CINGULIN IS A COMPONENT OF THE APICAL MEMBRANE INITIATION SITE THAT INDUCES EPITHELIAL CELL POLARIZATION AND LUMEN FORMATION by ANTHONY MANGAN B.S., St. John Fisher College, 2009 M.S., Rochester Institute of Technology, 2012 A thesis submitted to the faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements of the degree of Doctor of Philosophy Molecular Biology Program 2017 This thesis for the Doctor of Philosophy degree by Anthony Mangan Has been approved for the Molecular Biology Program by James McManaman, Chair Moshe Levi Jeffrey Moore Chad Pearson Rui Zhao Rytis Prekeris, Advisor Date: 05-19-2017 ii Mangan, Anthony (Ph.D., Molecular Biology) Cingulin is a Component of the Apical Membrane Initiation Site that Induces Epithelial Cell Polarization and Lumen Formation Thesis directed by Professor Rytis Prekeris ABSTRACT Epithelial cells are structurally and functionally polarized to transport specific molecules while maintaining a trans-epithelial barrier. Additionally, epithelial cells coordinate their polarization with neighboring cells to form an apical lumen, a key step in the establishment of epithelial tissue architecture, and thereby function. Recent work from several laboratories including ours identified Rab11 and its binding protein FIP5 as major components that regulate apical endosome transport during apical lumen formation. I demonstrate that Rab11/FIP5- containing endosomes (FIP5-endosomes) mediate the formation of apical lumen by targeted delivery of apical lumen proteins. Using MDCK cells in a 3D tissue culture system, it was also shown that targeting of FIP5-endosomes to the apical membrane initiation site (AMIS) is a key step for the formation and expansion of an apical lumen in epithelial cysts. Furthermore, midbody formation during telophase is the first symmetry-breaking event that determines the site of lumen formation between two epithelial cells. Despite recent advances in our understanding of the mechanisms mediating apical lumen formation, many questions regarding the initiation of lumen formation remain unanswered. Here, I also focus on the identification of the machinery that mediates AMIS establishment and FIP5-endosome targeting during apical lumen formation. A new FIP5- interacting protein, Cingulin, is identified. Cingulin is a tight junction protein that localizes to the AMIS and is an ideal tether to mediate FIP5-endosome targeting during apical lumen formation. The machinery mediating Cingulin recruitment to the midbody during late telophase was also 3 analyzed and it demonstrates that both microtubule binding and actin networks are required for establishing the site of the apical lumen. I have also shown that Cingulin binds to microtubule C- terminal tails (CTTs) and that this binding is likely regulated by glutamylation of the tubulin tails. I completed immunoprecipitation, immunofluorescence, and proteomics analysis of synchronized epithelial cells in telophase and identified components of the WAVE/SCAR complex as putative regulators of Cingulin recruitment to the midbody. Since Rac1 is known to activate the WAVE/SCAR complex, it was demonstrated that Rac1 is also present at the midbody and that Rac1 activation is required for Cingulin recruitment to the midbody during apical lumen formation. Additionally, the formation of actin flares at the midbody during late telophase were observed and these actin flares may initiate cell polarization and apical lumen formation during epithelial morphogenesis. This data supports a combinatorial role of microtubules and actin in the coordination and regulation of the apical membrane initiation site and forming lumen. The form and content of this abstract are approved. I recommend its publication. Approved: Rytis Prekeris 4 TABLE OF CONTENTS CHAPTER I. POLARIZED PROTEIN TRANSPORT AND LUMEN FORMATION DURING EPITHELIAL TISSUE MORPHOGENESIS Abstract .……….….…………………………………………………………………………………… .......... 1 Introduction ………….………………………….…………………………………………………… ............. 1 Polarization of Individual Epithelial Cells …..……………………………………………………..2 Polarity complexes and actin cytoskeleton .……...…………………………………3 Polarized membrane traffic .…………………………...…………………………………..5 Apical Lumen Formation .……………………………………………………………………… ........... 8 In vitro models of de novo apical lumen formation ………………………………8 Mechanisms of de novo apical lumen formation during epithelial tissue morphogenesis …………………………………………………………………………………..13 The mechanisms of lumen extension and coalescence ……………………….16 Summary and Future Objectives ..……………………………………………………………… ........ 17 II. SPATIOTEMPORAL DYNAMICS OF FIP5 AND CINGULIN DURING APICAL LUMEN FORMATION Abstract .……….….…………………………………………………………………………………… ......... 24 Introduction ………….………………………….…………………………………………………… ............ 