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Laboratory of Cellular and Molecular Biology, National Institute on Aging-Intramural Research Program, National Institutes of Health, Baltimore, Maryland 21224
Received 2 December 2005/ Returned for modification 27 December 2005/ Accepted 3 January 2006
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ABSTRACT |
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INTRODUCTION |
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TIA-1 and TIAR have been proposed to play a general role as translational repressors (1, 2). In the absence of stress, eukaryotic translation initiation factor 2 (eIF2)-GTP-tRNAMet ternary complexes are available in the cell, and a canonical preinitiation complex (comprising eIF1, eIF2, eIF3, eIF5, and the 40S ribosomal subunit) forms at the 5' end of capped mRNAs. Following the recognition of an initiation codon, the 60S subunit is assembled, displacing the initiation factors and forming a functional ribosome to initiate translation. In cells exposed to a variety of environmental stress agents, phosphorylation of eIF2
by a family of kinases (PKR, PERK, GCN2, and HRI) prevents the exchange of GTP, thereby lowering the concentration of the eIF2-GTP-tRNAMet ternary complex (recently reviewed in reference 19). Under conditions of cellular damage, TIAR and TIA-1 have been postulated to function as translational repressors by associating with eIF1, eIF3, and the 40S ribosomal subunit and forming noncanonical preinitiation complexes that are translationally inactive (2). The self-aggregating properties of TIA-1 and TIAR were further proposed to facilitate the accumulation of the translationally inactive preinitiation complexes into discrete cytoplasmic foci called stress granules (SGs). Given the presence of RBPs that influence mRNA stability (such as tristetraprolin and HuR) and translation (TIA proteins) at SGs, these foci have been hypothesized to function as dynamic sites of mRNA triage during stress, wherein molecular decisions are made regarding the composition of mRNA ribonucleoprotein (RNP) complexes and their subsequent engagement with the translation or degradation machineries (22, 24).
A related but distinct role for TIA-1 and TIAR has been suggested to specifically mediate the translational silencing of ARE-containing mRNAs, including those that encode tumor necrosis factor alpha (TNF-), matrix metalloproteinase 13 (MMP13), cyclooxygenase 2, and ß2-adrenergic receptor (12, 18, 20, 45). For example, TIA-1 was demonstrated to inhibit the translation of TNF- (2, 35), as lipopolysaccharide-stimulated macrophages that either lacked or expressed TIA-1 displayed similar steady-state levels of total TNF- mRNA, but the relative abundance of the TNF- mRNA in the actively translating polyribosomes was severalfold higher in TIA-1–/– macrophages than in wild-type macrophages, selectively elevating TNF- production (35). Similarly, a recent search for global TIA-1 target mRNAs revealed that TIA-1 suppressed the translation of specific subsets of bound transcripts (30). According to Anderson and coworkers, the specific translational suppression of ARE-containing TIA-1/TIAR target mRNAs is likely based upon the increased chance that these RBPs will assemble a translationally silent complex on such mRNAs (3).
Recently, we sought to identify TIAR target mRNAs at a transcriptome-wide level. During the course of these studies, we observed that TIAR silencing resulted in a global increase in protein translation. In addition, the marked decline in protein biosynthesis seen in control populations following exposure to low-level UVC (short-wavelength UV light) irradiation was largely relieved in the TIAR-silenced populations, wherein UVC-irradiated cells displayed significantly elevated translation rates. The low dose of UVC irradiation employed here failed to significantly increase the phosphorylation levels of eIF2, in keeping with the dose dependence of the UVC-triggered phosphorylation of eIF2 (14, 44). Subsequent analyses to elucidate global subsets of TIAR target mRNAs, based on the immunoprecipitation of RNP complexes from colon cancer cells, revealed the putative association of TIAR with many mRNAs encoding translation factors and other proteins involved in translation. We directly examined these RNP interactions and investigated the consequent influence of TIAR on global protein translation. Based on our findings, we propose that the TIAR-mediated inhibition of translation factor expression contributes to implementing a translationally repressed state following stress stimulation.
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MATERIALS AND METHODS |
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Northern and Western blot analyses.
For Northern blot analysis, oligonucleotides complementary to EIF4E2, EEF1B2, or 18S (CAGGACGTACCATGTGGCTATAAAACCTCCAGAACTGCTCC, CCCACTGGAACTAGTTTAGATGAGCCCCAGACTAAGCCGT, and ACGGTATCTGATCGTCTTCGAACC, respectively) were end labeled using [-32P]dATP and terminal transferase, while a c-Myc cDNA fragment was labeled using random primers, [-32P]dATP, and Klenow enzyme. The resulting signals were visualized with a PhosphorImager (Molecular Dynamics).
