Nucleotide regulation of dynein controls the t-force in the flagellar beat cycle.

 

Charles B. Lindemann, Kathleen A. Lesich and Dominic W. Pelle

 

Department of Biological Sciences, Oakland University, Rochester, MI 48309 U.S.A.

 

Several studies on the dynein motor protein strongly suggest that the multiple nucleotide binding sites on the AAA domain of the heavy chain play a role in regulating the microtubule binding affinity of the dynein stalk (Silvanovich et al., 2003, Mol. Biol. Cell, 14:1355; Kon et al., 2004, Biochem. 43:11266; Numata et al., 2008, Biochem. Soc. Trans., 36:131). Specifically, the residence of ADP at the non-catalytic nucleotide binding sites has been postulated to increase the binding affinity of the dynein stalk (Inoue and Shingyoji, 2007, Cell Motil. Cytoskeleton, 64:690) and make the motor more processive (Cho et al., 2008, J. Biol. Chem., 283:25839). We have studied the effects of 1mM and 4mM ADP on the beat cycle of Triton X-100 extracted bull sperm models reactivated with 0.1 mM ATP. We have found a highly significant (p< 0.0001) increase in the maximal curvature developed in the P-bend when ADP is present as compared to ATP only controls. From our measured values for the stiffness of a passive bull sperm flagellum we have calculated the torque that must be present to create the P-bends observed at the switch-point of the beat. Once the active torque acting within the flagellum is known, the t-force acting on the doublets can be calculated. The t-force at the switch-point of the beat is dramatically higher in the presence of ADP, averaging 2.9 nN/micron for cells in 4mM ADP as compared to 0.9 nN/micron in control cells. The Geometric Clutch computer model predicts that an increase in the binding affinity of dynein to its binding sites on the B-subtubule will result in an increased t-force at the switching event of the P-bend. This is because in the Geometric Clutch view of how switching occurs, a greater t-force is required to detach the dyneins from the B-subtubule if the dyneins hang on more tightly. Our experimental findings on reactivated sperm can be simulated in the Geometric Clutch computer model of a bull sperm by increasing the adhesion of the dyneins to their adjacent doublet. These results suggest that in real flagella the microtubule binding affinity at the dynein stalk corresponds to the parameter called the “bridge adhesion” in the computer model. This modeling parameter is a measure of how difficult it is to pull the dyneins away from the adjacent doublet. Motor proteins that can hang on tightly while performing their role in transporting cargo are commonly referred to as “processive” motors. Our findings support the concept that ADP makes the dynein motors more processive during the flagellar beat, consistent with the molecular studies of the dynein heavy chain. It also suggests that when the dyneins are more processive, more t-force develops at the switching event in the beat cycle. This is readily understandable in the context of the Geometric Clutch hypothesis. More t-force will be required to increase the separation between the doublets and terminate the action of the motors when more of the motors are tightly bound. The behavior we observe in real flagella is consistent with a switching mechanism in the beat cycle based on a balance of dynein adhesion and t-force. The concept that bridge adhesion in the Geometric Clutch hypothesis is the same physical entity as the binding affinity of the dynein stalk for the B-subtubule is conceptually a significant touchstone. It makes it possible to incorporate the dynein microtubule binding affinity, which is an experimentally identified property of the dynein molecule, into a theoretical framework for flagellar and ciliary beating.