The Role of Polarity Protein Angiomotin (AMOT) in the Human Placenta by Abby Patricia Farrell A thesis submitted in conformity with the requirements for the degree of Master of Science Institute of Medical Science University of Toronto © Copyright by Abby Farrell 2018 The Role of Polarity Protein Angiomotin (AMOT) in the Human Placenta Abby Patricia Farrell Master of Science Institute of Medical Science University of Toronto 2018 Abstract Angiomotin (AMOT) is a scaffolding protein involved in cell polarity regulation, cell migration and early embryo lineage differentiation, yet its biological significance in the human placenta remains unknown. I hypothesized that AMOT controls trophoblast cell polarity and migration, and is further regulated by transforming growth factor beta (TGF) signalling and upstream oxygen tension. AMOT localization to extravillous trophoblast (EVT) cells, corroborated by AMOT 80 overexpression increasing JEG3 cell migration rate, supports a role for AMOT in EVT migration. TGF1/3 treatment decreased AMOT protein levels and redistributed AMOT from the tight- junction to cytoplasmic F-actin in JEG3 cells. TGF1/3 also prompted a novel association between AMOT 80 and Partitioning Defective Protein-6. Similarly, low oxygen exposure negatively regulated AMOT levels and localization. Furthermore, Jumonji C Domain Containing Protein-6 (JMJD6), an oxygen sensor, was discovered to positively regulate AMOT via lysyl hydroxylation. Finally, AMOT levels were found markedly reduced in preeclampsia, a disease characterized by aberrant TGF signalling and chronic hypoxia. In conclusion, this study reveals AMOT is a mediator of TGF and oxygen signalling to regulate trophoblast migration in the human placenta. ii Table of Contents Table of Contents ......................................................................................................................... iii Acknowledgments ........................................................................................................................ vi List of Abbreviations .................................................................................................................. vii List of Tables ..................................................................................................................................x List of Figures ............................................................................................................................... xi Chapter 1 Introduction..................................................................................................................1 Introduction .................................................................................................................................1 1.1 Angiomotin (AMOT) .........................................................................................................2 1.1.1 Discovery and Structure ...........................................................................................2 1.1.2 Role of AMOT in Hippo Pathway Signalling..........................................................6 1.1.3 Role of AMOT in Cell Migration ............................................................................8 1.2 Cell Polarity Regulation ....................................................................................................9 1.2.1 Tight Junctions .........................................................................................................9 1.2.2 Polarity Protein Complexes ...................................................................................10 1.2.3 Role of Cell Polarity in Proliferation, Migration and Invasion .............................11 1.2.4 AMOT as a Novel Regulator of Cell Polarity .......................................................12 1.3 Human Placenta Development........................................................................................16 1.3.1 Trophoblast Differentiation ...................................................................................17 1.3.2 TGF signalling pathways .....................................................................................22 1.3.3 Role of TGFβ Signalling in Trophoblast Differentiation ......................................26 1.4 Preeclampsia .....................................................................................................................29 1.4.1 Altered Trophoblast Differentiation in preeclampsia ............................................30 1.4.2 Impairments in oxygen sensing in preeclampsia ...................................................32 1.4.3 JMJD6: a novel oxygen sensor and regulator in the placenta ................................33 1.5 Rationale, Hypothesis and Objectives ...............................................................................37 Chapter 2 Materials and Methods..............................................................................................39 Materials and Methods ..............................................................................................................39 2.1 Human Placenta Tissue Collection ....................................................................................39 2.2 JEG3 Human Choriocarcinoma Cell Culture ....................................................................41 2.3 In vitro treatments in JEG3 cells ........................................................................................42 2.3.1 Transforming Growth Factor- (TGF) Treatment ...............................................42 2.3.2 SB-431542 Treatment in JEG3 cells......................................................................42 iii 2.3.3 Minoxidil Treatment ..............................................................................................43 2.3.4 Low Oxygen (3% O2) Treatment in JEG3 cells.....................................................43 2.4 Plasmid DNA Constructs for Overexpression Studies ......................................................43 2.4.1 Plasmid DNA Transfection ....................................................................................45 2.5 siRNA Transfections ..........................................................................................................45 2.6 Wound Healing Assay .......................................................................................................46 2.7 Time-Lapse Live Cell Imaging ..........................................................................................46 2.8 Western Blot Analysis .......................................................................................................47 2.9 Antibodies ..........................................................................................................................49 2.10 Immunoprecipitation (IP)...................................................................................................50 2.11 Immunohistochemistry (IHC) ............................................................................................51 2.12 Immunofluorescence (IF) ...................................................................................................53 2.13 Proximity Ligation Assay ..................................................................................................56 2.14 RNA Isolation, cDNA conversion and Quantitative-PCR .................................................57 2.15 In vitro JMJD6 Hydroxylation Reaction............................................................................58 2.16 MALDI-TOF Mass Spectrometry......................................................................................59 2.17 Statistical analysis ..............................................................................................................60 Chapter 3 Results .........................................................................................................................61 Results .......................................................................................................................................61 3.1 AMOT exhibits distinct temporal and spatial expression patterns during human placenta development.........................................................................................................61 3.2 AMOT is regulated by TGF signalling pathway .............................................................67 3.2.1 AMOT resides at tight junction, cytoplasm and protruding edge of JEG3 cells ...67 3.2.2 TGF1/3 ligand treatment reduces AMOT 130 and 80 protein levels ..................68 3.2.3 TGF1/3 treatment promotes subcellular redistribution of AMOT ......................68 3.3 AMOT 130 is regulated by Smad-dependent TGF pathway ...........................................74 3.4 TGFβ promotes AMOT redistribution in migrating cells ..................................................78 3.5 AMOT 80 promotes JEG3 cell migration ..........................................................................79 3.6 Novel AMOT/Par6 interaction and its regulation by TGFβ ..............................................84 3.7 PDZ and coiled-coil binding domains are important for AMOT and Par6 interaction .....89 3.8 AMOT promotes dissolution of RhoA at the tight junction ..............................................93 3.9 AMOT protein levels and distribution is disrupted in preeclampsia .................................96 3.9.1 AMOT and Par6 interaction is impaired in preeclampsia .....................................97 iv 3.10 AMOT protein levels and localization