Small teams of myosin Vc motors coordinate their stepping for efficient cargo transport on actin bundles

Elena B Krementsova,Ken'Ya Furuta,Kazuhiro Oiwa,Kathleen M Trybus,M Yusuf Ali

doi:10.1074/jbc.m117.780791

Abstract

Myosin Vc (myoVc) is unique among vertebrate class V myosin isoforms in that it requires teams of motors to move continuously on single actin filaments. Single molecules of myoVc cannot take multiple hand-over-hand steps from one actin-binding site to the next without dissociating, in stark contrast to the well studied myosin Va (myoVa) isoform. At low salt, single myoVc motors can, however, move processively on actin bundles, and at physiologic ionic strength, even teams of myoVc motors require actin bundles to sustain continuous motion. Here, we linked defined numbers of myoVc or myoVa molecules to DNA nanostructures as synthetic cargos. Using total internal reflectance fluorescence microscopy, we compared the stepping behavior of myoVc versus myoVa ensembles and myoVc stepping patterns on single actin filaments versus actin bundles. Run lengths of both myoVc and myoVa teams increased with motor number, but only multiple myoVc motors showed a run-length enhancement on actin bundles compared with actin filaments. By resolving the stepping behavior of individual myoVc motors with a quantum dot bound to the motor domain, we found that coupling of two myoVc motors significantly decreased the futile back and side steps that were frequently observed for single myoVc motors. Changes in the inter-motor distance between two coupled myoVc motors affected stepping dynamics, suggesting that mechanical tension coordinates the stepping behavior of two myoVc motors for efficient directional motion. Our study provides a molecular basis to explain how teams of myoVc motors are suited to transport cargos such as zymogen granules on actin bundles.

Highlights

Myosin Vc is unique among vertebrate class V myosin isoforms in that it requires teams of motors to move continuously on single actin filaments
By resolving the stepping behavior of individual Myosin Vc (myoVc) motors with a quantum dot bound to the motor domain, we found that coupling of two myoVc motors significantly decreased the futile back and side steps that were frequently observed for single myoVc motors
To establish that the motion of the motor is not affected by attachment to the DNA origami, a single processive myosin Va (myoVa) motor was attached to a DNA nanotube that contained one binding site

Summary

Introduction

Myosin Vc (myoVc) is unique among vertebrate class V myosin isoforms in that it requires teams of motors to move continuously on single actin filaments. Myosin Vc, which is predominantly expressed in glandular tissues such as pancreas, colon, and stomach, was the only vertebrate class V isoform to be kinetically characterized as non-processive, implying that it would be unable to move cargo continuously as a single motor [2,3,4] This observation was surprising because myoVc is known to be involved in transport of secretory vesicles to the apical membrane [4]. It was recently shown that in the exocrine pancreas, parallel bundles of actin filaments nucleated by formins at the plasma membrane are the tracks on which zymogen granules are trafficked [5] Consistent with this biological observation, we recently showed that actin bundles are the required track for small ensembles of myoVc, attached to a quantum dot, to continuously move at physiologic ionic strength (150 mM KCl) [6]. The movement of the labeled scaffold was tracked, so that the stepping patterns of individual myoVc heads in the ensemble could not be moni-

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Conclusion