Name: analyzing cell surface adhesion remodeling in response to mechanical tension using magnetic beads
Uploaded: 2017-03-08T15:00:00
Description: Watch this Scientific Journal Video about Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads at JoVE.com

C.G. is supported by grants from the Agence National de la Recherche (ANR-13-JSV1-0008), from the European Union Seventh Framework Programme (Marie Curie Career Integration n&#730;8304162) and from European Research Council (ERC) under European Union&#39;s Horizon 2020 research and innovation program (ERC Starting Grant n&#730;639300).

1. Ligand Conjugation to Magnetic Beads
Note: Ligand conjugation is performed using superparamagnetic tosyl-activated beads with a 2.8 &#956;m diameter (stock solution concentration 108 beads/mL, 30 mg beads/mL). The following protocol is based on samples of approximately 2 x 105 cells, which correspond to MRC-5 cells grown to 80% confluency in a 60 mm tissue culture plate. Adjust the volume of beads and reagents accordingly if using plates of different sizes or cells at di...

<ol>
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The method described here constitutes a straightforward approach to apply tension to cell surface adhesion receptors and allow their subsequent purification. However, some steps are critical to perform efficient adhesion purification and potential optimization can be done depending on the targeted adhesion receptors. We present potential issues the user may encounter below.
We used 2.8 &#956;m diameter magnetic beads but larger beads can be used such as 4.5 &#956;m diameter. However, bead diam...

In metazoa, mechanical tension directs tissue development and homeostasis through the regulation of a myriad of cellular processes such as proliferation, differentiation and survival 1,2. Mechanical tension can arise from the extracellular matrix or can be generated by adherent cells, which sample their extracellular environment through the actomyosin contractile machinery that pulls onto extracellular matrix and probes its rigidity through tension-sensitive molecules. In response to tension, mechanosensitive adhesion proteins undergo conformational changes that trigger complex signaling cascades. In turn, these signaling pathways orchestrate a mechanoresponse encompassing proliferation, differentiation and survival that adjusts the cellular behavior to the extracellular environment. Such processes can be settled in a short-term time period (seconds to minutes) to quickly feed back onto the loop of mechanotransduction by modifying the mechanosensitive structures. For instance, integrin-based adhesions reinforce in response to tension through Rho GTPase-mediated cytoskeletal remodeling 3,4,5. In parallel, other signaling pathways are activated over hours and days to control genetic programs that eventually impact cell fate 6. Whereas, many studies have highlighted the effect of matrix stiffness on cell determinism and disease development 1,2 , the precise molecular mechanisms of adhesion-mediated mechanotransduction still remain elusive.
Various approaches have been developed to study the effects of cell-generated forces or external forces on cell behavior, including flow systems, fluorescence resonance energy transfer (FRET)-tension sensors 7,8, compliant substrates 9, magnetic tweezers, optical tweezers 10 and atomic force microscopy (AFM) 11. Here we present a protocol using superparamagnetic beads to characterize mechanotransduction pathways in response to tensional forces applied to specific adhesion receptors. Superparamagnetic beads are particles that reversibly magnetize when placed in a magnetic field. Once coated with a ligand for a specific receptor, these beads provide a powerful tool to study the effects of extracellular force application. This method has been validated by several studies 3,5,12-17 and present the advantage to largely facilitate biochemical analysis on adherent cells. Using similar collagen-coated magnetic beads followed by biochemical analysis, early work reported an increase in protein tyrosine phosphorylation and RhoA activation in response to tension 5,18,19. The method described below has also been used with fibronectin (FN)-coated beads to characterize the signaling pathways downstream from tension applied to integrins 3. In this study, Guilluy et al. showed that tension activates RhoA through the recruitment of the two guanine nucleotide exchange factors (GEFs), LARG and GEF-H1, to integrin adhesion complexes. Since that, other studies have shown that GEF-H1 is recruited to adhesion complexes in response to cell-generated tension using different methods 20,21, demonstrating the robustness of the methodology described here. As a result, activated RhoA was shown to promote adhesion reinforcement, through cytoskeletal remodeling. This system was also used to explore tension applied to cell/cell adhesion receptors. Application of forces onto magnetic beads coated with the extracellular domain of E-cadherin induced an increase in vinculin recruitment similarly to integrin associated adhesion complexes 12. Collins and colleagues observed that application of tension to PECAM-1 promotes integrin and RhoA activation 13. Another experimental approach using magnetic beads is the study of tension applied to isolated nuclei. Using beads coated with antibodies against the nuclear envelope protein nesprin-1, nuclear envelope complexes were purified to show that they are dynamically regulated in response to mechanical tension 22. These results support the powerfulness of this method in the study of mechanotransduction pathways. Moreover, while flow or traction force systems stimulate general cellular processes, magnetic beads specifically target a cell adhesion receptor by using either receptor ligands 3 or monoclonal antibodies against cell surface receptor 13,15.
Another advantage of this method is the isolation of adhesion complexes through a straightforward ligand affinity purification procedure. It is well known that addition of ligand-coated beads to cells binds adhesion receptors and induces the recruitment of several adhesion proteins23. Further application of forces to ligand-coated magnetic beads turns these adhesion complexes into macromolecular platforms that mediates various tension-dependent signaling pathways4,24. Cell lysis followed by bead concentration using a magnet permits the isolation of the adhesion platforms. Other methods used to purify adhesion complexes have already been used in adherent cells. They combine chemical crosslinking to conserve protein-protein interactions and a cell lysis step by detergent and shear flow or sonication 20,21,25,26,27,28. The final step is the collection of the resulting ventral plasma membranes containing the adhesion complexes. Unlike these methods, magnetic beads allow a greater purification level of cell adhesion complexes by selectively targeting a specific family of adhesion receptors. Magnetic beads have already been used to purify adhesion complexes in non-adherent cells attached to ligand-coated microbeads29,30. The method described below mimics biological situations where force is applied for a short sustained period (seconds to minutes). Therefore, it provides a powerful tool for investigating both the molecular composition of purified adhesion complexes and the downstream mechanosensitive-signaling pathways.
Here we present a detailed experimental protocol for using magnetic beads to apply tensional forces to adhesion surface proteins. A permanent neodymium magnet is placed on top of the culture dish surface. The pole face of the magnet is placed at a height of 6 mm so that the force on a single 2.8 &#956;m magnetic bead is constant (about 30-40 pN)31. The duration of tension stimulation is determined by the operator depending on the molecule of interest and its time-scale of activation. Cells are finally lysed, adhesion complexes are purified by beads separation using a magnet and biochemical analyses are processed. This protocol includes the preparation of ligand-coated superparamagnetic beads, and the application of tension through magnet followed by biochemical analyses. Additionally, we provide a representative sample of data demonstrating that tension applied to integrin-based adhesions induces adhesion remodeling and alters protein tyrosine phosphorylation.

