PhD Research Project: Cleavage of the protrusion regulator, Vav2, directs cell migration during wou

Healing delays affect 200,000 UK patients a year, often result in limb amputation, and are caused by defects in the migration of skin fibroblasts. Upon injury to healthy skin, resident skin fibroblasts are activated and cluster at the wound shoulder where they contract the defect. Failures in fibroblast migration cause healing delays to the extent that fibroblast senescence is one of the two hallmarks of chronic wounds. Therefore understanding how migration of fibroblasts towards wound signals is regulated is critical. The cytoskeletal regulator, Rac1, both drives and guides migration by stimulating localised membrane protrusion. A key question is how fibroblasts recognise wound signals and polarise protrusion so that migration to the wound site is efficient. Rac1 itself is activated by exchange factors such as Vav2, in response to extracellular changes in the wound environment. We have preliminary evidence that Vav2 mediates the migratory response during healing and that Vav2 is cleaved, immediately after activation, thus ensuring that protrusion signals are transient, making cells responsive to changing migration cues. This project will identify the Vav2 cleavage site and determine how that cleavage event affects decision making as fibroblasts migrate.

This project will use mass spectrometry to map the site of Vav2 cleavage in response to wound signals, so that cleavage-resistant mutants and pre-cleaved fragments can be generated. The mutants will be used to investigate the effect of cleavage on Rac1 regulation in cell-based assays and FRET experiments using Rac1 activity probes. Following characterisation of the effect of cleavage on Rac1, the mechanism will be investigated, thus determining how Vav2 regulation is linked to the extracellular events that occur upon wounding. Of critical importance will be the investigation of how Vav2 cleavage affects migration. 3D matrices that resemble the skin will be generated and used to investigate the effect of Vav2 cleavage on migration persistence, while micropatterned surfaces will be used to test the effect on decision-making when faced with alternative paths during migration. Together these experiments will resolve the mechanism by which protrusion is regulated and migration directed during healing.

Finally, the project will link to our ongoing clinical investigations. We are in the process of developing ultrasonic therapies that restore wound closure in healing-defective animals and patients. The major limitations to clinical translation of the work are the gaps in our understanding of how Rac1 activation is regulated during healthy healing. The progress in this PhD project will aid in predicting the healing prognosis of patients, including identification of groups that need and would respond well to intervention. The effect of the Vav2 cleavage mutants on Rac1 activation in response to ultrasonic stimuli will be tested, thus ensuring that the project connects molecular mechanism to therapeutic outcome and that the student gains understanding of therapeutic development.

Funding Notes

Applications from self-funded students or students with secured funding are also welcome.
Entry requirements
First class or upper second 2(i) in a relevant subject. To formally apply for a PhD Studentship, you must complete the University's application form using the following link: http://www.sheffield.ac.uk/bms/prospective_pg/how_to_apply
*All applicants should ensure that both references are uploaded onto their application as a decision will be unable to be made without this information*.

References

1) Roper, Williamson, Bally, Cowell, Brooks, Stephens, Harrison, Bass. (2015) Ultrasonic stimulation of mouse skin reverses the healing delays in diabetes and aging by activation of Rac1. J Invest Dermatol. 135: 2842.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4902130/
2) Williamson, Hammond, Bergen, Roper, Feng, Rendall, Race, Bass. (2014) A coronin-1C/RCC2 complex guides mesenchymal migration by trafficking Rac1 and controlling GEF exposure. J Cell Sci. 127: 4292.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4179493/
3) Bass, Williamson, Nunan, Humphries, Byron, Morgan, Martin, Humphries. (2011) A syndecan-4 hair trigger initiates wound healing through caveolin- and RhoG-regulated integrin endocytosis. Dev Cell. 21: 681.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3202633/

https://www.shef.ac.uk/bms/research/bass/area

Bass, Roach, Morgan, Mostafavi-Pour, Schoen, Muramatsu, Mayer, Ballestrem, Spatz, Humphries. (2007) Syndecan-4-dependent Rac1 regulation determines directional migration in response to the extracellular matrix. J Cell Biol. 177:527.

Kasolang, Dwyer Joyce, Ahmad (2013) PZT transducer design and pulsing optimization for film thickness and viscosity measurement. Sensors and Actuators, A: Physical, 203:386.

Brockett, Harper, Williams, Isaac, Dwyer-Joyce, Jin, Fisher (2008) The influence of clearance on friction, lubrication and squeaking in large diameter metal-on-metal hip replacements. J Mater Sci Mater Med 19:1575