This protocol describes the stimulation of cultured fibroblasts with low-intensity pulsed ultrasound, which drives focal adhesion formation and Rac1 activation by mimicking engagement of the transmembrane matrix receptor, syndecan-4. This approach allows investigation of a successful clinical technique at the cellular level, thereby providing opportunities for refinement of the therapy.
In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from regulation of cell migration and proliferation, to secretion and even differentiation. The signals underlying each of these complex processes arise from the molecular interactions of extracellular matrix receptors on the surface of the cell. Integrins are the prototypic receptors and provide a mechanical link between extracellular matrix and the cytoskeleton, as well as initiating some of the adhesion-dependent signaling cascades. However, it is becoming increasingly apparent that additional transmembrane receptors function alongside the integrins to regulate both the integrin itself and signals downstream. The most elegant of these examples is the transmembrane proteoglycan, syndecan-4, which cooperates with α5β1-integrin during adhesion to fibronectin. In vivo models demonstrate the importance of syndecan-4 signaling, as syndecan-4-knockout mice exhibit healing retardation due to inefficient fibroblast migration1,2. In wild-type animals, migration of fibroblasts toward a wound is triggered by the appearance of fibronectin that leaks from damaged capillaries and is deposited by macrophages in injured tissue. Therefore there is great interest in discovering strategies that enhance fibronectin-dependent signaling and could accelerate repair processes.
The integrin-mediated and syndecan-4-mediated components of fibronectin-dependent signaling can be separated by stimulating cells with recombinant fibronectin fragments. Although integrin engagement is essential for cell adhesion, certain fibronectin-dependent signals are regulated by syndecan-4. Syndecan-4 activates the Rac1 protrusive signal3, causes integrin redistribution1, triggers recruitment of cytoskeletal molecules, such as vinculin, to focal adhesions4, and thereby induces directional migration3. We have looked for alternative strategies for activating such signals and found that low-intensity pulsed ultrasound (LIPUS) can mimic the effects of syndecan-4 engagement5. In this protocol we describe the method by which 30 mW/cm2, 1.5 MHz ultrasound, pulsed at 1 kHz (Fig. 1) can be applied to fibroblasts in culture (Fig. 2) to induce Rac1 activation and focal adhesion formation. Ultrasound stimulation is applied for a maximum of 20 minutes, as this combination of parameters has been found to be most efficacious for acceleration of clinical fracture repair6. The method uses recombinant fibronectin fragments to engage α5β1-integrin, without engagement of syndecan-4, and requires inhibition of protein synthesis by cycloheximide to block deposition of additional matrix by the fibroblasts., The positive effect of ultrasound on repair mechanisms is well documented7,8, and by understanding the molecular effect of ultrasound in culture we should be able to refine the therapeutic technique to improve clinical outcomes.
In this protocol we describe the method by which a treatment that is normally applied to human patients can be used in cell-based experiments. The ultimate goal is to understand the molecular mechanism of ultrasound action so that the therapy can be refined. In this protocol, we use mouse embryonic fibroblasts (MEFs) as a model cell system, but ultrasound has been also been found to be effective in primary human foreskin fibroblasts 5, mesenchymal stem cells, osteoblasts and chondrocytes 6. We use Rac1 activation as an example biochemical assay, but the method could be used equally to test regulation of protein phosphorylation or formation of protein complexes by immunoprecipitation. For the immunofluorescent example we demonstrate an effect ultrasound on focal adhesion formation, but redistribution of any molecule could be examined. For example, one could test recruitment of cytosolic factors to the plasma membrane, colocalisation of proteins in trafficking vesicles or examine the effect of ultrasound on mitotic spindle organization. So far we have confined ourselves to testing fibronectin-dependent pathways, by the effect of ultrasound on cells on collagen, or in the presence of growth factors could be tested to build up a picture of how ultrasound affects cell behavior in a complex environment that more closely resembles an in vivo situation. A greater challenge will be to test the effect of ultrasound using time-lapse imaging, where the presence of the emitter blocking the light path and vibrations from the ultrasound present additional technical obstacles. However, the fact that therapeutic application requires only 20 minutes of stimulation per day suggests that analysis of cell behavior after a burst of stimulation may well be productive.
The authors have nothing to disclose.
This work was supported by Wellcome Trust grant 088419 to MDB and sponsorship by Smith & Nephew UK Ltd.
Name of the reagent | Company | Catalogue number | Comments (optional) |
sulpho-m-maleimidobenzoyl-N-hydrosuccinimide ester | FisherPerbio | PN22312 | 25 mM stocks dissolved in water can be stored at -20°C |
6-well tissue culture plate | Corning | 3516 | Plastic from other companies can coat poorly |
13-mm glass coverslip | Scientific Laboratory Supplies Ltd.SLS | MIC3336 | Glass from other companies can coat poorly |
PBS | Sigma | D8537 | |
PBS, Ca2+ Mg2+ | Sigma | D8662 | |
50K integrin ligand (fibronectin fragment) | Construct description and preparation in 9, 11 | Alternative matrix ligands could be used to interrogate other pathways | |
BSA | Sigma | A3059 | |
Cycloheximide | Sigma | C7698 | Can be stored as 10mg/ml stock in water |
DMEM/25 mM HEPES | Sigma | D6171 | |
Exogen 4000+ | Smith & Nephew Inc | ||
Ultrasound coupling gel | Smith & Nephew Inc | ||
SAFHS indicator | Smith & Nephew Inc | ||
hVIN-1 | Sigma | V9264 | |
DyLight 488-conjugated anti-mouse | Stratech Scientific | 715-485-150 | |
TRITC-labelled phalloidin | Sigma | P1951 | |
Prolong Gold antifade reagent | Invitrogen | P36930 | |
cOmplete protease inhibitor (EDTA free) | Roche | 056 489 001 | 100× stock made up from tablet stored at -20°C |
PAK-glutathione agarose beads | Construct description and preparation in 10 | ||
Glycerol | Fisher | G/0650/17 | |
Hepes | Apollo Scientific | BI8181 | Make 1M stock and pH to 7.4 |
NaCl | Fisher | S/0160/65 | |
NP40 | Sigma | I3021 | |
Sodium Deoxycholate | Sigma | D6750 | |
EGTA | Sigma | E4378 | |
EDTA | Sigma | E5134 |