Insight into the role of the keratin cytoskeleton during collective epithelial cell migration

Expression of the type II keratins 6a and 6b (K6a and K6b), normally confined to ectoderm-derived epithelial appendages, is rapidly and robustly induced in wound-proximal keratinocytes after injury to stratified epithelia and in various ex vivo culture paradigms. Genetic ablation of both Krt6 isoforms, a and b, consistently results in enhanced keratinocyte migration in ex vivo culture (Wong and Coulombe, 2003; Rotty and Coulombe, 2012), providing a unique opportunity to explore the role of keratins during epithelial cell migration. The laboratory previously reported that, under normal conditions, K6a/K6b proteins may suppress keratinocyte migration by interacting with Src kinase and impacting its membrane partitioning and activity status (Rotty and Coulombe, 2012). This thesis aims to further characterize how K6a/K6b impacts the collective migration of skin keratinocytes. We show that the increased migration of Krt6a/Krt6b null keratinocytes likely is a cell-autonomous property that result from a combination of increased speed, more coordinated movement, and alteration in both cell-matrix and cell-cell adhesion. Interactions between keratinocyte and different extracellular matrix (ECM) proteins have a distinct impact on the speed and directionality of Krt6a/Krt6b null skin keratinocytes, which otherwise exhibit an increased rate of focal adhesion disaasembly. Desmosome-mediated cell-cell adhesion is disrupted with the absence of K6a/K6b, and this is accompanied by weakened cell-cell adhesive strength, reduced levels and mis-localization of desmoplakin, a key components of desmosomes, into the nucleus. We also show that overexpressing desmoplakin partially normalizes the increased migratory potential of Krt6a/Krt6b null keratinocytes. We conclude that K6a/K6b proteins normally suppress keratinocyte migration in part by stabilizing desmoplakin at cell-cell adhesion. Thus, wound-induced keratin proteins such as the K6 isoforms impact the migration properties in a way that is profound and pleiotropic. This thesis also focused on exploring the cellular roles of K6a/K6b, relative to that of their main interacting partner, the type I keratin 16 (K16), in keratinocyte cultures ex vivo. We show that the loss of K6a/K6b or K16 had both similar and opposite effects on keratinocyte biology. Both K6a/K6b and K16 conferred a modest degree of protection against apoptosis, suppressed collective cell migration, dampened the expression of danger-associated molecular patters (DAMPs; also known as “alarmins”), and positively regulated Akt activity. However, the expression of epidermal differentiation-related markers, K1 and K10, were increased in the absence of K6a/K6b, but decreased in the absence of K16. Moreover, Krt6a/Krt6b null keratinocytes exhibit weakened cell-cell adhesive strength, while the latter is strengthened in Krt16 null keratinocytes. Taken at face value, these findings point to functional synergies as well as clear differences with regards to the cellular influence of the type I and type II moieties of a keratin pairing