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Midkine (MDK) and Pleiotrophin (PTN) are small heparin-binding cytokines with closely related structures. The Drosophila genome harbours two genes encoding members of the MDK/PTN family of proteins, known as miple1 and miple2. We have investigated the role of Miple proteins in vivo, in particular with regard to their proposed role as ligands for the Alk receptor tyrosine kinase (RTK). Here we show that Miple proteins are neither required to drive Alk signaling during Drosophila embryogenesis, nor are they essential for development in the fruit fly. Additionally we show that neither MDK nor PTN can activate hALK in vivo when ectopically co-expressed in the fly. In conclusion, our data suggest that Alk is not activated by MDK/PTN related growth factors Miple1 and Miple 2 in vivo.

Meiosis is a specialized cell division used by diploid organisms to form haploid gametes for sexual reproduction. Central to this reductive division is repair of endogenous DNA double-strand breaks (DSBs) induced by the meiosis-specific enzyme Spo11. These DSBs are repaired in a process called homologous recombination using the sister chromatid or the homologous chromosome as a repair template, with the homolog being the preferred substrate during meiosis. Specific products of inter-homolog recombination, called crossovers, are essential for proper homolog segregation at the first meiotic nuclear division in budding yeast and mice. This study identifies an essential role for the conserved Structural Maintenance of Chromosomes (SMC) 5/6 protein complex during meiotic recombination in budding yeast. Meiosis-specific smc5/6 mutants experience a block in DNA segregation without hindering meiotic progression. Establishment and removal of meiotic sister chromatid cohesin are independent of functional Smc6 protein. smc6 mutants also have normal levels of DSB formation and repair. Eliminating DSBs rescues the segregation block in smc5/6 mutants, suggesting that the complex has a function during meiotic recombination. Accordingly, smc6 mutants accumulate high levels of recombination intermediates in the form of joint molecules. Many of these joint molecules are formed between sister chromatids, which is not normally observed in wild-type cells. The normal formation of crossovers in smc6 mutants supports the notion that mainly inter-sister joint molecule resolution is impaired. In addition, return-to-function studies indicate that the Smc5/6 complex performs its most important functions during joint molecule resolution without influencing crossover formation. These results suggest that the Smc5/6 complex aids primarily in the resolution of joint molecules formed outside of canonical inter-homolog pathways., Author Summary Most eukaryotic cells are diploid, which means that they contain two copies of each chromosome – one from each parent. In order to preserve the chromosome number from generation to generation, diploid organisms employ a process called meiosis to form gametes containing only one copy of each chromosome. During sexual reproduction, two gametes (sperm and eggs in mammals) fuse to form a zygote with the same chromosome number as the parents. This zygote will develop into a new organism that has genetic characteristics unique from, but still related to, both parents. The reduction of chromosome number and the reshuffling of genetic traits during meiosis depend on the repair of naturally occurring DNA breaks. Improper break repair during meiosis may block meiosis altogether or form genetically instable gametes, leading to fertility problems or defects in the offspring. The study presented here demonstrates the importance of the evolutionarily conserved Smc5/6 protein complex in upholding the integrity of meiotic repair processes. Our results show that cells deficient in components of the Smc5/6 complex lead to inviable meiotic products. Cells lacking functional Smc5/6 complex are unable to direct DNA repair to the proper template and accumulate abnormal repair intermediates, which inhibit the reductive division.

The cohesin complex, which is essential for sister chromatid cohesion and chromosome segregation, also inhibits resolution of sister chromatid intertwinings (SCIs) by the topoisomerase Top2. The cohesin-related Smc5/6 complex (Smc5/6) instead accumulates on chromosomes after Top2 inactivation, known to lead to a buildup of unresolved SCIs. This suggests that cohesin can influence the chromosomal association of Smc5/6 via its role in SCI protection. Using high-resolution ChIP-sequencing, we show that the localization of budding yeast Smc5/6 to duplicated chromosomes indeed depends on sister chromatid cohesion in wild-type and top2-4 cells. Smc5/6 is found to be enriched at cohesin binding sites in the centromere-proximal regions in both cell types, but also along chromosome arms when replication has occurred under Top2-inhibiting conditions. Reactivation of Top2 after replication causes Smc5/6 to dissociate from chromosome arms, supporting the assumption that Smc5/6 associates with a Top2 substrate. It is also demonstrated that the amount of Smc5/6 on chromosomes positively correlates with the level of missegregation in top2-4, and that Smc5/6 promotes segregation of short chromosomes in the mutant. Altogether, this shows that the chromosomal localization of Smc5/6 predicts the presence of the chromatid segregation-inhibiting entities which accumulate in top2-4 mutated cells. These are most likely SCIs, and our results thus indicate that, at least when Top2 is inhibited, Smc5/6 facilitates their resolution., Author Summary When cells divide, sister chromatids have to be segregated away from each other for the daughter cells to obtain a correct set of chromosomes. Using yeast as model organism, we have analyzed the function of the cohesin and the Smc5/6 complexes, which are essential for chromosome segregation. Cohesin is known to hold sister chromatid together until segregation occurs, and our results show that cohesin also controls Smc5/6, which is found to associate to linked chromatids specifically. In line with this, our analysis points to that the chromosomal localization of Smc5/6 is an indicator of the level of entanglement between sister chromatids. When Smc5/6 is non-functional, the resolution of these entanglements is shown to be inhibited, thereby preventing segregation of chromatids. Our results also indicate that DNA entanglements are maintained on chromosomes at specific sites until segregation. In summary, we uncover new functions for cohesin, in regulating when and where Smc5/6 binds to chromosomes, and for the Smc5/6 complex in facilitating the resolution of sister chromatid entanglements.