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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2013

Duck hepatitis B virus covalently closed circular DNA appears to survive hepatocyte mitosis in the growing liver

Nucleos(t)ide analogues that inhibit hepatitis B virus (HBV) DNA replication are typically used as monotherapy for chronically infected patients. Treatment with a nucleos(t)ide analogue eliminates most HBV DNA replication intermediates and produces a gradual decline in levels of covalently closed circular DNA (cccDNA), the template for viral RNA synthesis. It remains uncertain if levels of cccDNA decline primarily through hepatocyte death, or if loss also occurs during hepatocyte mitosis. To determine if cccDNA survives mitosis, growing ducklings infected with duck hepatitis B virus (DHBV) were treated with the nucleoside analogue, Entecavir. Viremia was suppressed at least 10{sup 5}-fold, during a period when average liver mass increased 23-fold. Analysis of the data suggested that if cccDNA synthesis was completely inhibited, at least 49% of cccDNA survived hepatocyte mitosis. However, there was a large duck-to-duck variation in cccDNA levels, suggesting that low level cccDNA synthesis may contribute to this apparent survival through mitosis. - Highlights: • The hepatitis B virus nuclear template is covalently closed circular DNA (cccDNA). • cccDNA was studied during liver growth in duck hepatitis B virus infected ducks. • Virus DNA replication and new cccDNA synthesis were inhibited with Entecavir. • At least 49% of cccDNA appeared to survive hepatocyte mitosis. • Low level virus DNA synthesis may contribute to survival of cccDNA through mitosis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Detection of large extracellular silver nanoparticle rings observed during mitosis using darkfield microscopy

During studies on the absorption and interactions between silver nanoparticles and mammalian cells grown in vitro it was observed that large extracellular rings of silver nanoparticles were deposited on the microscope slide, many located near post-mitotic cells. Silver nanoparticles (AgNP, 80nm), coated with citrate, were incubated at concentrations of 0.3 to 30 μg/ml with a human-derived culture of retinal pigment epithelial cells (ARPE-19) and observed using darkfield and fluorescent microscopy, 24 h after treatment. Approximately cell-sized extracellular rings of deposited AgNP were observed on the slides among a field of dispersed individual AgNP. The mean diameter of 45 nanoparticles circles was 62.5 +/-12 microns. Ring structures were frequently observed near what appeared to be post-mitotic daughter cells, giving rise to the possibility that cell membrane fragments were deposited on the slide during mitosis, and those fragments selectively attracted and retained silver nanoparticles from suspension in the cell culture medium. These circular structures were observable for the following technical reasons: 1) darkfield microscope could observe single nanoparticles below 100 nm in size, 2) a large concentration (10 8 and 10 9 ) of nanoparticles was used in these experiments 3) negatively charged nanoparticles were attracted to adhesion membrane proteins remaining on the slide from mitosis. The observation of silver nanoparticles attracted to apparent remnants of cellular mitosis could be a useful tool for the study of normal and abnormal mitosis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Water droplet excess free energy determined by cluster mitosis using guided molecular dynamics

Atmospheric aerosols play a vital role in affecting climate by influencing the properties and lifetimes of clouds and precipitation. Understanding the underlying microscopic mechanisms involved in the nucleation of aerosol droplets from the vapour phase is therefore of great interest. One key thermodynamic quantity in nucleation is the excess free energy of cluster formation relative to that of the saturated vapour. In our current study, the excess free energy is extracted for clusters of pure water modelled with the TIP4P/2005 intermolecular potential using a method based on nonequilibrium molecular dynamics and the Jarzynski relation. The change in free energy associated with the “mitosis” or division of a cluster of N water molecules into two N/2 sub-clusters is evaluated. This methodology is an extension of the disassembly procedure used recently to calculate the excess free energy of argon clusters [H. Y. Tang and I. J. Ford, Phys. Rev. E 91, 023308 (2015)]. Our findings are compared to the corresponding excess free energies obtained from classical nucleation theory (CNT) as well as internally consistent classical theory (ICCT). The values of the excess free energy that we obtain with the mitosis method are consistent with CNT for large cluster sizes but for the smallest clusters, the results tend towards ICCT; for intermediate sized clusters, we obtain values between the ICCT and CNT predictions. Furthermore, the curvature-dependent surface tension which can be obtained by regarding the clusters as spherical droplets of bulk density is found to be a monotonically increasing function of cluster size for the studied range. The data are compared to other values reported in the literature, agreeing qualitatively with some but disagreeing with the values determined by Joswiak et al. [J. Phys. Chem. Lett. 4, 4267 (2013)] using a biased mitosis approach; an assessment of the differences is the main motivation for our current study.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2016

