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bioRxiv · 10.1101/068163

A FLIC motility assay reveals myosin-6 coordination limited by actin filament buckling

Agata K Krenc·Jagoda J Rokicka·Ronald S Rock
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Abstract

Teams of myosin motors carry out intracellular transport and contract the actin cytoskeleton. To fully understand the behavior of multi-myosin ensembles we need to know the properties of individual myosins and the mode of interaction between them. Current models of the interactions within the myosin complex treat the actin filament as a stiff rod, not contributing to the regulation of collective myosin dynamics. Here, we present data suggesting that force transduction through the actin filament is an important element of interaction within myosin-6 ensembles in vitro. Multiple myosin-6s coordinate their steps if they are separated by a short (and therefore high-force bearing) segment of actin. The measurements were performed using Fluorescence Interference Contrast Microscopy (FLIC) to measure small changes in the height of fluorescently labeled actin. Using FLIC, we assign the positions of myosins in a gliding filament assay geometry and measure their attachment time to actin. We also identify actin segments that are buckled or under tension. We show that myosin-6 holds actin about 10 nm above the surface. However, due to asynchronous myosin stepping, frequent buckles up to about 60 nm high appear. The buckle lifetime decreases as the distance between the myosin-6s is reduced, a sign of inter-motor coordination. Our data are consistent with coordinated stepping of closely spaced myosins, but uncoordinated motility with widely separated myosins where buckles can form. These features would be expected to operate on myosins in the cell, where motor spacing may vary considerably depending on the target organelle.\n\nSIGNIFICANCE STATEMENTMyosins are molecular motors that carry out intracellular transport. Interactions between the myosins are crucial for understanding their function. Using Fluorescence Interference Contrast (FLIC) microscopy we characterized the interaction between multiple myosin-6 motors immobilized to the surface of a slide and pulling the same actin filament. Our results point towards coordination of myosin steps as a mechanism governing the behavior of a multi-myosin complex. We also demonstrated the unique application of FLIC microscopy for highly parallel identification and measurement of single myosin motors in a gliding filament format. These features of FLIC enable a robust study of collective myosin dynamics.

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Agata K Krenc, Jagoda J Rokicka, Ronald S Rock. 2016-08-06. A FLIC motility assay reveals myosin-6 coordination limited by actin filament buckling. https://doi.org/10.1101/068163

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