UBC Theses and Dissertations
Functions of kindlin-1 in human keratinocytes and its immunolocalization in human oral mucosal tissues Petricca, Giorgio Maximillian
The primary protein structure of the kindlin-1 polypeptide reveals a number of features predicting that the function of this molecule relates to anchorage of the actin cytoskeleton to the plasma membrane. Kindlin-1 has C-terminal homology to talin and N-terminal homology to filopodin. Kindlin-1 also possesses a centrally-located pleckstrin homology (PH) domain and two regions of homology with the FERM (filopodin and ezrin/radixin/moesin) domain. Interestingly, kindlin-1 is homologue of Mig-2, a focal adhesion protein, which recruits migfilin, an actin-binding protein, to ECM-cell adhesions. Cells probe, respond to, and remodel the ECM using integrin-actin cytoskeleton adhesion complexes. Identification of the molecular components of cell-cell and cell-ECM adhesions and the structural determinants that control their localizations to these two distinct structures are therefore of critical importance to our understanding of tissue morphogenesis, cell growth, and differentiation in various biological processes including cancer, wound healing, and development. In order to increase our understanding of how kindlin-1 localizes to these ECM-cell adhesions and how knockdown of kindlin-1 expression alters keratinocyte cell behaviour, human and Kindler syndrome oral mucosa was stained for immunolocalization of kindlin-1 and siRNAmediated gene silencing was used to knockdown it's expression in HaCaT cells. Normal human oral palatal and gingival mucosa demonstrated localization of kindlin-1 and two of its potential binding partners, migfilin and paxillin, to the basement membrane zone (BMZ). Only migfilin and paxillin immunolocalized to blood vessels. In junctional epithelium from a non-periodontally diseased tooth, migfilin, paxillin, and kindlin-1 immunolocalized to the BMZ of the external basal lamina (EBL) and the internal basal lamina (IBL) zone against the tooth with ILK-1 and Mig-2 also localizing to the IBL zone. Both immunolocalizations suggest a possible interaction between paxillin, migfilin, and kindlin-1 and possibly a role in maintaining the integrity of the epithelial attachment against a tooth. In HaCaT cells, actin filaments terminated at sites of migfilin and kindlin- 1 focal adhesions and knockdown of kindlin-1, via siRNA-mediated gene silencing, resulted in decreased localization of kindlin-1 and some of it's potential binding partners, ILK-1, Mig-2, paxillin, and actin. Knockdown of kindlin-1 was confirmed by RT-PCR analysis (approximately 70%) and resulted in significant reduction in HaCaT cell spreading on different ECM matrices, migration during in vitro wounding, and proliferation when grown in serum. To conclude, the present study provides sufficient evidence to suggest that a loss of kindlin-1 likely results in altered keratinocyte cell adhesion at the level of the basement membrane, which clinically results in skin fragility and photosensitivity as well as aggressive periodontitis.