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

Abstract

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.

BDNF-induced local protein synthesis and synaptic plasticity

“…b-actin mRNA contains a cis-acting element 54-nucleotide long in the 3 0 UTR, which is recognized by ZBP1 (zipcode-binding protein 1) and is essential for the localization of the transcript in the cytoplasm of cultured chick embryo fibroblasts (Kislauskis et al, 1994;Ross et al, 1997) and in neurites and growth cones of cultured chick forebrain neurons (Zhang et al, 2001). The complex ZBP1-b-actin mRNA accumulates in dendritic spines upon synaptic activity (Tiruchinapalli et al, 2003). In developing neurons the localization of ZBP1-b-actin mRNA in growth cones, which is coupled to localized b-actin translation, critically regulates growth cone navigation by mediating responses to external cues (Lin and Holt, 2007).…”

Multiplexed Dendritic Targeting of α Calcium Calmodulin-dependent Protein Kinase II, Neurogranin, and Activity-regulated Cytoskeleton-associated Protein RNAs by the A2 Pathway

“…The stars indicate dendritic targeting measurements that are different from untreated cells with Ͼ99.6% confidence by Fisher's exact test (Fisher, 1922). (Tiruchinapalli et al, 2003), were predominantly segregated into separate granules, with ϳ0.2 overall correlation due to occasional misassembly of actin RNA into ␣CaMKII RNA granules and vice versa.…”

Ubiquitin ligase TRIM3 controls hippocampal plasticity and learning by regulating synaptic γ-actin levels

“…Dynamic actin remodeling contributes to the structural changes observed during synaptic plasticity (Matsuzaki et al, 2004;Okamoto et al, 2007) and plays a key role in membrane insertion, clustering, and internalization of postsynaptic receptors (Allison et al, 1998;Charrier et al, 2006), all of which are essential to the expression of functional plasticity, i.e., long-term potentiation (LTP) or long-term depression (Malinow and Malenka, 2002;Choquet and Triller, 2003;Shepherd and Huganir, 2007). Calcium entry into dendritic spines triggers intracellular signaling mechanisms that favor F-actin over G-actin, including direct calcium-dependent changes in actin polymerization and depolarization rates (Bergeron et al, 2010), local translation of actin (Tiruchinapalli et al, 2003), posttranslational modification of actin (Karakozova et al, 2006), and recruitment of regulatory actin-binding proteins (Schubert et al, 2006). Two isoforms of actin are constitutively expressed in neurons: β-actin and γ-actin (Rubenstein, 1990).…”