Grant: Discovery Grants Program - Individual

The outer most layer of the skin is called the epidermis which provides all mammals with protection against environmental insults. During the self-renewal of the epidermis that occurs every 2-4 weeks, keratinocytes must undergo a well-orchestrated series of events that involve cell proliferation, differentiation, migration, and programmed cell death. Long-term Objective: To accommodate the dynamic turnover of the epidermis, adhesive junctions (tight, adherens and desmosomes) and communication junctions (gap) found between contacting keratinocytes are destroyed and rebuilt in a highly regulated fashion. Adhesive and gap junctions share some common building blocks that facilitates their interplay and crosstalk that we proposed is critical for skin maintenance and barrier function. Our long-term goal is to fully understand how both adhesive and communication junctions work in harmony to orchestrate the renewal of the epidermis. Short-term Objective: Rodent epidermis contains at least six connexins (Cx26, Cx30, Cx30.3, Cx31, Cx31.1 and Cx43; termed hereon as "keratinocyte connexins") that are temporally and spatially expressed to form a complex array of gap junctions between contacting keratinocytes. Connexins in their canonical form allow the regulated and direct cell-cell passage of metabolites but may also have non-canonical roles where functional hemichannels at the cell surface engage in paracrine signalling or crosstalk with constituents of adhesive junctions to impact epidermis integrity and barrier function. Our short-term goal is to interrogate and determine how each keratinocyte connexin is deployed in keratinocytes to regulate the homeostasis of the epidermis. Hypothesis and Approach: It is our hypothesis that keratinocyte connexins have unique cellular life cycles and use both canonical and non-canonical functions to exquisitely regulate epidermal homeostasis and barrier function. Our studies will focus on differentiation-competent rat epidermal keratinocytes that exhibit robust features of being, a) amenable to the ectopic expression of untagged and tagged wildtype and functionally-limited keratinocyte connexins, b) ideal to investigate the connexin interactome and crosstalk with adhesive junction proteins, c) resilient to high-resolution live cell imaging and approaches to assess the life cycle of connexins, d) suitable for assessing both connexin hemichannel and intercellular channel function, and e) appropriate for determining the impact of connexins on the phenotype of keratinocytes and organotypic epidermis formation. Significance: The resistance of skin to environmental insults is largely established by life-sustaining barrier function provided by adhesive junctions that form between communication-competent keratinocytes. We are uniquely poised to investigate the mechanisms underpinning how adhesive and communication networks work together to accommodate the self-renewal of the epidermis.