Plasmonic nanoparticles (NPs), predominantly gold (AuNPs), are easily internalised into cells and commonly employed as nanosensors for reporter-based and reporter-free intracellular SERS applications. While AuNPs are generally considered non-toxic to cells, many biological and toxicity studies report that exposure to NPs induces cell stress through the generation of reactive oxygen species (ROS) and the upregulated transcription of pro-inflammatory genes, which can result in severe genotoxicity and apoptosis. Despite this, the extent to which normal cellular metabolism is affected by AuNP internalisation remains a relative unknown along with the contribution of the uptake itself to the SERS spectra obtained from within so called ‘healthy’ cells, as indicated by traditional viability tests. This work aims to interrogate the perturbation created by treatment with AuNPs under different conditions and the corresponding effect on the SERS spectra obtained. We characterise the changes induced by varying AuNP concentrations and medium serum compositions using biochemical assays and correlate them to the corresponding intracellular reporter-free SERS spectra. The different serum conditions lead to different extents of nanoparticle internalisation. We observe that changes in SERS spectra are correlated to an increasing amount of internalisation, confirmed qualitatively and quantitatively by confocal imaging and ICP-MS analysis, respectively. We analyse spectra and characterise changes that can be attributed to nanoparticle induced changes. Thus, our study highlights a need for understanding condition-dependent NP-cell interactions and standardisation of nanoparticle treatments in order to establish the validity of intracellular SERS experiments for use in all arising applications.

Myogenic differentiation in the C2C12 myoblast model system reflects a concerted and controlled activation of transcription and translation following the exit of cells from the cell cycle. Previously we have shown that the mTORC1 signaling inhibitor, RAD001, decreased protein synthesis rates, delayed C2C12 myoblast differentiation, decreased p70S6K activity but did not affect the hypermodification of 4E-BP1. Here we have further investigated the modification of 4E-BP1 during the early phase of differentiation as cells exit the cell cycle, using inhibitors to target mTOR kinase and siRNAs to ablate the expression of raptor and rictor. As predicted, inhibition of mTOR kinase activity prevented p70S6K, 4E-BP1 phosphorylation and was associated with an inhibition of myogenic differentiation. Surprisingly, extensive depletion of raptor did not affect p70S6K or 4E-BP1 phosphorylation, but promoted an increase in mTORC2 activity (as evidenced by increased Akt Ser473 phosphorylation). These data suggest that an mTOR kinase-dependent, but raptor-independent regulation of downstream signaling is important for myogenic differentiation.

During cell spreading, mammalian cells migrate using lamellipodia formed from a large dense branched actin network which produces the protrusive force required for leading edge advancement. The formation of lamellipodia is a dynamic process and is dependent on a variety of protein cofactors that mediate their local regulation, structural characteristics and dynamics. In the present study, we show that mRNAs encoding some structural and regulatory components of the WAVE [WASP (Wiskott-Aldrich syndrome protein) verprolin homologous] complex are localized to the leading edge of the cell and associated with sites of active translation. Furthermore, we demonstrate that steady-state levels of ArpC2 and Rac1 proteins increase at the leading edge during cell spreading, suggesting that localized protein synthesis has a pivotal role in controlling cell spreading and migration.

Translation mechanisms at different stages of the cell cycle have been studied for many years, resulting in the dogma that translation rates are slowed during mitosis, with cap-independent translation mechanisms favored to give expression of key regulatory proteins. However, such cell culture studies involve synchronization using harsh methods, which may in themselves stress cells and affect protein synthesis rates. One such commonly used chemical is the microtubule de-polymerization agent, nocodazole, which arrests cells in mitosis and has been used to demonstrate that translation rates are strongly reduced (down to 30% of that of asynchronous cells). Using synchronized HeLa cells released from a double thymidine block (G 1/S boundary) or the Cdk1 inhibitor, RO3306 (G 2/M boundary), we have systematically re-addressed this dogma. Using FACS analysis and pulse labeling of proteins with labeled methionine, we now show that translation rates do not slow as cells enter mitosis. This study is complemented by studies employing confocal microscopy, which show enrichment of translation initiation factors at the microtubule organizing centers, mitotic spindle, and midbody structure during the final steps of cytokinesis, suggesting that translation is maintained during mitosis. Furthermore, we show that inhibition of translation in response to extended times of exposure to nocodazole reflects increased eIF2α phosphorylation, disaggregation of polysomes, and hyperphosphorylation of selected initiation factors, including novel Cdk1-dependent N-terminal phosphorylation of eIF4GII. Our work suggests that effects on translation in nocodazole-arrested cells might be related to those of the treatment used to synchronize cells rather than cell cycle status.

