Dr Mark Bass
Room: B2 05 Florey building
Brief career history
Fibroblast migration during wound healing: signalling from extracellular matrix receptors to Rho-family GTPases.
Fibroblast migration during wound healing: signalling from extracellular matrix receptors to Rho-family GTPases
However, these processes become less efficient with age and risk factors such as diabetes, obesity or smoking, eventually leading to the formation of chronic wounds that include pressure ulcers, venous leg ulcers and diabetic foot ulcers.
The two hallmarks of a chronic wound are a chronic inflammatory response as the skin tries unsuccessfully to deal with infection and failure by fibroblasts to proliferate and migrate. The fact that scars are usually a fraction of the size of the original wound demonstrates very simply the importance of fibroblast migration and wound contraction, and improving this process is the core objective of our work.
Our laboratory investigates the activation of fibroblasts upon wounding, and the mechanisms by which migration is directed by regulation of the Rho family GTPases, especially the protrusion regulator, Rac1. Our work ranges from the investigation of signalling networks in single cells, through the analysis of in vivo wound healing models, to the development of therapies for treatment of patients.
The work can be divided into three main areas:
1) Regulation of membrane protrusion by Rho-family GTPases
Cell migration requires cycles of protrusion at the leading edge and contraction within the cell body, driven by the activation of Rac1 and RhoA respectively. The appearance of fibronectin in wounded tissue triggers cycles of Rac1 and RhoA activity in fibroblasts by engagement of the fibronectin sensor, syndecan-4.
We are examining how syndecan-4 regulates and coordinates Rac1 and RhoA signals by combining traditional biochemistry (A) with live cell imaging and FRET-based analysis (B). Perturbation of components of the syndecan-4 signalling chain by RNAi is revealing that syndecan-4 synchronises the activation/inhibition of Rac1 and RhoA signals and more importantly localises protrusion. By examining the migration of cells through complex fibrillar matrices, which are structurally similar to skin (C), we find that sydecan-4-directed GTPase regulation is necessary for persistent migration along matrix fibers.
A) Activation of Rac1 upon engagement of syndecan-4 by fibronectin, detected by pull-down assay.
2) Cooperation between extracellular matrix receptors regulates focal adhesion dynamics
Membrane protrusion must be coordinated with formation and dissolution of focal adhesions for migration to occur. We are investigating the regulation of integrin trafficking by syndecan-4 using atomic force microscopy to measure adhesive strength (D+E) and TIRF to follow removal of β1-integrin from the adhesion plane upon engagement of syndecan-4 (F). We are also using mass spectrometry to identify key trafficking regulators, and through this approach uncovering key roles for sorting nexins in integrin trafficking (G).
D) Single cells captured on the cantilever of an atomic force microscope can be brought into contact with an extracellular matrix before withdrawal of the cell to measure strength of adhesion.
3) Regulation of Rac1 by matrix receptors regulates cell migration in vivo and allows the development of healing therapies
The translation of our findings to in vivo healing models, and subsequently patient therapies is a crucial aspect of our work. The consequences of disrupting Rac1 signalling are that migration becomes less efficient, leading to delays in developmental and healing processes, and can be demonstrated in both fish (H) and mammalian (I) models.
We are investigating techniques to reverse healing defects by activating Rac1. Our most notable advances have come from the use of low-intensity pulsed ultrasound to stimulate fibroblast migration (Video protocol). (J) We find that skin wounds heal more slowly in diabetics (green curve) than healthy individuals (orange curve).
However, normal healing can be restored by daily treatments with ultrasound (pink line). The effect of ultrasound can be seen at the cellular level in biopsies as the number of brown fibroblasts recruited to an ultrasound-treated diabetic wound far exceeds that recruited to an untreated wound (K).
H) Knockdown of the Rac1 trafficking molecule, coronin 1C causes misalignment of pharyngeal arches in the developing zebrafish, due to compromised neural crest migration.
By combining this range of approaches to address the mechanism of fibroblast migration during wound healing, we move closer to developing some promising therapies, and bringing them into mainstream clinical use.
Undergraduate and postgraduate taught modules
Postgraduate PhD Opportunities
1. Cleavage of the protrusion regulator, Vav2, directs cell migration during wound healing
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.
