Photosensitive inhibition of motility of actin filaments covalently conjugated with Cy3 dyes

Hatori, K., Kimijima, H.

Department of Bio-System Engineering, Yamagata University



Abstract
In order to examine the functional modulation of actin molecules, both chemical modification and labeling of actin molecules were simultaneously carried out by the conjugation with amino-reactive dyes. Cy3-NHS was employed as the chemicals in this study. We found that the motility of actin filaments on myosin molecules was inhibited by the conjugation of Cy3-NHS to them over a 3-fold molar ratio. In the case of conjugation at 2-fold excess, Cy3-conjyugated actin filament (Cy3-F-actin) moved, but after exposure to excitation light stalled in a short duration. The duration time of movement was decreased with an increase of filament length. The stalling (or the decrease of velocity) occurred suddenly, indicating a cooperative manner of motility. We also measured the dissociation rate of Cy3-F-actin from a single HMM molecule in the absence of ATP. Dissociation rate constants for Cy3-F-actin and for intact one were 0.06 s-1 and 0.03 s-1, respectively.
Inhibition of motility of Cy3-F-actin was extensively suppressed by adding dithiothreitol (DTT) in the solution during the observation, which is likely to act as an antioxidant. In the absence of DTT, the duration time was below 1 sec. On the other hand, in the presence of 100 mM DTT, the duration time was increased above 10 sec. This result indicates that local oxidation of actin molecules may affect the motile function of actomyosin complex. We are attempting to identify the Cy3-conjugated sites in actin molecules by a mass spectrometry.