In response to environmental stress, cytoplasmic mRNAs aggregate to form stress granules (SGs). SGs have mainly been studied indirectly using protein markers, but the real-time behavior of endogenous mRNAs in SGs remains uncertain. Here, we visualized endogenous cytoplasmic poly(A)+ mRNAs in living mammalian cells using a linear antisense 2′-O-methyl RNA probe. In arsenite-stressed cells, endogenous mRNAs aggregated in granules that colocalized with SGs marked by TIA-1–GFP. Moreover, analysis of mRNA dynamics using fluorescence recovery after photobleaching showed that approximately one-third of the endogenous mRNAs in SGs was immobile, another one-third was diffusive, and the remaining one-third was in equilibrium between binding to and dissociating from SGs, with a time constant of approximately 300 seconds. These dynamic characteristics of mRNAs were independent of the duration of stress and microtubule integrity. Similar characteristics were also observed from fos mRNA labeled with an antisense 2′-O-methyl RNA probe. Our results revealed the behavior of endogenous mRNAs, and indicated that SGs act as dynamic harbors of untranslated poly(A)+ mRNAs.
In eukaryotic cells, regulation of translation plays an important role in protein synthesis. When cells are exposed to various types of environmental stress, such as oxidative stress, heat shock or UV exposure, translation is reprogrammed and untranslated mRNAs are recruited to cytoplasmic foci called stress granules (SGs) (Anderson and Kedersha, 2008; Buchan and Parker, 2009). SGs do not have membranes, and their typical dimensions are 1–2 μm. Electron microscopy shows that SGs have a fibrillo-granule structure with a low level of compaction (Souquere et al., 2009). SGs typically contain stalled mRNAs, 40S ribosomal subunits, a number of translational initiation factors (such as eIF2α and eIF3) and RNA binding proteins (e.g. TIA-1 and PABP) (Souquere et al., 2009; Kimball et al., 2003; Mazroui et al., 2006; Kedersha et al., 2005; Kedersha et al., 1999). The assembly of SGs, which starts with the detachment of ribosomes from mRNA transcripts (Kedersha et al., 2005; Kedersha et al., 1999; Wek et al., 2006), requires the self-aggregation and RNA binding ability of TIA-1 and TIAR (Gilks et al., 2004; Kedersha et al., 1999). Microtubules also contribute to the formation of SGs (Fujimura et al., 2009; Nadezhdina et al., 2010). These findings indicate the involvement of SGs in the regulation of translation.
Although the SG is generally regarded as the site where mRNAs are remodeled for reinitiation, degradation or storage (Anderson and Kedersha, 2002; Anderson and Kedersha, 2009; Buchan and Parker, 2009), its functions have not been clarified. The dynamics of SG components have been examined to determine the physiological significance of SGs. Previous studies using fluorescence recovery after photobleaching (FRAP) suggest that some protein components of SGs rapidly shuttle in and out. For instance, TIA-1–GFP in SGs is completely mobile and is quite rapidly replaced within several seconds (Kedersha et al., 2000), whereas PAPB-I–GFP (Kedersha et al., 2000) and CPEB1–GFP (Mollet et al., 2008) are not completely mobile in SGs, although they also have high exchange rates. These GFP-fused proteins have revealed kinetic information of SG components in living cells that suggest SGs are highly dynamic sites. However, the real-time behavior of mRNAs, the main components of SGs, in stressed cells is still unclear, which limits a deeper comprehension of SG functionality.
Mollet et al. investigated the kinetics of MS2–GFP-tagged β-gal mRNA in SGs and found that it is also mostly mobile and that residence time in SGs is even shorter than that in the cytoplasm (Mollet et al., 2008). These kinetic data of a tagged mRNA do not support the hypothesized role of SGs as storage or refuge sites for mRNA. If mRNAs could not remain there, it would be difficult to explain the necessity of recruiting mRNAs to SGs. However, these results might not truly reflect the dynamics of endogenous mRNAs during stress, because MS2–GFP-tagged mRNAs have a different structure from native mRNA. Therefore, it is very important to target endogenous mRNAs and to elucidate their real-time behavior under stress to further understand the role of SGs.