MiR-509-3-5p causes aberrant mitosis and anti-proliferative effect by suppression of PLK1 in human lung cancer A549 cells

MicroRNAs (miRNAs) are potent post-transcriptional regulators of gene expression and play roles in DNA damage response (DDR). PLK1 is identified as a modulator of DNA damage checkpoint. Although down-regulation of PLK1 by certain microRNAs has been reported, little is known about the interplay between PLK1 and miR-509-3-5p in DDR. Here we have demonstrated that miR-509-3-5p repressed PLK1 expression by targeting PLK1 3′-UTR, thereby causing mitotic aberration and growth arrest of human lung cancer A549 cells. Repression of PLK1 by miR-509-3-5p was further evidenced by over-expression of miR-509-3-5p in A549, HepG2 and HCT116p53{sup −/−} cancer cells, in which PLK1 protein was suppressed. Consistently, miR-509-3-5p was stimulated, while PLK1 protein was down-regulated in A549 cells exposed to CIS and ADR, suggesting that suppression of PLK1 by miR-509-3-5p is a component of CIS/ADR-induced DDR pathway. Flow cytometry and immunofluorescence labeling showed that over-expression of miR-509-3-5p in A549 induced G2/M arrest and aberrant mitosis characterized by abnormal bipolar mitotic spindles, condensed chromosomes, lagging DNA and chromosome bridges. In addition, over-expression of miR-509-3-5p markedly blocked A549 cell proliferation and sensitized the cells to CIS and ADR treatment. Taken together, miR-509-3-5p is a feasible suppressor for cancer by targeting PLK1. Our data may provide aid in potential design of combined chemotherapy and in our better understanding of the roles of microRNAs in response to DNA damage. - Highlights: • MiR-509-3-5p represses PLK1 expression by targeting PLK1 3ГЉВ№-UTR. • Expression of miR-509-3-5p is induced and PLK1 repressed upon DNA damage. • Overexpression of miR-509-3-5p induces G2/M arrest and aberrant mitosis. • MiR-509-3-5p inhibits cell proliferation and sensitizes cells to DNA damage agents.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Cycling with BRCA2 from DNA repair to mitosis

Genetic integrity in proliferating cells is guaranteed by the harmony of DNA replication, appropriate DNA repair, and segregation of the duplicated genome. Breast cancer susceptibility gene BRCA2 is a unique tumor suppressor that is involved in all three processes. Hence, it is critical in genome maintenance. The functions of BRCA2 in DNA repair and homology-directed recombination (HDR) have been reviewed numerous times. Here, I will briefly go through the functions of BRCA2 in HDR and focus on the emerging roles of BRCA2 in telomere homeostasis and mitosis, then discuss how BRCA2 exerts distinct functions in a cell-cycle specific manner in the maintenance of genomic integrity. - Highlights: • BRCA2 is a multifaceted tumor suppressor and is crucial in genetic integrity. • BRCA2 exerts distinct functions in cell cycle-specific manner. • Mitotic kinases regulate diverse functions of BRCA2 in mitosis and cytokinesis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Lamin A reassembly at the end of mitosis is regulated by its SUMO-interacting motif

