Mitotic spindle perturbations

Abstract

Microtubules are major components of the cytoskeleton and form the bipolar spindle apparatus during mitosis. The mitotic spindle consists of highly dynamic microtubule polymers that are under constant modulation, controlled by multiple motor proteins and microtubule-associated proteins. This tight spatiotemporal regulation of MT-dynamics within the spindle is essential for the fidelity of chromosome segregation and proper positioning of the mitotic spindle. The first part of this thesis describes mechanisms of spindle positioning in 2D cultured cells grown on adhesive micropatterns. In chapter 2, we demonstrated that dynein recruitment to the cortex occurs through two distinct pathways; one that originates from the extracellular environment and is regulated by the actin cytoskeleton and another one that is intracellular and microtubule-dependent. We further showed that perturbation in astral microtubule dynamics results in erroneous deposition of dynein at the cortex. In chapter 3, we uncovered a direct link between chromosome alignment errors and spindle orientation defects. We demonstrate that Plk1 is a negative regulator of cortical LGN, a key recruitment factor of dynein, and that the kinetochore-pool of Plk1 on misaligned chromosomes delocalizes LGN from cortical sites in its proximity, thereby inducing spindle misorientation. As proper regulation of MT dynamics and faithful mitotic outcome is important for cell survival, the mitotic spindle is also considered an important target in cancer therapy. Tumor cell killing is achieved by drug-mediated perturbations of MT dynamics and promotion of aberrant mitoses, which would terminate their cell cycle progression. Despite their initial anti-tumor activity, the clinical efficacy of many of such chemotherapeutics is often limited due to drug resistance. In the second part of this thesis, we have explored the possible role of alterations in specific tubulin isotype expression in taxol resistance. In chapter 4, we showed that induced overexpression of TUBB3 in cultured cells results in a minor decrease in their sensitivity to taxol. However, we also demonstrated that a functional role of TUBB3 is not generally applicable, as TUBB3 knockdown did not alter cell sensitivity to taxol in multiple cancer cell lines. Furthermore, we found that TUBB3 expression can be dynamically regulated unrelated to a taxol resistance phenotype. Thus, while TUBB3 expression can affect the cellular response to taxol, TUBB3 levels are not always indicative of how cells will respond to taxol.