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Bipolar spindle formation beyond Eg5 : A study on antagonists and synergists of the molecular motor Eg5 during mitotic spindle formation

Bipolar spindle formation beyond Eg5 : A study on antagonists and synergists of the molecular motor Eg5 during mitotic spindle formation

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FLORIAN, Stefan, 2011. Bipolar spindle formation beyond Eg5 : A study on antagonists and synergists of the molecular motor Eg5 during mitotic spindle formation

@phdthesis{Florian2011Bipol-15220, title={Bipolar spindle formation beyond Eg5 : A study on antagonists and synergists of the molecular motor Eg5 during mitotic spindle formation}, year={2011}, author={Florian, Stefan}, address={Konstanz}, school={Universität Konstanz} }

deposit-license 2011-08-31T10:13:36Z eng 2011 Cell division is an essential process which was prerequisite for the development of life and is key for both development of complex organisms and reproduction in general. The accuracy of this process, which is a part of mitosis, depends on the mitotic spindle, a bipolar, dynamic structure which segregates sister chromatids and thus separates the genomes of the two developing daughter cells. Its shape is defined by the arrangement of polar filaments called microtubules. Mitotic defects can result in cell death but also, if missed by cellular control systems, in the development of cancer. The tetrameric motor protein Eg5 is believed to separate spindle poles by cross-linking and pushing apart microtubules emanating from opposite poles and overlapping at the spindle midzone. Depletion or inhibition of Eg5 causes the formation of star-shaped, morphologically monopolar spindles. The function of the mitotic spindle has received a lot of attention in cell biology for the last 100 years leading to a very detailed characterization of the role of its individual substructures or proteins in spindle formation. During the last two decades, the advent of novel technologies like RNA interference, high-throughput sequencing and microscopy resulted in significant growth of our knowledge about this process. Although existing ideas about how individual elements might cooperate and the development of an integrated concept of bipolar spindle formation are evolving rapidly, we still struggle to keep pace with the experimental results made possible by technological progress. In this work, we aimed to better understand the interplay of Eg5 with its synergists and antagonists during spindle formation by searching for perturbations allowing bipolar spindles to form in absence of Eg5 activity.<br /><br />During preliminary studies for the project, we find that the most frequent approach to quantify the fraction of defective spindles forming after experimental perturbations, counting of their frequency in fixed immunofluorescence microscopy samples, results in fundamentally wrong conclusions. They are caused by the spindle assembly checkpoint, which arrests malformed spindles in mitosis until their defects can be repaired, and thus leads to their preferential accumulation in mitosis. We prove both the resulting distortion in fixed samples as well as the potentially false results using both mathematical modeling and proof-of-concept experiments.<br /><br />Therefore focusing on a rigorous live-cell-imaging approach, we identify a new pathway of bipolar spindle formation which does not depend on Eg5 motor activity. Upon slight perturbations of microtubule dynamics using either low doses of the microtubule drug nocodazole or depleting the protein TOGp, functional bipolar spindles assemble despite inhibition of Eg5. This process is accompanied by an increase in microtubule nucleation in the vicinity of chromatin. This is the first time that chromosomal nucleation is directly linked to centrosome separation. Moreover, we show that this is a fully redundant, self-sufficient and Eg5-independent pathway which suggests that current bipolarization models, collectively relying on Eg5 as a pivotal force behind pole separation, will have to be adjusted.<br /><br />Furthermore, because most current models rely on fixed samples as the main source of experimental evidence, these results will have to be revalidated using live-cell imaging. Our results might also have implications for the ongoing development of chemotherapeutic treatment regimens involving Eg5 inhibitors which are currently under evaluation for cancer treatment because of their antiproliferative effect. Because microtubule drugs (similar to nocodazole) are also used in such protocols, combination therapies involving both may be less effective than predicted.<br /> 2013-08-07T22:25:03Z Florian, Stefan Bipolar spindle formation beyond Eg5 : A study on antagonists and synergists of the molecular motor Eg5 during mitotic spindle formation Florian, Stefan

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

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