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Motor protein-based active transport is essential for mRNA localization and local translation in animal cells, yet how mRNA granules interact with motor proteins remains poorly understood. Using an unbiased yeast two–hybrid screen for interactions between murine RNA-binding proteins (RBPs) and motor proteins, here we identified protein interaction with APP tail-1 (PAT1) as a potential direct adapter between zipcode-binding protein 1 (ZBP1, a β-actin RBP) and the kinesin-I motor complex. The amino acid sequence of mouse PAT1 is similar to that of the kinesin light chain (KLC), and we found that PAT1 binds to KLC directly. Studying PAT1 in mouse primary hippocampal neuronal cultures from both sexes and using structured illumination microscopic imaging of these neurons, we observed that brain-derived neurotrophic factor (BDNF) enhances co-localization of dendritic ZBP1 and PAT1 within granules that also contain kinesin-I. PAT1 is essential for BDNF-stimulated neuronal growth cone development and dendritic protrusion formation, and we noted that ZBP1 and PAT1 co-locate along with β-actin mRNA in actively transported granules in living neurons. Acute disruption of the PAT1–ZBP1 interaction in neurons with PAT1 siRNA or a dominant-negative ZBP1 construct diminished localization of β-actin mRNA but not of Ca(2+)/calmodulin-dependent protein kinase IIα (CaMKIIα) mRNA in dendrites. The aberrant β-actin mRNA localization resulted in abnormal dendritic protrusions and growth cone dynamics. These results suggest a critical role for PAT1 in BDNF-induced β-actin mRNA transport during postnatal development and reveal a new molecular mechanism for mRNA localization in vertebrates.

Recently RNA localization has been appreciated as an essential post-transcriptional mechanism to program local proteome composition and function. Although RNA has been visualized using diverse techniques, the use of the bacteriophage MS2 method to encode genetically fluorescent RNA has revolutionized the study of RNA dynamics in living cells. Here, I highlight the strength of MS2 compared to other techniques, and how further evolution of this system will enable the visualization of RNA in the context of complex live-cell dynamics. Although the generation of MS2-fluorescence resonance energy transfer (FRET) and MS2-bifluorescence complementation (BiFC) will require further development, it has the potential to increase significantly the signal-to-noise ratio, which is the major obstacle to rapid live-cell imaging of RNA.

Autism and its highly variable symptomology were the themes of the 23rd Annual International Symposium of the Center for the Study of Gene Structure and Function at Hunter College in New York City, held 15 January 2010. The meeting explored the extensive research on autism from several perspectives-integrating research on genetics, neuroscience, and behavior-from researchers presenting new and innovative approaches to understanding the autism spectrum. Early diagnosis, intervention, and genetics were major themes because they are seen as essential areas in which progress is needed before the rise in numbers of cases of autism throughout the world, which some describe as approaching an epidemic, can be stemmed. Several genetic, neurobiological, and behavioral markers of autism have been identified that may ultimately provide the basis for early identification, and that presently define the key areas requiring intensive intervention.

Localization of beta-actin messenger RNA to sites of active actin polymerization modulates cell migration during embryogenesis, differentiation and possibly carcinogenesis. This localization requires the oncofetal protein ZBP1 (Zipcode binding protein 1), which binds to a conserved 54-nucleotide element in the 3'-untranslated region of the beta-actin mRNA known as the 'zipcode'. ZBP1 promotes translocation of the beta-actin transcript to actin-rich protrusions in primary fibroblasts and neurons. It is not known how the ZBP1-RNA complex achieves asymmetric protein sorting by localizing beta-actin mRNA. Here we show that chicken ZBP1 modulates the translation of beta-actin mRNA. ZBP1 associates with the beta-actin transcript in the nucleus and prevents premature translation in the cytoplasm by blocking translation initiation. Translation only occurs when the ZBP1-RNA complex reaches its destination at the periphery of the cell. At the endpoint of mRNA transport, the protein kinase Src promotes translation by phosphorylating a key tyrosine residue in ZBP1 that is required for binding to RNA. These sequential events provide both temporal and spatial control over beta-actin mRNA translation, which is important for cell migration and neurite outgrowth.

