FRET and mechanobiology

Abstract

Since the development of green fluorescent protein (GFP) and other fluorescent proteins (FPs) with distinct colors, genetically-encoded probes and biosensors have been widely applied to visualize the molecular localization and activities in live cells. In particular, biosensors based on fluorescence resonance energy transfer (FRET) have significantly advanced our understanding of the dynamic molecular hierarchy at subcellular levels. These biosensors have also been extensively applied in recent years to study how cells perceive the mechanical environment and transmit it into intracellular molecular signals (i.e. mechanotransduction). In this review, we will first provide a brief introduction of the recent development of FPs. Different FRET biosensors based on FPs will then be described. The last part of the review will be dedicated to the introduction of examples applying FRET biosensors to visualize mechanotransduction in live cells. In summary, the integration of FRET technology and the different cutting-edge mechanical stimulation systems can provide powerful tools to allow the elucidation of the mechanisms regulating mechanobiology at cellular and molecular levels in normal and pathophysiological conditions.

Fig. 1

Upper panel: a schematic drawing of a recombinant gene containing cDNA of a target molecule fused to that of GFP. Lower panel: the protein production of the recombinant gene in the upper panel allows the observation of the recombinant protein with excitation and emission wavelengths of GFP at 488 and 510 nm, respectively.

Fig. 2

The top panel shows the purified FPs under white light and the bottom panel demonstrates the fluorescence of these FPs. Image courtesy of R. Y. Tsien.

Fig. 3

(A) The excitation and emission spectra of a typical FRET pair: CFP as the donor and YFP as the acceptor. The broken lines represent the excitation spectra and the solid lines represent the emission spectra of CFP and YFP. The spectra curves of CFP and YFP are color-coded with cyan and yellow, respectively. The shaded red area represents the overlap between the CFP emission and the YFP excitation. (B) The cartoon shows that the FRET efficiency between a typical FRET pair, ECFP as the donor and EYFP as the acceptor, is mainly dependent on the distance and the relative orientation between the donor and acceptor.

Fig. 4

(A) A cartoon scheme depicting the activation mechanism of the Src biosensor. When Src kinase is inactive, ECFP and Citrine are positioned proximal to each other and have strong FRET. The excitation of the biosensor at 433 nm results in the emission from Citrine at 527 nm. When Src kinase is activated to phosphorylate the substrate peptide in the biosensor, the biosensor will undergo a conformational change and separate Citrine from ECFP, thus resulting in the decrease of FRET. The excitation of the biosensor at 433 nm then leads to the emission from ECFP at 476 nm. Hence, the FRET efficiency or ECFP/Citrine emission ratio of the biosensor represents the activation status of Src kinase. (B) HeLa cells transfected with the monomerized Src biosensor were stimulated with EGF (50 ng ml⁻¹), washed with serum-free medium (washout). “−EGF” and “+EGF” represent the images before and after EGF stimulation. Color images represent the ECFP/Citrine emission ratio images of the monomeric Src biosensor in HeLa cells in response to EGF or washout. The color scale bar on the left corresponds to the level of ECFP/Citrine emission ratio, with cold colors indicating low Src activity and hot colors indicating high Src activity. These images are adapted from Wang et al., 2005.

Fig. 5

(A) A cartoon scheme depicting the procedure of the laser-tweezer-traction experiment. A laser light is guided by an objective lens to focus o.-centered to exert force on a fibronectin-coated bead, which is adhered on a HUVEC and coupled to the cytoskeleton through integrin-ligation. (B) FRET response of a cell with clear directional wave propagation away from the site of mechanical stimulation introduced by laser-tweezers. The color bar on the left indicates ECFP/Citrine emission ratios, with cold color representing low ratios and hot color representing high ratios. The pink arrow represents the site of force application and the force direction. The white arrows point to the front edge of activated Src wave. This figure is adapted from Wang et al., 2005.

Fig. 6

(A) Stress-induced Src activation is rapid and EGF-induced Src activation is slow. Here is the time course of normalized CFP/YFP emission ratio, an index of Src activation in response to mechanical or soluble growth factor EGF stimulation. A local stress was applied to integrins using a 4-μm RGD-coated magnetic bead (17.5 Pa for 3 s step function). Src is activated at ~100 ms after stress but is not activated until ~12–20 s after EGF. n = 12 cells for +σ (stress), n = 8 cells for +EGF (40 ng ml⁻¹). Error bars represent SEM. (B) Src activation is stress-magnitude dependent. Note that the stress threshold for Src activation appears to be ~1.8 Pa and the Src activation response is nonlinear. n = 12 cells for stress of 17.5 Pa; 4 for 8.8 Pa; 4 for 3.5 Pa; 4 for 1.8 Pa; 3 for 0.7 Pa. Error bars represent SEM. This figure is reproduced from Na et al. with permission.

Fig. 7

Rapid (<300 ms) and strong Src activation (red/yellow spots) co-localizes with regions of large microtubule deformation (white arrows). A local stress was applied to integrins using a 4-μm magnetic bead (17.5 Pa for 3 s step) and Src activation was measured by CFP-YFP Src FRET. Then an oscillatory stress (17.5 Pa at 0.3 Hz) was applied for~10 s and microtubule deformation was quantified by tracking mCherry tubulin displacements induced by stress. For clarity, microtubule displacements <15 nm were omitted. Blue circle, bead–cell contact area (~6 μm²); blue arrow, bead movement direction; N = nucleus. The focal plane was ~1.5 μm above the cell base. Three other cells showed similar behaviors. 80% of strong Src activation co-localizes with regions of large microtubule deformation (>15 nm). In contrast, only ~10% of strong Src activation co-localizes with regions of large F-actin deformation. Reproduced from ref. with permission.