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Fibroblast migration during wound healing: regulation of integrins and Rho family GTPases by the fibronectin receptor syndecan-4
The appearance of fibronectin in wounded tissue triggers the migration of fibroblasts by engagement of the fibronectin sensor, syndecan-4. Upon recruitment to a wound, fibroblasts contract the matrix to reduce wound area within a matter of hours. We are investigating the mechanisms by which fibroblast migration is initiated and guided, starting at the molecular level and working up to whole organism healing models and therapeutic regimes. Our investigations follow three major routes:
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. We are examining how syndecan-4 coordinates these 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. More importantly, we are now investgating the roles that these signals play during developmental migration and find that localisation of GTPase signals by these mechanisms directs migration to the extent that neural crest derivatives are mislocalised in their absence.
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).
- The translation of our findings to in vivo healing models, and subsequently patient therapies is a crucial aspect of our work. We have established proceedures for the investigation of low-intensity pulsed ultrasound on cell adhesion. This therapy is already proving efficacious in fracture healing and as we improve our understanding of how the signal works, we hope to improve the therapy and bring it into mainstream clinical use.
Video protocol of the investigation of the affect of ultrasound on adhesion signalling
Roz Williamson, James Roper, Becky Brooks
Steinberg F, Heesom KJ, Bass MD, Cullen PJ. (2012) Sorting nexin-17 protects integrins from degradation by sorting between lysosomal and recycling pathways. Journal of Cell Biology. 197(2): 219-230.
Bass MD, Williamson RC, Nunan RD, Humphries JD, Byron A, Morgan MR, Martin P, Humphries MJ. (2011) A syndecan-4 hair trigger initiates wound healing through caveolin- and RhoG-regulated integrin endocytosis. Developmental Cell. 21: 681-693.
Humphries JD, Byron A, Bass MD, Craig SE, Pinney JW, Knight D, Humphries MJ. (2009) Proteomic analysis of integrin-associated complexes identifies RCC2 as a dual regulator of Rac1 and Arf6. Science Signaling. 2: ra51.
Bass MD, Morgan MR, Roach KA, Settleman J, Goryachev AB, Humphries MJ. (2008) p190RhoGAP is the convergence point of adhesion signals from a5ß1 integrin and syndecan-4. Journal of Cell Biology. 181:1013-1026.
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