SummaryExperience refines synaptic connectivity through neural activity-dependent regulation of transcription factors. Although activity-dependent regulation of transcription factors has been well described, it is unknown whether synaptic activity and local, dendritic regulation of the induced transcripts are necessary for mammalian synaptic plasticity in response to transcription factor activation. Neuronal depolarization activates the myocyte enhancer factor 2 (MEF2) family of transcription factors that suppresses excitatory synapse number. We report that activation of metabotropic glutamate receptor 5 (mGluR5) on the dendrites, but not cell soma, of hippocampal CA1 neurons is required for MEF2-induced functional and structural synapse elimination. We present evidence that mGluR5 is necessary for synapse elimination to stimulate dendritic translation of the MEF2 target gene Arc/Arg3.1. Activity-regulated cytoskeletal-associated protein (Arc) is required for MEF2-induced synapse elimination, where it plays an acute, cell-autonomous, and postsynaptic role. This work reveals a role for dendritic activity in local translation of specific transcripts in synapse refinement.

Neurotrophin regulation of actin-dependent changes in growth cone motility may depend on the signaling of beta-actin mRNA transport. Formation of an RNP complex between the beta-actin mRNA zipcode sequence and Zipcode Binding Protein 1 (ZBP1) was required for its localization to growth cones. Antisense oligonucleotides to the zipcode inhibited formation of this RNP complex in vitro and the neurotrophin-induced localization of beta-actin mRNA and ZBP1 granules. Live cell imaging of neurons transfected with EGFP-ZBP1 revealed fast, bidirectional movements of granules in neurites that were inhibited by antisense treatment, as visualized by FRAP analysis. NT-3 stimulation of beta-actin protein localization was dependent on the 3'UTR and inhibited by antisense treatment. Growth cones exhibited impaired motility in the presense of antisense. These results suggest a novel mechanism to influence growth cone dynamics involving the regulated transport of mRNA.

The localization and translation of messenger RNA (mRNA) within dendrites is a specialized form of gene expression which enables neurons to locally manage information flow and provides a mechanism for the spatiotemporal regulation of structural synapse plasticity that contributes to learning and memory. The delivery of mRNA from the nucleus into dendrites involves complex formation between cis-acting elements and numerous trans-acting proteins, which display dynamic movements and play essential roles in the maintenance of several forms of synaptic plasticity in the mammalian brain. The diverse composition of these posttranscriptional operons is presented in the context of activity-regulated dendritic mRNA transport with regard to their role in synaptic plasticity and defects in their regulation that result in neuronal disease. An example of the analysis of rapid dendritic transport in living hippocampal neurons is presented that shows calcium/calmodulin-dependent protein kinase II (CaMKII)-alpha mRNA movements at rates tenfold greater than previously estimated using improved imaging techniques.

The fragile X mental retardation protein (FMRP) is a selective RNA-binding protein that regulates translation and plays essential roles in synaptic function. FMRP is bound to specific mRNA ligands, actively transported into neuronal processes in a microtubule-dependent manner, and associated with polyribosomes engaged in translation elongation. However, the biochemical relationship between FMRP–microtubule association and FMRP–polyribosome association remains elusive. Here, we report that although the majority of FMRP is incorporated into elongating polyribosomes in the soluble cytoplasm, microtubule-associated FMRP is predominantly retained in translationally dormant, polyribosome-free messenger ribonucleoprotein (mRNP) complexes. Interestingly, FMRP–microtubule association is increased when mRNPs are dynamically released from polyribosomes as a result of inhibiting translation initiation. Furthermore, the I304N mutant FMRP that fails to be incorporated into polyribosomes is associated with microtubules in mRNP particles and transported into neuronal dendrites in a microtubule-dependent, 3,5-dihydroxyphenylglycine-stimulated manner with similar kinetics to that of wild-type FMRP. Hence, polyribosome-free FMRP–mRNP complexes travel on microtubules and wait for activity-dependent translational derepression at the site of function. The dual participation of FMRP in dormant mRNPs and polyribosomes suggests distinct roles of FMRP in dendritic transport and translational regulation, two distinct phases that control local protein production to accommodate synaptic plasticity.

