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Bundling of Cytoskeletal Actin by the Formin FMNL1 Contributes to Celladhesion and Migration

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Bundling of Cytoskeletal Actin by the Formin FMNL1 Contributes to Celladhesion and Migration Bundling of cytoskeletal actin by the formin FMNL1 contributes to celladhesion and migration Item Type Dissertation Authors Miller, Eric Rights Attribution-NonCommercial-NoDerivatives 4.0 International Download date 27/09/2021 05:11:17 Item License http://creativecommons.org/licenses/by-nc-nd/4.0/ Link to Item http://hdl.handle.net/20.500.12648/1760 Bundling of cytoskeletal actin by the formin FMNL1 contributes to cell adhesion and migration Eric W. Miller A Dissertation in the Department of Cell and Developmental Biology Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Graduate Studies of State University of New York, Upstate Medical University Approved ______________________ Dr. Scott D. Blystone Date______________________ i Table of Contents Title Page-------------------------------------------------------------------------------------------------------i Table of Contents-------------------------------------------------------------------------------------------ii List of Tables and Figures------------------------------------------------------------------------------vi Abbreviations----------------------------------------------------------------------------------------------viii Acknowledgements--------------------------------------------------------------------------------------xiii Thesis Abstract-------------------------------------------------------------------------------------------xvi Chapter 1: General Introduction-----------------------------------------------------------------------1 Introduction------------------------------------------------------------------------------------------2 Metastasis--------------------------------------------------------------------------------------------3 History and Current Significance-----------------------------------------------------3 Metastatic Cascade----------------------------------------------------------------------4 Breast Cancer---------------------------------------------------------------------------------------4 Five Subtypes-----------------------------------------------------------------------------4 Breast Cancer Metastasis--------------------------------------------------------------6 General Description of Metastatic Initiation----------------------------------------7 Cellular Locomotion-------------------------------------------------------------------------------9 Introduction--------------------------------------------------------------------------------9 History-------------------------------------------------------------------------------------10 Polarization-------------------------------------------------------------------------------11 Protrusion and Adhesion Formation-----------------------------------------------12 Adhesion----------------------------------------------------------------------------------14 Rear Retraction-------------------------------------------------------------------------16 Cancer Cell Invasion-----------------------------------------------------------------------------16 EMT----------------------------------------------------------------------------------------16 ii Migration During Invasion------------------------------------------------------------17 Actin--------------------------------------------------------------------------------------------------19 G-Actin and F-Actin--------------------------------------------------------------------19 Formins---------------------------------------------------------------------------------------------21 Formin History---------------------------------------------------------------------------21 Formin Diversity and Subfamilies---------------------------------------------------22 General Formin Domain Structure-------------------------------------------------23 Formin Function-------------------------------------------------------------------------25 FMNL1----------------------------------------------------------------------------------------------28 Structure and Functions---------------------------------------------------------------28 Localization of FMNL1-----------------------------------------------------------------29 Binding Partners of FMNL------------------------------------------------------------29 FMNL Proteins in Cancer-------------------------------------------------------------30 Formins as Actin Bundling Proteins----------------------------------------------------------30 Detailed Formin History and Actin Bundle Organizations--------------------30 Saccharomyces Cerevisiae – Bnr1------------------------------------------------34 Schizosaccharomyces Pombe – Fus1 and Cdc12-----------------------------35 Dictyostelium discoideum – ForC---------------------------------------------------37 Drosophila melanogaster – Cappuccino and DAAM---------------------------38 Mammals – Dia2, DAAM1, FMNL1-3, FHOD1, Delphilin---------------------40 Arabidopsis thaliana – AFH1, AFH8, AFH14, AFH16-------------------------44 Oryza sativa – OsFH5 and OsFH16-----------------------------------------------47 Toxoplasma gondii – TgFRM1 and TgFRM2------------------------------------48 The WH2 Domain--------------------------------------------------------------------------------49 General Description--------------------------------------------------------------------49 Non-formin WH2-domain Containing Proteins----------------------------------50 iii Mammalian Formins and WH2 Domains-----------------------------------------51 Hypothesis/Signficance-------------------------------------------------------------------------52 References-----------------------------------------------------------------------------------------53 Chapter 2: Expression patterns of human formins and FMNL1 alternative splice isoforms across multiple cell types----------------------------------------------------------------74 Introduction-----------------------------------------------------------------------------------------75 Materials and Methods--------------------------------------------------------------------------78 Results---------------------------------------------------------------------------------------------101 Discussion----------------------------------------------------------------------------------------135 References---------------------------------------------------------------------------------------138 Chapter 3: Two highly conserved amino acid residues of the FMNL1 FH2 domain are required for actin binding-----------------------------------------------------------------------147 Introduction---------------------------------------------------------------------------------------148 Materials and Methods------------------------------------------------------------------------153 Results---------------------------------------------------------------------------------------------154 Discussion----------------------------------------------------------------------------------------157 References---------------------------------------------------------------------------------------162 Chapter 4: The carboxy-terminus of the formin FMNL1ɣ bundles actin to potentiate adenocarcinoma migration--------------------------------------------------------------------------165 Abstract--------------------------------------------------------------------------------------------166 Introduction---------------------------------------------------------------------------------------166 Materials and Methods------------------------------------------------------------------------170 Results---------------------------------------------------------------------------------------------179 Discussion----------------------------------------------------------------------------------------205 References---------------------------------------------------------------------------------------211 Chapter 5: General Discussion---------------------------------------------------------------------217 iv Introduction---------------------------------------------------------------------------------------218 FMNL1 and FMNL1 alternative splice isoform expression---------------------------219 2 conserved amino acid residues of FMNL1 are required for actin binding------224 The FMNL1ɣ C-terminus bundles actin to potentiate cancer migration-----------226 Concluding Remarks---------------------------------------------------------------------------237 References---------------------------------------------------------------------------------------239 v List of Tables and Figures Chapter 1: General Introduction Figure 1 – The molecular subtypes of breast cancer-------------------------------------7 Figure 2 – The steps involved in cell migration-------------------------------------------15 Figure 3 – Invasion, migration and intravasation into the circulatory system-----18 Figure 4 – Formin genes are located on a variety of chromosomes----------------23 Figure 5 – The FH2 domain moves processively with the barbed end of the elongating actin filament while the FH1 domain recruits profilin-actin monomer subunits for the growing barbed end--------------------------------------------------------27 Chapter 2: Expression patterns of human formins and FMNL1 alternative splice isoforms across multiple cell types Table 1 – The formin family of proteins---------------------------------------------------104 Figure 1 – Alignment of formin protein sequences-------------------------------------107 Figure 2 – Examples of qualitative PCR products for each formin ----------------108 Figure 3 – HeLa Cell RNA and Formin
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