Investigation of multiple-dynein transport of melanosomes by non-invasive force measurement using fluctuation unit \u03c7

Shin Hasegawa,Takashi Sagawa,Yasushi Okada,Kazuho Ikeda,Kumiko Hayashi

doi:10.1038/s41598-019-41458-w

Abstract

Pigment organelles known as melanosomes disperse or aggregate in a melanophore in response to hormones. These movements are mediated by the microtubule motors kinesin-2 and cytoplasmic dynein. However, the force generation mechanism of dynein, unlike that of kinesin, is not well understood. In this study, to address this issue, we investigated the dynein-mediated aggregation of melanosomes in zebrafish melanophores. We applied the fluctuation theorem of non-equilibrium statistical mechanics to estimate forces acting on melanosomes during transport by dynein, given that the energy of a system is related to its fluctuation. Our results demonstrate that multiple force-producing units cooperatively transport a single melanosome. Since the force is generated by dynein, this suggests that multiple dyneins carry a single melanosome. Cooperative transport has been reported for other organelles; thus, multiple-motor transport may be a universal mechanism for moving organelles within the cell.

Highlights

Cellular cargo is transported through the microtubule network of eukaryotic cells by motor proteins[1,2]
In wild-type worms, the distribution of χ was spread over several clusters, implying the existence of several force-producing units (FPUs); this in turn indicates that a single SVP was carried by multiple UNC-104 motors, which are the generators of force
We observed the aggregation of melanosomes transported by dynein in zebrafish melanophores by bright-field microscopy at a high recording rate (800 fps)

Summary

Introduction

Cellular cargo is transported through the microtubule network of eukaryotic cells by motor proteins[1,2]. In order to investigate force generated by motors acting on a single cargo, a non-invasive force measurement[11,12,13] was recently developed based on the fluctuation theorem of non-equilibrium statistical mechanics[17,18,19,20,21,22]. The position of a moving cargo can be obtained non-invasively by fluorescence microscopic observation of cells, and the fluctuation of its position is observed with high time resolution. Using this fluctuating motion, a fluctuation unit (χ) constructed based on the fluctuation theorem is calculated as a force indicator[11,12,13]. Triphosphatase that relieves the autoinhibition of the motor, which is critical for avoiding unnecessary consumption of ATP when the motor is not bound to an SVP23—had fewer FPUs than their wild-type counterparts[12]

Methods

Results

Conclusion