Live cell imaging of mechanotransduction

Abstract

Mechanical forces play important roles in the regulation of cellular functions, including polarization, migration and stem cell differentiation. Tremendous advancement in our understanding of mechanotransduction has been achieved with the recent development of imaging technologies and molecular biosensors. In particular, genetically encoded biosensors based on fluorescence resonance energy transfer (FRET) technology have been widely developed and applied in the field of mechanobiology. In this article, we will provide an overview of the recent progress of FRET application in mechanobiology, specifically mechanotransduction. We first introduce fluorescent proteins and FRET technology. We then discuss the mechanotransduction processes in different cells including stem cells, with a special emphasis on the important signalling molecules involved in mechanotransduction. Finally, we discuss methods that can allow the integration of simultaneous FRET imaging and mechanical stimulation to trigger signalling transduction. In summary, FRET technology has provided a powerful tool for the study of mechanotransduction to advance our systematic understanding of the molecular mechanisms by which cells respond to mechanical stimulation.

Figure 1.

Functional mechanism of FRET biosensors. (a) Intramolecular FRET: two target molecules/domains covalently fused together between a pair of donor and acceptor FPs can interact with each other upon stimulation to cause FRET changes. (b) Intermolecular FRET: two target molecules/domains are separately fused to donor and acceptor FPs. The two target molecules/domains can interact with each other upon stimulation and bring the donor and acceptor FPs together to cause FRET changes.

Figure 2.

Directional and long-range propagation of Src activation induced by mechanical force. (a) Laser-tweezer traction on the bead at the upper right corner of the cell (shown on the left) caused FRET responses.The colour images on the right represent the FRET ratio of Src biosensor before and after the mechanical stimulation. (b) FRET responses of a cell with clear directional wave propagation away from the site of mechanical stimulation. Adapted from Wang et al. (2005).

Figure 3.

Magnetic tweezers and FRET imaging. Magnetic tweezers consist of a pair of permanent magnets placed above the sample holder of an inverted microscope. The FRET biosensor linked with a magnetized particle was coated onto a piece of glass coverslip. The magnets produce a strong field gradient that is used to exert a force on the magnetic beads. When subjected to the magnetic field, the internal magnetic moment of beads will tend to align with the external field, resulting in a rotational or twisting force on the FRET biosensor. The force can be varied by modulating the position of the magnets relative to the bead.

Figure 4.

The flow system and FRET imaging. The system comprises a flow chamber to seed the cell, two reservoirs to form the hydrostatic pressure difference, a pump to circulate the flow medium and the tube system to connect all the compositions. A glass coverslip seeded with cells expressing FRET biosensors will form the floor of a flow channel, created by sandwiching a silicone gasket between the cover glass slide and an acrylic plate. Cells will be exposed to various shear stresses created by flows caused by a hydrostatic pressure difference between two reservoirs in the circulation system. The force of shear stress can be calculated as: τ_w = 6μQ/h²w, where μ is the fluid viscosity of the solution, Q is the flow rate, h is the channel height and w is the channel width. Hence, by adjusting the flow rate or the channel height, different levels of wall shear stress can be generated to impose on the cell surface with a high precision. The FRET signals of biosensors can then be monitored by the objective to detect the cell response upon flow stimulation.