Mechanical distortion of single actin filaments induced by external force: detection by fluorescence imaging.
Actin is a major component of the cytoskeleton that transmits mechanical stress in both muscle and nonmuscle cells. As the first step toward developing a "bio-nano strain gauge" that would be able to report the mechanical stress imposed on an actin filament, we quantitatively examined the fluorescence intensity of dyes attached to single actin filaments under various tensile forces (5-20 pN). Tensile force was applied via two optically trapped plastic beads covalently coated with chemically modified heavy meromyosin molecules that were attached to both end regions of an actin filament. As a result, we found that the fluorescence intensity of an actin filament, where 20% of monomers were labeled with tetramethylrhodamine (TMR)-5-maleimide at Cys(374) and the filamentous structure was stabilized with nonfluorescent phalloidin, decreased by approximately 6% per 10 pN of the applied force, whereas the fluorescence intensity of an actin filament labeled with either BODIPY TMR cadaverin-iodoacetamide at Cys(374) or rhodamine-phalloidin showed only an approximately 2% decrease per 10 pN of the applied force. On the other hand, spectroscopic measurements of actin solutions showed that the fluorescence intensity of TMR-actin increased 1.65-fold upon polymerization (G-F transformation), whereas that of BODIPY-actin increased only 1.06-fold. These results indicate that the external force distorts the filament structure, such that the microenvironment around Cys(374) approaches that in G-actin. We thus conclude that the fluorescent dye incorporated into an appropriate site of actin can report the mechanical distortion of the binding site, which is a necessary condition for the bio-nano strain gauge.
Shimozawa
Togo
T
Department of Physics, Faculty of Science and Engineering, Waseda University, Tokyo, Japan.
Ishiwata
Shin'ichi
S
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001696:Biomechanical Phenomena; D005456:Fluorescent Dyes / Q000378:metabolism; D008297:Male; D008853:Microscopy; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013314:Stress, Mechanical
D000199:Actins; D005456:Fluorescent Dyes