In this study, SG function was explored by examining endogenous mRNAs in living cells. To visualize endogenous mRNAs, we used a linear antisense 2′-O-methyl RNA probe labeled with a fluorescent dye that can bind to native mRNA in a sequence-specific way (Molenaar et al., 2001; Molenaar et al., 2004; Ishihama et al., 2008; Okabe et al., 2011). A linear antisense probe was advantageous because of its ability to target native mRNA and its excellent hybridization kinetics in living cells (Okabe et al., 2011; Molenaar et al., 2001). Using this kind of probe, we directly visualized endogenous mRNAs in living cells and monitored the localization of mRNAs during the stress response in real time. Furthermore, we performed FRAP and inverse FRAP (iFRAP) to investigate the dynamics of endogenous mRNAs in SGs and to reveal the existence of mRNAs that remained in, and associated with, SGs. These observations are quite different from previous findings and support the hypothesis that SGs actively harbor mRNAs.
Endogenous mRNAs visualized using a linear antisense probe
To investigate the real time behavior of endogenous mRNAs in living cells during stress, we mainly targeted cytoplasmic poly(A)+ mRNAs, which are considered to be components of SGs (Kedersha et al., 2000; Kedersha et al., 1999; Souquere et al., 2009; Fujimura et al., 2009; Unsworth et al., 2010). A Cy3-labeled poly(U)22 2′-O-methyl RNA probe was used to detect poly(A)+ mRNAs in the cytoplasm (Fig. 1A). We chose 2′-O-methyl RNA as the backbone of the probe, because this artificial nucleic acid is resistant to nucleases in living cells and it has a high affinity for nucleic acids (Molenaar et al., 2001; Okabe et al., 2011). The poly(U)22 2′-O-methyl RNA probe has previously been used to detect nuclear poly(A)+ RNA (Molenaar et al., 2004; Ishihama et al., 2008). We further developed the probe for use with cytoplasmic mRNAs by combining the probe with streptavidin, which helps to prevent entry to the nucleus (Okabe et al., 2011). In our previous study, endogenous fos mRNA in SGs was selectively detected by observing fluorescence resonance energy transfer (FRET) from two linear antisense probes, which demonstrates that the poly(U)22 probe can be used to investigate endogenous poly(A)+ mRNAs in SGs (Okabe et al., 2011).
The poly(U)22 2′-O-methyl RNA probe was microinjected into the cytoplasm of living COS7 cells. As shown in Fig. 1B, fluorescence was mostly observed in the cytoplasm. To confirm the hybridization of probes and mRNAs in living cells, fluorescence correlation spectroscopy (FCS) was performed. FCS analysis can estimate the size of a molecule by quantitatively measuring its diffusion in living cells (Magde et al., 1972; Yoshida et al., 2001; Breusegem et al., 2006). When probes bind to mRNAs, diffusion time increases compared with that of unbound probes. The poly(A)18 probe, which has a different sequence but similar structure to the poly(U)22 probe, was used as a control. As can be seen in Fig. 1C, the poly(U)22 probe diffused more slowly than the poly(A)18 probe, which indicated its binding with poly(A)+ mRNAs. Fluorescence autocorrelation functions obtained from FCS measurements in living cells showed that 85% of the injected poly(U)22 probe bound to mRNAs. In arsenite-treated cells, the binding ratio in the cytoplasm outside SGs was 91% and that inside SGs was 88%. The injected poly(A)18 probe did not bind to mRNA. These FCS results demonstrated the hybridization between the probe and mRNAs, which confirmed that the fluorescence of the poly(U)22 probe represented endogenous poly(A)+ mRNAs.
