To identify sites of β-actin translation, β-actin promoter–driven constructs were generated to ensure the proper copy number of total actin transcripts. A TC tag (CCPGCC) was inserted at the N terminus, followed by a GFP fusion domain and the β-actin coding sequence, with or without the 3′UTR zipcode sequence. The construct that contains the β-actin 3′UTR and, thus, the zipcode will be referred to as the full-length (FL) construct, whereas the constructs that lacks the β-actin 3′UTR containing the zipcode will be referred to as the ΔZIP construct. After ReAsH staining, C2C12 cells transfected with the FL construct exhibited a staining pattern nearly identical to that of GFP–β-actin (). C2C12 myoblasts were used for these investigations based on their ability to target β-actin mRNA (Hill and Gunning, 1993
). In addition, these cells differentiate into muscle through fusion of cell contacts, a process that we were interested in investigating with regard to a potential role for localized translation at contacts.
Figure 1. ReAsH labeling of the TC motif in the absence of cycloheximide results in a pattern similar to GFP–β-actin. (A) Schematic representation of the time course of the procedure used in B and C. (B) Epifluorescence image of the distribution (more ...)
By using the concentration of nascent polypeptide chains at polysome sites, we expected to identify translation sites using ReAsH staining and the translation elongation inhibitor cycloheximide (). Cycloheximide is known to accumulate ribosomes and nascent polypeptide chains on mRNA (Godchaux et al., 1967
; Lodish, 1971
). ReAsH staining of C2C12 cells transfected with the FL construct resulted in the detection of discrete uniformly sized bright puncta generally localized at the cell periphery, even without cycloheximide (). Their distribution was similar to that of β-actin mRNA in mouse myoblast cells (Hill and Gunning, 1993
). Untransfected cells did not exhibit punctate staining (not depicted), indicating that the observed ReAsH puncta resulted specifically from the binding of dye to the transgene protein product. When cells containing the ΔZIP construct were stained with ReAsH, puncta were observed in the perinuclear region and very few were observed at the cell periphery, suggesting that the information contained within the zipcode region is responsible not only for the proper targeting of mRNA but also for proper targeting of the translation sites (). These data also demonstrate that translation sites can be detected after ReAsH staining by quick destaining and fixation () or by the use of cycloheximide during staining and destaining ().
Figure 2. ReAsH labeling reveals the sites of β-actin translation. Epifluorescence images of C2C12 cells containing the FL (B–D) or ΔZIP (F–H) construct stained with ReAsH. (A) Schematic representation of the time course of the procedure (more ...)
Intact polysomes are required for the detection of translation sites
To demonstrate that the puncta are a result of translation, we treated the cells with puromycin, a reagent known to dissociate ribosomes and nascent chains from mRNAs (Yarmolinsky and De la Haba, 1959
; Joklik and Becker, 1965
). As described, cells expressing the FL construct treated with cycloheximide and subsequently stained with ReAsH exhibited more numerous puncta throughout the cytoplasm than nontreated cells, consistent with the increased loading of nascent chains known to occur after this treatment (Singer and Penman, 1972
; ). In contrast, cells pretreated with puromycin, before cycloheximide, exhibited a decrease in the number of puncta, consistent with the dissociation of nascent chains known to occur after this treatment (). Quantification of the relative number of puncta/cell for the puromycin-treated cells compared with the cells with only cycloheximide showed an approximately sevenfold reduction (). These data indicate that intact polysomes are needed for the detection of the puncta after ReAsH staining and represent translation sites based on their sensitivity to translational inhibitors.
Figure 3. Disrupting polysome structure with puromycin prevents the detection of ReAsH sites. (A) Overlay image of cells stained with ReAsH in the presence of 100 μg/ml cycloheximide showing GFP–β-actin (green) and ReAsH staining (red) with (more ...)
Translation sites are transient in living cells
We would expect the nascent chains to “run off” after removing ReAsH. To investigate this, live cells were imaged after the removal of the ReAsH dye solution to determine the persistence of the puncta (). The translation sites were observed for ~5 min after ReAsH removal before the signal reached background levels (). Pretreatment with cycloheximide prolonged the persistence of the signal to >45 min (). These data suggest that the ReAsH-stained nascent chains continued to elongate because, in cells lacking cycloheximide, sites disappeared after a few minutes, consistent with the release of the nascent chains labeled with ReAsH and their replacement by nonlabeled nascent chains. The sites persisted for at least 45 min in the presence of cycloheximide because the nascent chains with bound ReAsH remained associated with polysomes because of the elongation block.
Figure 4. Prolonged imaging of translation sites requires inhibition of translational elongation. (A) A live ReAsH-stained C2C12 cell transfected with the FL construct has bright puncta for 5 min before the signal is no longer detectable because of “run (more ...)
The localization of mRNA, translation sites, and mature protein are spatially correlated at the cellular periphery and require the zipcode
Actively translating mRNA should colocalize with sites of ReAsH staining. Fluorescence in situ hybridization experiments were performed on C2C12 cells transfected with the FL or ΔZIP construct. When cells expressing the FL construct were hybridized with probes against the GFP coding sequence, mRNA was observed throughout the cell, including the periphery (). In contrast, cells containing the ΔZIP construct did not efficiently target GFP–β-actin mRNA to the cell periphery (). To compare the localization of the RNA in the two populations, the fluorescence intensity as a function of distance from the nucleus was compared among cells expressing either the FL construct or the ΔZIP construct (). For the population of cells with the FL construct, fluorescence extended to the cell periphery (, red line). In contrast, the ΔZIP construct was prevalent in the perinuclear region with a significant decrease at the cell periphery (, blue line).
