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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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