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C:\Documents and Settings\lproctor.ULL\Local Settings\Temporary Internet Files\OLK36\Copyright-thesis.doc Functional analysis of the cornified envelope precursors, periplakin and envoplakin. Karen Renee Groot Thesis presented in partial fulfilment of the degree of Doctor of Philosophy at University College London October 2004 l UMI Number: U593656 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U593656 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract Periplakin and envoplakin are components of the epithelial cornified cell envelope (CE). The CE is a crosslinked protein structure that forms beneath the plasma membrane of differentiating keratinocytes and is essential for epidermal barrier function. Periplakin and envoplakin belong to the plakin family of cytolinker proteins. Their N termini are thought to mediate their interaction with the plasma membrane. I found that the N terminus of envoplakin does not efficiently localise to desmosomes or microvilli, suggesting a dependence on periplakin for membrane localisation. I found that the first 133 amino acids of periplakin targets the protein to the apical plasma membrane. In a yeast two-hybrid screen, using this region as ‘bait’, a novel protein was identified and named kazrin. There are four alternatively spliced transcripts, encoding three proteins with different N termini. The interaction between periplakin and all three kazrin isoforms was confirmed by pull-down reactions. Kazrin was expressed in all layers of stratified squamous epithelia and was incorporated into the CEs of cultured keratinocytes. Kazrin localised to the cell periphery of differentiated cells, however it was more diffusely localised in cells of the basal layer. In stratified keratinocyte cultures kazrin partially colocalised with desmoplakin and periplakin at the desmosomes and periplakin at the interdesmosomal plasma membrane. In transient transfections, kazrin proteins localised predominantly to the apical plasma membrane of primary keratinocytes. The exogenous expression of kazrin proteins caused changes in keratinocyte cell shape and the actin cytoskeleton. I carried out in vivo analysis of the function of periplakin and envoplakin using mice lacking periplakin, envoplakin and involucrin expression. Triple knockout mice produced slightly larger CEs and had a slight delay in epidermal barrier formation relative to control mice. In future it would be interesting to establish the consequence of deleting the kazrin gene in mice. 2 To Mum and Dad 3 Table of Contents TITLE.................................................................................................................................................................................... 1 A b s t r a c t ........................................................................................................................................................................2 D e d i c a t i o n ................................................................................................................................................................... 3 T a b l e o f C o n t e n t s ................................................................................................................................................4 L is t if F i g u r e s ............................................................................................................................................................ 8 L is t o f T a b l e s ..........................................................................................................................................................11 A cknowledgements ...........................................................................................................................................12 CHAPTER 1. INTRODUCTION.................................................................................... 13 1.1 O rganisation o f t h e e p i d e r m i s .........................................................................................................13 1.2 K e r a t in intermediate f il a m e n t s .................................................................................................... 15 1.3 D e s m o s o m e s ..................................................................................................................................................... 16 1.3.1 Desmosomal cadherins..........................................................................................18 1.3.2 Plakoglobin and plakophilins.............................................................................. 19 1.3.3 Desmoplakin...........................................................................................................21 1.3.4 Changes in the protein composition of desmosomes during epidermal terminal differentiation...................................................................................................23 1.4 T h e c o r n if ie d c e l l e n v e l o p e a n d c o r n e o c y t e l ip id e n v e l o p e ..............................25 1.4.1 Early components of the CE and formation of the CE scaffold.......................28 1.4.2 Formation of the CLE............................................................................................32 1.4.3 CE scaffold reinforcement....................................................................................34 1.5 P l a k in f a m il y p r o t e i n s .......................................................................................................................... 3 8 1.5.1 Intermediate filament binding domain................................................................42 1.5.2 Periplakin linker subdomain interacting proteins............................................ 45 1.5.3 Actin and microtubule binding domains............................................................. 46 1.5.4 Rod domains...........................................................................................................47 1.5.5 The amino terminal plakin domains.................................................................... 49 1.6 A im s o f t h e s i s ..................................................................................................................................................51 CHAPTER 2. MATERIALS AND METHODS........................................................... 53 2.1 M o l e c u l a r B i o l o g y ...............................................................................................................................5 3 2.1.1 General solutions...................................................................................................53 2.1.2 DNA techniques...................................................................................................... 54 4 2.1.3 Vectors and primers.............................................................................................. 56 2.1.4 RNA techniques...................................................................................................... 61 2.2 Y e a s t t e c h n iq u e s ....................................................................................................... 61 2.2.1 Yeast