Figure 5. Tracking dynamic movements of single EGFP-ZBP1 granules in dendrites and spines. EGFP-ZBP1 granules are displayed in white for the higher contrast needed for particle tracking.

A, Transfected neurons showing granules at low magnification. ZBP1 granules from boxed dendritic region are analyzed in detail.

B, Higher magnification of this subregion revealed many moving granules. The trajectory of a granule moving rapidly in the retrograde (R, arrow) direction was tracked and analyzed frame by frame. The granule trajectory is shown as a heat map (granule moving from red to yellow to green to blue; see also video 2). Also tracked in this region was a rapidly moving anterograde granule (A, arrow). The granule trajectory is shown as a heat map (granule moving from red to yellow to green; see also video 3).

C, D, Montage of selected frames from the time lapse for retrograde (C) and anterograde (D) granules. Colored arrows correspond to the heat map trajectories displayed in B.

E, Histogram plots of instantaneous velocities (distance traveled between adjacent frames) for the retrograde granule.

F, Instantaneous velocities for the anterograde granule.

G, Average non-zero velocities of anterograde and retrograde granules (trajectories and frame-by-frame analysis for 15 granules that were analyzed). These average velocities included zero values where granules paused during a trajectory.

H, EGFP-ZBP1 granules frequently observed at sites of dynamic dendritic filopodia. Here a filopodia emerges after 9 sec of time lapse (green arrowhead).

I, EGFP-ZBP1 granule observed within a dendritic spine (red arrow). After 9 sec of time lapse a new granule emerges at the base of this spine (green). Granules were pseudo-colored (right panel) to compare pixels that contained ZBP1 in the first frame (red), both frames (yellow), or only in the second frame (green). ZBP1 granules present only in the second frame were attributed to movement of a new granule into the field of view.