Cell migration is a highly controlled essential cellular process, often dysregulated in tumour cells, dynamically controlled by the architecture of the cell. Studies involving cellular fractionation and microarray profiling have previously identified functionally distinct mRNA populations specific to cellular organelles and architectural compartments. However, the interaction between the translational machinery itself and cellular structures is relatively unexplored. To help understand the role for the compartmentalization and localized protein synthesis in cell migration, we have used scanning confocal microscopy, immunofluorescence and a novel ribopuromycylation method to visualize translating ribosomes. In the present study we show that eIFs (eukaryotic initiation factors) localize to the leading edge of migrating MRC5 fibroblasts in a process dependent on TGN (trans-Golgi network) to plasma membrane vesicle transport. We show that eIF4E and eIF4GI are associated with the Golgi apparatus and membrane microdomains, and that a proportion of these proteins co-localize to sites of active translation at the leading edge of migrating cells.

BACKGROUND INFORMATION:The spatial localization of translation can facilitate the enrichment of proteins at their sites of function while also ensuring that proteins are expressed in the proximity of their cognate binding partners.RESULTS:Using human embryonic lung fibroblasts and employing confocal imaging and biochemical fractionation techniques, we show that ribosomes, translation initiation factors and specific RNA-binding proteins localize to nascent focal complexes along the distal edge of migrating lamellipodia. 40S ribosomal subunits appear to associate preferentially with beta3 integrin in focal adhesions at the leading edges of spreading cells, with this association strongly augmented by a synergistic effect of cell engagement with a mixture of extracellular matrix proteins. However, both ribosome and initiation factor localizations do not require de novo protein synthesis.CONCLUSIONS:Taken together, these findings demonstrate that repression, complex post-transcriptional regulation and modulation of mRNA stability could potentially be taking place along the distal edge of migrating lamellipodia.

Here Budiman et al. (2009) demonstrate that the selective translation of selenocysteine-containing proteins can be regulated by the mutually exclusive binding of eIF4a3 and SECIS binding protein 2 (SBP2) to a cis-acting element in the 3' untranslated region (3'UTR) of the target mRNA.

Current accepted models suggest that hypophosphorylated 4E-binding protein (4E-BP1) binds to initiation factor 4E (eIF4E) to inhibit cap-dependent translation, a process readily reversed by its phosphorylation following activation of mammalian target of rapamycin (mTORC1) signalling. Myogenic differentiation in the C2C12 myoblast model system reflects a concerted and controlled activation of transcription and translation following the exit of cells from the cell cycle. Here we show that myogenic differentiation is associated with increased rates of translation, the up-regulation of both 4E-BP1 mRNA and protein levels and enhanced levels of eIF4E/4E-BP1 complex. Paradoxically, treatment of C2C12 myoblasts with an inhibitor of mTOR signalling (RAD001) which inhibits translation, promotes the hyperphosphorylation of 4E-BP1 on novel sites and prevents the increase in 4E-BP1 levels. In contrast, eIF4E appears to be under translational control with a significant delay between induction of mRNA and subsequent protein expression.

In activated monocytes, Interferon-γ modulates assembly of a heterotetrameric inhibitor of translation. This is responsive to signaling cascades promoting the induction and activation of death associated protein kinase (DAPK) and consequently zipper-interacting protein kinase (ZIPK). Now Mukhopadhyay et al., in a recent issue of Molecular Cell , show that the kinases themselves are regulated by the same translational silencing they promote thereby providing a negative feedback loop to limit late inflammatory gene expression.

