Automatisiertes Screening-Mikroskop
Final Report Abstract
We are interested in the process of chromosome segregation in higher eukaryotes. Specifically, our research focuses on the function and regulation of motor proteins involved in spindle assembly and chromosome congression, the regulation of cell cycle progression by ubiquitin-ligases and the coordination of karyokinesis (mitosis) with cytokinesis. For these studies we combine cell biological approaches in in human tissue culture cells and Xenopus egg extract with biochemical in vitro analyses. Studies on the function of the human kinesin Kif18A revealed that the motor protein has an additional, nonmotor microtubule binding site essential for the high processivity of Kif18A. Kif18A is a unique kinesin in that integrates both plus-end directed motility and microtubule depolymerization activity. Cells depleted of Kif18A have elongated spindles, hyperstable microtubules and severe defects in chromosome congression. Our in vitro analyses revealed that truncated Kif18A lacking the C-terminal microtubule binding site displays faster motility but lower processivity. In cells, the truncated form of Kif18A does not rescue the chromosome congression defect induced by depletion of endogenous Kif18A indicating that the high processivity of Kif18A is essential for its mitotic function. By screening a large compound collection we were able to identify a small molecule inhibitor of Kif18A. In vitro assays revealed that this compound inhibits Kif18A in an ATP- competitive but microtubule-uncompetitive manner. Future studies are required to characterize the mode of action of this inhibitor in cells. Studies on the process of bipolar spindle formation revealed that subtle changes in microtubule dynamics enables cells lacking the activity of Eg5 – a homotetrameric kinesin that uses its plus-end directed motility to push spindle poles apart – to assemble bipolar spindles. Thus, in the absence of Eg5 activity a subtle change of the dynamic behavior of microtubules is sufficient to assemble bipolar spindles. Notably, this process depends on the kinesin Kif15 which under normal conditions is dispensable for the establishment of spindle bipolarity. Given that alterations in microtubule dynamics are a common mechanism responsible for the resistance of tumor cells to microtubule poisons, e.g. vinblastine, our insights suggest that cells resistant to microtubule drugs might also be insensitive to drug-induced inhibition of Eg5. This observation might be important for the evaluation of the efficacy of Eg5 inhibitors which are currently tested in clinical phases. Our studies on the mechanism of spindle also revealed that the analysis of fixed cells is not appropriate to quantify the frequency of spindle phenotypes in cells. By combining a mathematical model with proof-ofconcept experiments we could demonstrate that the analysis of fixed samples is distorted due to the different life times of spindle phenotypes, i.e. defective but not normal spindles are delayed in mitotic progression because of the activity of the spindle assembly checkpoint. Without knowing the effect of a specific perturbation, e.g. chemical inhibitor treatment or siRNA-mediated depletion, on the life time of spindle phenotypes, fixed cell analyses result in wrong conclusions and, therefore, live-cell analyses have to be performed. In light of this insight, we revisited current models on spindle assembly and discovered that the mitotic push-pull model where Eg5 and dynein are direct antagonists in the mitotic spindle is not compatible with the cellular phenotypes we observed by live-cell imaging. For our studies on the regulation of cell cycle progression by ubiquitin ligases we focused on XErp1/Emi2, an inhibitor of the anaphase promoting complex / cyclosome (APC/C). Previously, we have shown that XErp1 is essential to mediate the metaphase II arrest of mature Xenopus eggs. Our latest results revealed that the APC/C can liberate itself from XErp1 inhibition by mediating the ubiquitylation of XErp1. As shown by our biochemical assays, ubiquitylation of XErp1 is accompanied by the dissociation of XErp1 from the APC/C resulting in APC/C activation. Thus, based on our data we suggest that the non-proteolytic ubiquitylation of XErp1 could contribute to the fast, switch-like activation of the APC/C at fertilization. In summary, our studies provided novel and unexpected insights into the process of mitotic and meiotic chromosome segregation, into the regulation of factors involved in these processes, and how cells can complement for defects in pathways involved in spindle assembly.
Publications
- (2009). BTB-1: The first small molecule inhibitor of the mitotic motor protein Kif18A. Angew. Chem. Int. Ed., 48, 9072-9076
Catarinella M, Grüner, T, Strittmatter, T, Marx, A, and Mayer, TU
(See online at https://doi.org/10.1002/anie.200904510) - (2010). Monastrol analogs: a synthesis of pyrazolopyridine, benzopyranopyrazolopyridine, and oxygen-bridged azolopyrimidine derivatives and their biological screening. Bioorg Med Chem Lett. 20, 4073-4076
Svetlik, J., Veizerová, L., Mayer, T.U., Catarinella, M.
(See online at https://dx.doi.org/10.1016/j.bmcl.2010.05.085) - (2011). A Non-motor Microtubule Binding Site is Essential for the High Processivity and Mitotic Function of Kinesin-8 Kif18A. PlosOne, 6, e27471
Mayr, M.I., Storch, M., Howard, J., and Mayer, T.U.
(See online at https://doi.org/10.1371/journal.pone.0027471) - (2011). A Versatile Framework for the Analysis of High-Throughput Screening Data. 8th International Workshop on Computational Systems Biology (WCSB), 57-60
Hamecher, J., Rieß, T., Bertini, E., Kozak, K., Kastl, J., Mayer, T.U., Merhof, D.
- (2011). Identification of Novel Quadruplex Ligands from Small Molecule Libraries by FRET-Based High-Throughput Screening. Chembiochem, 12, 1422-1426
Benz, A., Singh, V., Mayer, T.U., Hartig, J.S.
(See online at https://doi.org/10.1002/cbic.201100094) - (2011). Modulated microtubule dynamics enable Hklp2/Kif15 to assemble bipolar spindles. Cell Cycle, 10, 3533-3544
Florian, S. and Mayer, T.U.
(See online at https://doi.org/10.4161/cc.10.20.17817) - HiTSEE: A Visualization Tool for Hit Selection and Analysis in High-Throughput Screening Experiments. 1st IEEE Symposium on Biological Data Visualization (IEEE BioVis), pp. 95-102, 2011
Enrico Bertini, Hendrik Strobelt, Joachim Braun, Oliver Deussen, Ulrich Groth, Thomas U. Mayer, Dorit Merhof
(See online at https://doi.org/10.1109/BioVis.2011.6094053)
