Looking on the horizon; potential and unique approaches to developing radiation countermeasures for deep space travel.

Future lunar missions and beyond will require new and innovative approaches to radiation countermeasures. The Translational Research Institute for Space Health (TRISH) is focused on identifying and supporting unique approaches to reduce risks to human health and performance on future missions beyond low Earth orbit. This paper will describe three funded and complementary avenues for reducing the risk to humans from radiation exposure experienced in deep space. The first focus is on identifying new therapeutic targets to reduce the damaging effects of radiation by focusing on high throughput genetic screens in accessible, sometimes called lower, organism models. The second focus is to design innovative approaches for countermeasure development with special attention to nucleotide-based methodologies that may constitute a more agile way to design therapeutics. The final focus is to develop new and innovative ways to test radiation countermeasures in a human model system. While animal studies continue to be beneficial in the study of space radiation, they can have imperfect translation to humans. The use of three-dimensional (3D) complex in vitro models is a promising approach to aid the development of new countermeasures and personalized assessments of radiation risks. These three distinct and unique approaches complement traditional space radiation efforts and should provide future space explorers with more options to safeguard their short and long-term health.

Conserved function of Drosophila Fancd2 monoubiquitination in response to double-strand DNA breaks.

Fanconi anemia genes play key roles in metazoan DNA damage responses, and human FA mutations cause numerous disease phenotypes. In human cells, activating monoubiquitination of the Fanconi anemia protein Fancd2 occurs following diverse DNA damage stimuli. Monoubiquitinated Fancd2 forms nuclear foci to recruit additional repair factors. Fancd2 animal models to date have focused on molecular nulls or whole gene knockdown, leaving the specific in vivo role of monoubiquitination unclear. Using a point mutant in a conserved residue, we recently linked Drosophila Fancd2 monoubiquitination to a mitosis-specific DNA double-strand break response. In this context, we used CRISPR/Cas9 to generate the first animal model of an endogenous mutation in the conserved monoubiquitination site (fancd2K595R). Here, we expand upon our characterization of fancd2K595R. We also introduce and characterize additional Drosophila tools to study fancd2, including new mutant alleles and GFP-tagged rescue transgenes. Using these new reagents, we show the impact of Drosophila Fancd2 on organismal and cell viability, as well as on repair protein localization, in the presence or absence of double-strand breaks. These findings expand our understanding of Fanconi anemia gene function in vivo and provide useful reagents for DNA repair research.

Persistent DNA damage signaling and DNA polymerase theta promote broken chromosome segregation.

Cycling cells must respond to DNA double-strand breaks (DSBs) to avoid genome instability. Missegregation of chromosomes with DSBs during mitosis results in micronuclei, aberrant structures linked to disease. How cells respond to DSBs during mitosis is incompletely understood. We previously showed that Drosophilamelanogaster papillar cells lack DSB checkpoints (as observed in many cancer cells). Here, we show that papillar cells still recruit early acting repair machinery (Mre11 and RPA3) and the Fanconi anemia (FA) protein Fancd2 to DSBs. These proteins persist as foci on DSBs as cells enter mitosis. Repair foci are resolved in a stepwise manner during mitosis. DSB repair kinetics depends on both monoubiquitination of Fancd2 and the alternative end-joining protein DNA polymerase θ. Disruption of either or both of these factors causes micronuclei after DNA damage, which disrupts intestinal organogenesis. This study reveals a mechanism for how cells with inactive DSB checkpoints can respond to DNA damage that persists into mitosis.

Actin-dependent cytoplasmic streaming in C. elegans oogenesis.

Oocytes in the C. elegans gonad enlarge rapidly. During the stage of enlargement, they are transcriptionally quiescent, and it is not understood how they acquire large quantities of materials such as mRNA and protein. Enlarging oocytes are connected via cytoplasmic bridges to a large, younger population of transcriptionally active germ cells at various stages of mitosis and meiosis. We show here that there is a general streaming of gonad cytoplasm towards and into the enlarging oocytes, originating primarily from pachytene-stage germ cells. Because previous studies suggested that most or all of the pachytene germ cells have the potential to differentiate into oocytes, the pachytene cells appear to function transiently as nurse cells. Somatic gonadal cells that surround the germ cells do not appear essential for streaming. Instead, materials appear to be pulled into oocytes by forces generated either in, or adjacent to, the enlarging oocytes themselves. Streaming appears to be driven by the actomyosin cytoskeleton, although we show that populations of both microfilaments and microtubules are oriented in the direction of flow. Our study shows that oocyte enlargement in C. elegans differs significantly from that in Drosophila, where a small number of specialized nurse cells expel their contents into the enlarging oocyte.