Modification of proteins with small ubiquitin-related modifier (SUMO; SUMOylation) is involved in the regulation of various biological processes. Recent studies have demonstrated that noncovalent associations between SUMOylated proteins and co-operative proteins containing SUMO-interacting motifs (SIMs) are important for the spatiotemporal organization of many protein complexes. In this study, we demonstrate that interactions between lamin A, a major component of the nuclear lamina, and SUMO isoforms are dependent on one of the four SIMs (SIM3) resided in lamin A polypeptide in vitro. Live cell imaging and immunofluorescence staining showed that SIM3 is required for accumulation of lamin A on the chromosomes during telophase, and subsequent evaluation of a panel of deletion mutants determined that a 156-amino acid region spanning the carboxyl-terminal Ig-fold domain of lamin A is sufficient for this accumulation. Notably, mutation of SIM3 abrogated the dephosphorylation of mitosis-specific phosphorylation at Ser-22 of lamin A, which normally occurs during telophase, and the subsequent nuclear lamina reorganization. Furthermore, expression of a conjugation-defective SUMO2 mutant, which was previously shown to inhibit endogenous SUMOylation in a dominant-negative manner, also impaired the accumulation of wild type lamin A on telophase chromosomes. These findings suggest that interactions between SIM3 of lamin A and a putative SUMO2-modified protein plays an important role in the reorganization of the nuclear lamina at the end of mitosis. - Highlights: • Lamin A interacts with SUMO2 via a SUMO-interacting motif (SIM) in the Ig domain. • SIM3 of lamin A is responsible for chromosomal accumulation during telophase. • A 156-aa region spanning the Ig domain is sufficient for chromosomal accumulation. • Accumulation of lamin A is required for timely dephosphorylation on chromosomes. • A putative SUMO2-modified protein may mediate chromosomal accumulation of lamin A.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2012

The Cell Cycle Timing of Centromeric Chromatin Assembly in Drosophila Meiosis Is Distinct from Mitosis Yet Requires CAL1 and CENP-C

CENP-A (CID in flies) is the histone H3 variant essential for centromere specification, kinetochore formation, and chromosome segregation during cell division. Recent studies have elucidated major cell cycle mechanisms and factors critical for CENP-A incorporation in mitosis, predominantly in cultured cells. However, we do not understand the roles, regulation, and cell cycle timing of CENP-A assembly in somatic tissues in multicellular organisms and in meiosis, the specialized cell division cycle that gives rise to haploid gametes. Here we investigate the timing and requirements for CID assembly in mitotic tissues and male and female meiosis in Drosophila melanogaster, using fixed and live imaging combined with genetic approaches. We find that CID assembly initiates at late telophase and continues during G1 phase in somatic tissues in the organism, later than the metaphase assembly observed in cultured cells. Furthermore, CID assembly occurs at two distinct cell cycle phases during male meiosis: prophase of meiosis I and after exit from meiosis II, in spermatids. CID assembly in prophase I is also conserved in female meiosis. Interestingly, we observe a novel decrease in CID levels after the end of meiosis I and before meiosis II, which correlates temporally with changes in kinetochore organization and orientation. We also demonstrate that CID is retained on mature sperm despite the gross chromatin remodeling that occurs during protamine exchange. Finally, we show that the centromere proteins CAL1 and CENP-C are both required for CID assembly in meiosis and normal progression through spermatogenesis. We conclude that the cell cycle timing of CID assembly in meiosis is different from mitosis and that the efficient propagation of CID through meiotic divisions and on sperm is likely to be important for centromere specification in the developing zygote.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2011

Calpastatin is regulated by protein never in mitosis gene A interacting-1 (PIN1) in endothelial cells