For Western blot analysis, cytoplasmic lysates (20 µg) and nuclear lysates (10 µg) (prepared using digitonin, as previously described [26]), as well as whole-cell lysates (10 µg), were size separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. Blots were probed with monoclonal antibodies recognizing p-eIF2 (Cell Signaling), c-Myc (Sigma), ß-tubulin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or -tubulin (Santa Cruz Biotechnology); ß-actin (Abcam); or polyclonal antibodies recognizing eEF1B (ProteinTech Group, Inc.), eIF4E (Cell Signaling), TIAR, TIA-1, or hnRNP C1/C2 (Santa Cruz Biotechnology). No antibodies were available to detect eIF4A. Following incubation with the appropriate secondary antibodies, signals were detected with the ECL reagent (Amersham).
RT-PCR. RNA was purified from immunoprecipitation (IP) material or sucrose gradient fractions and was reverse transcribed using oligo(dT) and SSII RT (Invitrogen). The resulting material was amplified by either conventional PCR or quantitative ("real-time") PCR (qPCR) analysis using gene-specific primer pairs (see below); the resulting fragments were all 150 to 300 bp in length. An Applied Biosystems 7300 instrument and SYBR green PCR master mix (Applied Biosystems, Foster City, CA) were used to carry out the qPCR analysis.
Oligonucleotides used for conventional PCR- and qPCR-mediated detection of reverse-transcribed mRNAs in IP material and in pooled fractions from sucrose gradients. Oligonucleotides used for conventional PCR- and qPCR-mediated detection of reverse-transcribed mRNAs in IP material and in pooled fractions from sucrose gradients are as follows: GAGGACTTGTTGCGGAAACG and CCAAAGTCCAATTTGAGGCAGT for MYC mRNA, AAGCCAGATGTTGGCCATGAA and TGTTGTTTCCAAAATGTAAGTCACC for EIF3SE, GCTCAATCTCTGGGGGCTGA and CCTTCTTTTGCCTCAAAAAGTGAC for EIF4A1, GTGCCTCCACTGATGCAGGA and CCTAGGGCGAAGGTGGCTTT for EIF4EBP1, GAAGCTGGCGTCATCGGAGT and CTGCTGCGTGTCAGCCTGTT for EIF4EL3, CCGGCACACAAAGTGGGTTA and GACAGCTCATGTGTTGCCCTTT for E2F3, TCCGGTCAGGGTCAAGTCGT and CCCCCTCTGCCAATTCTGTG for EIF4A2, GCCGGAACCTCCTCAGCTCT and TTGTGGCAAGCCAGATGTCC for SUI1, TGCTTGTGGGATGCAGTGGT and ACTGGGAAGGGGTCCCTCAG for ARF3, CCAGCTCCTTGTTCCCAGGTT and CCGGTGAAGTCTCCCACGTT for ETF1, ATCTGGGGCCCATACTGGTT and GAGGGGGACAAGGCTGTAGAA for PLK, CCCAGGACCTCATCTCCAAA and GGAGGTAGAAAACAGATAAGGGAACA for STK12, CGTGGACTTCGTACCGCTTC and AAAACCACCGGGGATCTAGC for ENO1, ACTGCGCCCTTAACTGCATC and AGAAGCACCAAACGTGACCA for ID1, and CTCCCCTAGGCGCTGTTCTT and GAGGCAAGAGGAGGGGAGAA for SFN.
Binding assays: biotin pull-down assay and IP followed by reverse transcription (RT)-PCR. For biotin pull-down assays, PCR fragments containing the T7 RNA polymerase promoter sequence (T7) were used as a templates for in vitro transcription of the GAPDH 3'-untranslated region (3'UTR) and the MYC, EIF4A, EIF4E, and EEF1B coding region (CR) and 3'UTR using biotinylated CTP (below). Four micrograms of biotinylated transcripts was then incubated with 80 µg of total lysate for 30 min at 25°C, complexes were isolated with paramagnetic streptavidin-conjugated Dynabeads (Dynal), and the pull-down material was analyzed by Western blotting.
IP of RNP complexes of endogenous TIAR and endogenous mRNAs was carried out using methodologies previously described for other RNA-binding proteins (29, 40). In brief, 20 million RKO cells were collected per time point, and lysates were used for IP for 2 h at 25°C in the presence of excess (30 µg) IP antibody (either a goat polyclonal anti-TIAR antibody [C-18; Santa Cruz Biotechnology] or control goat immunoglobulin G [IgG] [BD Life Sciences]). RNA in the IP material was reverse transcribed and subjected to both conventional PCR and qPCR to assess the abundance of the products listed in Fig. 2 (the oligomer pairs are listed below).
