Defect of motile activity of actin filaments directly conjugated with fluorophores

Matsushita, S., Hatori, K.

Department of Bio-System Engineering, Yamagata University



Abstract
We observed the defect of sliding activity of actin filaments covalently conjugated with indocarbocyanine dye, IC3-OSu.
After actin filaments were treated with IC3-OSu at various molar ratio (1:3 to 1:6) for 60 min, actin filaments were transformed into monomeric actins by a dialysis against G-buffer. Consequentially actins were conjugated with IC3 at molar ratio from 1:2 to 1:6. IC3-conjugated actin monomers (IC3-actin) were polymerized into the filaments again by addition of KCl and phalloidin. When the actin filaments were observed under a fluorescent microscope, their length distribution was similar to that of intact actin filaments. However IC3-actin filaments at over 3 times molar ratio of IC3 to actin exhibited no sliding activity on myosin molecules in the presence of ATP, in spite of maintaining the ability to both polymerize and bind to myosin molecules. The motility was not recovered by changing the ionic strength or the hydrophobicity in the solution.
In order to examine the mechanical properties of the filaments the block copolymerized filaments of IC-3-actin and intact actin were prepared. They exhibited the sliding movement, but the velocity was decreased as the ratio of IC3-actin increased above 50%. The relation between the velocity and the ratio of IC3-actin was inconsistent with a Hill's hyperbolic curve.
The results show that the force generated on active components of a filament leads inactive components to exert the motile performance.