Figure 1. ZBP1 granules are localized in dendrites and beneath synapses of dendritic spines of cultured hippocampal neurons.

A, Double-label immunofluorescence detection of ZBP1 (red) and microtubule-associated protein MAP2 (blue) overlaid on DIC optics. ZBP1 was abundant in the cell body and distributed throughout dendrites in the form of granules, which frequently were observed to extend beyond the microtubule-rich region of the dendritic shaft and into spine-like protrusions [small box is enlarged (arrow)].

B, Triple-label fluorescence of ZBP1 (red), MAP2 (blue), and F-actin detected with phalloidin (green). ZBP1 granules were localized in the neck and head of spine-like structures (arrow, enlarged inset) and filopodia (arrowhead, enlarged inset).

C, Triple-label fluorescence of ZBP1 (red), synaptophysin (blue), and F-actin detected with phalloidin (green). Use of conventional digital imaging and color overlay show colocalization (arrows) of ZBP1 granules (red) with synaptophysin (blue) and phalloidin-labeled spines (green).

D-F, Three-dimensional reconstruction of a deconvolved z-series of the region shown in C depicts a track-like or clustered arrangement of several ZBP1 (red) granules in the dendritic shaft (see video 1). A cluster of ZBP1 granules also is observed on one side of a large spine (green) beneath a large presynaptic contact (blue) along the spine neck in a crevasse between the dendritic shaft and the bulbous spine head.

D, ZBP1 (red) and phalloidin (green) merged.

E, Synaptophysin (blue) and phalloidin (green) merged.

F, shows all three channels merged.