Visualizing myosin-actin interaction with a genetically-encoded fluorescent strain sensor

Abstract

Many proteins have been shown to undergo conformational changes in response to externally applied force in vitro, but whether the force-induced protein conformational changes occur in vivo remains unclear. To reveal the force-induced conformational changes, or strains, within proteins in living cells, we have developed a genetically encoded fluorescent "strain sensor," by combining the proximity imaging (PRIM) technique, which uses spectral changes of 2 GFP molecules that are in direct contact, and myosin-actin as a model system. The developed PRIM-based strain sensor module (PriSSM) consists of the tandem fusion of a normal and circularly permuted GFP. To apply strain to PriSSM, it was inserted between 2 motor domains of Dictyostelium myosin II. In the absence of strain, the 2 GFP moieties in PriSSM are in contact, whereas when the motor domains are bound to F-actin, PriSSM has a strained conformation, leading to the loss of contact and a concomitant spectral change. Using the sensor system, we found that the position of the lever arm in the rigor state was affected by mutations within the motor domain. Moreover, the sensor was used to visualize the interaction between myosin II and F-actin in Dictyostelium cells. In normal cells, myosin was largely detached from F-actin, whereas ATP depletion or hyperosmotic stress increased the fraction of myosin bound to F-actin. The PRIM-based strain sensor may provide a general approach for studying force-induced protein conformational changes in cells.

Fig. 1.

Optimization of GFP concatemer for PRIM. (A) Schematic drawing of GFP-29-cp174GFP on the basis of the structure of dimeric GFP (19). N and C in parentheses indicate original N and C terminus in the GFP structure. The figure was prepared with PyMOL (www.pymol.org). (B) Fluorescent excitation spectra of GFP-29-cp174GFP and an equal mixture of GFP and cp174GFP. The concentration of GFP-29-cp174GFP was 0.1 μM, and those of GFP and cp174GFP in the mixture were 0.1 μM. (C) Excitation ratios (490/390 nm) of GFP concatemers or monomeric derivatives. Numbers between GFP and GFP or cp174GFP indicate number of amino acid residues between the 2 GFP moieties. GFP or cp174GFP alone are monomeric GFP derivatives.

Fig. 2.

Characterization of a PRIM-based strain sensor using myosin-actin as a model system. (A) Domain structure of PriSSM-motor. The mutant proteins, PriSSM-GGG, PriSSM-ΔCMN, and PriSSM-ΔCMC are schematically represented. (B) Principle of the PRIM-based strain sensor using myosin-actin as a model system. (C) Fluorescent excitation spectra of PriSSM-motor. No, in the absence of F-actin and ATP; Actin, in the presence of 1 μM F-actin; Actin+ATP, in the presence of both F-actin and 1 mM ATP. (D) Excitation ratios (490/390 nm) of PriSSM-motor and effects of the mutations within the myosin motor domain. PriSSM-GGG, PriSSM-ΔCMN, and PriSSM-ΔCMC contain the mutations as shown in A. The mean and SD are shown for at least 3 measurements using 2 independent protein preparations.

Fig. 3.

Expression of myosin II-PriSSM fusion proteins in Dictyostelium cells. (A) Domain structures of Dictyostelium myosin II and its PriSSM-fusion proteins. (B) Development of fruiting bodies. Myosin-null cells (Left), myosin-null cells expressing wild-type myosin II (Center), and PriSSM-myosin (Right) were seeded onto bacterial lawns and allowed to develop for 7 days at 22° C.

Fig. 4.

Visualization of interaction between myosin II and F-actin in Dictyostelium cells. (A) Fluorescence excitation spectra of cells expressing PriSSM-myosin in the absence (Upper Left) or presence of 10 mM azide (Upper Right). The measured spectra were separated into the contributions from autofluorescence, the sensor in the presence of F-actin (PriSSM-motor actin), and the sensor in the presence of ATP (PriSSM-motor ATP), to be fitted with the sum of each component. (Lower Right) Excitation ratios (490/390 nm) of the sensor contributions in the absence (Buffer) or presence of 10 mM azide (Azide) or 350 mM sorbitol (Sorbitol). (B) Triton ghosts extracted from cells expressing PriSSM-myosin or PriSSM-myosinΔN before (No nucleotides) and after (+ATP) the addition of ATP. R_480/380 was color-encoded in intensity-modulated display mode according to the look-up table. (C) Living cells expressing PriSSM-myosin or PriSSM-myosinΔN in the absence (Buffer) or presence (DNP) of 200 μM DNP. R_480/380 was color-encoded as described above. (Scale bars: B and C, 10 μm.)