CELLULAR UPTAKE OF DNA NANOPARTICLES AND REGULATION OF CELL SURFACE NUCLEOLIN by XUGUANG CHEN Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation adviser: Dr. Pamela B. Davis, M.D., Ph.D. Department of Biochemistry CASE WESTERN RESERVE UNIVERSITY August, 2009 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of _____________________________________________________ candidate for the ______________________degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. Copyright © 2009 by Xuguang Chen All rights reserved TABLE OF CONTENTS Chapter 1. Introduction……………………………………………………………….....I Chapter 2. Cell surface nucleolin serves as receptor for DNA nanoparticles composed of PEGylated polylysine and DNA……………………………………..II Chapter 3. Regulation of cell surface expression of nucleolin by cell cycle dependent kinase Cdk1…………………………………………………………………………III Chapter 4. Gene delivery by DNA nanoparticles via lipid-raft mediated, dynamin- independent endocytosis…………………………………………………………...IV Chapter 5. Discussion and future directions……………………………………….....VI Bibliography………………………………………………………………………….....VI List of Tables……………………………………………………………………………VI List of Figures………………………………………………………………………….VII Abstract………………………………………………………………………………...XII Chapter 1. Introduction…………………………………………………………………1 Cystic fibrosis and CFTR……………………………………………………………..1 CFTR function and its regulation……………………………………………………..2 CFTR mutations and therapeutic strategies to correct CFTR defect………………….4 CF gene therapy and viral vectors………………….………………….……………...6 Nonviral gene therapy for CF………………….………………….…………………..9 Composition and stability of compacted DNA nanoparticles………………………..11 In vivo expression and immunogenicity of DNA nanoparticles……………………..13 Intracelluar trafficking of DNA nanoparticles………………….……………………17 i Nucleolin and its structure………………….………………….…………………….18 Phosphorylation and intracellular localization of nucleolin…………………………20 Intracellular function of nucleolin………………….………………….…………….22 Functions of cell surface nucleolin………………….………………….……………24 Association of nucleolin with cytoskeleton………………….………………………31 Cell cycle and cyclin dependent kinase (Cdk)….……………….…………………...33 Endocytosis and its regulation………………….………………….………………...34 Lipid raft and clathrin-independent endocytosis………………….………………….37 Means to study different forms of endocytosis………………….…………………...40 Summary………………….………………….………………….…………………...41 Chapter 2. Cell surface nucleolin serves as receptor for DNA nanoparticles composed of PEGylated polylysine and DNA………………….…………………43 Summary………………….………………….………………….………………….…...44 Introduction………………….………………….………………….…………………...45 Results………………….………………….………………….…………………………48 DNA nanoparticles localize to nucleolus after internalization………………………48 Nucleolin binds to DNA nanoparticles directly………………….…………………..49 Binding of nucleolin to unPEGylated and TFA nanoparticles………………………53 Binding of nucleolin to CK30 peptides………………….……………………………53 Nucleolin exists at the cell surface………………….………………….……………55 Uptake of DNA nanoparticles by HeLa and 16HBEo- cells………………………...57 Manipulating surface nucleolin affects expression from DNA nanoparticles………61 ii Purified nucleolin inhibits internalization of DNA nanoparticles…………………...64 Discussion………………….………………….………………….……………………..65 Materials and Methods………………….………………….…………………………..69 Reagents………………….………………….………………….……………………69 siRNA and Real-Time PCR………………….………………….…………………...69 Cell Cultures………………….………………….………………….……………….70 Purification of nucleolin………………….………………….………………………70 Surface Plasmon Resonance (SPR) ………………….………………….…………..70 Cell surface biotinylation and Western blot………………….………………………71 Fluorescence microscopy………………….………………….……………………...72 Labeling of DNA nanoparticles with rhodamine………………….…………………72 Luciferase reporter assay………………….………………….……………………...73 Calculation of surface areas of DNA nanoparticles and nucleolin…………………..73 Chapter 3. Regulation of cell surface expression of nucleolin by cell cycle dependent kinase Cdk1………………….………………….………………….……………….75 Summary………………….………………….………………….………………………76 Introduction………………….………………….………………….…………………...77 Results………………….………………….………………….………………….……...80 The N-terminus of nucleolin is required for its expression on the cell surface……...80 Intracellular localization of truncated nucleolin constructs………………………….82 Surface nucleolin is upregulated at the G2/M phase transition……………………...85 iii RO-3306 inhibits cell surface expression and threonine phoshphorylation of nucleolin………………….………………….