A linear antisense probe is also advantageous as it has a fast hybridization reaction with target mRNAs and is, therefore, applicable to real-time monitoring of endogenous mRNAs in living cells. In vitro evaluation of the poly(U)22 probe and mRNA binding in solution using fluorescence spectroscopy indicated that the poly(U)22 probe could bind to mRNAs within 10 seconds (at the concentration of 10 nM; supplementary material Fig. S1). Our previous study showed that fos antisense probe, a probe that could specifically bind to a single species of mRNA, hybridized with its target mRNA within 1 minute in living cells (Okabe et al., 2011). Because the poly(U)22 probe could hybridize nearly 25 times faster than the fos antisense probe (10 nM) in solution, the binding process of the poly(U)22 probe in living cells was estimated to be less than 1 minute. Thus, the poly(U)22 probe can be applied to monitor the rapid localization of endogenous poly(A)+ mRNAs in living cells during stress.
Real-time imaging of endogenous mRNAs during stress
Using the poly(U)22 probe, we monitored the aggregation of endogenous poly(A)+ mRNAs to SGs in living cells during stress. After stress was induced by 0.5 mM arsenite, the localization of cytoplasmic mRNAs was monitored for 60 minutes using epifluorescence microscopy (Fig. 2A). We observed that endogenous mRNAs rapidly aggregated (within 20 minutes) to form foci in response to stress (Fig. 2A, Ars 20 minutes). By 60 minutes after arsenite addition, ~10% of poly(A)+ mRNAs had accumulated in SGs. A quantitative analysis of mRNA aggregation in SGs was performed (Fig. 2B,C). The average size of the granules increased chronologically (Fig. 2B, filled circles), whereas the average number of the granules in each cell initially increased, but then began to decrease 30 minutes after the stress was induced (Fig. 2C, filled circles). These results indicated that there are two steps involved in mRNA accumulation in SGs: a number of small granules first form and then these gradually merge to become bigger SGs. Interestingly, SG formation in two steps has also been observed when microtubules were destroyed (Fujimura et al., 2009). In control experiments in the absence of stress, neither the size nor the number of granules changed over 60 minutes (Fig. 2B,C, open triangles). By contrast, cells injected with the poly(A)18 probe did not show changes despite being subjected to stress (Fig. 2D,E; supplementary material Fig. S2A).
Next, we expressed GFP-tagged TIA-1, which is a well-known protein component of SGs, to confirm that the granules in which mRNAs aggregated were SGs, and to compare the aggregation processes of the protein components and the endogenous mRNAs. After arsenite stress was induced, the accumulation of TIA-1–GFP and mRNAs was monitored simultaneously (Fig. 3A). The two kinds of fluorescence closely overlapped throughout the granule formation process. Also, the average size and number of SGs, indicated by TIA-1–GFP, showed similar changes to that observed for poly(A)+ mRNAs (Fig. 3B–E). These results showed that aggregation of mRNAs in SGs occurred simultaneously with that of TIA-1, indicating that before these two components were recruited to the visible SGs, they could have already started to interact in the cytoplasm. In control experiments, cells expressing GFP alone were induced with arsenite stress but accumulation of mRNA was not observed (supplementary material Fig. S2B).
By removing the arsenite stress we also observed the release of mRNAs from SGs that had formed in response to stress. In most cells (21 out of 22 cells), SGs were disassembled within 90 minutes after the removal of stress (supplementary material Fig. S3). During this process, small SGs appeared to dissolve earlier than large ones. Furthermore, it has been reported that mRNAs must be released from polysomes before being recruited to SGs (Kedersha et al., 2000; Mollet et al., 2008; Buchan et al., 2008). We treated cells with emetine before or after arsenite treatment. Emetine inhibits mRNA translation by binding to the 40S subunit of the ribosome (Jimenez et al., 1977). Our results showed that emetine treatment inhibited endogenous mRNAs aggregating to arsenite-induced SGs (supplementary material Fig. S4A). In addition, mRNAs that had accumulated in SGs under stress were gradually released as a result of emetine treatment (supplementary material Fig. S4B).
