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Microtubule nucleation sites in yeast consist of a ring of γ-tubulin small complexes and a slight excess of uncomplexed γ-tubulin.

SummaryWe report the mechanistic basis guiding the migration pattern of multiple nuclei in hyphae of Ashbya gossypii. Using electron tomography, we reconstructed the cytoplasmic microtubule (cMT) cytoskeleton in three tip regions with a total of 13 nuclei and also the spindle microtubules of four mitotic nuclei. Each spindle pole body (SPB) nucleates three cMTs and most cMTs above a certain length grow according to their plus-end structure. Long cMTs closely align for several microns along the cortex, presumably marking regions where dynein generates pulling forces on nuclei. Close proximity between cMTs emanating from adjacent nuclei was not observed. The majority of nuclei carry duplicated side-by-side SPBs, which together emanate an average of six cMTs, in most cases in opposite orientation with respect to the hyphal growth axis. Such cMT arrays explain why many nuclei undergo short-range back and forth movements. Only occasionally do all six cMTs orient in one direction, a precondition for long-range nuclear bypassing. Following mitosis, daughter nuclei carry a single SPB with three cMTs. The increased probability that all three cMTs orient in one direction explains the high rate of nuclear bypassing observed in these nuclei. The A. gossypii mitotic spindle was found to be structurally similar to that of Saccharomyces cerevisiae in terms of nuclear microtubule (nMT) number, length distribution and three-dimensional organization even though the two organisms differ significantly in chromosome number. Our results suggest that two nMTs attach to each kinetochore in A. gossypii and not only one nMT like in S. cerevisiae.

BackgroundGenome duplication has played a pivotal role in the evolution of many eukaryotic lineages, including the vertebrates. A relatively recent vertebrate genome duplication is that in Xenopus laevis, which resulted from the hybridization of two closely related species about 17 million years ago. However, little is known about the consequences of this duplication at the level of the genome, the epigenome, and gene expression.ResultsThe X. laevis genome consists of two subgenomes, referred to as L (long chromosomes) and S (short chromosomes), that originated from distinct diploid progenitors. Of the parental subgenomes, S chromosomes have degraded faster than L chromosomes from the point of genome duplication until the present day. Deletions appear to have the largest effect on pseudogene formation and loss of regulatory regions. Deleted regions are enriched for long DNA repeats and the flanking regions have high alignment scores, suggesting that non-allelic homologous recombination has played a significant role in the loss of DNA. To assess innovations in the X. laevis subgenomes we examined p300-bound enhancer peaks that are unique to one subgenome and absent from X. tropicalis. A large majority of new enhancers comprise transposable elements. Finally, to dissect early and late events following interspecific hybridization, we examined the epigenome and the enhancer landscape in X. tropicalis × X. laevis hybrid embryos. Strikingly, young X. tropicalis DNA transposons are derepressed and recruit p300 in hybrid embryos.ConclusionsThe results show that erosion of X. laevis genes and functional regulatory elements is associated with repeats and non-allelic homologous recombination and furthermore that young repeats have also contributed to the p300-bound regulatory landscape following hybridization and whole-genome duplication.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-017-1335-7) contains supplementary material, which is available to authorized users.

Nuclear migration during yeast karyogamy, termed nuclear congression, is required to initiate nuclear fusion. Congression involves a specific regulation of the microtubule minus end-directed kinesin-14 motor Kar3 and a rearrangement of the cytoplasmic microtubule attachment sites at the spindle pole bodies (SPBs). However, how these elements interact to produce the forces necessary for nuclear migration is less clear. We used electron tomography, molecular genetics, quantitative imaging, and first principles modeling to investigate how cytoplasmic microtubules are organized during nuclear congression. We found that Kar3, with the help of its light chain, Cik1, is anchored during mating to the SPB component Spc72 that also serves as a nucleator and anchor for microtubules via their minus ends. Moreover, we show that no direct microtubule-microtubule interactions are required for nuclear migration. Instead, SPB-anchored Kar3 exerts the necessary pulling forces laterally on microtubules emanating from the SPB of the mating partner nucleus. Therefore, a twofold symmetrical application of the core principle that drives nuclear migration in higher cells is used in yeast to drive nuclei toward each other before nuclear fusion.