Figure 5. Localization of the β-actin mRNA transgene correlates with the distribution of mature protein and translation sites. Fluorescence in situ hybridization experiments were performed on C2C12 cells transfected with the FL or ΔZIP construct (more ...)
Translation sites were also observed at the periphery of cells with the FL construct (, red line). In contrast, when the sites of translation for a population of cells expressing the ΔZIP transgene were analyzed as a function of relative distance from the nucleus, fluorescence at the cell periphery was not detected (, blue line). Likewise, the distribution of the GFP signal, representing synthesized protein, also showed asymmetric localization, similar to that observed for mRNA localization (, compare C and E). Thus, the zipcode appeared to be required for targeted β-actin mRNA to be translated at the cell periphery and β-actin protein to accumulate there.
It has been suggested that mRNAs are translated when associated with the cytoskeleton (Lenk et al., 1977
). To investigate whether the sites of translation were associated with the cytoskeleton, saponin-extracted C2C12 cells expressing the FL construct were stained with ReAsH. Fluorescence imaging confirmed that translation sites remained after detergent extraction, suggesting that they were associated with the detergent-resistant cytoskeleton (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200512137/DC1
). To further investigate the anchoring of β-actin translation sites, time-lapse imaging of C2C12 cells stained with ReAsH was performed (Video 1). Analysis of the position of the identified translation sites indicated that there was very little movement of these sites during the time course of the experiment, consistent with the hypothesis that translating mRNAs are anchored to the cytoskeleton. In contrast, directed movements of reporter mRNAs are measured at rates on the order of 1 μm/s, indicating that the translation sites are not a subset of directed mRNAs (Fusco et al., 2003
). Importantly, cells treated with the translational inhibitor cycloheximide for up to 3 h are able to target mRNA properly, suggesting that the lack of movement observed in the time-lapsed images was not due to an effect of the cycloheximide treatment on mRNA movements (Sundell and Singer, 1990
Nascent chain colocalization with mRNA at cell–cell contacts requires the zipcode and results in the local accumulation of newly synthesized protein
Overlay images of ReAsH and FISH images showed that colocalization between the red translation sites and green mRNAs were often near cell–cell contacts when the C2C12 cells contained the FL construct (, left). The majority of mRNAs not at cell contacts were not translating, suggesting that the contact between cells created an environment that enhanced localized translation (). In contrast, when contacting cells containing the ΔZIP construct were imaged, translation sites were not observed at cell contacts (, right). Therefore, targeting of translation sites to cell contacts required the zipcode.
Figure 6. Translation sites colocalize with their cognate mRNA near the sites of cell contact. ReAsH staining experiments were performed on live cells that were subsequently fixed and processed for FISH, showing a colocalization between the red translation puncta (more ...)
To investigate the fate of recently translated β-actin protein within the cell, pulse-chase staining of cells containing the FL or ΔZIP construct was performed, using FlAsH to bind up the unreacted TCs. After a short wash and a short pulse with ReAsH, the proteins synthesized during the ReAsH exposure could be evaluated (Gaietta et al., 2002
). Images obtained in the red and green channels were presented as a ratio to determine where the β-actin synthesized during the chase period had accumulated (high red/green ratio). When cells in sparse culture, containing the FL construct, were examined, the majority of β-actin protein synthesized during the chase period was found at the perinuclear region and the leading edge of the cell (, red). When cells containing the FL construct were investigated by pulse-chase staining as they contacted other cells, the majority of the newly synthesized β-actin was localized at the perinuclear region and at the site of the cell contacts (, red), consistent with the observed translation at these sites (). Time-lapse images of cells illustrated that newly synthesized β-actin protein accumulated at cell contacts (Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200512137/DC1
). In contrast, newly synthesized β-actin from the ΔZIP transgene was primarily at the perinuclear region but failed to localize to cell contacts (), consistent with the lack of translation sites there ().
Figure 7. Newly synthesized β-actin protein localizes to cell contacts and is zipcode dependent. Cells were pulse labeled with FlAsH and chased with ReAsH to follow newly synthesized protein. (A) Schematic representation of the time course of the procedure (more ...)
N-cadherin is lost from adherens junctions when β-actin translation is mislocalized
To assess the effect of mistargeting of β-actin translation on junction formation, we performed simultaneous ReAsH and N-cadherin staining on C2C12 cells. In contacting cells containing the FL transgene, which targets both translation sites and newly synthesized β-actin to the contact site, there was an enhancement of the amount of N-cadherin at the junction (). In contrast, contacting cells containing the ΔZIP transgene, which cannot target translation sites or newly synthesized β-actin, exhibit a decrease in the amount of N-cadherin at the junction (). When both populations were quantified and compared, there was an ~33% decrease in the strength of the N-cadherin signal at the cell contacts when β-actin translation sites were mistargeted ().
Figure 8. N-cadherin is lost from adherens junction when the translation of β-actin is mislocalized. (A) Indirect immunofluorescence image of N-cadherin staining of a cluster of C2C12 cells. The arrowheads point to the junction between a cell transfected (more ...)