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RNA
binding proteins may be important mediators of the activity-dependent
transport of mRNAs to dendritic spines of activated synapses. We used
fluorescence microscopy and digital imaging techniques applied to both
fixed and live cultured hippocampal neurons to visualize the localization
of the mRNA binding protein, zipcode binding protein 1 (ZBP1), and its
dynamic movements in response to KCl-induced depolarization at high spatial
and temporal resolution. With the use of immunofluorescence, image deconvolution,
and three-dimensional reconstruction, ZBP1 was localized in the form of
granules that were distributed in dendrites, spines, and subsynaptic sites.
KCl depolarization increased the dendritic localization of ZBP1 that was
not attributed to an increase in ZBP1 expression. Live cell imaging of
single cells before and after perfusion of KCl revealed the rapid and
directed efflux of ZBP1 granules from the cell body into dendrites in
a proximo-distal gradient. High-speed imaging of enhanced green fluorescence
protein-ZBP1 granules revealed rapid anterograde and retrograde movements
in dendrites as well as dynamic movements in dendritic spines. A population
of ZBP1 granules colocalized with beta-actin mRNA, and their spatial association
in dendrites was increased by KCl depolarization. The NMDA receptor antagonist
AP-5 impaired the dendritic localization of ZBP1 and beta-actin mRNA and
inhibited the KCl-induced transport of ZBP1. The activity-dependent trafficking
of ZBP1 and its dynamic movements within dendritic spines provide new
evidence to implicate RNA binding proteins as regulators of mRNA transport
to activated synapses in response to synaptic activity. |
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