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 videoenhanced 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 µm/s. The velocity histogram showed multiple peaks, which, when fit with Gaussians using a least squares minimization, yielded an average spacing of 0.57 µm/ 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.

Force–Velocity Curves of Motor Proteins Cooperating In Vivo

“…5). In previous studies, histograms of normalized vesicle velocities [37] or unscaled velocities [38,[42][43][44] revealed a similar pattern of peaks. These peaks indicate that the velocities of transported vesicles are constrained to quantized values.…”

“…These peaks indicate that the velocities of transported vesicles are constrained to quantized values. A likely hypothesis that has been proposed [37,[43][44][45] is that each peak represents a different number of motors actively pulling each vesicle through a viscous medium (as we will discuss below, this hypothesis requires the viscosity of the medium to be greater than about 0.1 Pa•s; a requirement that may not be met in, for example, a squashed-mount Drosophila embryo system [46]). Based on this hypothesis and our measured viscosity of 1.8 Pa•s, we assigned numbers of active motors to each of the constant velocity segments.…”

In vivo Multimotor Force–Velocity Curves by Tracking and Sizing Sub-Diffraction Limited Vesicles

“…26 Several of these studies reported multimodal profiles even in non-normalized velocity distributions. 5,24,26,29 This suggests that the sizes of tracked vesicles and the effective viscosities they experience were more uniform in these studies than what we measure in our NT2 cell system. Quantized velocities have also been reported in vitro from actin sliding at low myosin densities with the zero-load velocity believed to indicate number of active motors.…”

“…In our previous study of velocities in NT2 cells, we observed a similar multimodal pattern of regularly spaced peaks. 41 Other studies on vesicle velocities have observed quantized velocity distributions in cells such as rat PC12 cells, 14 in chick motor neurons, 5,29 in Drosophila, 24 and in melanosomes of Xenopus melanophores. 26 Several of these studies reported multimodal profiles even in non-normalized velocity distributions.…”

Intraneuronal vesicular organelle transport changes with cell population density in vitro

“…There is some precedence for changes in intracellular transport with DIV. For example, one study using the MEDIC technique showed that velocities of organelle transport in chick motor neurons slowed with increasing time in vitro [10]. Figure 2A also shows that even at 3-4 DIV, the pattern of increasing transport with increasing density begins to vaguely emerge as can be seen in the overlapping points seen at 20, 40, and 70 cells per mm 2 .…”