Previous observations of association of mRNAs and r ibosomes with subcellular structures highlight the importance of localised tr anslation within cells. However, little is known regarding associations bet ween eukaryotic translation initiation factors and cellular structures within t he cytoplasm of normally growing cells. Here we have used detergent-based cellular f ractionation methods coupled with immunofluorescence microscopy to investigate the su bcellular localisation of the eukaryotic initiation factors involved in recruitme nt of mRNA for translation in NIH3T3 fibroblasts. We have focussed on eIF4E, the mRNA cap-binding protein, the scaffold protein eIF4GI and poly(A) binding protein (PABP). Our data suggest that the bulk of these proteins ex ist in a soluble cytosolic pool, with only a sub-fraction tightly associated with cellula r structures. However, translation initiation factors engaged in active eIF4F complexe s w re more extensively sequestered in association with subcellular structu es. Immunofluorescence analysis reveals both a diffuse and a perinuclear distributi on of eIF4G, with the pernuclear staining pattern similar to that of the endoplasmic reticulum. eIF4E also shows both a diffuse staining pattern and a tighter perinuclear st in, partly coincident with vimentin intermediate filaments. For all three proteins we observed localisation to the lamellipodia of migrating cells in close proximity to ribosomes, microtubules, microfilaments and focal adhesions, with eIF4G and eIF4E at the periphery showing a similar staining pattern to the focal adhesion prot ein vinculin. Introduction Localised translation is increasingly recognise d as an important mechanism of delivering proteins to their sites of function with in cells (Carson et al., 1998; Jansen, 2001; Jockusch et al., 2003; Kloc et al., 2002), wi th complexes of mRNAs and associated proteins shown to interact with cytoskel etal networks to facilitate trafficking within the cell. Examples include mRNAs and other components of the protein synthesis machinery moving as granules in o ligodendrocytes (Carson et al., 1998; Jansen, 1999; Jansen, 2001), actin mRNA movin g to the leading edge of migrating fibroblasts (Chicurel et al., 1998; Farin et al., 2003), and staufen-mediated transport of oskar and bicoid mRNA to the appropriate poles of developing Drosophila embryos (Micklem et al., 2000). In addit ion, it has long been known that interaction of translating ribosomes with the endop lasmic reticulum plays a key role in directing proteins into the secretory pathway, a nd recent studies have elucidated further details of this mechanism (Lerner et al., 2 003; Nicchitta et al., 2005). Early work addressing possible links between pr otein synthesis and the cytoskeleton involved the fractionation of mammalia n cells using detergents to select for free or cytoskeleton-associated components. Gen tle lysis of cells in the presence of a non-ionic detergent (e.g. Triton X-100) releas ed a sub-fraction of the cellular ribosomes into the extract, most of which were inac tive 80S monomers (Lenk et al., 1977). Considerably more ribosomes were subsequentl y released if the pellet was extracted with the anionic detergent sodium deoxych olate (DOC). Generally, this fraction contained a much higher proportion of ribo somes in polysomes, suggesting that they were more active in protein synthesis (Bo nneau et al., 1985; Cervera et al., 1981; Lemieux and Beaud, 1982; Pramanik et al., 198 6; van Venrooij et al., 1981). The prevailing conclusion from these data, that tra nsl tion in vivo was mostly associated with the cytoskeleton, was reinforced by observations that ribosomes were released by treatment of cells with cytochalasin D (Lenk et al., 1977), and that protein synthesis was impaired in unattached cells (Farmer et al., 1983) or when the actin cytoskeleton was disrupted (Hudder et al., 2003; Or nelles et al., 1986; Stapulionis et al., 1997). Moreover, in vitro translation systems prepared from vertebrate cells under conditions that partially or wholly retained the cy toskeletal structure appear to have higher or more sustained protein synthetic activity than those prepared by conventional lysis (Biegel and Pachter, 1991; Negru tskii et al., 1994; Patrick et al., 1989). However, there are differing interpretations on the extent to which association of the translational apparatus with the endoplasmic re t ulum, rather than with the cytoskeleton, may contribute to these findings (Dan g et al., 1983; Lenk et al., 1977; van Venrooij et al., 1981; Hovland et al., 1996; Ra maekers et al., 1983). Indeed, evidence for the direct association of ribosomes an d translation factors with cytoskeletal components remains highly variable. Ri bosomes have been reported to associate with microtubules in sea urchin embryos ( Hamill et al., 1994) and with intermediate filaments in fibroblasts (Traub et al. , 1998), with elongation factor eEF1A recognised as a binding partner of both actin (Clore et al., 1996; Liu et al., 2002; Murray et al., 1996; Umikawa et al., 1998) an d tubulin (Moore and Cyr, 2000; Moore et al., 1998). eEF2 has also been identified as interacting with actin (Shestakova et al., 1991). Both eEF1A (Munshi et al ., 2001; Murray et al., 1996) and the release factor eRF3 (Valouev et al., 2002) have been reported to influence the organisation of the actin cytoskeleton, with eEF1A also involved in maintaining the localisation of β-actin mRNA in protrusions of migrating fibroblasts (Liu et al., 2002). For translational initiation factors, relativel y little is known about their association with cellular structures within the cytoplasm, alth ough in response to severe cellular stress several of them become sequestered with othe r prot ins and 40S ribosomal subunits in granules ((Cuesta et al., 2000; Kedersh a et al., 2005; Kedersha et al., 2001; Kim et al., 2005; Kimball et al., 2003). The larges t subunit (eIF3a, p 170, TIF32) of the multimeric initiation factor eIF3 has variously been reported to interact with an actin-associated protein (Palacek et al., 2001), wi th membranes via actin filaments (Pincheira et al., 2001), with microtubules (Hasek et al., 2000) and with intermediate filaments (Lin et al., 2001). A smaller subunit (eI F3g, p44) is suggested to be an anchor between the protein synthesis apparatus and the cytoskeleton in red blood cells (Hou et al., 2000). The poly (A) binding protein (P ABP) which associates with the initiation factor eIF4G (Prevot et al., 2003), has been shown to be localised to RNA granules on oligodendrocytes (Barbarese et al., 199 5), to stress granules (Kedersha and Anderson, 2002; Kedersha et al., 1999) and, int eres ingly, has been shown to colocalise with paxillin in the endoplasmic reticulum and at the leading edge of migrating fibroblasts (Woods et al., 2002). In addi tion, relocalisation of the cap recognition factor eIF4E during platelet activation from the membrane skeleton to the mRNA-rich cytoskeletal core has been shown to occur oncomitantly with a stimulation of protein synthesis, an event prevente d by disruption of the actin cytoskeleton (Lindemann et al., 2001). Moreover, in neuronal preparations, eIF4E was found to associate with two different actin net works in dendrites; one contained longer filaments easily disrupted by latrunculin A (lat A) while the other, located in dendritic spine heads consisted of a highly branche d network of shorter filaments enriched in granules containing mRNA and more resis tant to lat A (Smart et al., 2003). Following treatment with brain-derived neuro trophic factor (BDNF), the proportion of eIF4E in the dendritic spine heads wa increased, a change suggested to facilitate local translational activity. These observations raise important questions co cerning the topology of protein synthesis. A model whereby cellular translation lar gely involves localised components associated with cellular structures would be consis tent with earlier indications of “channelling” of aminoacyl-tRNAs into protein synth esis (Hudder et al., 2003; Negrutskii and Deutscher, 1991; Negrutskii and Deut scher, 1992; Negrutskii et al., 1994; Stapulionis and Deutscher, 1995; Stapulionis et al., 1997) and with the notoriously low translational activity of extracts derived from adherent cultured cells. To examine this we have focussed on the group of tr anslation initiation factors that interact with the mRNA 5’ cap as the first step in the recruitment of mRNAs for translation and examined the association of eIF4E, eIF4G and PABP with the major cytoskeletal networks. Our evidence suggests that, while the main pool of each of these proteins in cells is cytosolic, the majority of the eIF4F complex (eIF4E/4G/PABP) is compartmentalised, but not direc tly localised to either the actin or tubulin cytoskeletons. Rather, a significant pro portion of each of these proteins appears to be localised with the ER, with a smaller proportion observed at the leading edge of migrating cells. Materials and Methods Cell culture and treatments. NIH3T3 cells were cultured in DMEM (Invitrogen,UK) supplemented with 10% (v/v) foetal bovine serum (Labtech,UK) in a humidified atmosphere containing 5% CO 2 . In some experiments, as specified, microfilaments were disrupted by incubating cells w ith 2 μM Cytochalasin-D in ethanol for 1 hour and stress fibre formation was f acilitated by incubating cells with 25 μM lysophosphatidic acid (LPA) (Sigma, UK) for 1 hou r. Immunofluorescence microscopy. Coverslips were coated with 100 μg/ml poly-L lysine (Sigma,UK) in PBS and allowed to dry overnig ht. The coverslips were then washed twice in 1 ml PBS and coated with 100 μg/ml bovine fibronectin (Sigma, UK), incubated for 1 hour then washed once in PBS. 5 x 10 cells were seeded onto each 22 mm coverslip and allowed to grow for 24 hou rs. Cells were then washed once in 1 ml PBS at 37 C, then fixed in 4% paraformaldehyde/PBS for 20 min utes and pe