Highlights: Black-Right-Pointing-Pointer Depletion of PIN1 increases inhibitory effect of calpastatin against calpain in endothelial cells. Black-Right-Pointing-Pointer PIN1 associates with calpastatin. Black-Right-Pointing-Pointer PIN1, but not mutants, reduces the inhibitory activity of calpastatin in vitro. Black-Right-Pointing-Pointer Depletion of calpastatin shows that it is required for PIN1 depletion to reduce calpain activity. -- Abstract: The peptidyl-proline isomerase, protein never in mitosis gene A interacting-1 (PIN1) binds and isomerizes proteins phosphorylated on serine/threonine before a proline. It was previously found that depletion of PIN1 greatly increased induction of cyclooxygenase-2 and inducible nitric oxide synthase by lowering calpain activity in murine aortic endothelial cells (MAEC). Here we investigated the effect of PIN1 on the endogenous inhibitor of heterodimeric {mu}- and m-calpains, calpastatin. MAEC were transduced with small hairpin (sh) RNA to knock down PIN1 (KD) or an inactive Control shRNA. Cells were also treated with non-targeted double stranded small inhibitory RNA (siRNA) or siRNA designed to deplete calpastatin. Despite reducing calpain activity, PIN1 KD did not significantly affect the expression of {mu}- and m-calpains, or calpastatin, compared to Control shRNA. Instead, depletion of PIN1 increased the inhibitory activity of calpastatin. Calpastatin co-immunoprecipitated with endogenous PIN1 and was pulled down with glutathione-S-transferase (GST)-PIN1 fusion protein. Adding GST-PIN1 to KD cell extracts lacking PIN1 reduced calpastatin inhibitory activity. Substrate binding and catalytic domain mutants of PIN1 failed to do so. These results suggest that protein interaction and the proline isomerase functions of PIN1 are required for it to inhibit calpastatin. Furthermore, depletion of calpastatin raised calpain activity and reduced calpain inhibitory activity to similar levels in KD and Control MAEC, indicating that calpastatin is required for PIN1 depletion to lower calpain activity. Thus, PIN1 apparently restrains the ability of calpastatin to inhibit calpain, maintaining calpain activity in endothelial cells. PIN1 may act directly via phosphorylated serine/threonine-proline motifs in calpastatin, or indirectly via other PIN1 substrates that control calpastatin.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Prolyl isomerase Pin1 is required sperm production by promoting mitosis progression of spermatogonial stem cells

Highlights: • Pin1{sup −/−} mice had severe testicular atrophy. • Pin1{sup −/−} mice had less undifferentiated spermatogonia. • Pin1{sup −/−} and WT mice had similar cell counts of spermatogonial stem cells. • Pin1 promotes mitosis progression of spermatogonial stem cells. • Pin1 promotes differentiation of spermatogonial stem cell. A prolyl isomerase Pin1 deficient (Pin1{sup −/−}) male mice had severe testicular atrophy. We investigated the function of Pin1 in spermatogenesis by analyzing the Pin1{sup −/−} mice at reproductive age. Pin1{sup −/−} mice had lessαPLZF positive spermatogonia (undifferentiated spermatogonia) than wild type (WT). Nevertheless, the Pin1{sup −/−} testis contained approximately the same number of GFRα1 positive spermatogonia (SSCs in steady state) as the WT testis. Furthermore, degeneration of the spermatogenia appeared in seminiferous tubules of 10 months old Pin1{sup −/−} mouse testis, and abnormal shape GFRα1 positive spermatogonia were observed. In Pin1{sup −/−} spermatogonia, the ratio of the phospho-histone H3 positive cells (mitotic cells) in GFRα1-positive spermatogonia was higher than that of WT. These results suggest that Pin1 promotes the progression of the mitotic cell cycle of SSC in steady-state, which is required for the sperm production from SSCs.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Cell cycle-dependent SUMO-1 conjugation to nuclear mitotic apparatus protein (NuMA)

