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

Summary

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.

Abstract

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.

Introduction

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 pathways^4,24. 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 microbeads^29,30. 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 μm 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.

Protocol

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 different confluences. Use an amount of superparamagnetic beads in order to have 2 beads per cell. Therefore, 4 x 10⁵ beads are needed for a 60 mm plate.

1. Thoroughly resuspend the beads in the original vial by vortexing at least 30 s. Aliquot 40 µL of the resuspended superparamagnetic beads (corresponding to 4 x 10⁶ beads) to 1.5 mL microcentrifuge tube.
2. Place the tube on a magnetic separation stand in order to separate the magnetic beads from the solution. Discard the supernatant, remove tube from the magnetic separation stand and resuspend the beads in 1 mL 0.1 M Na-phosphate pH 7.4.
   1. Repeat wash step twice with 1 mL 0.1 M Na-phosphate pH 7.4.
3. After the final wash, resuspend the beads in 1 mL 0.1 M Na-Phosphate pH 7.4.
4. Combine 100 μg Bovine Fibronectin (FN) or any other cell adhesion receptor ligand to the 1 mL 0.1 M Na-phosphate pH 7.4 containing beads and mix by pipetting.
5. Repeat previous steps by replacing FN or any other specific ligand with BSA, poly-D-lysine or apo-transferrin for negative controls.
6. Optionally, for gel analysis, take a 10 μL aliquot of ligand solution for analysis of crosslinking efficiency and mix with an appropriate volume of concentrated Laemmli Sample Buffer.
7. Incubate beads with the ligand containing solution for 12-24 h at 37 °C on a rotor.
8. If bead aggregates appear after the reaction has occurred, sonicate (continuous 39 W power) for no more than 10-20 s.
9. Isolate the beads using magnetic separation stand, aspirate the remaining solution and add 1 mL of PBS/0.2% BSA pH 7.6 solution. Incubate for 1 h on rotor at 37 °C.
10. Wash beads using a magnet twice with 1 mL PBS/0.2% BSA pH 7.6. Resuspend beads in 1 mL PBS/0.2% BSA pH 7.6.
11. Sonicate beads if aggregates appear for no more than 20-30 s.
12. Optionally, remove a 10 μL aliquot to analyze coupling efficiency by western blot (mix with an appropriate volume of concentrated Laemmli Sample Buffer).
13. Proceed to cell assays or store beads at 4 °C for up to 1 month.
14. Optionally, run a SDS-PAGE gel and stain with Coomassie blue to analyze FN crosslinking to the magnetic beads.

2. Application of Tensional Forces on the Ligand-coated Beads Bound to Adhesion Receptors on the Dorsal Surface of Cells

1. Culture adherent cells on 60 mm tissue culture dish in appropriate growth medium (usually DMEM 4.5 g/L D-glucose supplemented with FCS 10%) until reaching 80% confluency.
2. Prepare non-denaturing non-ionic lysis buffer (20 mM Tris HCl pH 7.6, 150 mM NaCl, 2 mM MgCl₂, 0.1% NP-40) and chill microcentrifuge tubes.
3. Pellet the ligand-coated beads from section 1.10 and resuspend in 1 mL PBS/0.2% BSA pH 7.6. Add 100 μL of the ligand-coated beads solution to 5 mL of warm culture medium and vortex.
4. Aspirate medium in 60 mm culture dish and add 5 mL warm growth medium supplemented with beads. 
5. Incubate for 20 min under cell culture conditions (37 °C and 5% CO₂) to allow the beads to sediment and adhere to the cells. It is crucial to not exceed 20 min since beads may be internalized by phagocytosis.
6. Optionally, observe the beads under a light microscope using a 10X or 20X objective and check for bead adhesion by slightly shaking the dish to discriminate between attached and floating beads.
7. While keeping the culture dish in the incubator, swap the normal dish lid for a lid with a round 38 mm neodymium magnet attached on the upper face. The magnet is held in place by two smaller 13 mm neodymium magnets positioned on the lower face of the lid.  Since the magnets are very powerful, manipulate them carefully.
8. Incubate cells subjected to tension for the desired time points at cell culture conditions (37 °C and 5% CO₂). 
9. After treatment, place dish on ice and carefully aspirate the entire medium from the dish.
10. Add 300 μL of cell lysis buffer, and incubate for 10 min on ice. Collect the lysate using a cell scraper and transfer to a pre-chilled 1.5 mL microcentrifuge tube.
11. Pellet the magnetic beads using the magnetic separation stand and transfer the total cell lysate to a new pre-chilled 1.5 mL microcentrifuge tube. Store this fraction at -20 °C for further analysis.
12. Wash the beads 3 times with 1 mL ice-cold lysis buffer. Add 50 μL Laemmli Sample buffer to the bead pellet, mix by pipetting and boil at 95 °C for 5 min using a dry block heater. This fraction contains the isolated adhesion complexes. Proceed to biochemical analysis or store at -20 °C.
13. From the total cell lysate obtained after beads separation (about 300 μL), remove a 50 μL aliquot for western blot analysis.
    NOTE: The 250 μL left can be used for further biochemical studies such as protein-protein interaction studies (immunoprecipitation, GST pull-down assays) or GTPase activity experiments (changing the lysis buffer may be necessary depending on the GTPase of interest).

Results

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...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
Neodymium magnets (on the upper face of 60 mm dish)	K&J Magnetics, Inc	DX88-N52	grade N52 dimension: 1 1/2" dia. x 1/2" thick
Neodymium magnets (on the lower face of 60 mm dish)	K&J Magnetics, Inc	D84PC-BLK	grade N42 dimension: 1/2" dia. x 1/4" thick Black Plastic Coated
Dynabeads M280 Tosylactivated	Thermofisher	14203	superparamagnetic beads
DynaMag-2 Magnet	Thermofisher	12321D
Fibronectin	Sigma-Aldrich	F1141-5MG	Fibronectin from bovine plasma
Poly-D-Lysine	Sigma-Aldrich	P7280-5MG
Apo-Transferrin	Sigma-Aldrich	T1428-50MG	Bovine Apo-Transferrin
Bovine serum albumin	Sigma-Aldrich	A7906-500G
DMEM high glucose, GlutaMAX supplement, pyruvate	Life Technologies	31966-021	DMEM+GlutaMAX-I 500 ml
60*15 mm culture dish	Falcon	353004