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Organisation of microtubules and force-balance in metaphase spindles of HeLa and Ptk1 cells

During cell division, sister chromatids are segregated by mitotic spindle, a bipolar self-assembly of microtubules and associated proteins. Current models of mitotic spindle recognize three distinct subpopulations of microtubules: kinetochore, interpolar and astral microtubules. The role of antiparallel non-kinetochore microtubule bundles, termed bridging fibers, positioned under sister kinetochores in HeLa and Ptk1 cell lines, was analysed in this study. Using live-fluorescent imaging analysis it was shown that these bundles are present in differently labelled HeLa and Ptk1 cell lines with distinct thicknesses. The response of outermost spindle element to laser ablation in Ptk1 cell line was also analysed. Joint outward movement of bridging and k-fibers was observed suggesting connection into single mechanical unit. Decrease in inter-kinetochore distance was also observed after ablation. That decrease is inversely correlated with distance of the cut from the kinetochore. In addition, spindle element straightening after ablation was observed in all cell lines but with strongest straightening in HeLa cell line with thickest bridging fiber, demonstrating that response to ablation and thickness of bridging fiber are correlated. Finally, some of obtained results were compared with theoretical model of the HeLa metaphase spindle confirming robustness of the model by using some of our experimental data as inputs. In conclusion, obtained results demonstrate that bridging fiber is important structural component of metaphase spindle involved in balancing compressive and tensile forces in spindle.

Microtubules; metaphase spindle; cell division; bridging fiber

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