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(A*STAR) The role of desmosome regulation in development, wound healing and cancer


Project Description

The regulation of cell movement is a key factor in human development and disease. Desmosomes are intercellular junctions that give strength to normal tissues by binding their cells tightly together. Embryos lacking key desmosomal components die during development and aberrant desmosome function leads to skin blistering, heart disease, wound healing defects and cancer. During normal development, epidermal wound healing and cancer, cells undergo a plastic change known as epithelial-mesenchymal transition (EMT) that enables them to move. This necessitates the dynamic process of adhesion formation and down-regulation of desmosomes. It is not known how these processes occur but it needs to be clarified in order to understand normal development and to formulate treatments to aid wound healing and prevent the spread of cancer. To shed new light into desmosome regulation we propose to determine the composition of desmosomes during formation, down-regulation and the different adhesion states in normal and diseased conditions. For this we have established BioID mass spectrometry in the laboratory that will enable us to monitor the changes in desmosomal composition that mediate desmosome cell-cell adhesion. These experiments will be complemented with advanced fluorescence microscopy techniques including time-lapse fluorescence microscopy, super resolution microscopy and FRET that will help to visualise the dynamic processes of protein recruitment and protein interactions in time and space.
BioID and imaging will be used to study the molecular changes in different states of desmosomal adhesion varying from the initiation of cell-cell adhesion to strong tissue-like adhesion, through to adhesion weakening and reported engulfment. To do this, we have developed a culture model of EMT in which desmosomal adhesion in the cells appears to change in precisely the same way as during wound healing in vivo and cancer invasion. For BioID experiments we have designed desmosomal components tagged with BirA and for imaging studies we have coupled desmosomal components to GFP or fluorescent variants thereof.
As well as providing strong adhesion, desmosomes strengthen tissues by linking to the intermediate filament cytoskeleton, which is composed of keratin in epithelial cells. Keratin filaments may contribute to the regulation of desmosomal adhesion during EMT but the mechanism is not understood. In several human diseases including the severely debilitating condition epidermolysis bullosa simplex (EBS) congenital keratin mutations cause weakening or breakdown of keratin filaments and lead to skin blistering. In order to determine how the strength and integrity of keratin filaments regulates desmosomal adhesion, mutant keratins will be expressed in cells that assemble and disassemble desmosomes undergoing EMT and the consequent effect on desmosomal adhesion composition and dynamics will be determined by BioID and imaging. The project will thus provide a detailed analysis of how desmosomal adhesion is regulated and will have great relevance to our understanding of the regulation of cell motility in normal and disease processes.

Entry Requirements:
Applications should be submitted online and candidates should make direct contact with the Manchester supervisor to discuss their application directly. Applicants must have obtained, or be about to obtain, at least an upper second class honours degree (or equivalent) in a relevant subject.

Funding Notes

This project is available to UK/EU candidates. Funding covers fees (UK/EU rate) and stipend for four years. Overseas candidates can apply providing they can pay the difference in fees and are from an eligible country. Candidates will be required to split their time between Manchester and Singapore, as outlined on View Website.

As an equal opportunities institution we welcome applicants from all sections of the community regardless of gender, ethnicity, disability, sexual orientation and transgender status. All appointments are made on merit.

References

1) Berika M, Garrod D. Desmosomal adhesion in vivo. Cell Commun Adhes. (2014) 21:65-75.

2) Garrod, D.R., Berika, M.Y., Bardsley, W.F., Holmes, D, Tabernero, L. Hyper-adhesion in desmosomes: its regulation in wound healing and possible relationship to cadherins crystal structure. (2005) J. Cell Sci. 118: 5743-5754.

3) Mirjana Liovic, Mariella D'Alessandro, Marjana Tomic-Canic, Viacheslav N. Bolshakov, Stephanie E. Coats, E. Birgitte Lane. Severe keratin 5 and 14 mutations induce down-regulation of junction proteins in keratinocytes. Exp. Cell Res. 315 (2009) 2995–3003

For FRAP and FLAP imaging:

4) Atherton P, Stutchbury B, Wang DY, Jethwa D, Tsang R, Meiler-Rodriguez E, Wang P, Bate N, Zent R, Barsukov IL, Goult BT, Critchley DR, Ballestrem C. Vinculin controls talin engagement with the actomyosin machinery. Nat Commun. 2015 Dec 4;6:10038.
For BioID and the use of BirA in Mass Spectrometry:

5) Kyle J. Roux, Dae In Kim, Manfred Raida, and Brian Burke (2012). A promiscuous biotin ligase fusion protein identifyes proximal and interacting proteins in mammalian cells. JCB 196 No. 6 801–810

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