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

Abstract

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.

Figure 1

Retrograde GNP-endosomes under load. (A) Unidirectional trajectories of retrograde GNP-endosomes in axons captured by darkfield imaging are shown. The inset shows a snapshot of GNP-endosomes transporting in axons. Scale bars, 3 μm. (B) Gradual stalls and fast reversals (“jumps”) exhibited by GNP-endosomes in axons are shown. (C) The elastic tether model explains the endosome jumps as the gradual stalling and detachment of dyneins under load.

Figure 2

(A) Repeated stalls and detachments of a retrograde GNP-endosome under load. The black dotted line is the estimated location of the tether. (B) Histogram of endosome jump size, obtained as the maximum distance covered within each jump relative to the tether location, exhibits peak multiplicity of ∼120 nm. (C) Histogram of maximum frame velocity (V_maxf) within each jump exhibits peak multiplicity of ∼5.1 μm/s. (D) Sudden detachment recoil profile is shown. (E) Delayed detachment recoil profile is shown. (F) Sequential detachment recoil profile is shown. To see this figure in color, go online.

Figure 3

Single endosome statistics obtained from the multiple endosome jumps in Fig 2. (A) A depiction of the stall duration T_S, detachment duration T_D, and the recoil duration T_R, based on the minimal (q_min), maximal (q_max), and detachment (q_det) positions within the endosome jump is shown. (B) Histograms of q_min, q_max, and q_det positions are shown. The distributions of T_D, T_R, and T_S are shown in (C), (D), and (E), respectively. (F) The maximum frame velocity V_maxf within each successive jump is shown as a function of time (black markers). The moving average <V_maxf > (red line) shows the dynamic variation of detachment velocities over a span of minutes. (G) The autocorrelation of <V_maxf > is shown.

Figure 4

3D model for simulating the tethered endosome dynamics. (A) A schematic of the endosome with multiple dyneins in our 3D model is shown (see Methods and Supporting Material). (X, Y, Z) is the laboratory frame and (a, b, c) is the endosome frame of reference. (B) Random-fixed and clustered-fixed spatial distributions of dyneins on endosomes are shown. (C) Fluid distribution of dyneins on endosomes, which permits the dynein-endosome contacts to diffuse and slide under mechanical torque, is shown. To see this figure in color, go online.

Figure 5

High stall force model simulation with fluid distribution of dyneins on endosome (Table 1, row O). (A) Simulated trajectory of the tethered endosome exhibiting repeated stalls and detachments is shown. (B) Zooming into the endosome jumps shows key features like sudden, delayed, and sequential detachment profiles. The distributions of T_D, T_R, and T_S for the simulated endosome jumps are shown in (C), (D), and (E), respectively. (F) Histograms of q_min, q_max, and q_det positions for the simulated endosome jumps are shown. To see this figure in color, go online.

Figure 6

Dynamic clustering of dyneins on tethered endosomes shown by high stall force model simulation with fluid distribution of dyneins on 150 nm endosomes. (A) The spatial distribution of dyneins on endosome is shown by plotting the endosome body coordinates (polar versus azimuthal angles) for each dynein-endosome contact position. The initial distribution is shown in black (filled triangles), and the distribution after 10 min of stalls/detachments is shown in red (filled circles). D = 0.001 μm²/s (B), same as in (A), except D = 0.01 μm²/s. (C) Dynamic variation in motor detachment velocities (V_maxf as black markers and <V_maxf> as red line/dotted line) over a span of minutes from simulation is shown (Table 1, row P). The <V_maxf> shows a clear correlation with the average number of load sharing dyneins (blue line/dashed line). To see this figure in color, go online.