Desmosomal cadherins are a recent evolutionary innovation that make up the adhesive core of highly specialized intercellular junctions called desmosomes. Desmosomal cadherins, desmogleins and desmocollins, are related to the classical cadherins but their cytoplasmic domains are tailored for anchoring intermediate filaments instead of actin to sites of cell-cell adhesion. The resulting junctions are critical for resisting mechanical stress in tissues such as skin and heart. Desmosomal cadherins also act as signaling hubs that promote differentiation and facilitate morphogenesis, creating more complex and effective tissue barriers in vertebrate tissues. Interference with desmosomal cadherin adhesive and supra-adhesive functions leads to a variety of autoimmune, hereditary, toxin-mediated and malignant diseases. Here, I investigate mechanisms by which the differentiation-specific desmosomal cadherin, desmoglein-1 (Dsg1) is trafficked and stabilized on the plasma membrane in order to promote keratinocyte stratification.Sorting transmembrane cargo is essential for tissue development and homeostasis. However, mechanisms of transmembrane sorting and trafficking during the development and regeneration of the stratified epidermis are not well characterized. I describe a novel role for the endosomal trafficking complex, the retromer, in regulating epidermal differentiation and stratification. Through a BioID screen, we identified an interaction between the retromer component, VPS35, and desmosomal cadherin, Dsg1, and validated this interaction through proximity ligation assay and co-immunoprecipitation. My studies support a role for the retromer in promoting Dsg1 recycling away from the endo-lysosomal system to localize Dsg1 on the plasma membrane. When properly localized on the plasma membrane, Dsg1 recruits the actin nucleator complex Arp2/3 to junctions stimulating cortical actin remodeling, tension redistribution, and basal cell delamination. The mechanism by which Dsg1 regulates Arp2/3 activation to promote actin remodeling during keratinocyte stratification is unclear. I identified a phosphorylation site on Dsg1’s cytoplasmic domain that promotes Dsg1 interaction with Nck1 and Crk, which are SH2/SH3 adaptor proteins that facilitate Arp2/3 activation. We have preliminary evidence that suggests the expression of phospho-deficient Dsg1 disrupts Dsg1-mediated stratification. Additionally, I found that the pharmacological disruption of actin and Arp2/3 results in cytoplasmic accumulation of Dsg1, which suggests a feed-forward event where Dsg1 regulation of actin organization promotes Dsg1 plasma membrane stabilization during epidermal differentiation and stratification. By deepening our understanding of how Dsg1 is properly localized and stabilized on the plasma membrane to promote epidermal morphogenesis, we aim to aid in the development of strategies to restore Dsg1 localization and function in epidermal diseases. Here, we tested a small molecule chaperone, R55, which increases retromer stability and function. We found R55 enhanced retromer association with and plasma membrane localization of Dsg1 and a trafficking-deficient Dsg1 mutant that results in a systemic inflammatory condition called Severe dermatitis, multiple Allergies, and Metabolic wasting (SAM) syndrome. R55 also enhanced the ability of SAM-Dsg1 to induce stratification. We also investigated how Dsg1 disruption impacts downstream pathways that may contribute to pathogenesis. Using a Dsg1 knockout mouse and SAM patient skin, we found Dsg1 disruption is associated with increased glucose transporter 1 (GLUT1) plasma membrane localization and retromer association. GLUT1 is a well-known retromer cargo that regulates proliferation in basal keratinocytes and is upregulated in the inflammatory skin disease, psoriasis. Dsg1 overexpression results in the cytoplasmic accumulation of GLUT1 raising the possibility that the onset of Dsg1 expression during epidermal differentiation may initiate a switch in retromer cargo trafficking to decrease GLUT1 activity. Our work provides the first evidence for retromer function in epidermal regeneration, identifying it and GLUT1 as a potential therapeutic target for epidermal diseases.

Creator

• Hegazy, Marihan

DOI

• https://doi.org/10.21985/n2-8wbb-0j49

Subject

• Cellular biology

Language

• en

Alternate Identifier

• etdadmin_upload_944940
• http://dissertations.umi.com/northwestern:16344

Keyword

• Keratinocyte
• Desmosome
• Retromer chaperone
• Dsg1
• SAM syndrome
• Endosomal trafficking

Date created

• 2022-01-01

Resource type

• Dissertation

Rights statement

• In Copyright