The Mechanism of Dynein's Minus End Directionality and an Engineered Plus End Directed Dynein Motor

Abstract

Cytoplasmic dynein is responsible for nearly all minus-end directed transport along microtubules (MTs). However, little is known about how dynein achieves its directionality. Using an optical trap, we find that the interaction between the dynein microtubule binding domain (MTBD) and the MT is highly asymmetric. Release towards the plus-end is slow and force independent, whereas minus-end directed force greatly accelerates MT release. Motor directionality was reversed by exchanging the MTBD with a catalytically inactive kinesin motor domain that favors release towards the plus-end. High resolution assays revealed that this construct moves processively by taking 8-32 nm steps, similar to dynein. However, it stepped backward more frequently than dynein, which may be the result of its relatively low asymmetry in MT-release. To test if interhead tension alone is sufficient for motility, we generated dynein constructs that do not undergo nucleotide-dependent MT-release by attaching the N-terminal linker and the tip of the stalk of a dynein monomer to two artificial protein handles terminating with the dynein MTBD. This construct showed processive motility, suggesting that asymmetric MT-release in combination with tension generated by the motor domains is sufficient to drive minus-end directed motility. We propose that MTBD, not the ATPase ring, determines dynein directionality by asymmetry in MT release under tension and preferential rebinding to MTs when the stalk is tilted towards the plus end.