The dynamics of endogenous poly(A)+ mRNAs in SGs investigated by FRAP analysis
Previous studies have suggested that both GFP-tagged protein and GFP-tagged RNA are mostly mobile and exchange rapidly between SGs and the cytoplasm (Kedersha et al., 2000; Mollet et al., 2008). Our FRAP experiment also showed that TIA-1–GFP was mostly mobile (98%) in SGs (supplementary material Fig. S5). However, endogenous mRNAs might not behave in the same way as GFP-tagged protein and GFP-tagged RNA. To determine the dynamics of endogenous mRNAs in SGs, we performed a FRAP analysis (Fig. 4). Endogenous mRNAs bound with the poly(U)22 probe in a single SG were photobleached and the fluorescence recovery was recorded (Fig. 4A).
First, the diffusion of poly(A)+ mRNAs in the cytoplasm without stress was investigated and produced a fluorescence recovery of 90% (Fig. 4B). The time course of fluorescence recovery was fitted to a single exponential curve with a time constant of 38 seconds. We also performed the experiment in the cytoplasm of cells under stress; ~94% of poly(A)+ mRNAs in the cytoplasm were mobile and the time constant was 36 seconds (Fig. 4C). We noticed that fluorescence recovery was not 100% and we presumed that this might represent mRNAs that were bound to the cell cytoskeleton or to other cellular structures, such as processing bodies.
Next, FRAP of poly(A)+ mRNAs in a single SG was analyzed (Fig. 4A). As shown in Fig. 4D, only 66% of the fluorescence intensity recovered in SGs induced by a 60 minute treatment of arsenite stress. This result suggested that ~34% of the endogenous poly(A)+ mRNAs in SGs was immobile (unrecoverable part). Interestingly, two protein components of SGs, PAPB-I–GFP and CPEB1–GFP, are not completely mobile in SGs (Kedersha et al., 2000; Mollet et al., 2008). These results suggest that there is machinery in SGs that retains components during stress. Moreover, the time course of fluorescence recovery was biphasic, with a fast phase of 40 seconds (τ1) and a slow phase of 275 seconds (τ2). The proportion of τ1 was ~38% for all mRNAs in SGs, and that of τ2 was ~28%. The fast phase had a similar time constant to that of cytoplasmic poly(A)+ mRNAs. This could include mRNAs outside SGs, because epifluorescence microscopy also captures the cellular region above or below the plane of focus. In addition, examination of the ultrastructure of SGs by electron microscopy indicated that there is some interspace in their granular structure (Souquere et al., 2009). Therefore, the fast phase of recovery might also include cytoplasmic mRNAs passing through the SGs. The slow phase was not observed in the cytoplasm, but only appeared in SGs. This phase could represent either a fraction of poly(A)+ mRNAs that are slowly diffusing or a fraction that is binding to and dissociating from the SG machinery.
The dynamics of endogenous mRNAs in SGs is independent of the duration of stress treatment, microtubule integrity and the species of mRNA
Real-time imaging indicated that there were two steps in the assembly of SGs (Fig. 2), which raised the possibility that the status of mRNAs in SGs might be different depending on the stage of SG formation. Therefore, to test the mobility of mRNAs in early stage SGs, we investigated the dynamics of poly(A)+ mRNAs after 30 minutes of arsenite treatment (Fig. 5A). The results were similar to those following 60 minutes of arsenite treatment: 65% of overall fluorescence recovered, with biphasic time constants (τ1 and τ2 were 38 seconds and 299 seconds, respectively) and the remaining 35% was unrecovered. These data indicated that the dynamics of endogenous mRNAs in SGs does not depend on the duration of stress treatment.
