Visualizing myosin-actin interaction with a genetically-encoded fluorescent strain sensor.
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.
Iwai
Sosuke
S
Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan. sosuke@aist.go.jp
Uyeda
Taro Q P
TQ
D000199:Actins / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D004023:Dictyostelium / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009154:Mutation; D024462:Myosin Type II / Q000032:analysis / Q000378:metabolism*; D009218:Myosins; D013050:Spectrometry, Fluorescence
D000199:Actins; D049452:Green Fluorescent Proteins; D024462:Myosin Type II; D009218:Myosins