1School of Biochemistry, University of Bristol, 2Smith and Nephew
Roper, J., Harrison, A., Bass, M. D. Induction of Adhesion-dependent Signals Using Low-intensity Ultrasound. J. Vis. Exp. (63), e4024, doi:10.3791/4024 (2012).
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.
1. Coating Surfaces with Matrix Ligand
2. Preparation of Cells
3. Ultrasound Stimulation
4. Focal Adhesion Analysis by Immunofluorescence
5. Quantification of Rac1 Activation by Pull-down Assay
6. Representative Results
In this protocol we describe the induction of vinculin-stained focal adhesions and Rac1 activity by ultrasound. For the focal adhesion experiment, the baseline position is that fibroblasts spread on a ligand of α5β1-integrin do not form vinculin-containing adhesions (Fig. 3A) unless a second fibronectin receptor, syndecan-4, is engaged by addition of soluble ligand (Fig. 3B) 3,4. However, stimulation with ultrasound induces focal adhesion formation to the same extent as engagement of syndecan-4 (Fig. 3C), indicating that ultrasound stimulation can substitute for certain components of the fibronectin-dependent signaling pathway 5. The key hurdle with this protocol is eliminating focal adhesion formation in the absence of stimulant. Incomplete inhibition of protein synthesis by cycloheximide will allow matrix deposition that is sufficient for the cells to auto-stimulate. Overcrowding of cells can also lead to low-level focal adhesion formation and a poor response to ultrasound as crosstalk between cell-cell and cell-matrix contacts will complicate an experiment that is designed to isolate a single stimulus. As a development of the basic assay we show the same experiment, repeated with fibroblasts lacking syndecan-4 (Fig. 4). These fibroblasts still respond to ultrasound (Fig. 4C), but fail to respond to syndecan-4 ligand (Fig. 4B). The experiment using Sdc4 -/- fibroblasts demonstrates that ultrasound can indeed bypass the need for certain fibronectin receptors, and illustrates how the simple protocol can be developed to gain extra information about the signaling pathway.
For the biochemical experiment we demonstrate that ultrasound can induce activation of Rac1, using a pull-down assay that is an adaptation of the method described by Del Pozo et al. 10. Precipitation of active Rac1 0, 10, 30 and 60 minutes after initiation of ultrasound stimulation reveals that Rac1 activity peaks at 10-30 minutes, before returning to the baseline (Fig. 5). Blotting crude lysates for vinculin ensures equivalent loading between time points. The result resembles activation of Rac1 by engagement of syndecan-43, but is slightly more protracted than activation by fibronectin. Therefore 8-10 repeats are typically required to achieve significant data. Activation of Rac1 is responsible for commitment of cells to focal adhesion formation, and nascent focal adhesions start to form at 10-30 minutes, coinciding with Rac1 activation. Once initiated, those adhesions will continue to mature into the large adhesion plaques shown in Figures 3 and 4.
Figure 1. The ultrasound wave form. Ultrasound comprises an intermittent 1.5 MHz signal that due to low amplitude (30 mW/cm2, spatial average, temporal average) has no heating effect.
Figure 2. Schematic representation of the workflow for preparation and stimulation of cells with ultrasound.
Figure 3. Ultrasound induction of focal adhesion formation in fibroblasts. Fibroblasts were spread on the integrin-binding fragment of fibronectin (A) before stimulation with the syndecan-4-binding fragment of fibronectin (B) or ultrasound (C). Following 60-minute stimulation, cells were fixed, stained for vinculin and actin, and imaged by epifluorescence. Bar = 10 μm.
Figure 4. Ultrasound induction of focal adhesion formation in fibroblasts lacking syndecan-4. Sdc4 -/- fibroblasts were spread on the integrin-binding fragment of fibronectin (A) before stimulation with the syndecan-4-binding fragment of fibronectin (B) or ultrasound (C). Following 60-minute stimulation, cells were fixed, stained for vinculin and actin, and imaged by epifluorescence. Although Sdc4 -/- fibroblasts were fibronectin-insensitive, ultrasound still induced focal adhesion formation, indicating that ultrasound bypasses matrix receptor engagement. Bar = 10 μm.
Figure 5. Activation of Rac1 by ultrasound. Cells were stimulated for 0, 10, 30 and 60 minutes with 6 wells used per time point. The amount of GTP-Rac1 present in samples from a Rac1 pull-down assay time course was visualized by incubation with anti-Rac1 antibody on a western blot (top image). Quantification of the band intensity (average of 8-10 replicates) reveals increased Rac1 activity resulting from ultrasound signal at 10 and 30 minutes, before returning to baseline levels at 60 minutes (graph). Error bars represent s.e.m.
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.
Andrew Harrison is an employee of Smith & Nephew UK Limited.
This work was supported by Wellcome Trust grant 088419 to MDB and sponsorship by Smith & Nephew UK Ltd.
|sulpho-m-maleimidobenzoyl-N-hydrosuccinimide ester||Fisher Scientific||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||MIC3336||Glass from other companies can coat poorly|
|PBS, Ca2+ Mg2+||Sigma-Aldrich||D8662|
|50K integrin ligand (fibronectin fragment)||Construct description and preparation in 9, 11. Alternative matrix ligands could be used to interrogate other pathways|
|Cycloheximide||Sigma-Aldrich||C7698||Can be stored as 10mg/ml stock in water|
|DMEM/25 mM HEPES||Sigma-Aldrich||D6171|
|Exogen 4000+||Smith & Nephew Inc.|
|Ultrasound coupling gel||Smith & Nephew Inc.|
|SAFHS indicator||Smith & Nephew Inc.|
|DyLight 488-conjugated anti-mouse||Stratagene, Agilent Technologies||715-485-150|
|Prolong Gold antifade reagent||Invitrogen||P36930|
|cOmplete protease inhibitor (EDTA free)||Roche Group||056 489 001||100× stock made up from tablet stored at -20°C|
|PAK-glutathione agarose beads||Construct description and preparation in 10|
|Hepes||Apollo Scientific||BI8181||Make 1M stock and pH to 7.4|