Recently, it has been reported that microtubule integrity is required for the spatial movement of SGs, but is unnecessary for the formation of initial SG foci (Fujimura et al., 2009; Nadezhdina et al., 2010). We examined the influence of microtubule integrity on the mobility of mRNAs in SGs. Cells were treated with nocodazole to depolymerize microtubules (Fujimura et al., 2009) and then arsenite stress was induced (0.5 mM, 60 minutes). Unlike the untreated cells (Fig. 2), nocodazole-treated cells formed smaller SGs that rarely merged (supplementary material Fig. S6). Analysis of single photobleached SGs revealed that even though the microtubules were destroyed, 33% of the poly(A)+ mRNAs in the SGs remained immobile (Fig. 5B). The time constants were 37 seconds (τ1) and 278 seconds (τ2), and the proportion of τ2 was ~28%. Overall, the dynamics of endogenous mRNAs in SGs was independent of microtubule integrity.
To investigate whether a single species of mRNA also undergoes similar dynamics in SGs, we performed a FRAP experiment on a single species of mRNA (Fig. 5C). We visualized endogenous fos mRNA using a linear antisense 2′-O-methyl RNA probe that had been developed previously (Okabe et al., 2011) and its dynamics were analyzed in SGs. Thirty percent of the fos mRNA in SGs was immobile and the time course of fluorescence recovery was biphasic with τ1=31 seconds and τ2=345 seconds. The proportion of τ1 was ~29%, and that of τ2 was ~41%. These results indicated that endogenous fos mRNA behaves in a similar way to that of endogenous poly(A)+ mRNAs. Taken together, the dynamics of endogenous mRNAs in SGs is independent of the duration of stress treatment and the integrity of microtubules. Furthermore, similar dynamic behavior was also observed for a single species of mRNA (Fig. 5D).
A fraction of endogenous mRNAs in SGs undergoes binding to and dissociation from the SG machinery
The fast phase of fluorescence recovery could represent the free diffusion of mRNAs, whereas the slow phase could represent either the slow diffusion of mRNAs or mRNAs binding to and dissociating from the SG machinery. To determine the underlying processes of these two phases, we varied the size of the photobleached area when performing FRAP experiments. By changing the diameter of the photobleached spot, the time constant for freely diffusing mRNA should differ according to the spot size. By contrast, mRNA bound to the SG machinery should not be under such an influence. With the enlargement of the spot diameter (from 3 μm to 4.5 μm and 6 μm), τ1 increased accordingly, but τ2 remained almost unchanged (Fig. 6). These results indicated that τ1 represents free diffusion, whereas τ2, which was independent of the photobleached area, represents mRNAs binding to and dissociating from the SG machinery.
To further verify the significance of τ2, we performed iFRAP experiments on SGs (Fig. 7). The fraction of endogenous mRNAs in SGs during τ2 is in equilibrium between binding to and dissociating from the SG machinery; therefore, the slow phase identified by the FRAP experiment represented the binding process, and the iFRAP experiment should show the dissociation process. An entire cell injected with poly(U)22 probe was photobleached except for a single SG, and then the relative fluorescence intensity of the SG was recorded (Fig. 7A). As shown in Fig. 7B, 29% of the endogenous poly(A)+ mRNAs remained in the SG, which was in agreement with the FRAP experiment (34% immobile). Furthermore, the time course of relative fluorescence intensity was fitted to a single exponential curve with a time constant of 263 seconds. This time constant was almost the same as the slow phase in the FRAP experiment, which confirmed that a fraction of endogenous mRNAs in SGs are in equilibrium between binding to and dissociating from the SG machinery. The fast phase observed in the FRAP experiment was not present here because of a long photobleaching time (40 seconds).
