Fewer active motors per vesicle may explain slowed vesicle transport in chick motoneurons after three days in vitro

Abstract

Vesicle transport in cultured chick motoneurons was studied over a period of 3 days using motion-enhanced differential interference contrast (MEDIC) microscopy, an improved version of video-enhanced DIC. After 3 days in vitro (DIV), the average vesicle velocity was about 30% less than after 1 DIV. In observations at 1, 2 and 3 DIV, larger vesicles moved more slowly than small vesicles, and retrograde vesicles were larger than anterograde vesicles. The number of retrograde vesicles increased relative to anterograde vesicles after 3 DIV, but this fact alone could not explain the decrease in velocity, since the slowing of vesicle transport in maturing motoneurons was observed independently for both anterograde and retrograde vesicles. In order to better understand the slowing trend, the distance vs. time trajectories of individual vesicles were examined at a frame rate of 8.3/s. Qualitatively, these trajectories consisted of short (1-2 s) segments of constant velocity, and the changes in velocity between segments were abrupt (<0.2 s). The trajectories were therefore fit to a series of connected straight lines. Surprisingly, the slopes of theses lines, i.e. the vesicle velocities, were often found to be multiples of ~0.6 mum/s. The velocity histogram showed multiple peaks, which, when fit with Gaussians using a least squares minimization, yielded an average spacing of 0.57 mum/s (taken as the slope of a fit to peak position vs. peak number, R(2)=0.994). We propose that the abrupt velocity changes occur when 1 or 2 motors suddenly begin or cease actively participating in vesicle transport. Under this hypothesis, the decrease in average vesicle velocity observed for maturing motoneurons is due to a decrease in the average number of active motors per vesicle.

Fig. 1. DIC vs. MEDIC

In a traditional DIC image (left) the cell body and two processes are evident—but not the transported vesicles inside the two processes. In a MEDIC, or motion enhanced DIC, image of the same cell (right) moving vesicles are clearly seen (white arrows, see also Supplemental Information, Movie M1–Movie M3). A conspicuous difference in the number of filopodia on processes originating from the same cell, as is seen here, is typically observed in these chick motoneurons.

Fig. 2. Distance vs. time of a retrograde vesicle

Vesicle tracking data displays abrupt changes in velocity for most vesicles, including this one that traveled in the retrograde direction for 7.8 s (1.3 µm/s average speed). Inset: The values of χ²_red for fitting 2 to 11 straight-line segments indicate that 6 segments are optimal. Line segments are fit to the data by a least squares optimization (MatLab Optimization Toolbox).

Fig. 3. Histograms of average velocities of individual vesicles at 1 or 3 days in vitro

Each occurrence is the average velocity of one vesicle (N=290) as determined by the manual method. The average speeds in either direction (anterograde velocities are indicated by positive values and retrograde by negative values) decrease as chick motoneurons are cultured for 3 days. A shift toward more retrograde transport is apparent in day 3 relative to day 1 (see also Table 1).

Fig. 4. Histograms of average velocities for large and small vesicles

Each occurrence corresponds to the average velocity of one vesicle (N=179, manual method). Large vesicles (>0.8 µm diameter) move predominantly in the retrograde direction and move slower than small vesicles (<0.6 µm diameter). The mean velocities for the four populations shown in this histogram are: small anterograde = 2.5 µm/s; large anterograde = 2.0 µm/s; small retrograde = 1.8 µm/s; and large retrograde = 1.4 µm/s. The prevalence of retrograde transport is >4-fold higher for large vesicles than for small (large = 3.4-fold more retrograde than anterograde, small = 1.3 fold less retrograde than anterograde).

Fig. 5. Histogram of instantaneous retrograde velocities of individual vesicles

(frame rate = 8.3/s). Each occurrence corresponds to the slope of the best-fit line segment at each frame (N=2075) for vesicle trajectories on DIV 1, 2, and 3 obtained by the automated method (see Fig. 2). The histogram is binned at 1/6 µm/s intervals and fit (thick white curve) with 7 Gaussians (thin white curves) and one constraint (the first peak starts at 0 µm/s). The χ²_red for this fit was 3.4. Inset: A graph of the peak positions vs. peak index for the multi-Gaussian fit reveals a regular 0.59 µm/s peak spacing (R²=0.994).