i Basement Membrane Dynamics During Anchor Cell Invasion by Meghan Morrissey University PDF
Preview i Basement Membrane Dynamics During Anchor Cell Invasion by Meghan Morrissey University
Basement Membrane Dynamics During Anchor Cell Invasion by Meghan Morrissey University Program in Genetics and Genomics Duke University Date:_______________________ Approved: ___________________________ David Sherwood, Supervisor ___________________________ Amy Bejsovec ___________________________ Harold Erickson ___________________________ Daniel Kiehart ___________________________ Terry Lechler Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the University Program in Genetics and Genomics in the Graduate School of Duke University 2015 i v ABSTRACT Basement Membrane Dynamics During Anchor Cell Invasion by Meghan Morrissey University Program in Genetics and Genomics Duke University Date:_______________________ Approved: ___________________________ David Sherwood, Supervisor ___________________________ Amy Bejsovec ___________________________ Harold Erickson ___________________________ Daniel Kiehart ___________________________ Terry Lechler An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in University Program in Genetics and Genomics in the Graduate School of Duke University 2015 i v Copyright by Meghan Morrissey 2015 Abstract Basement membranes are a dense, sheet-‐‑like form of extracellular matrix that underlie epithelia and endothelia, and surround muscle, fat and Schwann cells. Basement membranes separate tissues and protect them from mechanical stresses. Although traditionally thought of as a static support structure, a growing body of evidence suggests that dynamic basement membrane deposition and modification instruct cell behavior and morphogenetic processes. In this thesis, I discuss how changes to basement membrane affect anchor cell (AC) invasion during C. elegans uterine vulval attachment. During AC invasion, the uterine AC breaches two juxtaposed basement membranes to contact the underlying vulval epithelium. Using live-‐‑cell imaging, genetics, molecular biology and electron microscopy I identify three modifications to the basement membrane that affect AC invasion. In Chapter 2, I describe a system for linking juxtaposed basement membranes to stably align or connect adjacent tissues. This adhesion system promotes rapid AC invasion and also regulates a more long-‐‑term connection between the uterine tissue and the hypodermal seam cell in the adult worm. Chapter 3 elucidates how the basement membrane component SPARC promotes cell invasion. As SPARC overexpression is correlated with cancer metastasis, this study aims to understand how SPARC overexpression promotes invasion in a pathological situation. In Chapter 4, I discuss preliminary data showing that the AC actively secretes iv laminin into the basement membrane targeted for invasion. I outline how future studies could elucidate the mechanism by which AC-‐‑derived laminin might promote cell invasion. Finally, Chapter 5 discusses conclusions and future directions for these studies. v Dedication Dedicated to my husband, John, for whole-‐‑heartedly supporting every decision I’ve made, regardless of its merits. vi Contents Abstract .......................................................................................................................................... iv Dedication ..................................................................................................................................... vi List of Tables ................................................................................................................................. xi List of Figures .............................................................................................................................. xii Acknowledgements ................................................................................................................... xiv 1. An active role for basement membrane assembly and modification in tissue sculpting 1 1.1 Introduction ....................................................................................................................... 1 1.2 Basement membrane deposition instructs cell polarization and tissue shape ........ 4 1.3 Collagen within the basement membrane constricts and contours tissues. ............ 8 1.4 Basement membrane linkages (B-‐‑LINKs) connect tissues ........................................ 15 1.5 Conclusions ..................................................................................................................... 21 2. B-‐‑LINK: A hemicentin, plakin and integrin-‐‑dependent adhesion system links tissues by connecting adjacent basement membranes ........................................................................ 23 2.1 Introduction ..................................................................................................................... 23 2.2 Results .............................................................................................................................. 26 2.2.1 A BM-‐‑BM linkage forms specifically under the AC prior to invasion. ............. 26 2.2.2 Hemicentin is present at the site of BM-‐‑BM adhesion ......................................... 31 2.2.3 BM-‐‑BM adhesion is hemicentin dependent .......................................................... 35 2.2.4 AC invasion is delayed when BM-‐‑BM adhesion is disrupted ............................ 36 2.2.5 VAB-‐‑10A (plakin) promotes BM-‐‑BM adhesion .................................................... 40 2.2.6 VAB-‐‑10A (plakin) anchors hemicentin punctae under the AC .......................... 51 vii 2.2.7 Integrin is required to assemble hemicentin punctae .......................................... 52 2.2.8 Hemicentin, VAB-‐‑10A and integrin mediate utse-‐‑seam cell connection .......... 57 2.3 Discussion ........................................................................................................................ 60 2.4 Acknowledgements ........................................................................................................ 