structured illumination investigations, Nup153 and Mad1 depletion led to alterations in NE architecture, characterised by a change of membrane curvature at nuclear pore complexes (NPCs) and an expansion of the spacing between inner and outer nuclear membranes. Nup153 depletion, but not Mad1 depletion, caused defects in interphase NPC assembly, with partial displacement of cytoplasmic nucleoporins and a reduction in NPC density. Taken together, our results suggest that Nup153 has separable roles in NE and NPC formation: in post-mitotic NE re-formation in concert with Mad1 and in interphase NPC assembly, independent of Mad1.

Molecular Contribution to Embryonic Aneuploidy and Genotypic Complexity During Initial Cleavage Divisions of Mammalian Development

ABSTRACTEmbryonic aneuploidy is highly complex, often leading to developmental arrest, implantation failure, or spontaneous miscarriage in both natural and assisted reproduction. Despite our knowledge of mitotic mis-segregation in somatic cells, the molecular pathways regulating chromosome fidelity during the error-prone cleavage-stage of mammalian embryogenesis remain largely undefined. Using bovine embryos and live-cell fluorescent imaging, we observed frequent micro-/multi-nucleation of anaphase lagging or mis-segregated chromosomes in initial mitotic divisions that underwent unilateral inheritance, re-fused with the primary nucleus, or formed a chromatin bridge with neighboring cells. A correlation between a lack of maternal and paternal pronuclei fusion (syngamy), multipolar cytokinesis, and uniparental genome segregation was also revealed and single-cell DNA-seq showed propagation of primarily non-reciprocal mitotic errors in embryonic blastomeres. Depletion of the mitotic checkpoint protein, BUB1B/BUBR1, resulted in micro-/multi-nuclei formation, atypical cytokinesis, chaotic aneuploidy, and disruption of the kinase-substrate network regulating mitotic progression and exit, culminating in embryo arrest prior to genome activation. This demonstrates that embryonic micronuclei sustain multiple fates, provides a mechanism for blastomeres with uniparental origins, and substantiates the contribution of defective checkpoint signaling and/or the inheritance of other maternally-derived factors to the high genotypic complexity afflicting preimplantation development in higher-order mammals.

Mitotic checkpoint protein Mad1 is required for early Nup153 recruitment to chromatin and nuclear envelope integrity

The nucleoporin Nup153 is a multifunctional protein and the mitotic checkpoint protein Mad1one of its many binding partners. The functional relevance of their interaction has remained elusive. Here, we have further dissected Nup153’s and Mad1’s interface and functional interplay. By in situ proximity ligation assays, we found that the presence of a nuclear envelope (NE) is prerequisite for the Nup153-Mad1 interaction. Time-lapse microscopy revealed that depletion of Mad1 delayed recruitment of Nup153 to anaphase chromatin, which was often accompanied by a prolongation of anaphase. Furthermore, as seen by electron microscopic and three-dimensional structured illumination investigations, Nup153 and Mad1 depletion led to alterations in NE architecture, characterised by a change of the membrane curvature at nuclear pore complexes (NPCs) and an expansion of the spacing between the inner and outer nuclear membranes. Nup153 depletion, but not of Mad1, caused defects in interphase NPC assembly with partial displacement of cytoplasmic nucleoporins and a reduction in NPC density. Together our results suggest that Nup153 has separable roles in NE and NPC formation: in post-mitotic NE reformation in concert with Mad1 and in interphase NPC assembly, independent of Mad1.SummaryThe mitotic checkpoint protein is required for Nup153 recruitment to anaphase chromatin and in turn post-mitotic, but not interphase nuclear pore complex assembly.

Prognostic value of mitotic checkpoint protein BUB3, cyclin B1, and pituitary tumor-transforming 1 expression in prostate cancer

The mitotic checkpoint protein BUB3, cyclin B1 (CCNB1) and pituitary tumor-transforming 1 (PTTG1) regulates cell division, and are sparsely studied in prostate cancer. Deregulation of these genes can lead to genomic instability, a characteristic of more aggressive tumors. We aimed to determine the expression levels of BUB3, CCNB1, and PTTG1 as potential prognostic markers of recurrence after radical prostatectomy. Protein levels were determined by immunohistochemistry on three formalin-fixed paraffin-embedded tissue sections from each of the 253 patients treated with radical prostatectomy. Immunohistochemistry scores were obtained by automated image analysis for CCNB1 and PTTG1. Recurrence, defined as locoregional recurrence, distant metastasis or death from prostate cancer, was used as endpoint for survival analysis. Tumors having both positive and negative tumor areas for cytoplasmic BUB3 (30%), CCNB1 (28%), or PTTG1 (35%) were considered heterogeneous. Patients with ≥1 positive tumor area had significantly increased risk of disease recurrence in univariable analysis compared with patients where all tumor areas were negative for cytoplasmic BUB3 (hazard ratio [HR] = 2.18, 95% confidence interval [CI] 1.41–3.36), CCNB1 (HR = 2.98, 95% CI 1.93–4.61) and PTTG1 (HR = 1.91, 95% CI 1.23–2.97). Combining the scores of cytoplasmic BUB3 and CCNB1 improved risk stratification when integrated with the Cancer of the Prostate Risk Assessment post-Surgical (CAPRA-S) score (difference in concordance index = 0.024, 95% CI 0.001–0.05). In analysis of multiple tumor areas, prognostic value was observed for cytoplasmic BUB3, CCNB1, and PTTG1.

Requirement of the Dynein-adaptor Spindly for mitotic and post-mitotic functions in Drosophila

Spindly is a mitotic checkpoint protein originally identified as a specific regulator of Dynein activity at the kinetochore. In metaphase, Spindly recruits the Dynein/Dynactin complex, promoting the establishment of stable kinetochore-microtubule interactions and progression into anaphase. While details of Spindly function in mitosis have been worked out in cultured human cells and in the C. elegans zygote, the function of Spindly within the context of an organism has not yet been addressed. Here we present loss- and gain-of-function studies of Spindly in Drosophila. We investigated the requirements of distinct protein domains for the localisation and function of Spindly. We find that knock-down of Spindly results in a range of mitotic defects in the female germ line and during cleavage divisions in embryogenesis. Overexpression of Spindly in the female germ line is embryonic lethal and results in altered egg morphology. To determine whether Spindly plays a role in post-mitotic cells we altered Spindly protein levels in migrating cells and found that ovarian border cell migration is sensitive to the levels of Spindly protein. Our study uncovers novel functions of the mitotic checkpoint protein Spindly in Drosophila.