Magnetic Tweezers in a Microplate Format
Summary
Here, we describe the use of a novel microplate assay to enable mechanical manipulation of biomolecules while performing ensemble biochemical assays. This is achieved using a microplate lid modified with magnets to create multiple static magnetic tweezers across the microplate.
Abstract
Mechanobiology describes how the physical forces and mechanical properties of biological material contribute to physiology and disease. Typically, these approaches are limited single-molecule methods, which restricts their availability. To address this need, a microplate assay was developed that enables mechanical manipulation while performing standard biochemical assays. This is achieved using magnets incorporated into a microplate lid to create multiple magnetic tweezers. In this format, force is exerted across biomolecules connected to paramagnetic beads, equivalent to a typical magnetic tweezer. The study demonstrates the application of this tool with FRET-based assays to monitor protein conformations. However, this approach is widely applicable to different biological systems ranging from measuring enzymatic activity through to the activation of signaling pathways in live cells.
Introduction
Mechanobiology focuses on understanding how the propagation of physical forces within and between cells regulates cellular activity1,2 and how this correlates with the organization and dynamics of both proteins and cells.
Single-molecule force measurements have revealed how force is used in biological systems, from single proteins to whole cells and tissues3,4,5,6,7. These challenging experiments require specialized equipment and technical expertise. Conversely, standard biochemical assays can be performed at higher throughput in readily available commercial equipment.
Here, the study describes a mechanobiology assay that enables magnetic tweezer-based manipulation and biochemical assays to be performed together8. Magnets are placed on a 3D printed microplate lid (Figure 1A–D), enabling the use of commercial plate readers for the assays. Force is applied across the biomolecule of interest by coupling the molecule to paramagnetic particles. The magnets then exert tension across the molecule. Altering the distance between the particles and magnets adjusts the exerted force across the biomolecule (Figure 1E).
We represent the use of this assay using the actin-based molecular motor, Myosin VI. Myosin VI is regulated by intramolecular backfolding9. Myosin VI has been shown to exist in an auto-inhibited state, whereby the binding of partner proteins, such as NDP52, triggers the unfolding of myosin VI10,11. To perform these assays, we will use a dual-labeled construct of the myosin VI tail domain with an N-terminal GFP and a C-Terminal RFP whereby backfolding of the protein generates Fluorescence Resonance Energy Transfer (FRET) between GFP and RFP. The N-terminus also carries a biotinylation tag to immobilize the protein on the surface. We use this assay in combination with FRET measurements to show how force can impact myosin VI back-folding.
Protocol
Representative Results
Discussion
This approach enables force-based measurements to be readily applied in a microplate using fluorescent plate readers. Importantly, this assay format assumes there is functional protein when it is bound to a surface. Therefore, prior knowledge is required before embarking on these measurements to ensure there is protein activity. It is also beneficial to make sure that the binding of molecules to the paramagnetic beads and surface is optimized for each system.
This concept can be modified to f…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Cancer Research UK (A26206), the MRC (MR/M020606/1), and the Royal Society (RG150801) for funding.
Materials
| 24 well glass-bottom microplate | Cellvis | P24-1.5H-N | Multiple sources are available. Unless needed, it is best to avoid treated surfaces and we use Imaging grade glass N1.5. |
| Anti-RFP antibody | Abcam | ab290 | Multiple sources are available but must ensure there is minimal reactivity with GFP. |
| Bench top light microscope | Optika | IM-3 | |
| Bench top Rotator | Cole-Palmer-Stuart | SB3 | |
| Biotin-BSA | Sigma Aldrich | A8549 | |
| CAD Software – Sketch Up Educator | Sketch Up | Alternative CAD softwares can be used. Users should ensure the file formats are compatiable with their 3D printer. | |
| Dynabeads Protein A | Fisher Scientific | 10746713 | 2.8 µm paramagnetic beads with recombinant Protein A |
| Impact contact adhesive | EVO-STIK | ||
| MagnaRack magnetic separation rack | ThermoFisher Scientific | CS15000 | Magnetic Isolator |
| NaCl | Fisher Scientific | 10316943 | |
| Neodymium N42 5mm cube Magnets | Supermagnete | W-05-N | |
| Plate Reader – ClarioStar | BMG Labtech | All plate reader systems can be used where measurements are possible from under the microplate. The magnet lid excludes standard measurements from above | |
| Streptavidin | Sigma Aldrich | 189730 | |
| Tris-HCl | Fisher Scientific | 10142400 | |
| Ultimaker PETG Filament | Ultimaker | ||
| Ultimaker S3 – 3D printer | Ultimaker |
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