Highlights: •NuMA is modified by SUMO-1 in a cell cycle-dependent manner. •NuMA lysine 1766 is the primary target site for SUMOylation. •SUMOylation-deficient NuMA induces multiple spindle poles during mitosis. •SUMOylated NuMA induces microtubule bundling. -- Abstract: Covalent conjugation of proteins with small ubiquitin-like modifier 1 (SUMO-1) plays a critical role in a variety of cellular functions including cell cycle control, replication, and transcriptional regulation. Nuclear mitotic apparatus protein (NuMA) localizes to spindle poles during mitosis, and is an essential component in the formation and maintenance of mitotic spindle poles. Here we show that NuMA is a target for covalent conjugation to SUMO-1. We find that the lysine 1766 residue is the primary NuMA acceptor site for SUMO-1 conjugation. Interestingly, SUMO modification of endogenous NuMA occurs at the entry into mitosis and this modification is reversed after exiting from mitosis. Knockdown of Ubc9 or forced expression of SENP1 results in impairment of the localization of NuMA to mitotic spindle poles during mitosis. The SUMOylation-deficient NuMA mutant is defective in microtubule bundling, and multiple spindles are induced during mitosis. The mitosis-dependent dynamic SUMO-1 modification of NuMA might contribute to NuMA-mediated formation and maintenance of mitotic spindle poles during mitosis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2012

Fully functional global genome repair of (6-4) photoproducts and compromised transcription-coupled repair of cyclobutane pyrimidine dimers in condensed mitotic chromatin

During mitosis, chromatin is highly condensed, and activities such as transcription and semiconservative replication do not occur. Consequently, the condensed condition of mitotic chromatin is assumed to inhibit DNA metabolism by impeding the access of DNA-transacting proteins. However, about 40 years ago, several researchers observed unscheduled DNA synthesis in UV-irradiated mitotic chromosomes, suggesting the presence of excision repair. We re-examined this subject by directly measuring the removal of UV-induced DNA lesions by an ELISA and by a Southern-based technique in HeLa cells arrested at mitosis. We observed that the removal of (6-4) photoproducts from the overall genome in mitotic cells was as efficient as in interphase cells. This suggests that global genome repair of (6-4) photoproducts is fully functional during mitosis, and that the DNA in mitotic chromatin is accessible to proteins involved in this mode of DNA repair. Nevertheless, not all modes of DNA repair seem fully functional during mitosis. We also observed that the removal of cyclobutane pyrimidine dimers from the dihydrofolate reductase and c-MYC genes in mitotic cells was very slow. This suggests that transcription-coupled repair of cyclobutane pyrimidine dimers is compromised or non-functional during mitosis, which is probably the consequence of mitotic transcriptional repression. -- Highlights: Black-Right-Pointing-Pointer Global genome repair of (6-4) photoproducts is fully active in mitotic cells. Black-Right-Pointing-Pointer DNA in condensed mitotic chromatin does not seem inaccessible or inert. Black-Right-Pointing-Pointer Mitotic transcriptional repression may impair transcription-coupled repair.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

γ-Taxilin temporally regulates centrosome disjunction in a Nek2A-dependent manner

Highlights: • γ-Taxilin co-localizes with Nek2A at the centrosome during interphase. • γ-Taxilin interacts with Nek2A in yeast two-hybrid and pull-down assays. • γ-Taxilin protein expression levels decrease at the onset of mitosis. • γ-Taxilin depletion induces immature splitting of centrosomes. • The immature splitting of centrosomes induced by γ-taxilin depletion depends on Nek2A. Never in mitosis A-related kinase 2A (Nek2A), a centrosomal serine/threonine kinase, is involved in mitotic progression by regulating the centrosome cycle. Particularly, Nek2A is necessary for dissolution of the intercentriole linkage between the duplicated centrosomes prior to mitosis. Nek2A activity roughly parallels its cell cycle-dependent expression levels, but the precise mechanism regulating its activity remains unclear. In this study, we found that γ-taxilin co-localized with Nek2A at the centrosome during interphase and interacted with Nek2A in yeast two-hybrid and pull-down assays and that γ-taxilin regulated centrosome disjunction in a Nek2A-dependent manner. γ-Taxilin depletion increased the number of cells with striking splitting of centrosomes. The precocious splitting of centrosomes induced by γ-taxilin depletion was attenuated by Nek2A depletion, suggesting that γ-taxilin depletion induces the Nek2A-mediated dissolution of the intercentriole linkage between the duplicated centrosomes nevertheless mitosis does not yet begin. Taken together with the result that γ-taxilin protein expression levels were decreased at the onset of mitosis, we propose that γ-taxilin participates in Nek2A-mediated centrosome disjunction as a negative regulator through its interaction with Nek2A.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2019