In eukaryotic cells, aggregation of untranslated mRNAs and associated proteins to SGs is a key process in the modulation of translation during stress. This study is the first to directly investigate SGs by monitoring endogenous poly(A)+ mRNAs in living cells using a linear antisense 2′-O-methyl RNA probe. Our previous study showed that a linear antisense probe could selectively bind to a specific endogenous mRNA in the cytoplasm (Okabe et al., 2011). In this study, we monitored the localization of endogenous cytoplasmic mRNAs during SG formation and captured their real-time behavior in the SGs to explore their physiological role in response to stress. Our FCS experiments showed that most of the poly(U)22 probe (~90%) bound to endogenous poly(A)+ mRNAs.
The dynamics of endogenous mRNAs revealed by FRAP analysis indicated that one-third of poly(A)+ mRNAs in SGs were immobile. This suggests that there should be machinery that retains mRNAs in SGs during stress. Furthermore, a fraction of poly(A)+ mRNAs, which was considered to be transiently associated with the SG machinery, was detected by both FRAP and iFRAP analysis. Approximately 30% of poly(A)+ mRNAs in SGs were in equilibrium between binding to and dissociating from SGs with a time constant of ~300 seconds. Furthermore, mRNAs that had accumulated in SGs under stress were gradually released after emetine treatment, which blocks rebinding of cytoplasmic mRNAs to SGs. We also investigated the mechanism by which endogenous mRNAs bind to the SG machinery. First, we revealed that the association of endogenous mRNAs with the SG machinery is independent of the maturity of SGs, and, interestingly, it can also be observed for a single species of mRNA. These results indicate that the harboring mRNAs in SGs is crucial for SG function. Second, the kinetics of endogenous poly(A)+ mRNAs we measured were found to be different from those of MS2–GFP-tagged mRNAs (Mollet et al., 2008). Although MS2–GFP-tagged mRNAs were recruited to SGs, they did not remain there and showed no re-association with the SG machinery. This implies that both endogenous and GFP-tagged mRNAs can be recruited, but possibly they can be distinguished by the machinery in SGs, and hence the MS2–GFP-tagged mRNAs failed to remain in SGs.
Previous studies on SG protein components suggested that they undergo a highly dynamic shuttling in and out (Kedersha et al., 2000; Mollet et al., 2008). Our results for TIA-1–GFP in SGs were consistent with this view. Taken together with our data showing that mRNA movement in SGs was considerably restricted, the highly dynamic kinetics of these protein components might indicate many mRNAs from the cytoplasm being recruited to SGs. This phenomenon – that RNA binding proteins showed different kinetics from endogenous mRNAs – has also been observed in the nucleus: PAPB–GFP moves in and out of speckles faster than poly(A)+ RNA (Molenaar et al., 2004). The persistence of endogenous mRNAs in SGs supports the hypothesis that SGs might function as a triage or a storage site of mRNAs during stress (Anderson and Kedersha, 2002; Anderson and Kedersha, 2008; Buchan and Parker, 2009). Moreover, the endogenous mRNA binding to and dissociating from the SG machinery might also indicate a role of SGs as a remodeling site, where mRNAs are processed, followed by exchange with unprocessed mRNAs.
Another interesting feature of SGs is gathering together and merging during assembly, the physiological significance of which remains unclear. In our study, the kinetics of mRNAs in SGs before and after merging did not differ. These data indicate that SGs can function without merging with each other. Possibly, the advantage of SG merging might be to condense their components to enhance reaction efficiency.
In conclusion, we successfully monitored the dynamic distribution of endogenous cytoplasmic poly(A)+ mRNAs in living mammalian cells under stress using a linear antisense probe. The FRAP and iFRAP data suggest that a portion of the poly(A)+ mRNAs in SGs are immobile and that a portion are in equilibrium between association with and dissociation from SGs. We suggest that the retention of mRNAs in SGs might contribute to translational regulation and that it might ensure enough time for mRNAs to be remodeled for re-initiation of translation, degradation or storage. Determining the details of the SG machinery requires further investigation; however, our observations of endogenous mRNAs provide a new angle for future studies of SGs and of the regulation of translation during stress. In addition to cytoplasmic mRNAs, small RNAs and argonaute protein (the protein that associates with microRNAs) are also enriched in SGs during stress. Moreover, localization of argonaute protein to SGs depends on the presence of microRNAs (Leung et al., 2006). Therefore, microRNA is implicated in having an important role in the reprogramming of mRNA translation in SGs (Leung and Sharp, 2007; Leung and Sharp, 2010; Leung et al., 2006). Our results indicate that endogenous mRNAs harbored in SGs are regulated in a particular way. Clarification of the dynamics of endogenous mRNAs in SGs will provide a new approach to reveal the function of microRNAs in response to stress.
