Investigating a keratin-endoplasmic reticulum contact site in skin disease

Summary: Abstract Body: Keratins are epithelial-specific intermediate filament proteins that provide mechanical resistance to environmental stressors in tissues such as the epidermis. Epidermolysis bullosa simplex (EBS), a skin blistering disease, is caused by mutations in keratins 5 and 14 (KRT5/ KRT14). Prior studies have reported activation of endoplasmic reticulum (ER) stress and inflammatory pathways in EBS cell culture. However, the mechanisms linking keratin dysfunction to activated stress responses in EBS remain poorly understood. We first wanted to determine the association between the ER and keratin using electron and fluorescence microscopy in A431 epithelial cells. We found that peripheral ER tubules are in close proximity to keratin filaments and form paired arrangements at desmosome cell-cell junctions. Focused ion beam scanning electron microscopy (FIB-SEM) reveals intricate associations of ER tubules with keratin filaments at points of contact we term the keratin-ER contact site. Further, fluorescence live-cell microscopy demonstrates that keratin filaments stabilize ER membrane. Disruption of keratin filaments by expression of an EBS-causing aggregation mutant, KRT14R125C, leads to changes in ER morphology, converting ER tubules at the cell periphery to ER sheets. Lastly, FIB-SEM datasets reveal that keratin filaments are present within nanometers of ER-mitochondria contact sites. To determine the effects of keratin disruption on mitochondrial organization, we labelled cells with MitoView dye. In KRT14R125C-expressing cells, mitochondria are more peripheral, co-localizing with ER sheets, suggesting that ER morphological domains govern mitochondrial positioning. Our results demonstrate that keratin filaments regulate the stability and organization of the ER network in epithelial cells. Further, these studies suggest that keratin disruption in EBS alters the organization of other organelles via its effects on ER morphology, potentially leading to organelle dysfunction. Navaneetha Krishnan Bharathan¹, William Giang¹, Jesse Aaron², Satya Khuon², Teng-Leong Chew², Andrew Kowalczyk¹ 1. Dermatology, Penn State College of Medicine, Hershey, PA, United States. 2. Howard Hughes Medical Institute Janelia Research Campus, Ashburn, VA, United States. Epidermal Structure and Barrier Function