VCP maintains nuclear size by regulating the DNA damage-associated MDC1–p53–autophagy axis in Drosophila

The maintenance of constant karyoplasmic ratios suggests that nuclear size has physiological significance. Nuclear size anomalies have been linked to malignant transformation, although the mechanism remains unclear. By expressing dominant-negative TER94 mutants in Drosophila photoreceptors, here we show disruption of VCP (valosin-containing protein, human TER94 ortholog), a ubiquitin-dependent segregase, causes progressive nuclear size increase. Loss of VCP function leads to accumulations of MDC1 (mediator of DNA damage checkpoint protein 1), connecting DNA damage or associated responses to enlarged nuclei. TER94 can interact with MDC1 and decreases MDC1 levels, suggesting that MDC1 is a VCP substrate. Our evidence indicates that MDC1 accumulation stabilizes p53A, leading to TER94K2A-associated nuclear size increase. Together with a previous report that p53A disrupts autophagic flux, we propose that the stabilization of p53A in TER94K2A-expressing cells likely hinders the removal of nuclear content, resulting in aberrant nuclear size increase.

151 Multipolar zygotic divisions result in multinuclear and anuclear blastomeres in cattle

The mammalian zygotic cleavage is expected to result in two mononuclear blastomeres. However, zygotes undergoing multipolar divisions resulting in direct cleavage into three or four cells are frequently observed in bovine and human embryonic development and have been associated with decreased euploidy rates of resulting blastocysts and a lower pregnancy rate (Somfai et al. 2010 J. Reprod. Dev. 56, 200-207; https://doi.org/10.1262/jrd.09-097a; Zhan et al. 2016 PLoS ONE 11, 1-19; https://doi.org/10.1371/journal.pone.0166398; Sugimura et al. 2017 J. Reprod. Dev. 63, 353-357; https://doi.org/10.1262/jrd.2017-041). Therefore, multipolar zygotic divisions may underly genetic abnormalities by aberrant segregation of the chromosomal material resulting in multinucleated or anuclear blastomeres. These abnormal blastomeres have been observed in human cleavage-stage embryos (Nogueira et al. 2000 Fertil. Steril. 74, 295-298; https://doi.org/10.1016/s0015-0282(00)00642-7; Chatzimeletiou et al. 2006 Hum. Reprod. 20, 672–682; https://doi.org/10.1093/humrep/deh652), but the prevalence in bovine embryos and the direct association with the multipolar division in both bovine and human embryos remains unknown. We hypothesised that anuclear and multinuclear blastomeres also occur in bovine embryos, and we aimed to unravel the link between multipolar zygotic divisions and genome segregation errors by determining the nuclear blastomere content in a bovine model. Therefore, oocytes from 5 cows were matured and fertilized in vitro by the same bull according to our standard in vitro production procedure (Wydooghe et al. 2014 Reproduction 148, 519-529). The first cleavage was monitored the by time-lapse imaging. Forty-three blastomeres from 22 bipolar zygotic divisions, and 65 blastomeres from 20 multipolar zygotic divisions were collected immediately after the first cleavage, using pronase to isolate the individual blastomeres. The area of each blastomere was measured and the number of nuclei was determined after fixation and staining with Hoechst 33342. Generalized mixed effect models were built to identify the effect of the type of cleavage (bipolar vs. multipolar) on the number of nuclei (mononuclear vs. anuclear or multinuclear) in the blastomeres. Linear mixed models were built to determine the effect of the type of cleavage and the nuclear content on the size of the blastomeres. Embryos presented a greater number of blastomeres with a normal nuclear content (92.6 ± 0.4%) after a bipolar cleavage compared with multipolar division (73.2 ± 0.7%; P=0.03). Moreover, blastomeres presented a 28% larger blastomere area (P<0.001) after bipolar division compared with multipolar division. Notably, anuclear blastomeres tended to be smaller than multi- and mononuclear blastomeres (P=0.09 for both), while no difference was found between mono and multinucleated blastomeres (P=0.84). In conclusion, this is one of the first reports on the association between nuclear blastomere content in bovine embryos and the dynamics of the first zygotic division. Even though sample size was limited, these results confirm the hypothesised link between multipolar division and abnormal genome segregation as determined by multinuclear and anuclear blastomeres in the resulting blastomeres. Therefore, multipolar cell divisions at the zygotic division may underly at least some of the genetic abnormalities observed in embryos at early development.