Materials and Methods
COS7 cells were cultured in DMEM (Gibco, Carlsbad, CA, USA), supplemented with 10% fetal calf serum (Gibco) at 37°C in 5% CO2. Before use, cells were transferred to 2 ml medium without Phenol Red (Gibco) in 35 mm glass culture dishes (ASAHI Techno GLASS, Tokyo, Japan). During all experiments, cells were maintained at 37°C on the microscope stage, using a stage plate heater (TOKAI HIT, Fujinomiya, Japan) and a microscope objective lens heater (TOKAI HIT).
Plasmid and reagent treatments
The TIA-1–GFP plasmid was provided by Paul Anderson (Harvard Medical School, USA). COS7 cells were transfected with the plasmid using FuGENE 6 (Roche Diagnostics, Indianapolis, IN) and cultured for 18 hours before use.
To induce arsenite stress, cells were incubated with 0.5 mM sodium arsenite (NaAsO2; Sigma-Aldrich, Steinheim, Germany) for 60 minutes. For microtubule depolymerization, nocodazole (Sigma-Aldrich) was used at 2 μg/ml for 2 hours before stress was induced and was maintained after stress induction (Fujimura et al., 2009).
Preparation of 2′-O-methyl RNA probe
A poly(U)22 2′-O-methyl RNA probe was synthesized by Japan Bio Services (Saitama, Japan) as a 22 mer with Cy3 at its 5′ end and biotin at its 3′ end. A Cy3-labeled poly(A)18 probe was used as a control and was prepared in the same way. A 2′-O-methyl linear antisense probe specifically detecting fos mRNA (5′-Cy3-UCUAGUUGGUCUGUCUCCGC–biotin-3′) was designed as previously described (Okabe et al., 2011) and was purchased from FASMAC (Kanagawa, Japan). Streptavidin was allowed to react with the probes at room temperature for 20 minutes to bind to the 3′ biotin to prevent the probes from accumulating in the nucleus.
Probes were dissolved in microinjection buffer containing 80 mM KCl, 10 mM K2PO4 and 4 mM NaCl pH 7.2 and were then filtered using an Ultrafree-MC filter (Millipore, Billerica, MA). The poly(U)22 probe was used at a final concentration of 3 μM, except for FCS under stress conditions, when the probe was used at 1 μM because 3 μM resulted in too many fluorescent molecules in the SGs to be accurately measured by FCS. The poly(A)18 probe was also prepared at 3 μM. The filtered probe solution was microinjected into the cytoplasm of cells. Microinjection was performed using a FemtoJet injector (Eppendorf, Hamburg, Germany) equipped with a micromanipulator (Eppendorf) and a glass capillary microneedle (Femtotips II; Eppendorf). The injection pressure was ~20 hPa. After microinjection, cells were observed with a phase contrast microscope (IX 70; Olympus, Tokyo, Japan) and obviously damaged cells were excluded from further analysis. All analyses were performed at least 10 minutes after microinjection to ensure that the hybridization between the probes and the mRNAs had achieved equilibrium.
