Abstract Adherens junctions formed by E-cadherin adhesion complexes play central roles in the organisation and apical-basal polarisation of both mammalian and insect epithelia. Here we investigate the function of the components of the E-cadherin adhesion complex in the Drosophila midgut epithelium, which establishes polarity by a different mechanism from other fly epithelia and has an inverted junctional arrangement, in which the adherens junctions lie below the septate junctions. Unlike other epithelial tissues, loss of E-cadherin, Armadillo (β-catenin) or α-catenin has no effect on the polarity or organisation of the adult midgut epithelium. This is not due to redundancy with N-cadherin, providing further evidence that the midgut polarises in distinct way from other epithelia. However, E-cadherin ( shg ) and armadillo mutants have an expanded septate junction domain and a smaller lateral domain below the septate junctions. Thus, E-cadherin adhesion complexes limit the basal extent of the septate junctions. This function does not appear to depend on the linkage of E-cadherin to the actin cytoskeleton because α-catenin mutants do not significantly perturb the relative sizes of the septate and sub-septate junction domains.
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.
// Mihoko A. Tame 1, * , Anna G. Manjón 1, * , Daria Belokhvostova 1 , Jonne A. Raaijmakers 1 and René H. Medema 1 1 Division of Cell Biology and Cancer Genomics Center, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands * Co-first author Correspondence to: René H. Medema, email: r.medema@nki.nl Keywords: taxol, resistance, microtubule, βIII-tubulin, CRISPRa Received: November 30, 2016 Accepted: April 22, 2017 Published: May 10, 2017 ABSTRACT Microtubules are cellular targets for a variety of anticancer therapies because of their critical function in mitosis. Taxol belongs to a class of microtubule targeting agents that suppresses microtubule dynamics and interferes with the functioning of the mitotic spindle, thereby effectively blocking cell cycle progression of rapidly proliferating tumor cells. Despite its antitumor activity, drug resistance remains a common obstacle in improving its overall clinical efficacy. Previous studies have shown that the expression of a specific β-tubulin isotype, βIII-tubulin/TUBB3, is dysregulated in drug-refractory tumors. However, whether enhanced TUBB3 expression is directly involved in promoting taxol resistance remains a subject of debate. Here, we have used several approaches to assess the functional relation of TUBB3 overexpression and taxol resistance. First, we generated a number of taxol-resistant cell lines, to find that TUBB3 expression was elevated in a resistant cell line (RPE-20) derived from untransformed retinal pigment epithelial (RPE) cells, but the abundance of TUBB3 remained unchanged in four other cell lines after taxol treatment. However, although RPE-20 cells displayed enhanced TUBB3 levels, we find that simultaneous up-regulation of the P-glycoprotein (P-gP) drug-efflux pump is responsible for the resistance to taxol. Indeed, we could show that TUBB3 levels were dynamically regulated upon taxol exposure and withdrawal, unrelated to the resistance phenotype. Next, we generated cell lines in which we could induce robust overexpression of TUBB3 from its endogenous locus employing the CRISPRa system. We demonstrate that solely enhancing TUBB3 expression results in a very minor decrease in the sensitivity to taxol. This was further substantiated by selective depletion of TUBB3 in a series of breast cancer cell lines expressing high levels of TUBB3. We find that TUBB3 depletion had a minimal effect on the sensitivity to taxol in one of these cell lines, but had no effect in all of the others. Based on these findings we propose that TUBB3 overexpression can only marginally affect the sensitivity to taxol in cultured cell lines.
Cytoplasmic dynein is recruited to the cell cortex in early mitosis, where it can generate pulling forces on astral microtubules to position the mitotic spindle. Recent work has shown that dynein displays a dynamic asymmetric cortical localization, and that dynein recruitment is negatively regulated by spindle pole-proximity. This results in oscillating dynein recruitment to opposite sides of the cortex to center the mitotic spindle. However, although the centrosome-derived signal that promotes displacement of dynein has been identified, it is currently unknown how dynein is re-recruited to the cortex once it has been displaced. Here we show that re-recruitment of cortical dynein requires astral microtubules. We find that microtubules are necessary for the sustained localized enrichment of dynein at the cortex. Furthermore, we show that stabilization of astral microtubules causes spindle misorientation, followed by mispositioning of dynein at the cortex. Thus, our results demonstrate the importance of astral microtubules in the dynamic regulation of cortical dynein recruitment in mitosis.
