Activity-Dependent Trafficking and Dynamic Localization of Zipcode Binding Protein 1 and β-Actin mRNA in Dendrites and Spines of Hippocampal Neurons

KCl-induced localization of ZBP1 granules in dendrites is dependent on NMDA receptor activity. A, Triple-label fluorescence detection of ZBP1 (red), synaptophysin (blue), and phalloidin (green). The colored boxes overlaid on top of the dendrites show schematically how the dendrite was divided into three regions of interest (proximal, middle, and distal) for performing quantitative digital imaging analysis of pixel intensities with IPLab software (Scanalytics). These three regions have been enlarged at the bottom of each panel by using the same color-coded border. All images were acquired with shorter exposure times than those used in Figure 1, such that the ZBP1 signal in dendrites was sparse and undetectable in distal regions of unstimulated neurons.B, The addition of 20 mm KCl to the culture medium for 15 min increased ZBP1 levels in all three dendritic regions as compared with unstimulated neurons. After KCl treatment ZBP1 fluorescence was now apparent in distal regions. C, Histogram of mean ZBP1 immunofluorescence intensities for each treatment; 45 dendrites, one per neuron, were analyzed from three experiments. KCl elicited a statistically significant increase in ZBP1 levels (mean 19.8% increase) as compared with nonstimulated control neurons. The NMDA receptor antagonist AP-5 completely inhibited the KCl response. Exposure of neurons to AP-5 alone for 15 min decreased ZBP levels (mean 12.6% decrease), which was not increased by KCl treatment in the presence of AP-5. Bars show group mean fluorescence intensity/area ± SEM ;*p < 0.01, two-tailed Mann–Whitney t test. Black asterisks denote significance as compared with control untreated neurons. Red asterisks denote significant difference between AP-5+KCl as compared with KCl treatment.

Western blot analysis of ZBP1 expression.A, KCl treatment for 15 min did not have a statistically significant effect on ZBP1 levels (normalized band intensities from three experiments). Densitometric ratio of ZBP1 to β-tubulin from the unstimulated neurons was scaled to 100%. ZBP1 levels from neurons treated with KCl, cycloheximide (CHX), KCl plus cycloheximide, or KCl plus APV showed <10% fluctuation in normalized intensities (which were not statistically significant). B, One representative Western blot for ZBP1 and β-tubulin (normalized control) is shown as an example.

Rapid stimulation of EGFP-ZBP1 granule transport into dendrites visualized in live neurons after KCl depolarization. Neurons were transfected with EGFP-ZBP1 and imaged live before and after perfusion of 20 mm KCl containing culture medium. A short time lapse (10 frames) was acquired at t = 0, 5, 15, and 30 min after perfusion of KCl. A, EGFP-ZBP1 signal in proximal dendrite (arrow) from frame 1. Intensity was thresholded so no signal was apparent at distal sites (arrowhead). Fluorescence intensity was displayed as a heat-map (inset).A′, Frame 10 just before KCl perfusion shows comparable signal to Frame 1. B, At 5 min after KCl perfusion there is a marked increase in EGFP-ZBP1 levels at proximal site (arrow; note warmer colors via the heat map). Some weak signal is apparent at a distal site (arrowhead). C, D, EGFP-ZBP1 signals continue to increase at 15 and 30 min time points. E, Plot of timed average fluorescence intensities over time for proximal, middle, and distal dendritic regions of the cell imaged inA–D. Each of the four time lapses is shown in a different color (t = 0, 5, 15, and 30 min).F, The effects of KCl perfusion-induced increase in ZBP1 localization were analyzed in three live neurons, and the average fluorescence intensities in each region were plotted over time in KCl.

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, available at www.jneurosci.org). 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, available at www.jneurosci.org). 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.

KCl-induced localization of β-actin mRNA granules. A, Localization of β-actin mRNA granules in dendrites of an unstimulated neuron. B, Increased localization of β-actin mRNA in dendrites 15 min after KCl stimulation. C, Exposure to the NMDA receptor antagonist AP-5 reduced the localization of β-actin mRNA in dendrites.D, Inhibition of NMDA receptor activation with AP-5 in the presence of KCl did not impair the depolarization-induced localization of β-actin mRNA in dendrites. E, Quantitative analysis of mean fluorescence intensities from 45 neurons per treatment demonstrated a significant increase in ZBP1 levels after KCl treatment in proximal, middle, and distal dendritic regions. Treatment of AP-5 alone significantly decreased the localization of β-actin mRNA as compared with control unstimulated neurons but did not inhibit the KCl-induced increase in β-actin mRNA. Bars show group means ± SEM; p ≤ 0.05*, two-tailed Mann–Whitney t test. Black asterisks denote significance as compared with control untreated neurons. Red asterisks denote comparison of AP-5- and AP-5+KCl-treated neurons.