Microinjection, epifluorescence microscopy and FRAP were performed on an inverted microscope (IX 70, Olympus) using a 60× oil immersion objective lens (UplanApo, NA 1.40; Olympus). Images were taken using a cooled CCD camera (ORCA-ER, Hamamatsu Photonics, Shizuoka, Japan), quantitatively analyzed using AQUA-Lite ver. 10 (Hamamatsu Photonics) and further compiled using Adobe Photoshop ver. 6.0. For epifluorescence microscopy, Cy3 was excited with a green solid-state laser (532 nm, 100 mW Compass 315M-100; Coherent, Santa Clara, CA) of 1.4 mW. A dichroic mirror (565LP; Chroma Technology, Rockingham, VT) and an emission filter (610/75M; Chroma Technology) were used. GFP was excited with a blue laser (488 nm, 30 mW, Sapphire 488-30; Coherent) of 1.1 mW. A dichroic mirror (Q505LP; Chroma Technology) and an emission filter (HQ535/50x; Chroma Technology) were used.
In FRAP experiments, COS7 cells were microinjected with a poly(U)22 probe, followed by 60 minutes of arsenite treatment (0.5 mM) to induce the assembly of SGs. After that, a single SG was photobleached in a circle of 3 μm diameter at maximum power of the green laser (28.5 mW) for 10 seconds. Fluorescence recovery was followed for 10 minutes. The cytoplasm outside SGs was also bleached using the same conditions, with or without stress. For the FRAP experiment on TIA-1–GFP SGs, COS7 cells were transfected with the plasmid and cultured for 18 hours before treatment with arsenite for 60 minutes. After that, a single SG was photobleached in a circle of 3 μm in diameter at maximum power of the blue laser (11.1 mW) for 20 seconds, and the fluorescence recovery was followed for 1 minute. Fluorescence recoveries were recorded using epifluorescence microscopy. Pre-bleach, bleach and post-bleach steps were unlinked so that FRAP and epifluorescence microscopy had to be switched manually. Therefore, some recovery had already occurred when recording was started. Background was determined from the average fluorescence intensities outside the fluorescently labeled cells, and was subtracted. The ratio of the fluorescence intensity of the photobleached spot to that of the whole cell was calculated for each time point [F(t)spot/F(t)whole cell], and the fluorescence recovery ratio was determined as the ratio of each time point to the initial time point before photobleaching [F(0)spot/F(0)whole cell] (Eqn 1). The time course of fluorescence recovery was fitted by Eqn 2 as previously reported (Darzacq et al., 2007). Fitting was performed using KaleidaGraph 3.6 (Synergy Software, Reading, PA, USA).
The fluorescence correlation spectroscopy (FCS) system consisted of an inverted microscope (IX70, Olympus) equipped with a water immersion objective lens (UplanApo, 60×, NA 1.20; Olympus), a green laser (532 nm, COMPASS 415M, Cohrent), an avalanche photodiode (SPCM-AQA-14PC, PerkinElmer Optoelectronics, Fremont, CA) and a digital autocorrelator (ALV-6010/160, ALV-GmbH, Langen, Germany). A pinhole 30 μm in diameter was set in front of the photodiode.
Cells were injected with Cy3-labeled probes and the spots to be measured were in cytoplasmic space outside or inside SGs. The focal point was fixed where maximum fluorescence intensity along the z-axis was observed. Samples were excited by the green laser (1.56 μW), and the fluorescence from the detection spot was separated by an emission filter (Q560LP, Chroma Technology). The fluorescence autocorrelation function [G(τ)] was measured for 10 seconds, repeated three times, and was fitted by Eqn 4 using Origin software (Origin Lab, Northampton, MA).
We thank Paul Anderson (Brigham and Women's Hospital, Boston, MA) for kindly providing the GFP-TIA-1 plasmid. We also thank Yanting Song for helping to operate the FRAP system.
This work was supported by a Grant-in-Aid for Young Scientists from the Ministry of Education, Culture, Sports, Science and Technology of Japan [21770162 to K.O.]; the Japan Society for the Promotion of Science (JSPS) through its Funding Program for World-Leading Innovation R&D in Science and Technology (FIRST Program) [T.F.].