25 3D Time-Lapse Method …………………………………………………………………………………..26 Materials …………………………………………………………………………… ........... 26 Detailed instructional method ……………………………….…………………………..27 Results …………………………………………………………………………………………………………….30 5 Midbody and central spindle microtubules mediate AMIS formation and FIP5-endosome transport during telophase ……………………………………….30 Discussion ……………………………………………………………………………………………………….31 III. CINGULIN AND ACTIN MEDIATE MIDBODY-DEPENDENT APICAL LUMEN FORMATION DURING POLARIZATION OF EPITHELIAL CELLS Abstract .……….….…………………………………………………………………………………… ........ 38 Introduction ………….………………………….…………………………………………………… ............ 38 Results …………………………………………………………………………………………………………….40 Cingulin is a FIP5 binding protein concentrated at the AMIS ………… ......... 40 CGN is required for single apical lumen formation ……………………………..42 The WAVE/Scar complex is present at the AMIS …………………………………43 Active Rac1 is required for AMIS formation at the midbody ……………….44 CGN binding to tubulin mediates targeting to the midbody ……………….49 Discussion ……………………………………………………………………………………………………….52 Methods ………………………………………………………………………………………………………...55 Plasmids and antibodies ………………………………………………………………… .... 55 CGN knock-out by CRISPR/Cas9 in MDCK cells …………………………………...56 Immunoprecipitation and proteomic analysis …………………………………….56 Protein expression and purification ……………………………………………………57 Glutathione bead pull-down assays …………………………………………………...58 Cell culture …………………………………………………………………………………………59 Immunofluorescent, time-lapse, and FRET microscopy ………………………59 MDCK cell lines …………………………………………………………………………………..60 Rac1 inhibition studies ……………………………………………………………………….60 6 Yeast tubulin purification …………………………………………………………………..61 Microtubule binding assays ……………………………………………………………….61 Western blots ……………………………………………………………………………………62 IV. CONCLUSIONS AND FUTURE DIRECTIONS Conclusions ………………………………………………………………………………………………………….92 Future Directions …………………………………………………………………………………………………94 References ………………………………………………………………………………………………………………………..97 vii LIST OF FIGURES Figure 1.1 The proteins that establish and maintain epithelial cell polarity. 1.2 Cavitation (A) and Hallowing (B) models of apical lumen formation in vitro. 1.3 The role of the midbody during apical membrane initiation site (AMIS) formation and apical endosome recruitment. 1.4 Model depicting apical lumen coalescence during the formation of the zebrafish intestinal lumen. 2.1 Apical membrane initiation site (AMIS) formation around the midbody at late telophase mediates FIP5-endosome targeting during lumen formation. 2.2 FIP5-endosomes deliver apical proteins along central spindle microtubules to the midbody-associated AMIS. 2.3 Figure 2.3. Proposed models of FIP5-endosome trafficking and apical lumen initiation. 3.1 Cingulin (CGN) is an AMIS-associated FIP5-binding protein. 3.2 CGN is a FIP5-interacting protein. 3.3 Cingulin is required for the formation of a single apical lumen. 3.4 CGN knock-down affects apical lumen formation. 3.5 CGN depletion affects actin cytoskeleton in MDCK cells. 3.6 Components of the WAVE/Scar complex and branched actin filaments are present at the midbody during lumen formation. 3.7 Midbody and Cingulin associated actin flares. 3.8 Rac1 is activated at the AMIS. 8 3.9 Rac1 inhibition affects midbody-associated AMIS formation. 3.10 Rac1 is required for apical lumen formation. 3.11 Rac1 is required for the gp135 targeting during apical lumen formation. 3.12 Rac1 is required for the formation of a single apical lumen. 3.13 Electrostatic interactions mediate CGN binding to microtubule C-terminal tails (CTTs). 3.14 CGN binds to tubulin C-terminal tail domains. 3.15 Generation of CGN knock-out in MDCK cells using CRISPR/Cas9. 3.16 Mutation of basic patch disrupts subcellular CGN targeting. 3.17 CGN interaction with midbody microtubules and Rac1-induced branched actin cytoskeleton is required for AMIS formation and apical lumen initiation. 3.18 Uncropped scans of western blots. 9 CHAPTER I POLARIZED PROTEIN TRANSPORT AND LUMEN FORMATION DURING EPITHELIAL 1 TISSUE MORPHOGENESIS Abstract One of the major challenges in biology is to explain how complex tissues and organs arise from the collective action of individual polarized cells. The best-studied model of this process is the cross talk between individual epithelial cells during their polarization to form multi-cellular epithelial lumens during tissue morphogenesis. Multiple mechanisms of apical lumen formation have been proposed. One of the most widely accepted is that new epithelial lumens form from pre-existing polarized epithelial structures by wrapping epithelial sheets of budding new epithelial branches. However, a de novo lumen formation mechanism has recently emerged as an