64 3. SPARC promotes cell invasion by decreasing levels of type IV collagen in the basement membrane ................................................................................................................... 66 3.1 Introduction ..................................................................................................................... 66 3.2 Results .............................................................................................................................. 71 3.2.1 SPARC promotes anchor cell invasion in multiple mutant backgrounds ........ 71 3.2.2 SPARC promotes invasion through a direct interaction with type IV collagen ............................................................................................................................................... 75 3.2.3 SPARC overexpression decreases levels of type IV collagen at the BM ........... 78 3.2.4 SPARC functions extracellularly to reduce type IV collagen levels and promote invasion ................................................................................................................................ 79 3.2.5 SPARC overexpression slows collagen recovery in the BM ............................... 82 3.3 Discussion ........................................................................................................................ 84 3.4 Acknowledgements ........................................................................................................ 87 4. AC-‐‑derived laminin promotes cell invasion ....................................................................... 88 4.1 Introduction ..................................................................................................................... 88 4.2 Results .............................................................................................................................. 90 4.2.1 The AC expresses laminin just prior to AC invasion ........................................... 90 4.2.2 Laminin expression is regulated by FOS-‐‑1A ......................................................... 91 viii 4.2.3 Reducing laminin expression specifically in the uterine tissue delays AC invasion ................................................................................................................................ 93 4.3 Discussion ........................................................................................................................ 94 5. Discussion ................................................................................................................................ 96 5.1 B-‐‑LINKs in other contexts ............................................................................................. 96 5.2 Uncovering more B-‐‑LINK regulators .......................................................................... 99 5.3 Understanding extracellular trafficking and regulation of type IV collagen ...... 103 5.4 Concluding remarks ..................................................................................................... 105 Appendix A ................................................................................................................................ 107 A.1 Chapter 2 Materials and Methods ............................................................................. 107 A.1.1 Strains and Culture Conditions ............................................................................ 107 A.1.2 Light Microscopy, Image Acquisition, Processing and Analysis .................... 107 A.1.3 Electron Microscopy .............................................................................................. 108 A.1.4 Landmark photobleaching and tissue shifting .................................................. 108 A.1.5 Uterine-‐‑vulval tissue separation .......................................................................... 109 A.1.6 Statistical Analysis ................................................................................................. 109 A1.7 Targeted screen and RNA interference ................................................................ 109 A.1.8 Construction of vab-‐‑10a::GFP fusions .................................................................. 111 A.2 Chapter 3 Materials and Methods ............................................................................. 112 A.2.1 C. elegans strain and culture information ............................................................ 112 A.2.2 Microscopy, image acquisition, processing, and analysis ................................ 112 A.2.3 Analysis of AC invasion ........................................................................................ 113 ix A.2.4 Generation of SPARC overexpression lines ....................................................... 114 A.2.5 Quantitative Real-‐‑Time PCR ................................................................................ 114 A.2.6 RNA interference .................................................................................................... 115 A.2.7 Immunocytochemistry ........................................................................................... 115 A.2.8 Construction of vesicle markers ........................................................................... 116 A.2.9 Statistical Analysis ................................................................................................. 116 A.3 Chapter 4 Materials and Methods ............................................................................. 116 A.3.1 Light Microscopy, Image Acquisition, Processing and Analysis .................... 116 A.3.2 Analysis of AC invasion ........................................................................................ 117 A.3.3 RNA interference .................................................................................................... 117 A.3.4 Construction of lam-‐‑1>NLS::GFP ......................................................................... 118 References ................................................................................................................................... 119 Biography ................................................................................................................................... 136 x
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