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A multidisciplinary approach to cell division: From human oocyte to synthetic biology

Periodic Reporting for period 2 - DivIDe (A multidisciplinary approach to cell division: From human oocyte to synthetic biology)

Reporting period: 2018-04-01 to 2020-03-31

Cell division is a fundamental process for the development, growth and tissue maintenance and renewal of all organisms. There are two type of cell divisions, mitosis (producing two identical daughter cells) and meiosis (producing female and male gametes). Mistakes occurring during cell division may have dramatic consequences for the development and health of the organisms that may lead to cell death, infertility and cancer. The process of cell division is therefore critical for the life and health of higher organism. In fact, the cell relies on highly complex regulatory networks involving specific sets of genes and proteins as well as error correction mechanisms to avoid mistakes in chromosome segregation during mitosis and meiosis.
DivIDE was a European training network of international researchers that aimed to address the mechanisms underlying cell division through an interdisciplinary approach combining molecular biology, biophysics, engineering, mathematical modelling, clinical embryology, and drug discovery. The network had two main objectives. First, training a new generation of molecular engineers with interdisciplinary and intersectoral skills to address cell division from a wide technological perspective. Second, to develop interconnected research projects addressing the functional role of post-translational modifications of the microtubules as well as essential protein complexes and mechanisms occurring during mitosis and meiosis.
Eleven young researchers addressed cell division from a different perspective using complementary techniques, approaches and experimental model systems. Ivan, Jijumon., Evi and Pragya studied tubulin post-translational modifications. Using human tissue culture cells and zebra fish embryos Ivan found that spindle microtubule polygltuamylation is essential for faithful chromosome segregation during mitosis. Jijumon managed to purify, tubulin from mammalian cell lines with a set of precisely defined posttranslational modifications, and directly measure how these modifications control the interactions with a panel of microtubule-associated proteins. Evi focused on the enzymes that create tubulin posttranslational modifications in the meiotic spindle of human oocytes finding specific differences in expression between oocytes cultured in vitro and matured in vivo. Pragya aimed at identifying inhibitors of Tubulin Tyrosine Ligase Like (TTLL) 4, an enzyme, that polyglutamylates tubulin a modification involved in neuronal maturation and metastatic progression. She managed to set up a novel pipeline for High Throughput Screening of TTLLs identifying and validating small molecule inhibitors of a chemical compound library comprising 190.000 compounds.
Christel, Ennio and Alejandra studied the topology of protein assemblies within the spindle. Christel successfully purified some of the main human components of the dynein complex to perform in vitro experiments of spindle assembly. Ennio purified Dynactin subcomplexes to study the silencing of Spindle Assembly Checkpoint, and studied the crucial role of the putative adaptor and cargo binding protein Spindly in this process. Alejandra studied the mechanisms regulating minus-end dynamics of spindle microtubules focusing on the characterization of a recently identified protein complex that associates specifically with the K-fiber microtubule minus-end during mitosis. In collaboration with Robert visualised through tomography how this complex alters K-fibers of spindles assembled in silenced cells.
Ana, Robert, Manuel, and Pablo focused on the visualization and the quantitative analysis of cell division. While studying the differences between meiosis and mitosis in the spindle dynamics and chromosome segregation, Ana found the proteins responsible for spindle elongation during meiosis. Robert obtained the first full 3D reconstructions of metaphase and anaphase spindles in a human cell line. These reconstructions are complemented by detailed quantitative descriptions of microtubule organization within these spindles. Manuel used mathematical modelling to explain how motor proteins, dynamic filaments, and microtubules assemble to form an elongating spindle. He found why longer spindles elongate faster. Pablo developed a microfluidic device based on the Cherry Temp device that allows thermalizing and perfusing with different buffers a cell sample in the microscope. Further, Pablo started to develop an Artificial Intelligence based classifier to better predict the effect of specific targets on the cell decision.
As a main training and dissemination activity, the DivIDE fellows organized the conference “From Pole to Pole” in Barcelona. The fellows that were located hundreds of kilometres away across Europe had to team up to decide on the scientific content and the speakers list, as well as establish the needed logistics, the budgeting of expenses, scouting for sponsors and outreaching the event to manage the conference etc. This was an intensive learning process during the months prior to this event and also during the event itself. The conference was a big success and all fellows acknowledged that the event was mind opening.
Results obtained in this network, together with first published reports suggest that their role is to adapt the microtubule network to its specific functions. It appears that these regulatory functions lead to subtle but important changes in microtubule behaviour, and might thus be ideally leading to late-onset diseases like cancer and neurodegeneration. The 3D reconstructions of HeLa cells spindle that are available now will provide the grounds to further test the effect of microtubule polyglutamylation on the ultrastructural organization of the spindles.
Our findings regarding the mitotic protein NuMA has provided new insight into the molecular mechanism of spindle poles formation during cell division. This improves our understanding of an important mechanism required for spindle stability and correct cell division.
Work in this consortium also led to ground-breaking theoretical models of mitotic spindles, and to a development of the computational tools used to analyse these models. Such progress is essential to ultimately understand complex biological processes.
The development of a new technology(microfluidics device) and of development of therapeutic drugs (patentable small molecules) has significant exploitable potential. Indeed, the TTLL inhibitors developed can be used as chemical probes to develop new drugs for the treatment of cell cycle related diseases (e.g. cancers) or neurodegenerative diseases (e.g. Parkinson). The microfluidic technology goes beyond the current state of the art as it improves the understanding of the temperature usage as a controllable switch/trigger to decipher complex biological phenomena. This advance will most likely raise the interest of the research community in the coming decades.
Overall, DiviDE shows our society that scientific innovation and basic research are complementary and can have direct impact on our daily life.
Importantly, all the fellows highly valued the training offered within DiviDE because it offered additional technical and soft skills and an ideal niche to network and collaborate with people within and outside academia.