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

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...