<table border="0" cellpadding="0" cellspacing="0" width="822">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Neodymium magnets (on the upper face of 60 mm dish)</td>
			<td style="width:115px;">K&#38;J Magnetics, Inc</td>
			<td style="width:103px;">DX88-N52</td>
			<td style="width:375px;">grade N52 dimension: 1 1/2&#34; dia. x 1/2&#34; thick</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Neodymium magnets (on the lower face of 60 mm dish)</td>
			<td style="width:115px;">K&#38;J Magnetics, Inc</td>
			<td style="width:103px;">D84PC-BLK</td>
			<td>grade N42 dimension: 1/2&#34; dia. x 1/4&#34; thick Black Plastic Coated&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Dynabeads M280 Tosylactivated</td>
			<td style="width:115px;">Thermofisher</td>
			<td style="width:103px;">14203</td>
			<td>superparamagnetic beads&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">DynaMag-2 Magnet</td>
			<td style="width:115px;">Thermofisher</td>
			<td style="width:103px;">12321D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Fibronectin&#160;</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:103px;">F1141-5MG</td>
			<td>Fibronectin from bovine plasma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Poly-D-Lysine</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:103px;">P7280-5MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Apo-Transferrin</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:103px;">T1428-50MG</td>
			<td>Bovine Apo-Transferrin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:103px;">A7906-500G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">DMEM high glucose, GlutaMAX supplement, pyruvate&#160;</td>
			<td style="width:115px;">Life Technologies</td>
			<td style="width:103px;">31966-021</td>
			<td>DMEM+GlutaMAX-I 500 ml&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">60*15 mm culture dish</td>
			<td style="width:115px;">Falcon</td>
			<td style="width:103px;">353004</td>
			<td></td>
		</tr>
	</tbody>
</table>

analyzing cell surface adhesion remodeling in response to mechanical tension using magnetic beads

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both force-sensing molecules at adhesion sites, and force-dependent transcription factors that regulate lineage-specific gene expression and drive phenotypic outputs. However, the signaling networks converting mechanical tension into biochemical pathways have remained elusive. To explore the signaling pathways engaged upon mechanical tension applied to cell surface receptor, superparamagnetic microbeads can be used. Here we present a protocol for using magnetic beads to apply forces to cell surface adhesion proteins. Using this approach, it is possible to investigate not only force-dependent cytoplasmic signaling pathways by various biochemical approaches, but also adhesion remodeling by magnetic isolation of adhesion complexes attached to the ligand-coated beads. This protocol includes the preparation of ligand-coated superparamagnetic beads, and the application of define tensile forces followed by biochemical analyses. Additionally, we provide a representative sample of data demonstrating that tension applied to integrin-based adhesion triggers adhesion remodeling and alters protein tyrosine phosphorylation.

The schematic of the technique is illustrated in Figure 1a. Following ligand conjugation, magnetic beads are incubated with cells for 20 min, and then a permanent magnet is used to apply tensile forces of about 30-40 pN for various amount of time. Figure 1b shows 2.8 &#181;m FN-coated magnetic beads bound to MRC5 cell adhesion receptors.
The wash steps of superparamagnetic beads after cell lysis are crucial and determine the degree of purification. A minimum o...

Watch this Scientific Journal Video about Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads at JoVE.com

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Cell surface adhesions are central in mechanotransduction, as they transmit mechanical tension and initiate the signaling pathways involved in tissue homeostasis and development. Here, we present a protocol for dissecting the biochemical pathways that are activated in response to tension, using ligand-coated magnetic microbeads and force application to adhesion receptors.

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both ...

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