We developed a nonradioactive fluorescence-activated cell sorting-based assay, called surface sensing of translation (SUnSET), which allows the monitoring and quantification of global protein synthesis in individual mammalian cells and in heterogeneous cell populations. We demonstrate here, using mouse dendritic and T cells as a model, that SUnSET offers a technical alternative to classical radioactive labeling methods for the study of mRNA translation and cellular activation.

In this study, the principles of surface sensing of translation (SUnSET) were used to develop a nonradioactive method for ex vivo and in vivo measurements of protein synthesis (PS). Compared with controls, we first demonstrate excellent agreement between SUnSET and a [(3)H]phenylalanine method when detecting synergist ablation-induced increases in skeletal muscle PS ex vivo. We then show that SUnSET can detect the same synergist ablation-induced increase in PS when used in vivo (IV-SUnSET). In addition, IV-SUnSET detected food deprivation-induced decreases in PS in the heart, kidney, and skeletal muscles, with similar changes being visualized with an immunohistochemical version of IV-SUnSET (IV-IHC-SUnSET). By combining IV-IHC-SUnSET with in vivo transfection, we demonstrate that constitutively active PKB induces a robust increase in skeletal muscle PS. Furthermore, transfection with Ras homolog enriched in brain (Rheb) revealed that a PKB-independent activation of mammalian target of rapamycin is also sufficient to induce an increase in skeletal muscle PS. Finally, IV-IHC-SUnSET exposed the existence of fiber type-dependent differences in skeletal muscle PS, with PS in type 2B and 2X fibers being significantly lower than that in type 2A fibers within the same muscle. Thus, our nonradioactive method allowed us to accurately visualize and quantify PS under various ex vivo and in vivo conditions and revealed novel insights into the regulation of PS in skeletal muscle.

In heat-stressed (HS) tomato (Lycopersicon peruvianum) cell cultures, the constitutively expressed HS transcription factor HsfA1 is complemented by two HS-inducible forms, HsfA2 and HsfB1. Because of its stability, HsfA2 accumulates to fairly high levels in the course of a prolonged HS and recovery regimen. Using immunofluorescence and cell fractionation experiments, we identified three states of HsfA2: (i) a soluble, cytoplasmic form in preinduced cultures maintained at 25°C, (ii) a salt-resistant, nuclear form found in HS cells, and (iii) a stored form of HsfA2 in cytoplasmic HS granules. The efficient nuclear transport of HsfA2 evidently requires interaction with HsfA1. When expressed in tobacco protoplasts by use of a transientexpression system, HsfA2 is mainly retained in the cytoplasm unless it is coexpressed with HsfA1. The essential parts for the interaction and nuclear cotransport of the two Hsfs are the homologous oligomerization domain (HR-A/B region of the A-type Hsfs) and functional nuclear localization signal motifs of both partners. Direct physical interaction of the two Hsfs with formation of relatively stabile hetero-oligomers was shown by a two-hybrid test in Saccharomyces cerevisiae as well as by coimmunoprecipitation using tomato and tobacco whole-cell lysates.

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Increased Tau protein amyloidogenicity has been causatively implicated in several neurodegenerative diseases, collectively called tauopathies. In pathological conditions, Tau becomes hyperphosphorylated and forms intracellular aggregates. The deletion of K280, which is a mutation that commonly appears in patients with frontotemporal dementia with Parkinsonism linked to chromosome 17, enhances Tau aggregation propensity (pro-aggregation). In contrast, introduction of the I277P and I308P mutations prevents β-sheet formation and subsequent aggregation (anti-aggregation). In this study, we created a tauopathy model by expressing pro- or anti-aggregant Tau species in the nervous system of Caenorhabditis elegans. Animals expressing the highly amyloidogenic Tau species showed accelerated Tau aggregation and pathology manifested by severely impaired motility and evident neuronal dysfunction. In addition, we observed that the axonal transport of mitochondria was perturbed in these animals. Control animals expressing the anti-aggregant combination had rather mild phenotype. We subsequently tested several Tau aggregation inhibitor compounds and observed a mitigation of Tau proteotoxicity. In particular, a novel compound that crosses the blood-brain barrier of mammals proved effective in ameliorating the motility as well as delaying the accumulation of neuronal defects. Our study establishes a new C. elegans model of Tau aggregation-mediated toxicity and supports the emerging notion that inhibiting the nucleation of Tau aggregation can be neuroprotective.

