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An Unconventional Role for the Septate Junctions and Gliotactin in Cell Division

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An Unconventional Role for the Septate Junctions and Gliotactin in Cell Division AN UNCONVENTIONAL ROLE FOR THE SEPTATE JUNCTIONS AND GLIOTACTIN IN CELL DIVISION by KRISTI CHARISH B.Sc. (Molecular Biology and Biochemistry), Simon Fraser University, 2004 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Zoology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) July 2011 © Kristi Charish, 2011 Abstract The focus of this thesis is to investigate the integration of cell division with the septate junction domain in the Drosophila imaginal wing disc epithelium. Columnar epithelia of the imaginal wing disc exhibit complex architecture due to an elaborate series of junctions that are found throughout the membrane. During cell division, these junctions are maintained while new junctions are established; however, their role and influence during mitosis is unclear. This thesis shows that the septate junctions are essential for cytokinesis and Gliotactin at the tricellular junctions is necessary to localize cell division to the septate junction domain, and illustrates a unique role for Gliotactin and the septate junctions outside their classic role of maintaining a permeability barrier. The septate junctions are basolaterally localized transmembrane junctions required in epithelial cells to form a permeability barrier. Although the septate junctions are formed by a large protein complex, this thesis only investigates the three core SJ proteins, NeurexinIV (NrxIV), Coracle (Cor), and Neuroglian (Nrg). Gliotactin (Gli), a Drosophila Neuroligin homologue, is a septate junction associated protein concentrated at the tricellular junction (TCJ), which is necessary to maintain the septate junction permeability barrier. Loss of any of the septate junction proteins, or Gliotactin, leads to structural disruption of the septate junctions and loss of the permeability barrier in a wide range of epithelial derived tissues. Chapter two examines the process of cell division in epithelial cells of the wing imaginal disc with respect to the septate junctions and tricellular junction. Chapter three looks at the role of Gliotactin in maintaining the plane of cell division within the septate junction domain, and chapter four shows that the septate junctions are necessary for ingression furrow stability and the association of the contractile ring with the membrane during late cytokinesis. This work ii demonstrates a novel role for the septate and tricellular junctions during mitosis in Drosophila, which has implications for the role of tight junctions in vertebrate cells. iii Table of Contents Abstract ......................................................................................................................................... ii Table of Contents ......................................................................................................................... iv List of Tables ............................................................................................................................... vi List of Figures ............................................................................................................................. vii List of Abbreviations ................................................................................................................... xi Acknowledgements ..................................................................................................................... xii I. INTRODUCTION ................................................................................................................... 1 II. CELL DIVISION IN POLARIZED EPITHELIA CELLS OF THE DROSOPHILA IMAGINAL WING DISC ........................................................................................................ 60 Introduction ............................................................................................................................ 60 Materials and Methods ........................................................................................................... 62 Results .................................................................................................................................... 64 Discussion .............................................................................................................................. 77 III. GLIOTACTIN IS ESSENTIAL TO MAINTAIN THE PLANE OF CELL DIVISION IN POLARIZED EPITHELIA CELLS OF THE IMAGINAL WING DISC .................. 111 Introduction .......................................................................................................................... 111 iv Materials and Methods ......................................................................................................... 113 Results .................................................................................................................................. 116 Discussion ............................................................................................................................ 125 IV. THE SEPTATE JUNCTIONS ARE NECESSARY FOR THE COMPLETION OF CYTOKINESIS ....................................................................................................................... 145 Introduction .......................................................................................................................... 145 Materials and Methods ......................................................................................................... 148 Results .................................................................................................................................. 151 Discussion ............................................................................................................................ 161 V. GENERAL DISCUSSION ................................................................................................ 182 REFERENCES ........................................................................................................................ 196 v List of Tables I. Introduction Table 1.1: Drosophila septate junction proteins and functions ................................................... 57 Table 1.2: The Drosophila septate junction and tricellular junction proteins ............................. 59 vi List of Figures I. Introduction Figure 1.1: Drosophila septate and tricellular junctions ............................................................. 35 Figure 1.2: The permeability barrier is disrupted in septate junction and tricellular junction mutants ........................................................................................................................................ 37 Figure 1.3: Components of the septate junctions and their functions ......................................... 39 Figure 1.4: The tricellular tight junction ..................................................................................... 41 Figure 1.5: Gliotactin and the tricellular junction ....................................................................... 44 Figure 1.6: Mitosis ...................................................................................................................... 46 Figure 1.7: The spindle establishes the cleavage plane .............................................................. 49 Figure 1.8: Progression of the ingression furrow through cytokinesis ....................................... 51 Figure 1.9: Establishing apical basal polarity ............................................................................. 53 Figure 1.10: The Drosophila 3rd instar imaginal wing disc ........................................................ 55 II. Cell division in polarized epithelia cells of the Drosophila imaginal wing disc Figure 2.1: Localization of cell division markers in polarized epithelia .................................... 88 Figure 2.2: The septate junctions and cell division..................................................................... 91 Figure 2.3: Initiation of the cleavage furrow during telophase ................................................... 93 vii Figure 2.4: Progression of the cleavage furrow during telophase .............................................. 96 Figure 2.5: The centrosomes migrate apically during telophase ................................................ 99 Figure 2.6: Polo kinase associates with key structures throughout mitosis in columnar epithelial cells ........................................................................................................................................... 102 Figure 2.7: The contractile ring localizes to the septate junctions during cytokinesis ............. 104 Figure 2.8 Zipper/myosin II has a distinct localization pattern in columnar epithelial cells .... 106 Figure 2.9: The adherens junctions form during telophase C ................................................... 108 Figure 2.10: Squamous peripodial epithelial cells show same division of the contractile rings in telophase as the columnar epithelial cells .................................................................... 110 III. Gliotactin is essential to maintain the plane of cell division in polarized epithelial cells of the imaginal wing disc Figure 3.1: Null clones for Gliotactin fail to undergo cell division .......................................... 132 Figure 3.2: Expression of septate junction RNAi in the imaginal wing disc leads to a disruption in the septate junction associated protein Gliotactin ..............................................
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