……………………………………….89 Threonine to glutamate mutations of Cdk sites increase surface expression of nucleolin………………….………………….………………….……………………92 CK2 phosphorylation has little impact on the cell surface expression of nucleolin…94 Effect of extracellular pH, EDTA and K+ on the association of nucleolin to the cell membrane….……………….……………….……………….……………….………96 Discussion….……………….……………….……………….……………….………..100 Materials and Methods….……………….……………….……………….…………..104 Reagents….……………….……………….……………….……………………….104 Cell culture and transfection….……………….……………….……………….…..104 Construction of truncated nucleolin-GFP fusion proteins….………………………105 Site-directed mutagenesis of nucleolin….……………….…………………………106 Detection of cell surface proteins by biotinylation….……………….……………..107 Fluorescent microscopy….……………….……………….……………….……….107 Cell cycle synchronization by double thymidine block….……………….………...107 Subcellular fractionation of HeLa cells….……………….……………….………..108 Immunoprecipitation of endogenous and GFP-tagged nucleolin….……………….108 Transfection of DNA nanoparticles….……………….……………….……………109 Release of cell surface nucleolin….……………….……………….……………….109 Chapter 4. Gene delivery by DNA nanoparticles via lipid-raft mediated, dynamin- independent endocytosis….……………….……………….……………………...110 iv Summary….……………….……………….……………….……………….…………111 Introduction….……………….……………….……………….………………………112 Results….……………….……………….……………….……………….……………116 Transfection of DNA nanoparticles is saturable….……………….………………..116 DNA nanoparticles enter polarized 16HBEo- cells from the apical surface….……117 Cell surface nucleolin is found only on the apical surface of 16HBEo- cells….…..118 Transfection of DNA nanoparticles depends on the integrity of lipid rafts….……..119 Surface nucleolin appears in clusters on the plasma membrane….………………...121 Nucleolin exists in lipid rafts….……………….……………….…………………..122 DNA nanoparticles are also found in lipid raft fractions….……………….……….126 Transfection of DNA nanoparticles does not require dynamin….…………………129 Transfection of DNA nanoparticles depends on microtubules but not actin filaments….……………….……………….……………….……………….………130 Discussion….……………….……………….……………….……………….………..132 Materials and Methods….……………….……………….……………….………….136 Reagents and plasmids….……………….……………….……………….………..136 Cell culture….……………….……………….……………….……………………136 Transfection of DNA nanoparticles and reporter luciferase assay….……………...136 Fitting of the suturation curve of nanoparticle transfection….……………………..137 Cell surface biotinylation….……………….……………….……………….……...137 Immunoprecipitation and Western blot….……………….………………………...138 Immunocytochemistry and electron microscopy….……………….……………….138 Fluorescence microscopy….……………….……………….……………….……...139 v Isolation of lipid rafts by sucrose density gradient….……………….……………..140 Amplification and visualization of plasmid DNA from gradient fractions….……..140 Chapter 5. Discussion and future directions….……………….…………………….142 Non-viral gene therapy for CF….……………….……………….…………………142 Application of DNA nanoparticles in CF….……………….………………………145 Interaction of DNA nanoparticles and nucleolin….……………….……………….147 Dissecting the interaction of nanoparticles with nucleolin using SPR……...……...149 Diverse use of SPR technology….……………….……………….………………...152 Detecting cell surface nucleolin in vivo….……………….……………….………..156 Targeting cell surface nucleolin using DNA nanoparticles….……………………..159 Endocytic pathway(s) employed by DNA nanoparticles….……………….……….162 Fluorescent labeling of DNA nanoparticles for real-time tracking….……………..164 Molecular partners of nucleolin and DNA nanoparticles….……………….………169 Phosphorylation and regulation of cell surface nucleolin….……………………….174 Summary….……………….……………….……………….……………….……...176 Bibliography….……………….……………….……………….……………….……..178 List of Tables Chapter 1. Introduction 1.1 Extracellular ligands for cell surface nucleolin….……………….……………...30 1.2 Examples of different endocytic pathways and their inhibitors….………………42 vi Chapter 3. Regulation of cell surface expression of nucleolin by cell cycle dependent kinase Cdk1 3.1 DNA oligos for generating truncated nucleolins….……………….…………...105 3.2 DNA oligos for mutating Cdk sites to glutamate (E)…..………………………106 3.3 DNA oligos for mutating Cdk sites to alanine (A)…..…………………………106 3.4 DNA oligos for mutating CK2 sites to alanine….……………….……………..107 List of Figures Chapter 1. Introduction 1.1 Hypothesized structure of CFTR….……………….……………….……………..2 1.2 Cellular barriers for in vivo gene transfer….……………….……………………..9 1.3 Transmission electron micrographs of compacted DNA nanoparticles in saline..12 1.4