NESPRIN-2G AND CELL-MATRIX ADHESIONS INFLUENCE CELL MIGRATION DURING SKIN WOUND HEALING By ALEXANDRA VICTORIA WOYCHEK A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY WASHINGTON STATE UNIVERSITY School of Molecular Biosciences MAY 2019 © Copyright by ALEXANDRA VICTORIA WOYCHEK, 2019 All Rights Reserved © Copyright by ALEXANDRA VICTORIA WOYCHEK, 2019 All Rights Reserved To the Faculty of Washington State University: The members of the Committee appointed to examine the dissertation of ALEXANDRA VICTORIA WOYCHEK find it satisfactory and recommend that it be accepted. Ryan Driskell, Ph.D., Co-Chair Jonathan Jones, Ph.D., Co-Chair Kwanhee Kim, Ph.D. Michael Konkel, Ph.D. Gary Wayman, Ph.D. ii ACKNOWLEDGMENT I would like to thank those who have aided my research projects. My advisor Dr. Jonathan Jones has provided invaluable feedback regarding experimental designs and composition of written documents. Dr. Susan Hopkinson has provided valuable assistance in the design and implementation of molecular biology experiments. iii NESPRIN-2G AND CELL-MATRIX ADHESIONS INFLUENCE CELL MIGRATION DURING SKIN WOUND HEALING Abstract by Alexandra Victoria Woychek, Ph.D. Washington State University May 2019 Co-Chairs: Ryan Driskell and Jonathan Jones Human skin is essential for separating internal organs from environmental, physical, and biological insults. The skin is composed of two layers, the epidermis and dermis, which are composed of primarily keratinocytes and fibroblasts, respectively. The epidermis prevents water loss and invasion of pathogens while the dermis plays a supportive role and gives skin its stretchy property. Damage to either of these layers initiates wound healing in which the cells of the epidermis and dermis migrate into the area to restore skin function. Wound healing in both layers begins with the infiltration of migrating cells into the wound. Cell migration is mediated by a variety of proteins, including the nucleus, actin cytoskeleton, and cell-matrix adhesions. The nucleus/actin cytoskeleton interaction is important in nuclear positioning and proper orientation during cell migration. Cell-matrix adhesions allow cells to adhere to the underlying matrix and to migrate across this matrix during cell migration. Here, we explore two cell-matrix adhesions: focal adhesions (FAs) and hemidesmosomes (HDs). We show that loss of a nuclear protein, nesprin-2G, leads to an abnormal actin cytoskeleton and FA formation in primary mouse fibroblasts. This loss of FA formation leads to inability of the knockout cells to tug on their iv underlying matrix to generate traction forces used during cell migration. Consequently, the loss of nesprin-2G results in decreased cell migration compared to wildtype fibroblasts. However, these results are not replicated in keratinocytes. The formation of these adhesions is crucial, but it is also important to understand how these protein components assemble. To better understand this mechanism, we explored the role of the α6 integrin message in α6β4 integrin localization to HDs. We show that a sequence within the 3’ untranslated region (UTR) of the α6 integrin message is required for localization of α6β4 integrin to HDs in A549 cells. Sequences lacking this 3’UTR allow the formation of α6β4 integrin, but it is not localized to HDs. Taken together, these results imply that adhesion formation is a tightly regulated processes which is imperative for cell attachment and migration. v TABLE OF CONTENTS Page ACKNOWLEDGMENT................................................................................................................ iii ABSTRACT ................................................................................................................................... iv LIST OF TABLES ........................................................................................................................ vii LIST OF FIGURES ..................................................................................................................... viii CHAPTER ONE: INTRODUCTION ..............................................................................................1 CHAPTER TWO: NESPRIN-2G KNOCKOUT KERATINOCYTES DISPLAY NORMAL CELL MIGRATION AND PROTEIN EXPRESSION .................................................................54 CHAPTER THREE: NESPRIN-2G KNOCKOUT FIBROBLASTS EXHIBIT REDUCED MIGRATION, CHANGES IN FOCAL ADHESION COMPOSITION, AND REDUCED ABILITY TO GENERATE TRACTION FORCES ......................................................................72 CHAPTER FOUR: THE α6 INTEGRIN 3’UTR REGULATES LOCALIZATION OF THE α6β4 INTEGRIN HETERODIMER ............................................................................................104 CHAPTER FIVE: CONCLUSIONS AND FUTURE DIRECTIONS ........................................127 vi LIST OF TABLES Page CHAPTER 3 3.1: Primer sets used for genotyping..............................................................................................92 3.2: Primer set used for message presence.....................................................................................92 3.3: Quantification of FA characteristics .......................................................................................92 3.4: Quantification of FA characteristics after MG132 incubation ...............................................93 vii LIST OF FIGURES Page CHAPTER 1 1.1: Skin structure ..........................................................................................................................36 1.2: Full- and partial-thickness wounds in human skin .................................................................37 1.3: Nesprins at the nuclear envelope ............................................................................................38 1.4: Cell-matrix adhesions protein complexes ...............................................................................39 CHAPTER 2 2.1: Confirmation of KO keratinocytes .........................................................................................66 2.2: Cell motility of WT and KO keratinocytes .............................................................................67 2.3: Immunofluorescence staining of WT and KO keratinocytes ..................................................69 2.4: Flow cytometric analyses of integrin surface expression of WT and KO keratinocytes ........71 CHAPTER 3 3.1: Nesprin-2G knockout fibroblasts exhibit abnormal FAs ........................................................94 3.2: Nesprin-2G knockout fibroblasts exhibit reduced FA protein levels and altered FA message levels ..............................................................................................................96 3.3: Nesprin-2G knockout fibroblasts show decreased collective cell migration in a scratch assay and single cell motility .............................................................................................97 3.4: Nesprin-2G knockout fibroblasts generate less traction forces (TFs) than their wildtype counterparts .....................................................................................................................98 S3.1: Verification of nesprin-2G knockout ....................................................................................99 S3.2: MG132 incubation restores FA staining in knockout cells ................................................101 viii S3.3: Nesprin-2G knockout affects nuclear staining and transmembrane actin-associated nuclear (TAN) line formation .....................................................................................................103 CHAPTER 4 4.1: The α6 integrin 3’UTR contains a zipcode which localizes the α6β4 integrin heterodimer to cell-matrix adhesions ..............................................................................................................120 4.2: Overexpression of the α6 integrin 3’UTR impacts α6β4 integrin adhesions in transduced A549 cells ....................................................................................................................................122 4.3: The α6 integrin 3’UTR is responsible for α6β4 integrin heterodimer formation in iHEKs .123 S4.1: α6-GFP protein products interact with β4 integrin and are soluble regardless of whether their mRNAs possess the α6 integrin 3’UTR ..............................................................................125 CHAPTER 5 5.1: Representative REMSA ........................................................................................................136 ix CHAPTER ONE: INTRODUCTION Overview Skin, the outermost layer of the body, maintains a barrier between the body and environment and is thus exposed to physical, environmental, and biological insults. The two skin layers, namely the epidermis and dermis, have different functions to prevent invasion from the environment. The epidermis acts as a barrier to prevent water loss and the infiltration of pathogens, whereas the dermis has a supportive function that gives skin its “stretchy” quality. Damage to either of these layers can disrupt normal functioning. Epithelial cells respond to such damage by infiltrating the wound to re-establish the barrier function of the skin by means of a process that involves both the proliferation and migration of living cells in the surrounding area. Within the dermis, cells secrete factors that are responsible for filling the open wound area. In both skin layers, wound healing is