Dynamic Clustering of Dyneins on Axonal Endosomes: Evidence from High-Speed Darkfield Imaging.

One of the fundamental features that govern the cooperativity of multiple dyneins during cargo trafficking in cells is the spatial distribution of these dyneins on the cargo. Geometric considerations and recent experiments indicate that clustered distributions of dyneins are required for effective cooperation on micron-sized cargos. However, very little is known about the spatial distribution of dyneins and their cooperativity on smaller cargos, such as vesicles or endosomes <200 nm in size, which are not amenable to conventional immunostaining and optical trapping methods. In this work, we present evidence that dyneins can dynamically be clustered on endosomes in response to load. Using a darkfield imaging assay, we measured the repeated stalls and detachments of retrograde axonal endosomes under load with <10 nm localization accuracy at imaging rates up to 1 kHz for over a timescale of minutes. A three-dimensional stochastic model was used to simulate the endosome motility under load to gain insights on the mechanochemical properties and spatial distribution of dyneins on axonal endosomes. Our results indicate that 1) the distribution of dyneins on endosomes is fluid enough to support dynamic clustering under load and 2) the detachment kinetics of dynein on endosomes differs significantly from the in vitro measurements possibly due to an increase in the unitary stall force of dynein on endosomes.