Determination of the mechanisms of desmosome loss during EMT

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.

Desmosomes are cell-cell junctions linked to intracellular intermediate filaments (IF). They adopt hyper-adhesion that assists tissues, e.g. epidermis and heart muscle, to resist shear, but restricts cell movement. In development, wound repair and cancer spread, epithelial-mesenchymal transition (EMT) enables movement. EMT involves junction downregulation, but how desmosomes are downregulated is unknown. Pilot data show cells engulf whole desmosomes during EMT, consistent with electron microscopy analyses of wounds and cancer, but how it occurs is unclear as IF lack motor activity. Pilot data indicate engulfment involves actin and protein kinase C alpha (PKC), and suggest crosstalk with actin-linked adherens junctions (AJ). The switch from hyperadhesion to Ca2+-dependence precedes desmosome downregulation. Pilot fluorescence recovery after photobleaching (FRAP) suggests this involves "loosening" of structure, consistent with EM studies.
The major question of this study will be: How are desmosomes downregulated during EMT?

The two questions that we will address are:
1) What dynamic changes accompany desmosome downregulation?
We will use FRAP and photoactivation to study mobility of desmosomal components, and FRET and superresolution microscopy to study desmosome associations with AJ and actin regulators. We will use proximity biotinylation (Bio-ID) in conjunction with mass spectrometry to determine the protein associations of desmosomes during downregulation.

2) What is the mechanism of desmosome downregulation?
Pilot data have indicated regulatory roles for plakoglobin and plakophilin-1. Retroviral protein expression will be used to determine the protein domains and phosphorylation sites that are involved. Phosphoproteomic analysis will identify phosphorylation sites in these and other plaque proteins.

Insights from these studies will ultimately lead to novel strategies to prevent diseases and further our understanding of development.

The proposed project combines biological, physical, and medical aspects, is concerned with the refinement of cutting edge techniques and investigates molecular mechanisms potentially relevant to development, pathology and medical treatment. Thus, there are a wide range of direct and indirect beneficiaries from the research:

(1) Biotechnology. Understanding how cells interact with each other and establishing methodologies that enable cellular responses to be directed will be of benefit for biotechnology research and industry, particularly for wound healing and possibly tissue engineering. Cell lines stably expressing GFP-tagged proteins may become valuable for the screening of materials and drugs affecting cellular behaviour. We expect a high potential impact in the biotechnology area and will actively search for relevant systems/companies to share our knowledge. The impact will be direct and mid-term.

(2) Pharmaceutical industry. Unravelling how desmosome regulation is involved in strengthening tissues will provide a starting point for the development of pharmaceutical products influencing cell to cell communication. Modulating cell responses to changing environments may promote regeneration. Thus, there is the potential to commercialise products used to modulate cell to cell adhesion. It will be direct and mid- to long-term.

(3) General public. Images generated from this project are colourful, intuitive, attractive and make the science more accessible. They are useful for engaging the public, and particularly children through school lectures, about science. We will further set up a website about "The Cell's Ability to Talk to Each Other" which will contain sections accessible to the lay person. This will focus on how disciplines can be integrated to deliver tangible benefits for society, in terms of finding new ways to understand and treat disease. Moreover, contributing to the successful treatment of tissue injuries has an enormous impact on general health. Promoting regeneration processes will improve life quality of thousands of people in the UK and beyond the borders. Furthermore, it will drastically reduce treatment costs, thus directly and indirectly impacting the healthcare system. The impact is indirect and mid- to long-term.

(4) Researchers of various backgrounds. Understanding cell adhesion processeses is highly relevant to biology and biophysics. It is known that cell to cell communication is involved in many physiological and pathological processes ranging from embryo formation to blistering diseases, wound healing defects and cancer, adding an impact on medical research. The application and refinement of cutting edge methods is particularly relevant to method developers and analysts. Accordingly, scientists working in any of those areas might be highly interested in the outcome of the project. The impact will be direct and immediate.

(5) Staff working on the project. Researchers will work interdisciplinary, interact with many scientists of different backgrounds and companies and creatively solve problems. They will further develop communication, problem solving and entrepreneurial skills and acquire new technical and IT skills, which will be useful in any later profession.