The epidermis is a multilayered epithelial tissue with a critical barrier function that regenerates through the proliferation of basal cells which exit the cell cycle and transit into suprabasal layers where they undergo differentiation and formation of the barrier. Regulating the differentiation and morphogenesis of the epidermis is a desmosomal cadherin called desmoglein-1 (Dsg1). Our lab previously showed that the proper positioning of newly expressed Dsg1 on the plasma membrane recruits the actin nucleator complex Arp2/3 to junctions where it stimulates cortical actin remodeling, tension redistribution, and basal cell delamination. To identify trafficking machinery that regulates Dsg1 delivery we conducted a Dsg1 and E-cadherin protein interaction screen. We identified VPS35, an essential component of an endosomal trafficking complex called the retromer, as a putative Dsg1-interacting partner that was not present in the E-cadherin screen. To test if endosomal trafficking is necessary for the delivery of Dsg1 to the plasma membrane, we inhibited endosomal trafficking with Primaquine, which resulted in a decrease of Dsg1, but not E-cadherin, localization on the plasma membrane and accumulation of Dsg1 in VPS35 positive endosomes. Depletion of VPS35 in primary human keratinocyte culture resulted in decreased Dsg1 plasma membrane localization and protein expression due to lysosomal degradation. Therefore, it is hypothesized that the retromer complex is necessary for the endosomal trafficking of Dsg1 to the plasma membrane (AIM1). I will investigate if the retromer regulates endosome to plasma membrane trafficking of Dsg1 by using biochemical and immunofluorescence recycling assays. Moreover, I will test if pharmacological enhancement of the retromer function with R55 compound is sufficient to enrich wild-type and a disease- associated trafficking deficient Dsg1 mutant on the plasma membrane. Delivery of Dsg1 to the plasma membrane promotes an Arp2/3-dependent increase in the concentration of cortical actin filaments. The role of Arp2/3-mediated actin remodeling during keratinocyte differentiation is unclear. Preliminary evidence suggests the pharmacological disruption of actin and Arp2/3 results in the internalization of Dsg1 and impairment in stratification, respectively. Considering initial recruitment of Dsg1 on the plasma membrane in committed basal cells promotes the enrichment of cortical actin, these data suggest a feed-forward event where Dsg1 regulation of actin organization promotes Dsg1 plasma membrane stabilization during epidermal differentiation and stratification (AIM2). I will explore the role of Arp2/3 and actin in regulating Dsg1 plasma membrane stabilization and internalization during different stages of keratinocyte differentiation. Additionally, I will investigate Arp2/3?s role in regulating Dsg1-mediated stratification. These experiments will deepen our understanding of how Dsg1 is properly localized and stabilized on the plasma membrane to promote epidermal morphogenesis, which in the future could aid in the development of strategies to restore Dsg1 localization and function in epidermal diseases.
Proper skin development, which is essential to protect against environmental and physical stress, requires an adhesive complex called the desmosome that facilitates cells sticking together. Disruption of the location of the adhesive component in desmosomes can result in a systemic and severe inflammatory skin condition. This project will investigate the mechanism by which the adhesive molecule is delivered and stabilized to the correct location to regulate skin development.
