Unbinding Force of Cytoplasmic Dynein

Abstract

Dynein is a molecular motor that moves toward the minus-end of microtubules using the chemical energy of ATP hydrolysis. Cytoplasmic dynein play roles in positioning the Golgi complex and other organelles in cells, movement of chromosomes, and positioning the mitotic spindles during mitosis. Force generation by a dynein molecule, thus, is one of the important factors for understanding molecular properties of dynein. To examine the binding mode between dynein and a microtubule, we measured the unbinding force of dynein in various nucleotide conditions. We used truncated C-terminal motor domain, thus we can eliminate possible effects of tail region and/or accessory proteins on the motor activity of dynein. Dynein with the biotin-tag was attached to avidin-coated polystyrene bead. The bead was trapped by optical tweezers, and the external load was imposed by moving the stage. When the stage was moved at 160nm/sec (loading rate is 5.9 pN/sec), the mean value of the unbinding force of strongly bound state of dynein was 5.7 - 6.6pN upon backward loading. The unbinding force was 20-35% smaller when force was applied to the minus end of microtubules. These data indicate that dynein unbinds from microtubules easily toward the minus end of microtubules to which dynein moves.