Table Of ContentBasement 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
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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
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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.
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Dedication
Dedicated to my husband, John, for whole-‐‑heartedly supporting every decision
I’ve made, regardless of its merits.
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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
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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
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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
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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
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Description:Dedicated to my husband, John, for whole-‐‑heartedly supporting every collagen or perlecan does not alter pharyngeal polarity (Graham et al.,
