REGULATION OF APICAL POLARITY COMPLEXES By - Deep Blue

255 Pages·2010·4.57 MB·English

by  Jay Pieczynski

REGULATION OF APICAL POLARITY COMPLEXES

By

Jay Nicholas Pieczynski

A dissertation submitted in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
(Biological Chemistry)
in the University of Michigan
2010

Doctoral Committee:

Professor Benjamin L. Margolis, Chair
Professor Eric R. Fearon
Professor Robert S. Fuller
Assistant Professor Daniel A. Bochar
Assistant Professor Hisashi Umemori
Acknowledgements

I would like to thank my mentor Dr. Ben Margolis for his guidance and support
throughout the process of earning my doctorate degree. He has taught me that being a
scientist means being your own toughest critic and to always remember that your work
represents who you are and what you have accomplished. I would also like to thank the
current and former members of the Margolis laboratory who made my time in the lab
very enjoyable. Each person past and present has brought their own personality to the lab
and contributed to the success of the group. It has been an honor to be associated with
truly gifted scientists, yet even better people. The work contained within this thesis is
also dedicated to my former mentors especially Michael Baxter and Dr. Todd Weaver.
Michael Baxter was a former teacher of mine growing up in Wisconsin and recognized
my potential as a scientist at a very early age. He taught me that science is not about how
intelligent you are, but asking the write questions. Dr. Todd Weaver was also very
instrumental in my scientific career, allowing me to begin my independent research
career during the first years of college. Dr. Weaver recognized my desire to always know
more, and challenged me to work outside the classroom in his laboratory for most of my
undergraduate career; an experience that I am forever indebted to.
Finally, I would like to thank my friends and family. I am blessed to have some
of the best friends that one could ask for and my success can only be attributed to having
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people back me up all the way. My brother Tad has been my best friend my entire life
and always encouraged me to be the best at whatever I do. My parents, Guy and Liz,
have always been there for me especially when times got tough. They taught me that
nothing in life is free and that hard work always eventually pays off. I am truly lucky to
have two of the best parents who always wanted more for their children than they had.

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Preface

Chapter 1 will be submitted as part of an invited review for the American Journal of
Physiology Renal Physiology. Chapter 2 was published in December of 2006 in the
Journal of Biological Chemistry and contains work completed by Dr. Samuel Straight and
Jay Pieczynski. Chapter 3 contains data that will be submitted as a manuscript to
Experimental Cell Research and is collaboration between Jay Pieczynski, Dr.
Sanjeevkumar Patel, and Dr. Peter Arvan. Chapter 4 contains work that will be published
in a future manuscript. Appendix A contains the full data sets of the microarray analysis
completed in Chapter 3. Appendix B contains data that was published in Schlüter et al. in
Molecular and Cellular Biology in September of 2009 and unpublished data related to
that manuscript.
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Table of Contents

Acknowledgements............................................................................................................ii 
Preface...............................................................................................................................iv 
List of Figures................................................................................................................viii 
List of Tables......................................................................................................................x 
List of Appendices............................................................................................................xi 
Chapter 1: Development and Maintenance of Renal Epithelial Cell Polarity.............1 
1.1 Introduction...........................................................................................................1 
1.2 Molecular Interactions Governing the Establishment of Mammalian Epithelial
Cell Polarity.................................................................................................................4 
1.3 Mammalian Polarity Complexes and Their Interactions.......................................7 
1.3.1 The Par Complex Overview...............................................................7 
1.3.2 Par3...................................................................................................10 
1.3.3 Par6...................................................................................................15 
1.3.4 aPKCζ..............................................................................................18 
1.3.5 The Scribble Complex Overview.....................................................20 
1.3.6 Scribble.............................................................................................20 
1.3.7 Lgl....................................................................................................22 
1.3.8 Dlg....................................................................................................24 
1.3.9 Crumbs complex Overview..............................................................27 
1.3.10 PALS1............................................................................................27 
1.3.11 PATJ and MUPP1..........................................................................29 
1.3.12 Crumbs3.........................................................................................32 
1.4 Molecular Mechanisms Establishing Renal Epithelial Cell Polarity..................35 
1.4.1 Establishing Renal Polarity: MET vs. EMT.....................................35 
1.4.2 Regulation and Maintenance of Renal Epithelial Polarity: A Trail of
Crumbs......................................................................................................37 
1.4.3 Renal Apico-Basal Epithelial Polarity in Context: A Link to
Nephropathies, Planar Cell Polarity, and Renal Cilia...............................42 
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1.5 Concluding Remarks...........................................................................................45