Mutations in two genes encoding the putative kinases LRRK2 and PINK1 have been associated with inherited variants of Parkinson disease. The physiological role of both proteins is not known at present, but studies in model organisms have linked their mutants to distinct aspects of mitochondrial dysfunction, increased vulnerability to oxidative and endoplasmic reticulum stress, and intracellular protein sorting. Here, we show that a mutation in the Caenorhabditits elegans homologue of the PTEN-induced kinase pink-1 gene resulted in reduced mitochondrial cristae length and increased paraquat sensitivity of the nematode. Moreover, the mutants also displayed defects in axonal outgrowth of a pair of canal-associated neurons. We demonstrate that in the absence of lrk-1, the C. elegans homologue of human LRRK2, all phenotypic aspects of pink-1 loss-offunction mutants were suppressed. Conversely, the hypersensitivity of lrk-1 mutant animals to the endoplasmic reticulum stressor tunicamycin was reduced in a pink-1 mutant background. These results provide the first evidence of an antagonistic role of PINK-1 and LRK-1. Due to the similarity of the C. elegans proteins to human LRRK2 and PINK1, we suggest a common role of both factors in cellular functions including stress response and regulation of neurite outgrowth. This study might help to link pink-1/PINK1 and lrk-1/LRRK2 function to the pathological processes resulting from Parkinson disease-related mutants in both genes, the first manifestations of which are cytoskeletal defects in affected neurons. Mutations in the Parkinson disease (PD)2 -related gene PINK1 have been associated with increased sensitivity to oxidative stress and mitochondrial dysfunction (1-4). Although a series of reports support a localization of PINK1 only in mitochondria (5-7), recent studies have shown that a portion of endogenous PINK1 is also distributed to the cytoplasm (8 -11). Notably, the cytoplasmic kinase activity and not the mitochondrial targeting of PINK1 seems to be prerequisite of its protective effects against mitochondrial stress (12).The GTPase-regulated kinase LRRK2, another gene associated with familial PD, has been linked to the biogenesis and regulation of vesicular transport (13,14). In support of this notion, the C. elegans lrk-1, the LRRK2 homologue, has recently been shown to be involved in synaptobrevin-associated vesicular transport (15). Moreover, the Dictyostelium homologue of LRRK2/LRK-1 proteins, GbpC, a cGMP-binding protein is required for the normal phosphorylation and cytoskeletal assembly of myosin (16). Based on this data, it had been anticipated that PINK1/PINK-1 and LRRK2/LRK-1 might be involved in distinct cellular functions, whereas pathological mutations in both genes, leading either to loss-of-function (PINK1) or gain-of-function (LRRK2), result in a similar phenotype, the loss of dopaminergic neurons.In the present study, we found a functional connection between their Caenorhabditits elegans homologues pink-1 and lrk-1. We demonstrate that loss of C. elega...

Considerable evidence indicates that the general blockade of protein synthesis prevents both the initial consolidation and the postretrieval reconsolidation of long-term memories. These findings come largely from studies of drugs that block ribosomal function, so as to globally interfere with both cap-dependent and -independent forms of translation. Here we show that intraamygdala microinfusions of 4EGI-1, a small molecule inhibitor of cap-dependent translation that selectively disrupts the interaction between eukaryotic initiation factors (eIF) 4E and 4G, attenuates fear memory consolidation but not reconsolidation. Using a combination of behavioral and biochemical techniques, we provide both in vitro and in vivo evidence that the eIF4E-eIF4G complex is more stringently required for plasticity induced by initial learning than for that triggered by reactivation of an existing memory. T he synthesis of new proteins within relevant neuronal circuits is widely agreed to be a basic requirement for long-term memory (LTM) storage. Translation is important for stabilizing active memories because it triggers the production of new proteins that are required for persistent molecular and synaptic changes during both consolidation (after learning) and reconsolidation (after memory reactivation). However, the role of translation in memory formation has been explored only in the context of overall cellular protein translation. There are at least two forms of protein synthesis that could in principle be exploited for either memory consolidation or reconsolidation. The primary mode of translation initiation requires formation of a multiprotein complex of eukaryotic initiation factors (eIFs) bound to the 5′ methylated-GTP cap of target mRNAs (1, 2). Specifically, the interaction between eIF4E and eIF4G facilitates eIF4A RNA helicase activity, recruitment of the 40S ribosomal subunit, scanning, and peptide elongation (3, 4). Molecules that block the formation of eIF4F (eIF4E + eIF4G + eIF4A), such as the endogenous regulator 4E-binding protein, which binds to and sequesters eIF4E, therefore effectively inhibits cap-dependent translation. Likewise, the small molecule, 4EGI-1, which selectively disrupts eIF4E-eIF4G interactions (eIF4F formation) in vitro (5), also inhibits cap-dependent translation. The second route that mRNAs can be translated occurs via internal ribosomal entry sites (IRES), which are unaffected by disruptions to the 5′ cap translation machinery, such as blockade of eIF4E-eIF4G interactions (5). A role for eIF4E-eIF4G interactions during hippocampal synaptic plasticity has been shown (6-8), but they have not yet been demonstrated for memory formation. The ability to dissociate mechanisms of translation control is relevant to the study of associative learning because little is known about the relative roles of cap-dependent and IRES-mediated translation in mammalian brain function. For example, there is evidence that an IRES mediates translation of fragile X mental retardation protein, a protein that is absent in ...

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