Nanoparticle-assisted optical tethering of endosomes reveals the cooperative function of dyneins in retrograde axonal transport.

Praveen D Chowdary,Daphne L Che,Bianxiao Cui,Moungi Bawendi,Ou Chen,Kanyi Pu,Luke Kaplan

doi:10.1038/srep18059

Abstract

Dynein-dependent transport of organelles from the axon terminals to the cell bodies is essential to the survival and function of neurons. However, quantitative knowledge of dyneins on axonal organelles and their collective function during this long-distance transport is lacking because current technologies to do such measurements are not applicable to neurons. Here, we report a new method termed nanoparticle-assisted optical tethering of endosomes (NOTE) that made it possible to study the cooperative mechanics of dyneins on retrograde axonal endosomes in live neurons. In this method, the opposing force from an elastic tether causes the endosomes to gradually stall under load and detach with a recoil velocity proportional to the dynein forces. These recoil velocities reveal that the axonal endosomes, despite their small size, can recruit up to 7 dyneins that function as independent mechanical units stochastically sharing load, which is vital for robust retrograde axonal transport. This study shows that NOTE, which relies on controlled generation of reactive oxygen species, is a viable method to manipulate small cellular cargos that are beyond the reach of current technology.

Highlights

This highlights that the collective function of dyneins is generic over a wide range of cargo sizes and is vital for the long-distance retrograde axonal transport in neurons
We took advantage of a novel phenomenon termed nanoparticle-assisted optical tethering of endosomes (NOTE) to reveal the collective function of dyneins on retrograde endosomes in axons
Our results indicate that the endosomes can recruit up to 7 active dyneins and on average ~4 leading dyneins share the load. This number we report for retrograde endosomes is higher than the average number (1–5 dyneins) observed by immunoblotting of purified neuronal vesicles[25] and immunostaining of prion protein vesicles in axons[26]

Summary

Introduction

We report a novel phenomenon termed nanoparticle-assisted optical tethering of endosomes (NOTE) that made it possible to study the collective function of dyneins on retrograde axonal endosomes in live neurons. The retrograde transport velocities of Alexa-WGA (Mean ± SD, 1.88 ± 1.1 μ m/s), QD-WGA (1.90 ± 1.2 μ m/s) and INP-WGA (1.8 ± 0.7 μ m/s) endosomes are comparable and the endosome run velocity distribution ranged from 0.25 to as high as 5 μ m/s (Supplementary Fig. S4).

Results

Conclusion