The Dynamics and Proximitome of Desmosomes during Adhesion Maturation

Abstract

Multicellular organisms rely on stable cell-cell and cell-extracellular matrix connections for structural integrity and cell communication. Desmosomes are cell-cell junctions, which are indispensable for the mechanical resistance of the myocardium and epithelial tissues, such as the skin. The primary function of desmosomes is to serve as bridges for the intermediate filament networks and stably attach neighbouring cells. Many studies of disease conditions, including dermatoses and myopathies, have revealed the importance of the desmosome-intermediate filament scaffold and the remarkable stability and plasticity of the desmosomal complex. The contextual adaption and downregulation of desmosomal adhesion strength were demonstrated in vivo when cells transition from an epithelial to a more motile mesenchymal phenotype, for example, during embryogenesis and at wound edges. In vitro desmosomes acquire a resistance to the chelation of calcium, known as hyper-adhesion, depending on the duration of the cell culture. However, the underlying mechanisms of desmosome maturation and downregulation are largely unknown. This study sought to understand how the adhesion state of desmosomes influences the mobility and interactors of individual desmosomal components in Madin Darby canine kidney cells to uncover further their roles in desmosomes. Using fluorescent live-cell imaging to determine desmosomal protein dynamics, I found that desmosomes consist of two contrasting protein moieties. A very stable moiety comprised the desmosomal receptors, desmoglein 2 and desmocollin 2a, the cytoplasmic linker plakoglobin and the intermediate filament-binding desmoplakin, whereas plakophilin 2a was highly mobile and continuously exchanged from desmosomes and the cytoplasm. Both moieties become more stable as desmosomes acquire hyper-adhesion, yet plakophilin 2a was persistently dynamic. Furthermore, a tissue culture model was developed to study desmosome downregulation that revealed desmosomes were internalised as whole double-plaque-bearing complexes by one or other of the pair of adjacent cells in an actomyosin-dependent manner. In the second part of the thesis, proximity-based proteomics and mass spectrometry were used to asses the interactomes of desmocollin 2a, plakoglobin and plakophilin 2a, in Ca2+-dependent and hyper-adhesive desmosomes. Known and unknown proximal interactors, such as other desmosomal components and signalling proteins like heat shock proteins, were discovered and their potential roles discussed. Furthermore, the individual desmosomal components were in close vicinity to a multitude of unique preys, reflecting their distinct roles beyond desmosomal adhesion. We found many changes within the proximitomes concerning the maturation state of desmosomes. Lastly, I demonstrated that inhibiting the actomyosin network with a Rho-associated serine/threonine kinase 1 inhibitor resulted in a severely reduced proximitome, particularly nuclear proteins, of plakophilin 2a, suggesting its translocation from desmosomes to the nucleus was inhibited. This work demonstrates that desmosomes are organised in moieties, a stable one forming a stable axis to the intermediate filament network and a dynamic one allowing continuous signalling from and to desmosomes. Both moieties are pivotal for desmosome function in providing reliable yet flexible cell-cell adhesion. The partial desmosomal proximitome significantly advances our knowledge of the specific roles of key desmosomal components and underscores a paramount role for desmosomes in epithelial maturation. Together, these data provide a repository for future work to find explanations and remedies for abnormal desmosome function and disease conditions.

Date of Award	1 Aug 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	David Garrod (Supervisor) & Christoph Ballestrem (Supervisor)