Deciphering the Regulation of Cytoplasmic Dynein's Attachment to Microtubules

Abstract

Cytoplasmic dynein is a homodimeric microtubule (MT) motor responsible for most MT minus-end-directed transport in eukaryotes. Its function is critically important to both health and disease.;Unlike other cytoskeletal motor proteins, dynein has four active AAA+ adenosine triphosphatases (ATPases; AAA1-4) per motor domain ("head"), separated from the MT-binding domain by a coiled-coil "stalk." Dynein MT-affinity and ATPase activities are interdependent, and the stalk configuration affects both functions. How are ATPase activity and MT binding coordinated as dynein "walks" along MTs?;Leading and trailing dynein heads likely experience tension in opposite directions. Previous work suggests that the motor's bond to the MT is modulated by force and that tension may affect how the motor steps. We therefore hypothesized that the oppositely-directed tension experienced by the two heads regulates their MT binding differentially. To test this, we measured forces required to unbind single recombinant Saccharomyces cerevisiae dynein heads from MTs. We describe the development and application of this assay, including construction of a multicolor force-fluorescence microscope and optimization of glass surface chemistry for optical trapping studies on MTs.;In the absence of nucleotide, we find that dynein binding to MTs is weaker under forward (MT minus-end-directed) tension than under rearward tension. Thus, tension may promote trailing head detachment while keeping the leading head bound, thereby contributing to the directionality of dynein and its ability to bear force. Further, we find that nucleotides modify this intrinsic behavior. Adenosine diphosphate (ADP) at AAA1 may strengthen MT binding, while at AAA3, ADP induces a previously undescribed, weak MT-binding state that reduces the asymmetry of dynein's response to tension. Adenosine triphosphate (ATP) binding to AAA1 weakens MT binding in both directions, but ATP binding affinity may be diminished when tension is applied to the dynein tail/linker. We interpret possible implications of these results in the context of the mechanochemical cycle of dynein dimers.;This work provides a foundation for dissecting the role of force in dynein mechanochemistry. Our results suggest that intramolecular tension is important for regulating dynein motility and MT attachment, and highlight the importance of considering multiple AAA+ subunits in models for dynein mechanochemistry.