Analysis of Gliotactin mediated microtubule organization during tricellular junction development
Evolutionary Cell and Developmental Biology (Zoology)
Final Report Abstract
The initial hypothesis that microtubule organization at the tricellular junction (TCJ) in the wing imaginal disc epithelium of Drosophila is mediated by Gliotactin (Gli) was experimentally rejected, as the preliminary results from the host lab could not be reproduced in this study. However, I developed a novel approach to visualize TCJs and pleated septate junctions (pSJ) in 3D by combining Lanthanum impregnation, prior to chemical fixation, with TEM tomography. The use of this technique allowed me to validate the current TCJ model in Drosophila, which so far was mainly based on experiments performed in amphipods, and furthermore to study TCJs in more detail. I could show that tricellular plugs (TCPs) are geometric structures and elucidated how these plugs are connected to the plasma membrane and to the pSJs. In addition, TCPs were found to be vertically connected to their adjacent neighbours. They exhibit different conformations, namely open and closed, which allows or denies the entrance of Lanthanum, respectively. These conformations appear to be formed randomly and further investigation is required to elucidate their specific function. Based on novel observations in this study, regarding the change of structure of pSJ along the bicellular junctions towards the TCJs, I propose a model in which the organization of the pSJ core complex increases gradually towards the TCJs. The hypothesized enhancement in organization results in an increasing rigidity, which is a putative result of the condensation of the pSJ core complexes. In the functional analysis of TCJs, the down regulation of Gli showed a reduced number of leading strands compared to the control. Additionally, detectable leading strands appear to be more distant to TCJs. The structure of TCJs, determined by detectability of TCPs, was also slightly reduced upon down regulation of Gli. The functional analysis of Anakonda (Aka) using RNAi mediated knockdown revealed a pronounced reduction of TCPs and leading strands compared to the control but also compared to the downregulation of Gli. These results indicate an involvement of Aka in TCJ formation, whereas Gli appears to act as a mediator and does not seem to be as important for the assembly of TCPs as Aka. However, these experiments require further validation, especially in a broader cellular context to assay the overall structure of TCJs and pSJs between different cells. In summary, the presented findings, based on the newly developed TCJ visualization method in combination with the morphological and functional analysis of TCJ proteins, provide novel insights into the structure and organization of TCJs as well as pSJs in Drosophila.