The function of local protein synthesis in synaptic plasticity and its dysregulation in fragile X syndrome (FXS) is well studied, however the contribution of regulated mRNA transport to this function remains unclear. We report a function for the fragile X mental retardation protein (FMRP) in the rapid, activity-regulated transport of mRNAs important for synaptogenesis and plasticity. mRNAs were deficient in glutamatergic signaling-induced dendritic localization in neurons from Fmr1 KO mice, and single mRNA particle dynamics in live neurons revealed diminished kinesis. Motor-dependent translocation of FMRP and cognate mRNAs involved the C terminus of FMRP and kinesin light chain, and KO brain showed reduced kinesin-associated mRNAs. Acute suppression of FMRP and target mRNA transport in WT neurons resulted in altered filopodia-spine morphology that mimicked the FXS phenotype. These findings highlight a mechanism for stimulus-induced dendritic mRNA transport and link its impairment in a mouse model of FXS to altered developmental morphologic plasticity.

RNA localization is one mechanism to temporally and spatially restrict protein synthesis to specific subcellular compartments in response to extracellular stimuli. To understand the mechanisms of mRNA localization, a number of methods have been developed to follow the path of these molecules in living cells including direct labeling of target mRNAs, the MS2-GFP system, and molecular beacons. We review advances in these methods with the goal of identifying the particular strengths and weaknesses of the various approaches in their ability to follow the movements of mRNAs from transcription sites to translation sites.

Fragile X syndrome is caused by the absence of the fragile X mental-retardation protein (FMRP), an mRNA-binding protein, which may play important roles in the regulation of dendritic mRNA localization and/or synaptic protein synthesis. We have recently applied high-resolution fluorescence imaging methods to document the presence, motility and activity-dependent regulation of FMRP granule trafficking in dendrites and spines of cultured hippocampal neurons. In this study, we show that FMRP granules distribute to F-actin-rich compartments, including filopodia, spines and growth cones during the staged development of hippocampal neurons in culture. Fragile X mental-retardation protein granules were shown to colocalize with ribosomes, ribosomal RNA and MAP1B mRNA, a known FMRP target, which encodes a protein important for microtubule and actin stabilization. The levels of FMRP within dendrites were reduced by disruption of microtubule dynamics, but not by disruption of F-actin. Direct measurements of FMRP transport kinetics using fluorescence recovery after photobleaching in living neurons showed that microtubules were required to induce the mGluR-dependent translocation into dendrites. This study provides further characterization of the composition and regulated trafficking of FMRP granules in dendrites of hippocampal neurons.

Fragile X syndrome is caused by the absence of the mRNA-binding protein Fragile X mental retardation protein (FMRP), which may play a role in activity-regulated localization and translation of mRNA in dendrites and at synapses. We investigated whether neuronal activity and glutamatergic signals regulate trafficking of FMRP and its encodingFmr1mRNA into dendrites or at synapses. Using high-resolution fluorescence and digital imaging microscopy in cultured hippocampal neurons, FMRP andFmr1mRNA were localized in granules throughout dendrites and within spines. KCl depolarization rapidly increased FMRP andFmr1mRNA levels in dendrites. Metabotropic glutamate receptor (mGluR) activation, in particular mGluR5 activation, was necessary for localization of FMRP into dendrites. Blockade of either PKC or internal calcium prevented mGluR-dependent localization of both FMRP andFmr1mRNA in dendrites. The activity-dependent localization of FMRP was not dependent on protein synthesis. Fluorescence recovery after photobleaching analysis of live neurons transfected with enhanced green fluorescent protein–FMRP revealed increased granule trafficking in response to KCl depolarization. In contrast to its dendritic localization, mGluR activation diminished FMRP, but notFmr1mRNA, localization at synapses. These results demonstrate regulation of FMRP andFmr1mRNA trafficking in dendrites and synapses in response to specific glutamatergic signals.

Since the identification of the FMR1 gene basic research has been focused on the molecular characterization of the FMR1 gene product, the fragile X mental retardation protein (FMRP). Recent developments in fragile X research have provided new insights and knowledge about the physiological function of FMRP in the cell and the nerve cell in particular. Currently, compelling evidence suggests a role for FMRP in the transport/translation of dendritically localized mRNAs. In addition, the identification of some of the target mRNAs of FMRP have led to an increased interest in the neurobiology of the syndrome. This review highlights the role of FMRP in dendritic mRNA transport/translation in relation to synaptic plasticity, a molecular mechanism implicated in learning and memory.

