Heterogeneous Motion of Secretory Vesicles in the Actin Cortex of Live Cells: 3D Tracking to 5-nm Accuracy

Abstract

Fluorescence microscopy tracks the three-dimensional motion of green fluorescent protein (GFP)-labeled large dense-core secretory vesicles (LDCVs) within the actin cortex of live PC12 cells. In this study, we achieve a 26-ms time resolution and a spatial accuracy of 5 nm or better in each dimension (one standard deviation in one dimension, σ1D). The resulting high-resolution trajectories reveal not only heterogeneity among vesicles but also heterogeneity within single-vesicle trajectories. As in earlier work, we observe three apparent groups of vesicles: the immobile, mobile, and directed-motion groups, but the distinctions among the groups are blurred. The directed trajectories exhibit segments with kinesin-like speed punctuated by pauses and changes in speed and direction. The immobile vesicles nearest the plasma membrane jump among sub-25-nm-diameter “mini-traps”. Comparison with microrheological data from entangled F-actin solutions suggests that the jumps may be caused by local remodeling of F-actin. Motion within a mini-trap is quantitatively modeled by a random walk in a parabolic restoring potential to yield single-trap restoring force constants of ∼0.04 pN/nm. As judged by mean-square displacement versus time, the mobile vesicles execute nearly free random walks in an elastic medium. We find no clear evidence of quasi-linear, directed motion in the mobile group. However, heterogeneity is evident in the distribution of frame-to-frame displacements, P(r), which requires a two-component fit. Evidently, mobile vesicles move by a combination of diffusion and motor-driven motion, with the direction changing rapidly as myosin-V crisscrosses the dense F-actin meshwork. The frequency of long frame-to-frame displacements of 25−70 nm suggests the presence of one or more myosin-V motors on the mobile vesicles. We argue that the motors on the immobile vesicles are less active or completely inactive. This suggests a regulatory mechanism for motor activity that may be related to the cell's ability to mobilize vesicles upon stimulation by Ca2+.