2. Localisation of Rac1 signals in chronic wound and cancer-associated fibroblasts
Healing defects are one of the largest current health challenges. Chronic leg ulcers affect 200,000 UK patients and cost the NHS £3.1 billion (3% of expenditure) annually, and the rise in linked risk factors such as age and diabetes mean that the challenge is increasing. Although not fatal, chronic wounds have a huge effect on quality of life as they cause chronic pain and frequently result in amputation of the limb. Therefore if we are to achieve lifelong health, it is essential that we understand the healing process and identify ways to reverse the defect when healing breaks down. In healthy individuals, dermal fibroblasts migrate to the wound bed, where they contract to draw the edges of the wound together, but in unhealthy individuals, defects in fibroblast migration are one of the two hallmarks of chronic wounds.
Our laboratory investigates the regulation of migratory signals induced by changes in the extracellular matrix that occur upon wounding. Directional fibroblast migration requires: a) activation of the protrusion signal, Rac1, at the front of the cell, but suppression at the rear, b) precise temporal regulation of protrusion signals to allow coordination with cell contraction to pull the cell body forward. We recently reported a Rac1-sequestering molecule, RCC2, and newer data have revealed a link between RCC2 and the transmembrane matrix sensor, syndecan-4. Genetic disruption of either RCC2 or syndecan-4 perturbs the directionality of fibroblast migration, and both have been demonstrated in vivo. RCC2-depletion in zebrafish compromises developmental migration, while syndecan-4-knockout in mice retards wound healing. Both have also been linked to cancer development, and whether this is due to a change in the cancer cells themselves, or cancer-associated fibroblasts remains to be seen.
This project will determine how signalling from syndecan-4 to RCC2 localises Rac1 signalling and thereby directs fibroblast migration. The composition of the RCC2 complex will be determined by combining syndecan-4 and RCC2 mutants with knockdown of other complex components, identified by mass spectrometry. The influence of those interactions on Rac1 signalling will be determined using cell-based signalling assays and FRET. Crucially you will test the effect of the pathways on fibroblast migration through synthetic skin mimics and genuine skin sections.
This project will also link to our ongoing investigations into the use of ultrasound to promote Rac1 activation in cells where matrix-dependent signalling is defective. We have demonstrated that ultrasound can overturn healing defects in diabetic and syndecan-4-knockout mice and are now testing whether it reverses senescence in both chronic wound and cancer-associated fibroblasts. The putative role of the syndecan-4/RCC2 pathway in both these instances suggests a link to ultrasound efficacy, meaning that this project will result in clinical impact within a few years.
For further information about these projects and how to apply, see our PhD Opportunities page:
- Williamson RC, Cowell CAM, Reville T, Roper JA, Rendall TCS & Bass MD (2015) Coronin-1C Protein and Caveolin Protein Provide Constitutive and Inducible Mechanisms of Rac1 Protein Trafficking. Journal of Biological Chemistry, 290(25), 15437-15449. View this article in WRRO
- Roper JA, Williamson RC, Bally B, Cowell CAM, Brooks R, Stephens P, Harrison AJ & Bass MD (2015) Ultrasonic Stimulation of Mouse Skin Reverses the Healing Delays in Diabetes and Aging by Activation of Rac1. Journal of Investigative Dermatology, 135(11), 2842-2851. View this article in WRRO
- Williamson RC, Cowell CAM, Hammond CL, Bergen DJM, Roper JA, Feng Y, Rendall TCS, Race PR & Bass MD (2014) Coronin-1C and RCC2 guide mesenchymal migration by trafficking Rac1 and controlling GEF exposure. Journal of Cell Science, 127(19), 4292-4307. View this article in WRRO
- Bass MD, Williamson RC, Nunan RD, Humphries JD, Byron A, Morgan MR, Martin P & Humphries MJ (2012) A Syndecan-4 Hair Trigger Initiates Wound Healing through Caveolin- and RhoG-Regulated Integrin Endocytosis. Developmental Cell, 23(5), 1081-1082.
- Bass MD, Roach KA, Morgan MR, Mostafavi-Pour Z, Schoen T, Muramatsu T, Mayer U, Ballestrem C, Spatz JP & Humphries MJ (2007) Syndecan-4–dependent Rac1 regulation determines directional migration in response to the extracellular matrix. The Journal of Cell Biology, 177(3), 527-538. View this article in WRRO