Centrosome assembly is important for mitotic spindle formation and if defective may contribute to genomic instability in cancer. Here we show that in somatic cells centrosome assembly of two proteins involved in microtubule nucleation, pericentrin and gamma tubulin, is inhibited in the absence of microtubules. A more potent inhibitory effect on centrosome assembly of these proteins is observed after specific disruption of the microtubule motor cytoplasmic dynein by microinjection of dynein antibodies or by overexpression of the dynamitin subunit of the dynein binding complex dynactin. Consistent with these observations is the ability of pericentrin to cosediment with taxol-stabilized microtubules in a dynein- and dynactin-dependent manner. Centrosomes in cells with reduced levels of pericentrin and gamma tubulin have a diminished capacity to nucleate microtubules. In living cells expressing a green fluorescent protein-pericentrin fusion protein, green fluorescent protein particles containing endogenous pericentrin and gamma tubulin move along microtubules at speeds of dynein and dock at centrosomes. In Xenopus extracts where gamma tubulin assembly onto centrioles can occur without microtubules, we find that assembly is enhanced in the presence of microtubules and inhibited by dynein antibodies. From these studies we conclude that pericentrin and gamma tubulin are novel dynein cargoes that can be transported to centrosomes on microtubules and whose assembly contributes to microtubule nucleation.

Pericentrin and gamma-tubulin are integral centrosome proteins that play a role in microtubule nucleation and organization. In this study, we examined the relationship between these proteins in the cytoplasm and at the centrosome. In extracts prepared from Xenopus eggs, the proteins were part of a large complex as demonstrated by sucrose gradient sedimentation, gel filtration and coimmunoprecipitation analysis. The pericentrin-gamma-tubulin complex was distinct from the previously described gamma-tubulin ring complex (gamma-TuRC) as purified gamma-TuRC fractions did not contain detectable pericentrin. When assembled at the centrosome, the two proteins remained in close proximity as shown by fluorescence resonance energy transfer. The three- dimensional organization of the centrosome-associated fraction of these proteins was determined using an improved immunofluorescence method. This analysis revealed a novel reticular lattice that was conserved from mammals to amphibians, and was organized independent of centrioles. The lattice changed dramatically during the cell cycle, enlarging from G1 until mitosis, then rapidly disassembling as cells exited mitosis. In cells colabeled to detect centrosomes and nucleated microtubules, lattice elements appeared to contact the minus ends of nucleated microtubules. Our results indicate that pericentrin and gamma-tubulin assemble into a unique centrosome lattice that represents the higher-order organization of microtubule nucleating sites at the centrosome.

Trans isomers of dietary fatty acids, generated during the commercial hydrogenation of unsaturated fats, may contribute to coronary heart disease (CHD) in humans by interfering with lipid metabolism. To examine this possibility in a fat-sensitive model, the Mongolian gerbil (Meriones unguiculatus) was used to compare the cholesterolemic and triglyceridemic potential of modest increments of trans fatty acids from partially hydrogenated soybean oil with other saturated fatty acids in the presence and absence of dietary cholesterol. Age-, dose-, and time-dependent effects were examined in weanling, 6-month-old, and 1-year-old gerbils. Although lipoprotein metabolism in weanling gerbils was initially refractory to trans fat, even as perturbations by saturated fatty acids were demonstrable, these gerbils eventually (after 16 weeks) developed a trans-induced hypercholesterolemia that was intermediate between the response to 16:0 and 12:0 + 14:0. The hepatic and plasma 18:1/18:2 cholesteryl ester (CE) ratio was depressed by trans in a manner similar to saturated fatty acids. The 6-month-old gerbils readily developed hypertriglyceridemia but not hypercholesterolemia, again revealing a decrease in the plasma 18:1/18:2 CE ratio. The 1-year-old gerbils revealed a dose-related (0, 5, 10%en as trans) elevation in total cholesterol (TC), and especially triglycerides (TG), that was accentuated by 0.04% dietary cholesterol. Increases in plasma lipids were again accompanied by a significant decrease in the mass of hepatic esterified cholesterol, particularly 18:1-cholesteryl esters. Thus, dietary trans-fatty acids induce age-, time-, and dose-dependent modulations in gerbil plasma lipids associated with decreased 18:1 cholesteryl esters. Further investigation with gerbils may reveal mechanisms by which trans fat consumption disturbs lipoprotein metabolism.