Boczonadi, Veronika (2009) Elucidation of cytolinker function: periplakin-associated proteins in epithelial cells. Doctoral thesis, Durham University.
Periplakin is a cytolinker protein which participates in the barrier formation of the skin by playing part in the cornified envelope assembly of epidermal cells. Previous studies had identified peripłakin, envoplakin and involucrin as constituents of the cornified envelope, but gene-targeting studies had demonstrated that lack of the proteins individually or in any "double knock-out" combination did not disturb the epidermal differentiation or skin barrier function. In order to gain more information about periplakin, which is also expressed in simple epithelial cells, a stably transfected MCF-7 subclone overexpressing the HA-tagged periplakin N-terminus was generated. Co-immunoprecipitation was used for screening of protein complexes associated with the HA-tagged periplakin N-terminus to identify previously uncharacterised periplakin partners. Co-IP with anti-HA antibody and mass-spectrometry revealed a 500 kDa periplakin interacting protein, plectin and another protein around the size of 34 kDa identified as annexin A9. Endogenous periplakin co-localised with annexin A9 in the plasma membrane of MCF-7 cells and showed a similar staining pattern in newborn and adult mouse skin. Transient transfection of periplakin deletion constructs indicated that the first 133 amino acid residues are essential for the co- localisation with plectin at cell borders. Immunofluorescence analysis demonstrated that periplakin N-terminus and different plectin isoforms, such as plectin-1,-1 f and 1k, are co- localise at cell borders of MCF-7 epithelia and also co-localise with endogenous periplakin at suprabasal layers of the skin. Ablation of the plectin by siRNA transfection in HaCaT keratinocytes resulted in aggregation of periplakin into small clusters in the cytoplasm. Scratch wounded MCF-7 epithelia expressing the N-terminal half of periplakin showed accelerated keratin re-organisation which was hampered by plectin downregulation. The role of periplakin and keratin IPs in the migration of simple epithelial cells was also investigated. Stable expression of periplakin C-terminus increased keratin bundling and Ser-431 phosphorylation of keratin 8 at the free wound edge, delaying wound closure. Depletion of periplakin or plectin by SİRNA transfection impaired wound closure, while simultaneous ablation of both proteins reduced the speed of wound healing even further. Knockdown of keratin 8 IPs with SİRNA resulted in the loss of desmoplakin at cell borders and the failure of different simple epithelial sheets migrating as a collective unit.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Faculty and Department:||Faculty of Science > Biological and Biomedical Sciences, School of|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||08 Sep 2011 18:32|