Previous observations of association of mRNAs and ribosomes with subcellular structures highlight the importance of localised translation. However, little is known regarding associations between eukaryotic translation initiation factors and cellular structures within the cytoplasm of normally growing cells. We have used detergent-based cellular fractionation coupled with immunofluorescence microscopy to investigate the subcellular localisation in NIH3T3 fibroblasts of the initiation factors involved in recruitment of mRNA for translation, focussing on eIF4E, the mRNA cap-binding protein, the scaffold protein eIF4GI and poly(A) binding protein (PABP). We find that these proteins exist mainly in a soluble cytosolic pool, with only a subfraction tightly associated with cellular structures. However, this "associated" fraction was enriched in active "eIF4F" complexes (eIF4E.eIF4G.eIF4A.PABP). Immunofluorescence analysis reveals both a diffuse and a perinuclear distribution of eIF4G, with the perinuclear staining pattern similar to that of the endoplasmic reticulum. eIF4E also shows both a diffuse staining pattern and a tighter perinuclear stain, partly coincident with vimentin intermediate filaments. All three proteins localise to the lamellipodia of migrating cells in close proximity to ribosomes, microtubules, microfilaments and focal adhesions, with eIF4G and eIF4E at the periphery showing a similar staining pattern to the focal adhesion protein vinculin.