Low Intensity Pulsed Ultrasound (LIPUS) is used clinically to promote wound healing. In vivo studies show that LIPUS is effective in a wide range of tissue types, and in vitro experiments show that multiple cell types respond to LIPUS stimulation. Despite this, there is no unifying mechanism of how LIPUS stimulation is sensed by cells, and it is unknown what the early signalling events are. The LIPUS signal is a mechanical one; therefore I hypothesised that mechanosensitive organelles, called focal adhesions, would be essential for the induction of cellular signalling events in response to this type of stimulation. Proteins within these structures (such as vinculin and talin) link the actin cytoskeleton to the extracellular matrix via integrins, and are known to be sensitive to mechanical forces, capable of generating intracellular signalling events in response to mechanical stimulation. The purpose of this work was to identify the early signalling events occurring within minutes of LIPUS stimulation; determine the molecular mechanisms behind such events; and to investigate whether such events require integrin-mediated adhesions. In the first part of the work, I established the use of live-cell imaging together with LIPUS stimulation to directly observe the cellular response. I determined rapid reorganizations of the actin cytoskeleton, which led to increased cell velocity. These effects were found to be Rac dependent, and, using FRET-based probes, I measured rapid increases in Rac activity occurring within minutes of LIPUS stimulation. The second part of this work identified an increase in the number of early endosomes in cells stimulated with LIPUS. This phenotype was also Rac dependent, as well as requiring the early endosomal regulator protein Rab5. In this chapter, I observed an increase in the association between Rac and Rab5 in response to LIPUS stimulation, and this contributes to Rac activation. Using substrates to block integrin-mediated adhesion, I determined that cell-matrix adhesions are required for the effects of LIPUS stimulation. Using vinculin-deficient cells, I determined that this mechanosensitive protein is vital for co-ordinating Rac activation in response to LIPUS. In particular, the actin binding tail is needed for mechanosensing of this LIPUS signal. In the final chapter I established the use of photoactivatable (PA) GFP to assess adhesion protein turnover. This technique was used to show that LIPUS stimulation directly affects the turnover of vinculin. Overall, this work shows that the mechanosensitive protein vinculin is crucial for sensing the mechanical stimulation provided by LIPUS, orchestrating downstream Rab5-mediated Rac activation to enhance cell motility.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:697803 |
| Date | January 2016 |
| Creators | Atherton, Paul |
| Contributors | Ballestrem, Christoph ; Millard, Thomas |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/the-role-of-ultrasound-in-wound-healing(94a0808e-f676-4431-a415-582525f24674).html |
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