Chapter 2: Mammalian Lin-7 Stabilizes Polarity Complex Proteins.........................47 
2.1 Introduction.........................................................................................................47 
2.2 Materials and Methods........................................................................................49 
2.2.1 DNA Constructs...............................................................................49 
2.2.2 Cell Culture and Transfection..........................................................50 
2.2.3 Calcium Switch Assay and Transepithelial Electrical Resistance
Measurement.............................................................................................51 
2.2.4 Antibodies........................................................................................52 
2.2.5 Immunoprecipitation and Immunoblotting......................................53 
2.2.6 CRB3 Peptide Beads........................................................................54 
2.2.7 Pulse-Chase Labeling.......................................................................54 
2.2.8 Immunostaining and Confocal Microscopy.....................................55 
2.2.9 Quantitative Real-time PCR.............................................................56 
2.3 Results.................................................................................................................57 
2.3.1 Lin-7 shRNA affects the formation of tight junctions.....................57 
2.3.2 Lin-7 shRNA affects expression of MAGUK family members.......61 
2.3.3 The L27 domain of Lin-7C is essential for correct tight junction
formation...................................................................................................63 
2.3.4 Reexpression of PALS1 can reverse the tight junction defect seen in
Lin-7 knockdown cells..............................................................................66 
2.3.5 Lin-7 expression affects stability of PALS1 in polarized MDCKII
cells............................................................................................................73 
2.4 Discussion...........................................................................................................75

Chapter 3: Slug Represses Crumbs3 in Epithelial Cells..............................................78 
3.1 Introduction.........................................................................................................78 
3.2 Materials and Methods........................................................................................81 
3.2.1 Cell lines...........................................................................................81 
3.2.2 DNA Constructs...............................................................................82 
3.2.3 Antibodies and Immunostaining......................................................83 
3.2.4 Microarray and Statistics..................................................................84 
3.2.5 qRT-PCR..........................................................................................84 
3.2.6 ChIP Assays.....................................................................................85 
3.3 Results.................................................................................................................87 
3.3.1 FRTL5 thyroid epithelial cells are lacking polarized epithelial
markers......................................................................................................87 
3.3.2 Altered transcription of junction components in FRTL5 cells.........89 
3.3.3 Snai2 is upregulated in FRTL5 cells................................................92 
3.3.4 Slug expression leads to polarity defects in MDCKII cells.............93 
3.3.5 Slug-V5 directly represses Crb3 in MDCKII cells..........................97 
3.4 Discussion...........................................................................................................99 
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Chapter 4: Regulation of Epithelial Polarity by Endocytosis of Crb3a...................105 
4.1 Introduction.......................................................................................................105 
4.2 Materials and Methods......................................................................................108 
4.2.1 Cell Lines and Plasmids.................................................................108 
4.2.2 Antibodies and Immunofluorescence Staining...............................109 
4.2.3 3-Dimensional Tissue Culture Cyst Assays...................................109 
4.2.4 Imaging/Live-cell Imaging/FRAP..................................................110 
4.2.5 Cell treatments and Modified Pulse chase experiments.................111 
4.2.6 Quantitative Western Blot Analysis...............................................111 
4.2.7 Biotinylation Assay........................................................................112 
4.3 Results...............................................................................................................113 
4.3.1 Efficient repopulation of Crb3a in polarized MDCKII cells..........113 
4.3.2 Inducible GFP-Crb3a system.........................................................115 
4.3.3 GFP-Crb3a is not recycled in G phase MDCKII cells..................119 
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4.3.4 GFP-Crb3a can be isolated from tissue culture media...................122 
4.3.5 TSG101 knockdown increases Crb3a protein levels......................124 
4.3.6 GFP-Crb3a Half-life is independent of epithelial morphology......129 
4.3.7 Crb3a lateral mobility as measured by FRAP is constant regardless
of cellular localization.............................................................................138 
4.4 Discussion.........................................................................................................141

