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We examine the effect of cargo-motor linkage stiffness on the mechanobiological properties of the molecular motor myosin VI. We use the programmability of DNA nanostructures to modulate cargo-motor linkage stiffness and combine it with high-precision optical trapping measurements to measure the effect of linkage stiffness on the motile properties of myosin VI. Our results reveal that a stiff cargo-motor linkage leads to shorter step sizes and load-induced anchoring of myosin VI, while a flexible linkage results in longer steps with frequent detachments from the actin filament under load. Our findings suggest a novel regulatory mechanism for tuning the dual cellular roles of the anchor and transporter ascribed to myosin VI.

Stiffness of Cargo–Motor Linkage Tunes Myosin VI Motility and Response to Load