Separating Ciliary Beating Force into Dynein-Driven Force and Axoneme-Intrinsic Bending Force By 3-D Tracking Microscopy

Abstract

Highly coordinated dyneins generate asymmetric ciliary beating composed of clearly distinguished effective and recovery strokes. We herein aim to understand the mechanism of asymmetric beating of a single cilium by combining multiple optical-microscope techniques: 3-D tracking; optical tweezers; DIC adjusted to the different focal planes. A fluorescent bead was attached to the tip of the surface-immobilized mouse-tracheal cilium that was reactivated with a 100 µM ATP-containing solution, and trapped at various points of the beating stroke to measure the 3-D force (F). Next, after the solution was replaced with 100 µM ATP and 50 µM Vanadate, we measured 2-D structural bending energy in a relaxed state at the same cilium. With the estimation of the 3-D force driven by dyneins (FD) by subtraction of the effect of bending energy from F, the following were obtained: 1) FD periodically changed in the range from −75 to 35 pN, which is about 10 times larger than the force of viscous drag. 2) As the trapping point was displaced from the position of post-effective stroke, maximum values of FD (FDM) of the effective stroke increased from 3 to 35 pN and FDM of the recovery stroke increased from −1 to −75 pN, suggesting that the change in the force depends on the bending deformation of the cilium. 3) Under the trapping condition, in spite of the small curvature change, the direction of FD was spontaneously switched from the recovery direction to the effective direction, and vice versa. This result supports that FD in the effective and the recovery stroke direction, were generated by the different dynein-bridge sets on each side of the axoneme.