Regulation of protein kinase Cδ Nuclear Import and Apoptosis by Mechanistic Target of Rapamycin Complex-1

Inactivation of the protein complex ‘mechanistic target of rapamycin complex 1’ (mTORC1) can increase the nuclear content of transcriptional regulators of metabolism and apoptosis. Previous studies established that nuclear import of signal transducer and activator of transcription-1 (STAT1) requires the mTORC1-associated adaptor karyopherin-α1 (KPNA1) when mTORC1 activity is reduced. However, the role of other mTORC1-interacting proteins in the complex, including ‘protein kinase C delta’ (PKCδ), have not been well characterized. In this study, we demonstrate that PKCδ, a STAT1 kinase, contains a functional ‘target of rapamycin signaling’ (TOS) motif that directs its interaction with mTORC1. Depletion of KPNA1 by RNAi prevented the nuclear import of PKCδ in cells exposed to the mTORC1 inhibitor rapamycin or amino acid restriction. Mutation of the TOS motif in PKCδ led to its loss of regulation by mTORC1 or karyopherin-α1, resulting in increased constitutive nuclear content. In cells expressing wild-type PKCδ, STAT1 activity and apoptosis were increased by rapamycin or interferon-β. Those expressing the PKCδ TOS mutant exhibited increased STAT1 activity and apoptosis; further enhancement by rapamycin or interferon-β, however, was lost. Therefore, the TOS motif in PKCδ is a novel structural mechanism by which mTORC1 prevents PKCδ and STAT1 nuclear import, and apoptosis.

Mechanisms of nuclear content loading to exosomes

Exosome cargoes are highly varied and include proteins, small RNAs, and genomic DNA (gDNA). The presence of gDNA suggests that different intracellular compartments contribute to exosome loading, resulting in distinct exosome subpopulations. However, the loading of gDNA and other nuclear contents into exosomes (nExo) remains poorly understood. Here, we identify the relationship between cancer cell micronuclei (MN), which are markers of genomic instability, and nExo formation. Imaging flow cytometry analyses reveal that 10% of exosomes derived from cancer cells and <1% of exosomes derived from blood and ascites from patients with ovarian cancer carry nuclear contents. Treatment with genotoxic drugs resulted in increased MN and nExos both in vitro and in vivo. We observed that multivesicular body precursors and exosomal markers, such as the tetraspanins, directly interact with MN. Collectively, this work provides new insights related to nExos, which have implications for cancer biomarker development.

PO-095 Effects of dynamic foot stimulation on GSK-3 beta signaling pathway in rat soleus muscle under hindlimb unloading

Objective It is known that under simulated microgravity GSK-3 beta phosphorylation (Ser 9) is decreased [Mirzoev et al., 2016] which is associated with the activation of its kinase activity. GSK-3 beta activation may lead to NFATc1 export from myonuclei resulting in a slow-to-fast myosin shift in soleus muscle and may cause a decrease in muscle oxidative capacity and protein synthesis [Glass, 2003, Theeuwes et al, 2017]. It was demonstrated that GSK-3 beta Ser 9 phosphorylation is decreased in response to nitric oxide synthase inhibition [Martins et al., 2011]. Under unloading conditions, the content of NO as well as nitric oxide synthase is attenuated in rat soleus muscle [Tidball et al., 1998; Lomonosova et al, 2011]. Dynamic foot stimulation (DFS) of the soles of the feet results in an increase of neuromuscular activation [Muller et al., 2005] of the lower limb muscles and prevents nitric synthase content decrease during 7-day exposure to dry immersion [Moukhina et al, 2004; Shenkman et al., 2004]. The aim of our study was to analyze the effect of rat dynamic foot stimulation during early unloading on GSK-3 beta phosphorylation and some of its downstream targets. Methods Male Wistar rats were randomly assigned to vivarium control, 1-day unloading and 1-day unloading with DFS. The pressure stimulation protocol mimicked the normal animal walking (104 mm Hg pressure, 4 Hz frequency and 250 ms signal duration) for a total of 20 min followed by a 10-min rest interval for 4 hours. Results We found that 1-day unloading caused a significant decrease (p˂ 0.025) in NFATc1 nuclear content as well as slow myosin (MHC I (beta) isoform) mRNA expression. In the 1-day unloading group, the level of glycogen synthase 1 phosphorylation (Ser 641), which is a direct GSK-3 beta target, was significantly higher than that in the control group, although a decrease in GSK-3 beta Ser 9 phosphorylation in the 1-day unloaded group was not statistically significant compared to the control group. However, in the DFS-treated unloaded rats the level of GSK-3 beta phosphorylation was significantly higher than in the untreated unloaded rats. NFATc1 nuclear content, slow myosin mRNA expression and glycogen synthase 1 phosphorylation (Ser 641) did not differ from the control group. The glycogen content in soleus muscles of both unloaded groups was higher than in the control group, which is in accordance with previous studies [Henriksen and Tischler, 1988], but in DFS subjected unloaded group the glycogen content was higher compared to the untreated unloaded group. Conclusions Thus, we found that dynamic foot stimulation during 1-day hindlimb unloading leads to GSK-3 beta inactivation and prevention of both NFATc1 myonuclei export and the decrease in MyHC I beta expression. The study was supported by the Russian Foundation for Basic Research grant #17-29-1029 and Basic Research Program of the IBMP, RAS. The authors express their deep gratitude to Prof. Inessa B. Kozlovskaya for useful recommendations concerning the design of the study.