Mechanisms of desmosome down-regulation during epithelial to mesenchymal transition

Abstract

Desmosomes are the principal intercellular adhesive links in tissues that undergo substantial mechanical stress, e.g. skin. Their down-regulation is a key step during epithelial to mesenchymal transition (EMT), during which cells become more migratory. However, the precise mechanism of desmosome down-regulation/ internalisation is unknown. Therefore, the first goal was to set up assays to study desmosome internalisation: i) an HGF assay to induce EMT and ii) the calcium chelation assay. Live-cell imaging confirmed internalisation of half- desmosome after calcium chelation. In contrast and to our surprise, HGF induced the internalisation of whole desmosomes. Interestingly, this finding is strikingly similar to published in vivo data from carcinomas and wound edges. Correlative light electron microscopy and immunostainings revealed that all major desmosomal proteins were co-localising at whole internalised desmosomes. Live-cell imaging of whole desmosomes engulfment and data showing keratin disruption suggested that forces play a role in cell separation and internalisation of the complexes. However, the lack of motor activity along keratin filaments that could induce forces led to the hypothesis of a crosstalk of the actomyosin machinery with the desmosomal complexes. Experiments with actomyosin inhibitors revealed a crucial role of actomyosin in desmosome internalisation. Further experiments showed that the actin cytoskeleton influenced intercellular adhesive strength, demonstrating the importance of a cross-talk between actomyosin and desmosome regulation. To gain insight into the molecular regulation I analysed the composition of whole internalised desmosomes and the potential cross- talk with the actin cytoskeleton and actin-linked cell-cell junction proteins. Surprisingly, β-catenin was found significantly enriched in proximity of internalised desmosomes, suggesting a potential interaction. To elucidate the molecular mechanisms of the actomyosin crosstalk with desmosome, I depleted cells of proteins that could provide the potential link of desmosomes to the actomyosin machinery (α-catenin, β-catenin). While α-catenin knockdown disturbed desmosome localisation, interesting findings showed that β-catenin knockdown significantly decreased internalisation in the HGF assay. Overall, this work revealed the importance of actin and actomyosin during desmosome internalisation and further supports the idea of a cross-talk between desmosomes and actin. Intriguing data suggest a key role for β-catenin during desmosome internalisation, potentially by linking the desmosome complex to the actin cytoskeleton.

Date of Award	1 Aug 2018
Original language	English
Awarding Institution	The University of Manchester
Supervisor	David Garrod (Supervisor) & Christoph Ballestrem (Supervisor)