Feasibility of using computed tomography texture analysis parameters as imaging biomarkers for predicting risk grade of gastrointestinal stromal tumors: comparison with visual inspection

Purpose: To evaluate the feasibility of using computed tomography texture analysis (CTTA) parameters for predicting malignant risk grade and mitosis index of gastrointestinal stromal tumors (GISTs), compared with visual inspection. Method and materials: CTTA was performed on portal phase CT images of 145 surgically confirmed GISTs (mean size: 42.9 ± 37.5 mm), using TexRAD software. Mean, standard deviation, entropy, mean of positive pixels (MPP), skewness, and kurtosis of CTTA parameters, on spatial scaling factor (SSF), 2–6 were compared by risk grade, mitosis rate, and the presence or absence of necrosis on visual inspection. CTTA parameters were correlated with risk grade. Diagnostic performance was evaluated with receiver operating characteristic curve analysis. Enhancement pattern, necrosis, heterogeneity, calcification, growth pattern, and mucosal ulceration were subjectively evaluated by two observers. Results: Three to four parameters at different scales were significantly different according to the risk grade, mitosis rate, and the presence or absence of necrosis (p < 0.041). MPP at fine or medium scale (r = − 0.547 to − 393) and kurtosis at coarse scale (r = 0.424–0.454) correlated significantly with risk grade (p < 0.001). HG-GIST was best differentiated from LG-GIST by MPP at SSF 2 (AUC, 0.782), and kurtosis at SSF 4 (AUC, 0.779) (all p < 0.001). CT features predictive of HG-GIST were density lower than or equal to that of the erector spinae muscles on enhanced images (OR 2.1; p = 0.037; AUC, 0.59), necrosis (OR, 6.1; p < 0.001; AUC, 0.70), heterogeneity (OR, 4.3; p < 0.001; AUC, 0.67), and mucosal ulceration (OR, 3.3; p = 0.002; AUC, 0.62). Conclusion: Using TexRAD, MPP and kurtosis are feasible in predicting risk grade and mitosis index of GISTs. CTTA demonstrated meaningful accuracy in preoperative risk stratification of GISTs.

62 RADIOLOGY AND NUCLEAR MEDICINE↗
DOE OSTI2013

Mitotic phosphorylation of VCIP135 blocks p97ATPase-mediated Golgi membrane fusion

Highlights: •VCIP135 is mitotically phosphorylated on Threonine-760 and Serine-767 by Cdc2. •Phosphorylated VCIP135 does not bind to p97ATPase. •The phosphorylation of VCIP135 inhibits p97ATPase-mediated Golgi membrane fusion. -- Abstract: In mammals, the Golgi apparatus is disassembled early mitosis and reassembled at the end of mitosis. For Golgi disassembly, membrane fusion needs to be blocked. Golgi biogenesis requires two distinct p97ATPase-mediated membrane fusion, the p97/p47 and p97/p37 pathways. We previously reported that p47 phosphorylation on Serine-140 and p37 phosphorylation on Serine-56 and Threonine-59 result in mitotic inhibition of the p97/p47 and the p97/p37 pathways, respectively [11,14]. In this study, we show another mechanism of mitotic inhibition of p97-mediated Golgi membrane fusion. We clarified that VCIP135, an essential factor in both p97 membrane fusion pathways, is phosphorylated on Threonine-760 and Serine-767 by Cdc2 at mitosis and that this phosphorylated VCIP135 does not bind to p97. An in vitro Golgi reassembly assay revealed that VCIP135(T760E, S767E), which mimics mitotic phosphorylation, caused no cisternal regrowth. Our results indicate that the phosphorylation of VCIP135 on Threonine-760 and Serine-767 inhibits p97-mediated Golgi membrane fusion at mitosis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2013

