Final Report Summary - MECHANICUS (The mechanics of nuclear division and positioning) SCIENTIFIC SUMMARY:During development of an embryo, the genetic material is multiplied several fold to generate the embryonic tissue that later specified. Multiplication involves several cell division events, so called mitotic divisions. In higher organisms, the division is accompanied by the physical division of the cell membrane. The cell membrane acts as a physical barrier and identifies each cell as a biochemical unit. In other species, such as insects, cell membrane division does not occur, and the genetic material is copied and distributed in the same cell until many thousands of copies exist. In addition, many egg cells are ten to a hundred times larger than the genetic material. From a physical point of view, this is a challenging task for the embryo as it has to maintain the genomic units, the nuclei, well separated and ordered. We know that when nuclei are distributed irregularly the embryo prematurely aborts development. In this project, we have elucidated the physical mechanisms that guarantee the nuclear separation. First, we found that the microtubule cytoskeleton of the embryo cell as well as the organiser of this cytoskeleton, the centrosomes, are essential for the spatial organisation – the transport, precise positioning and separation of nuclei – in a unicellular embryo with multiple nuclei. As the name suggests, the centrosomes are the central structure from which microtubules grow outwards, forming a cytoskeletal structure called aster. Every nucleus is associated with one or two asters. These asters are transporting the nucleus within the cell between nuclear divisions. Second, by analysing mutants of the fruit fly Drosophila melanogaster, we found that these microtubule asters alone, in the absence of a nucleus, self-organise into a matrix with the same spatial regularity and period, suggesting that the microtubule cytoskeleton is the spatial organiser of nuclei in the embryo, and that the nuclei themselves do not actively contribute much to this mechanical process. Third, we resolved the mechanical nature for matrix assembly; we show evidence for the existence of a repulsive force between neighbouring asters, and this force inversely scales with the distance between asters. In a system of multiple asters this distance-dependent force results in a dynamic equilibrium of nuclear positioning, whereby any perturbation leads to a brief, dynamic adjustment and a new equilibrium with recovered spatial period. Fourth, in Drosophila melanogaster we have identified two genes to be essential for organising the microtubule cytoskeleton into a regular matrix, which is necessary for the biophysical process of nuclear positioning to work. These genes are encoding microtubule binding proteins that cross-link single microtubules and modulate their length. Because of these properties, these proteins are mechanically coupling microtubules from neighbouring asters, preventing these asters from approaching each other. We hypothesise that part of the repulsive force described above is generated by the same proteins.We foresee that the identified mechanism is conserved not only for all insects, which represent the majority of animals, but also in higher organisms and important for the development of embryos that are comparatively large.COMMENTS ON PROJECT MANAGEMENT:We have only used Drosophila melanogaster as model system throughout the project.A detailed description of the method that enabled all the studies of this project has been published as a chapter of a renowned book series of cell biological methods. The results on essential genes for nuclear separation have been published as a PhD thesis and accepted by the Instituto de Tecnologia Quimica e Biologica Antonio Xavier, Univeridade Nova de Lisboa. All described accomplishments and results are currently assembled into two manuscripts for peer-review publication. The first manuscript will report on the mechanics of microtubule aster-based nuclear separation, while the second manuscript will highlight the genes that were found essential for assembling the cytoskeletal matrix.During the second reporting period, the fellow interrupted his research activity for the last six months of 2017. The reason was a severe accident that happened to his spouse during horse riding leading to severe spinal cord injury. The incident forced the fellow to accompany his spouse during the majority of time in hospitals and rehabilitations centres, and not being present at the host institute. Nevertheless, the postdoc and the PhD students who worked in the framework of the project, and whose salary was paid by the project, continued their activity and the fellow supervised them remotely. Naturally, this has led to considerable delays in the project reporting phase, most importantly the production of manuscripts for peer-reviewed publication. The host institute has fully supported the fellow’s decision of priority.WEBSITE:http://www.igc.gulbenkian.pt/itelley Documenti correlati Figure
