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 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.

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, the....

1. Ligand Conjugation to Magnetic Beads

Note: Ligand conjugation is performed using superparamagnetic tosyl-activated beads with a 2.8 μm 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.......

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 µm 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.......

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 μm diameter magnetic beads but larger beads can be used such as 4.5 μm diameter. However, bead diam.......

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˚8304162) and from European Research Council (ERC) under European Union's Horizon 2020 research and innovation program (ERC Starting Grant n˚639300).