Determination of the mechanisms of desmosome loss during EMT

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Cells in stress-exposed tissues, e.g. heart muscle and the coverings of body surfaces, are bound together by tiny, rivet-like structures called desmosomes. Aberrant function of these structures causes diseases such as sudden heart failure, defective wound healing, cancer spread and certain blistering diseases of the skin and oral cavity. Some of these conditions are among the most common causes of morbidity and death, while others are rare but extremely unpleasant, difficult to treat and can be fatal. Desmosomes are also important for normal development, where they stabilise developing tissues. It is therefore essential to understand how desmosome function is regulated.We have shown that an important factor contributing to the toughness of tissues is that desmosomes exhibit a highly adhesive state known as hyper-adhesion. Hyper-adhesion is important for tissue strength, but also locks cells together, thus restricting their movement. During wound healing, epidermal cells migrate to close the wound, their migration being triggered by wounding. The invasive spread of cancer cells also requires cell migration and in development cell movement generates the correct architecture of tissues. In order to move, cells need to reduce the degree of adhesion between them. We have shown that on wounding desmosomes rapidly lose hyper-adhesion, becoming more weakly adhesive. However, this weakening of adhesion may not be sufficient to permit the adequate movement. Instead, cells may need to lose some or all of their desmosomes. How they do this is not understood.Some evidence from electron microscopy studies of cancers and wounds suggests cells may be able to engulf whole desmosomes and therefore become stuck together more loosely. This is evident because desmosomes have a characteristic, easily recognisable structure. Normally desmosomes appear at the junction between cells but these studies have shown whole desmosome inside cells, as though one cell has "eaten" the desmosome! However unlikely this seems we have now induced cell separation in culture and shown that they do indeed engulf whole desmosomes. This is exciting because it enables us to investigate the mechanism behind a process that occurs in normal and diseased tissues.Desmosome engulfment resembles a process called phagocytosis whereby cells of the immune system engulf extracellular particles, e.g. bacteria. Phagocytosis requires active contractile activity by the engulfing cell so as to surround the particle and draw it inside. This, in turn, requires the action of the cell's contractile apparatus, which depends upon filamentous proteins called actin and myosin, similar to those involved in muscle contraction. Desmosomes are not normally associated with these proteins but instead are internally linked to other filaments called intermediate filaments (IF). The IF are linked from cell-to-cell by desmosomes, forming a scaffold that gives great strength to tissues. However, IF possess no contractile activity. So if the filaments they attach to cannot contract, how are desmosomes engulfed? Our pilot studies suggest the cell's contractile apparatus is somehow involved in desmosome engulfment and a regulatory enzyme called protein kinase C (PKC), known to be involved in phagocytosis and actin regulation, participates. To understand the mechanism in greater detail we will use state-of the-art microscopy to study how desmosomes become associated with the contractile machinery as they switch from hyper-adhesion to engulfment. Also we will use mass spectrometry to identify novel proteins involved in the process and to determine the role of PKC. Finally, we will use molecular cell biology to determine the functions of the key proteins in detail.Our results will both further our understanding of normal development and provide a basis for new therapies for major health problems such as chronic wounds, skin blistering diseases and, potentially, for limiting the spread of cancer.