Mechanisms and functional consequences of impaired keratin network formation in genetic skin disorders

Lehmann, Sonja Maria; Spehr, Marc (Thesis advisor); Leube, Rudolf (Thesis advisor); Zimmer-Bensch, Geraldine Marion (Thesis advisor)

Aachen : RWTH Aachen University (2022, 2023)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2022

Abstract

One of the most important barriers of the body is the skin. Its outermost stratified layer, the epidermis, is mainly composed of differentiating keratinocytes. The integrity of the epidermis is to a large extent provided by structural elements such as the keratin intermediate filament cytoskeleton. The importance of keratin networks is emphasised by mutation-induced keratinopathies such as Epidermolysis bullosa simplex (EBS) or Pachyonychia congenita (PC), caused by mutations in keratins 5/14 and keratins 6/16/17, respectively. EBS is characterised by trauma-induced blister formation due to rupture of keratinocytes in the basal epidermal layer, which is caused by the disruption of the keratin filament network into granular structures. PC patients, on the other hand, display epidermal thickening with extreme hyperkeratosis in certain skin areas, such as the foot soles. This work aimed to gain further insight into the different pathogeneses, in particular to apply novel image analysis tools to quantitatively describe mutant EBS keratin granules, and to investigate whether autophagy of mitochondria, representing an essential step of epidermal differentiation, is disturbed in PC. Using live-cell microscopy of epithelial cells stably overexpressing fluorophore-tagged EBS-mutant keratins, an automated tracking routine was established. It allowed a detailed quantitative analysis of different parameters of mutant keratin granule dynamics. In particular, it was shown that the mutant granules are initially formed in the outermost lamellum of epithelial cells. Subsequently, they grow up to a plateau size, and are constitutively transported inwards with a velocity of approximately 0.5 µm/min. Most keratin granules display multiple fusion events with other granules during their lifetime before they rapidly disassemble at the boundary of the lamellum and the inner cytoplasm. Their transport highly resembles actin-dependent transport, and pharmacological inhibition of the actin motor protein non-muscle myosin II significantly reduced their dynamics. Fluorescence recovery after photobleaching experiments furthermore revealed that the granules rapidly exchange soluble keratins with the surrounding cytoplasm and within the granule itself. Thus, the formation of EBS-related keratin granules is based on liquid-liquid phase separation (LLPS). The kinase DYRK was investigated next, based on its ability to dissolve different LLPS condensates. Although a clear colocalisation of different overexpressed DYRK isoforms and mutant keratin granules was shown in patient-derived EBS keratinocytes, pharmacological inhibition of the kinases did not alter the percentage of granule-containing cells in keratinocyte cell clones stably overexpressing fluorophore-tagged mutant keratins. In the second part of this work, functional consequences of mutant keratins on mitochondrial integrity were examined. First, the mitochondrial age was determined by a fluorescent reporter in both EBS and PC keratinocytes. This revealed that PC cells contain increased amounts of overaged mitochondria, which was not the case for EBS. Furthermore, contact sites between mitochondria and the endoplasmic reticulum are reduced in PC. The expression of early mitophagy markers is not changed, but clearance of mitochondria is severely impaired in PC keratinocytes. Although they are able to form autolysosomes, these structures were shown to accumulate in PC. Assessment of lysosomal function revealed defective enzymatic capacity and the mitochondrial overaging phenotype could be mimicked by lysosomal pH modifications in healthy cells. Thus, it can be concluded that the process of autolysosomal recycling, which is essential for macroautophagy, is impaired in PC, which results in impaired mitophagy. Overall, this work established new image analysis tools which allowed a detailed quantification of mutant keratin dynamics, and elucidated that mutant keratins modulate mitophagy and autolysosomal recycling in PC keratinocytes.

Institutions

• Department of Biology [160000]
• Chemosensation Laboratory [163310]