Effects of dynein inhibitor on the number of motor proteins transporting synaptic cargos

Abstract

Synaptic cargo transport by kinesin and dynein in hippocampal neurons was investigated by noninvasively measuring the transport force based on nonequilibrium statistical mechanics. Although direct physical measurements such as force measurement using optical tweezers are difficult in an intracellular environment, the noninvasive estimations enabled enumerating force-producing units (FPUs) carrying a cargo comprising the motor proteins generating force. The number of FPUs served as a barometer for stable and long-distance transport by multiple motors, which was then used to quantify the extent of damage to axonal transport by dynarrestin, a dynein inhibitor. We found that dynarrestin decreased the FPU for retrograde transport more than for anterograde transport. This result indicates the applicability of the noninvasive force measurements. In the future, these measurements may be used to quantify damage to axonal transport resulting from neuronal diseases, including Alzheimer's, Parkinson's, and Huntington's diseases.

Figure 1

Synaptic cargo transport in the axon of a hippocampal neuron. Synaptic cargos (pink circles) labeled with GFP (green stars) undergo anterograde transport by kinesin and retrograde transport by dynein along microtubules (green cylinder). White dots in the fluorescence micrographs represent synaptic cargo. The force (F) is generated by the motor proteins and equivalent to the drag force, $\Gamma v$, at a CVS, where $v$ and $\Gamma$ represent the velocity and friction coefficient of moving cargo, respectively. A single cargo transported by multiple FPUs is shown(bottom). To see this figure in color, go online.

Figure 2

Fluctuation of position of a transported cargo. (A) Typical example of cargo position (X) over time. The boxed time interval is aCVS. (B) Magnified view of the time course (Fig. 2A) at a CVS. $\Delta X=X\left(t+\Delta t\right)-X\left(t\right)$. (C) Distribution $P\left(\Delta X\right)$ of $\Delta X$ for the cases in which $\Delta t$ = 10 ms (left), 50 ms (middle), and 100 ms (right). $P\left(\Delta X\right)$ fit a Gaussian distribution (black curve). (D) Example of $\chi$ (n = 1) for the CVS (Fig. 2A) calculated using Eq. 3 plotted as a function of $\Delta t$ (the thick curve). The thin curves represent$\chi$ calculated from different partial segments cut from the original CVS to estimate the error of $\chi$ in a bootstrapping manner (Materials and methods). After relaxation time (∼100 ms), $\chi$ reached a constant value. (E and F) Noting that $\chi =v/D$ (Eq. 3), where $D$ and $v$ are the diffusion coefficient and velocity of a cargo, respectively, $\Delta t$ dependence of $D$ and $v$ were also shown. To see this figure in color, go online.

Figure 3

Force index $\chi$ for anterograde transport. Shown here is$\chi$ as a function of $\Delta t$ for n different cargos in cells treated with (A) 0 μM dynarrestin (n = 131), (B) 100 μM dynarrestin (n = 92), and (C) 200 μM dynarrestin (n = 119) (left panels). Each color denotes a cluster (i.e., FPU). The number of clusters was determined by clustering analysis (described in Materials and methods) (middle panels). The number of clusters are displayed as a function of q, which is the sole parameter of the cluster analysis (right panels). The most probable cluster number was decided as the number valid for the wide range of q. To see this figure in color, go online.

Figure 4

Force index $\chi$ for retrograde transport. Shown here is$\chi$ as a function of $\Delta t$ for n different cargos in cells treated with (A) 0 μM dynarrestin (n = 116), (B) 100 μM dynarrestin (n = 102), and (C) 200 μM dynarrestin (n = 123) (left panels). Each color denotes a cluster (i.e., FPU). The number of clusters was decided by clustering analysis (described in Materials and methods) (middle panels). The right panels show the number of clusters as a function of q, which is the sole parameter of the cluster analysis. The most probable cluster number was decided as the number valid for the wide range of q. To see this figure in color, go online.

Figure 5

Relationship between $\chi$, velocity, and fluorescence intensity. (A) Number of the elements for each FPU, calculated from the results in Fig. 3 and 4, representing as probability density. (B) Mean values of $\chi$ as a function of dynarrestin concentration for anterograde (left) and retrograde (right) transport. Error bars represent standard error (SE). (C) Mean velocity (v) at the CVSs as a function of dynarrestin concentration for anterograde (left) and retrograde (right) transport. Error bars represent SE. (D) Relationship between $\chi$ in the case $\Delta t$ = 150 ms and $v$ in the absence of dynarrestin for the case of anterograde (left) and retrograde (right) transport. Each color of the dots denotes each cluster shown in Figure 3, Figure 4A, respectively. (E) Comparison among fluorescence intensities of all cargos observed, anterograde cargos belonging to six FPUs, and retrograde cargos belonging to six FPUs, respectively. To see this figure in color, go online.

Figure 6

The trajectories of χ for each FPU in the cases of anterograde (A) and retrograde (B) transport. The colored, black, and gray trajectories represent the cases of 0, 100, and 200 μM dynarrestin, respectively. The statistical analysis was performed between the sets of χ ($\Delta t$=10–150 ms) for the case of 0 μM dynarrestin and those for the case of 100 or 200 μM dynarrestin. To see this figure in color, go online.

Figure 7

$\langle {\chi }_{\Delta t=150\text{ms}}\rangle$ in the case of one retrograde FPU as a function of dynarrestin concentration. Error bars represent standard error (SE). The black fitting curve represents $0.071exp\left(-\left[\text{concentration}\right]/70\right)+0.12$. The data for 50 μM dynarrestin are shown in Fig. S2. To see this figure in color, go online.