The moyamoya disease susceptibility variant RNF213 R4810K (rs112735431) induces genomic instability by mitotic abnormality

Highlights: •Overexpression of RNF213 R4810K inhibited cell proliferation. •Overexpression of RNF213 R4810K had the time of mitosis 4-fold and mitotic failure. •R4810K formed a complex with MAD2 more readily than wild-type. •iPSECs from the MMD patients had elevated mitotic failure compared from the control. •RNF213 R4810K induced mitotic abnormality and increased risk of aneuploidy. -- Abstract: Moyamoya disease (MMD) is a cerebrovascular disease characterized by occlusive lesions in the Circle of Willis. The RNF213 R4810K polymorphism increases susceptibility to MMD. In the present study, we characterized phenotypes caused by overexpression of RNF213 wild type and R4810K variant in the cell cycle to investigate the mechanism of proliferation inhibition. Overexpression of RNF213 R4810K in HeLa cells inhibited cell proliferation and extended the time of mitosis 4-fold. Ablation of spindle checkpoint by depletion of mitotic arrest deficiency 2 (MAD2) did not shorten the time of mitosis. Mitotic morphology in HeLa cells revealed that MAD2 colocalized with RNF213 R4810K. Immunoprecipitation revealed an RNF213/MAD2 complex: R4810K formed a complex with MAD2 more readily than RNF213 wild-type. Desynchronized localization of MAD2 was observed more frequently during mitosis in fibroblasts from patients (n = 3, 61.0 ± 8.2%) compared with wild-type subjects (n = 6, 13.1 ± 7.7%; p < 0.01). Aneuploidy was observed more frequently in fibroblasts (p < 0.01) and induced pluripotent stem cells (iPSCs) (p < 0.03) from patients than from wild-type subjects. Vascular endothelial cells differentiated from iPSCs (iPSECs) of patients and an unaffected carrier had a longer time from prometaphase to metaphase than those from controls (p < 0.05). iPSECs from the patients and unaffected carrier had significantly increased mitotic failure rates compared with controls (p < 0.05). Thus, RNF213 R4810K induced mitotic abnormalities and increased risk of genomic instability.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Aurora A regulates the architecture of the Golgi apparatus

Highlights: • Knockdown or inhibition of Aurora A resulted in Golgi dispersal during interphase. • Overexpression of Aurora A led to tightly packed Golgi apparatus during interphase. • Inhibition of Aurora A induced Golgi dispersal after mitosis. • Knockdown or inhibition of Aurora A also led to disruption in integrity of centrosome and Golgi apparatus during interphase. The Golgi apparatus plays roles in cell polarity, directional cell migration, and bipolar spindle assembly, as well as the secretary pathway. In addition, recent studies have suggested the Golgi-dependent control of mitotic entry. We studied the role of the centrosomal kinase Aurora A in maintaining the Golgi apparatus. Knockdown of Aurora A resulted in Golgi dispersal during interphase. Golgi dispersal was also induced by a selective Aurora A inhibitor, MLN8237. Conversely, overexpression of Aurora A led to tightly packed Golgi apparatus during interphase. Knockdown or inhibition of Aurora A had little or no effect on Golgi vesiculation during mitosis. By synchronizing cell division, we studied whether mitosis was required to induce Golgi dispersal during interphase. Aurora A inhibition induced aberrant mitotic spindle and Golgi dispersal only after mitosis. However, the cells treated with the inhibitor MLN8237 at earlier cell cycle stages (wherein the cells remained undivided) had a normal Golgi architecture. Knockdown or inhibition of Aurora A also led to aberrant integrity of centrosome and Golgi apparatus during interphase. These results suggest that Aurora A activity is involved in the maintenance of Golgi architecture and the relationship between the Golgi apparatus and centrosome.

60 APPLIED LIFE SCIENCES↗