The spatial regulation of protein translation is an efficient way to create functional and structural asymmetries in cells. Recent research has furthered our understanding of how individual cells spatially organize protein synthesis, by applying innovative technology to characterize the relationship between mRNAs and their regulatory proteins, single-mRNA trafficking dynamics, physiological effects of abrogating mRNA localization in vivo and for endogenous mRNA labelling. The implementation of new imaging technologies has yielded valuable information on mRNA localization, for example, by observing single molecules in tissues. The emerging movements and localization patterns of mRNAs in morphologically distinct unicellular organisms and in neurons have illuminated shared and specialized mechanisms of mRNA localization, and this information is complemented by transgenic and biochemical techniques that reveal the biological consequences of mRNA mislocalization.

The physical manifestation of learning and memory formation in the brain can be expressed by strengthening or weakening of synaptic connections through morphological changes. Local actin remodeling underlies some forms of plasticity and may be facilitated by local β-actin synthesis, but dynamic information is lacking. In this work, we use single-molecule in situ hybridization to demonstrate that dendritic β-actin messenger RNA (mRNA) and ribosomes are in a masked, neuron-specific form. Chemically induced long-term potentiation prompts transient mRNA unmasking, which depends on factors active during synaptic activity. Ribosomes and single β-actin mRNA motility increase after stimulation, indicative of release from complexes. Hence, the single-molecule assays we developed allow for the quantification of activity-induced unmasking and availability for active translation. Further, our work demonstrates that β-actin mRNA and ribosomes are in a masked state that is alleviated by stimulation.

Microglia are the main immune cells of the brain, and under some circumstances they can play an important role in removal of fibrillar Alzheimer amyloid ␤ peptide (fA␤). Primary mouse microglia can internalize fA␤, but they do not degrade it efficiently. We compared the level of lysosomal proteases in microglia and J774 macrophages, which can degrade fA␤ efficiently, and we found that microglia actually contain higher levels of many lysosomal proteases than macrophages. However, the microglial lysosomes are less acidic (average pH of ϳ6), reducing the activity of lysosomal enzymes in the cells. Proinflammatory treatments with macrophage colony-stimulating factor (MCSF) or interleukin-6 acidify the lysosomes of microglia and enable them to degrade fA␤. After treatment with MCSF, the pH of microglial lysosomes is similar to J774 macrophages (pH of ϳ5), and the MCSF-induced acidification can be partially reversed upon treatment with an inhibitor of protein kinase A or with an anion transport inhibitor. Microglia also degrade fA␤ if lysosomes are acidified by an ammonia pulse-wash or by treatment with forskolin, which activates protein kinase A. Our results indicate that regulated lysosomal acidification can potentiate fA␤ degradation by microglia.

How RNA-binding proteins recognize specific sets of target mRNAs remains poorly understood because current approaches depend primarily on sequence information. In this study, we demonstrate that specific recognition of messenger RNAs (mRNAs) by RNA-binding proteins requires the correct spatial positioning of these sequences. We characterized both the cis-acting sequence elements and the spatial restraints that define the mode of RNA binding of the zipcode-binding protein 1 (ZBP1/IMP1/IGF2BP1) to the b-actin zipcode. The third and fourth KH (hnRNP K homology) domains of ZBP1 specifically recognize a bipartite RNA element comprised of a 59 element (CGGAC) followed by a variable 39 element (C/A-CA-C/U) that must be appropriately spaced. Remarkably, the orientation of these elements is interchangeable within target transcripts bound by ZBP1. The spatial relationship of this consensus binding site identified conserved transcripts that were verified to associate with ZBP1 in vivo. The dendritic localization of one of these transcripts, spinophilin, was found to be dependent on both ZBP1 and the RNA elements recognized by ZBP1 KH34.

Localization of mRNA is required for protein synthesis to occur within discrete intracellular compartments. Neurons represent an ideal system for studying the precision of mRNA trafficking because of their polarized structure and the need for synapsespecific targeting. To investigate this targeting, we derived a quantitative and analytical approach. Dendritic spines were stimulated by glutamate uncaging at a diffraction-limited spot, and the localization of single β-actin mRNAs was measured in space and time. Localization required NMDA receptor activity, a dynamic actin cytoskeleton, and the transacting RNA-binding protein, Zipcodebinding protein 1 (ZBP1). The ability of the mRNA to direct newly synthesized proteins to the site of localization was evaluated using a Halo-actin reporter so that RNA and protein were detected simultaneously. Newly synthesized Halo-actin was enriched at the site of stimulation, required NMDA receptor activity, and localized preferentially at the periphery of spines. This work demonstrates that synaptic activity can induce mRNA localization and local translation of β-actin where the new actin participates in stabilizing the expanding synapse in dendritic spines.single molecule | glutamate uncaging | β-actin | RNA localization | HaloTag S ubcellular localization of mRNA allows control of protein synthesis with respect to space and time (1). By sorting mRNAs to their respective compartments, neurons can regulate translation in response to extracellular signal at the place of protein function (2). Many mRNAs have been shown to be present in dendrites and axons (3), and efforts to characterize mRNA transport revealed that depolarization can lead to detectable increases in alpha calcium calmodulin kinase II (αCaMKII), BDNF, or β-actin mRNAs in dendrites (4-6). Likewise, studies in local translation have shown that dendrites can synthesize the necessary complement of proteins for synaptic plasticity (7). Furthermore, electron microscopy observations of polyribosomes within dendrites and synaptic spines have confirmed that translation occurs readily in neuronal subdomains far from the soma (8, 9). The development and use of fluorescent protein-based translation reporters were pivotal in visualizing local translational output within dendrites (10-12). However, missing from these findings was the high-resolution detection of spatial and kinetic events that result in dynamic repositioning of individual mRNAs and translated proteins within dendrites in response to locally defined input.Actin is the major cytoskeletal component of dendritic spines where filamentous actin (F-actin) dynamics confer motility and structural plasticity (13). Interestingly, the mRNA that encodes for the most abundant actin isoform in neurons, β-actin, is also present in dendrites in relatively large numbers (3). Similar to β-actin, the mRNAs for PSD-95 and αCaMKII (among many) also are found at high levels, with the latter considered one of the most abundant mRNAs in dendrites. Why such abundant synaptic proteins need ...