Local β-actin synthesis in growth cones of developing axons plays an important role in growth cone steering; however, the mRNA binding proteins required for this process are unknown. Here we used Zbp1/Imp1−/− mice to test the hypothesis that zipcode binding protein 1 (ZBP1) is required for the regulation of β-actin mRNA transport and local translation underlying growth cone guidance. To address the biological function of ZBP1, we developed a novel in vitro turning assay with primary cortical neuron balls having axons over 1mm in length and demonstrate that growth cones of mammalian neurons exhibit protein synthesis-dependent attraction to either netrin-1 or brain-derived neurotrophic factor (BDNF). Interestingly, this attraction is lost in Zbp1-deficient neurons. Furthermore, BDNF-stimulated β-actin mRNA localization was attenuated in Zbp1-deficient neurons, which impaired enrichment of β-actin protein in the growth cone. Finally, using a photoconvertible translation reporter, we found that ZBP1 is necessary for netrin-1 stimulated local translation of β-actin mRNA in axonal growth cones. Together, these results suggest that netrin-1 induced growth cone attraction required ZBP1 mediated local translation of β-actin mRNA, and therefore ZBP1 regulates protein synthesis dependent axon guidance. Thus, mRNA binding proteins regulating local translation can control spatio-temporal protein expression in response to guidance cues and directional cell motility.

The localization of specific mRNAs and their local translation in growth cones of developing axons has been shown to play an important mechanism to regulate growth cone turning responses to attractive or repulsive cues. However, the mechanism whereby local translation and growth cone turning may be controlled by specific mRNA-binding proteins is unknown. Here we demonstrate that brain-derived neurotrophic factor (BDNF) signals the Src-dependent phosphorylation of the ␤-actin mRNA zipcode binding protein 1 (ZBP1), which is necessary for ␤-actin synthesis and growth cone turning. We raised a phospho-specific ZBP1 antibody to Tyr396, which is a Src phos-

In neurons, specific mRNAs are transported in a translationally repressed manner along dendrites or axons by transport ribonucleic-protein complexes called RNA granules. ZBP1 is one RNA binding protein present in transport RNPs, where it transports and represses the translation of cotransported mRNAs, including β-actin mRNA. The release of β-actin mRNA from ZBP1 and its subsequent translation depends on the phosphorylation of ZBP1 by Src kinase, but little is known about how this process is regulated. Here we demonstrate that the ribosomal-associated protein RACK1, another substrate of Src, binds the β-actin mRNA/ZBP1 complex on ribosomes and contributes to the release of β-actin mRNA from ZBP1 and to its translation. We identify the Src binding and phosphorylation site Y246 on RACK1 as the critical site for the binding to the β-actin mRNA/ZBP1 complex. Based on these results we propose RACK1 as a ribosomal scaffold protein for specific mRNA-RBP complexes to tightly regulate the translation of specific mRNAs.

Ca2+ regulates numerous biological processes through spatiotemporal changes in the cytosolic Ca2+ concentration and subsequent interactions with Ca2+ binding proteins. The endoplasmic reticulum (ER) serves as an intracellular Ca2+ store and plays an essential role in cytosolic Ca2+ homeostasis. There is a strong need to develop Ca2+ sensors capable of real-time quantitative Ca2+ concentration measurements in specific subcellular environments without using natural Ca2+ binding proteins such as calmodulin, which themselves participate as signaling molecules in cells. In this report, a strategy for creating such sensors by grafting a Ca2+-binding motif into chromophore sensitive locations in green fluorescence protein is described. The engineered Ca2+ sensors exhibit large ratiometric fluorescence and absorbance changes upon Ca2+ binding with affinities corresponding to the Ca2+ concentrations found in the ER (Kd values range from 0.4 to 2 mM). In addition to characterizing the optical and metal binding properties of the newly developed Ca2+ sensors with various spectroscopic methods, we also examined the kinetic properties using stopped-flow spectrofluorimetry to ensure accurate monitoring of dynamic Ca2+ changes. The developed Ca2+ sensor was successfully targeted to the ER of mammalian cell lines to monitor Ca2+ changes occurring in this compartment in response to stimulation with agonists. We envision that this class of Ca2+ sensors can be modified further to measure the Ca2+ concentration in other cellular compartments, providing tools for studying the contribution of these compartments to cellular Ca2+ signaling.