Chapter 5: Conclusions, Perspectives, and Future Directions..................................151 
Appendices.....................................................................................................................158 
Bibliography...................................................................................................................213 

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List of Figures

Figure 1-1: Model of basic cubodial epithelial cells..........................................................3 
Figure 1-2: The conserved polarity complex interactome .................................................8 
Figure 1-3: Sizes and domains of conserved polarity proteins. .......................................9 
Figure 1-4: Identified regulators of Crumbs in Drosophila and mammalian systems.....41 
Figure 2-1: Lin-7 shRNA clones......................................................................................58 
Figure 2-2: Immunofluorescence (IF) staining of Lin-7 shRNA clones 19, 20, and 21..59 
Figure 2-3: Knockdown of Lin-7 effects tight junction formation but not expression of
tight junction structural proteins……………………………………………………...….60 
Figure 2-4: Lin-7 shRNA causes defects in tight junction formation during cell
polarization........................................................................................................................62 
Figure 2-5: Lin-7 shRNA reduces expression of MAGUK family members..................64 
Figure 2-6: Lin-7 shRNA effects expression of PATJ, but not Crb3 or Par polarity
complex proteins...............................................................................................................65 
Figure 2-7: Rescue of protein expression with murine mycLin-7 FL and mycLin-7N in
Lin-7 shRNA cell lines......................................................................................................67 
Figure 2-8: TER of murine mycLin-7 rescue constructs..................................................68 
Figure 2-9: IF of murine Lin-7 rescues............................................................................69 
Figure 2-10: Rescue of Lin-7 shRNA cells with mycPALS1..........................................70 
Figure 2-11: Expression of mycPALS1 can partially rescue TER profiles of Lin-7
shRNA cells.......................................................................................................................71 
Figure 2-12: Other Lin-7 L27 domain binding partners cannot rescue Lin-7 shRNA tight
junction defects..................................................................................................................72 
Figure 2-13: PALS1 protein degradation is increase in Lin-7 knockdown cells.............74 
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Figure 3-1: Altered expression of conserved polarity proteins and tight juction structural
protines in wild type FRTL5 thyrocytes............................................................................88 
Figure 3-2: PCR of Snai2 (Slug) transcripts from wild type FRTL5 cells.......................95 
Figure 3-3: Slug overexpression leads to EMT in MDCKII cells....................................96 
Figure 3-4: ChIP of Crb3a promoter with Slug-V5.........................................................98 
Figure 4-1: Expression and tracking of Den2-Crb3a in MDCKII cells.........................114 
Figure 4-2: The iGFP-Crb3a sytem.................................................................................117 
Figure 4-3: N and O-linked glycosylation of Crb3a.......................................................118 
Figure 4-4: GFP-antibody uptake in single cells. ..........................................................121 
Figure 4-5: Endocytosis of iGFP-Crb3a in fully polarized monolayers........................123 
Figure 4-6: Pharmacological inhibition of Crb3a degradation........................................125 
Figure 4-7: iGFP-Crb3a isolated for tissue culture media. ...........................................126 
Figure 4-8: Knockdown of TSG101 is MDCKII cells...................................................128 
Figure 4-9: 6 and 12 day-old TSG101 shRNA 1 cysts. .................................................130 
Figure 4-10: 2-cell stage of TSG101 shRNA cells. .......................................................131 
Figure 4-11: TSG101 shRNA in mEGFP-Crb3a cells....................................................132 
Figure 4-12: iGFP-Crb3a cells expressing Snail-Flag. .................................................133 
Figure 4-13: The half-life if iGFP-Crb3a.......................................................................135 
Figure 4-14: Half-life of iGFP-Crb3a with TGF-β. .....................................................136 
Figure 4-15: Apical identity and the maturation of iGFP-Crb3a. .................................137 
Figure 4-16: FRAP analyis of mEGFP-Crb3a cells. .....................................................139 
Figure 4-17: Model of Crb3a exocytosis and endocytosis.............................................140 

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List of Tables

Table 3-1: Polarity proteins differentially regulated in FRT cells relative to FRTL5
cells ...................................................................................................................................90
Table 3-2: Thyroid horome specific genes upregulated only in FRTL5 cells..................91 
Table 3-3: Annotated genes for EMT trancription factors in FRT cells relative to FRTL5
cells....................................................................................................................................94 

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