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Analysis of newly translated protein. To quantify whole-cell translation, 100,000 cells that were either left untreated or irradiated with 20 J/m2 UVC were incubated at various time points with 50 µCi L-[35S]methionine and L-[35S]cysteine (Easy Tag EXPRESS; NEN/Perkin-Elmer) per 60-mm plate for 20 min; radiolabel incorporation was then monitored by both size fractionation of lysates (by using SDS-PAGE followed by visualization of the transferred lysates with a PhosphorImager) and trichloroacetic acid (TCA) precipitation. To quantify the translation of specific proteins, newly translated c-Myc, eIF4E, and eEF1B were investigated by incubating 106 cells with 1 mCi L-[35S]methionine and L-[35S]cysteine per 60-mm plate for 20 min, whereupon cells were lysed using TSD lysis buffer (50 mM Tris [pH 7.5], 1% SDS, and 5 mM dithiothreitol). IP reactions were carried out using 300 µg of lysate per sample for 1 h at 4°C using appropriate antibodies and IgG as a control; the methodology was described previously (31, 32). Following extensive washes in TNN buffer (50 mM Tris [pH 7.5], 250 mM NaCl, 5 mM EDTA, 0.5% NP-40), the IP material was resolved by 12% SDS-PAGE, transferred onto PVDF filters, and visualized and quantified using a PhosphorImager (Molecular Dynamics).
cDNA array data for TIAR target transcripts in IP material. cDNA array data for TIAR target transcripts in IP material are available at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE1433.
Immunofluorescence. RKO cells cultured on coverslips were fixed in 2% paraformaldehyde and permeabilized in 0.4% Triton X-100 (each 15 min). After incubation in blocking buffer (1 h in phosphate-buffered saline containing 2% bovine serum albumin and 0.1% Tween 20), coverslips were incubated with either goat anti-TIAR or goat anti-TIA-1 (Santa Cruz Biotechnology) in blocking buffer (16 h, 1:100 dilution), washed with blocking buffer, and further incubated with Alexa Fluor 568 donkey anti-goat IgG (heavy plus light chains) (Molecular Probes) (1 h, 1:500 dilution). After washes with blocking buffer, coverslips were mounted in Vectashield (Vector Laboratories) and visualized with a Zeiss LSM410 confocal microscope. Representative photographs from three independent experiments are shown in the figures.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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phosphorylation in the inhibition of global protein translation, we hypothesized that the levels of phosphorylated eIF2 might differ between these transfection groups in a UVC-dependent manner. Unexpectedly, however, while eIF2 phosphorylation was readily detectable in these cultures, it did not appear to be substantially elevated in either culture, whether left unirradiated or exposed to 20 J/m2 UVC (Fig. 1C); these findings are in keeping with previous observations that exposure to low levels of UVC did not perceptibly increase eIF2 phosphorylation (>50 J/m2 was needed to attain measurable increases in eIF2 phosphorylation) (14, 44). Embryo fibroblasts derived from either wild-type or TIAR knockout mice also revealed no increase in eIF2 phosphorylation following 20 J/m2 UVC irradiation (see Fig. S2 in the supplemental material). These findings suggested that TIAR was capable of influencing global protein translation without an overt increase in eIF2 phosphorylation and prompted efforts to identify endogenous TIAR target mRNAs. TIAR specifically associates with mRNAs encoding translation factors and translation-related proteins. First, we immunoprecipitated TIAR RNP complexes from RKO cell lysates and performed microarray-based identification of TIAR target mRNAs (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE1433). These experiments revealed the putative association of TIAR with many transcripts encoding translation factors and other proteins involved in translation; as anticipated, these transcripts contained AREs in their 3'UTRs (Fig. 2A; see Table S1 in the supplemental material). Figure 2B depicts the levels of TIAR, as detected by Western blotting of the IP material (lysate input was even; see Fig. S3 in the supplemental material). To validate these interactions, the presence of putative TIAR target mRNAs in the IP material was tested by performing RT-PCR amplification using gene-specific primers. As shown, the PCR products corresponding to TIAR "target mRNAs" were considerably more abundant in the TIAR IP lanes than in the IgG lanes (Fig. 2C), supporting the specificity of the interaction between TIAR and such mRNAs within TIAR RNP complexes. Furthermore, control mRNAs that were not TIAR targets ("nontarget mRNAs") revealed no amplification in either IP (as with PLK and ID1) or showed background amplification that was comparable between the two IP groups (as seen with GAPDH and STK12, which were found as low-level contaminating mRNAs).
TIAR contributes to the UVC-imposed translational repression of target mRNAs. The interaction of TIAR with target transcripts was further investigated after exposure of cells to UVC, a stimulus that triggered the formation of SGs (2, 23). SGs appeared in the cytoplasm by 1 h after UVC irradiation of RKO cells, were largest by 3 h, and virtually disappeared by 6 h (Fig. 3A; see Fig. S4 in the supplemental material). The TIAR-related protein TIA-1 has been reported to undergo dynamic shuttling into and out of SGs (21), and TIAR is likely subject to similar subcellular transport. However, whole-cell TIAR as well as overall TIAR in the cytoplasm and the nucleus appeared unchanged following UVC irradiation (Fig. 3B).
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10% of TIAR remaining in the silenced cultures (Fig. 1A) was insufficient to recapitulate all of the binding activity seen in the presence of normal TIAR levels (Fig. 4A). As expected, little or no product was amplified in the IgG IP lanes, indicating that there was minimal background contamination of mRNA in the IP material (Fig. 5A). The reduction in cellular and SG-associated TIAR signals was also readily detectable by immunofluorescence (Fig. 5B). TIAR siRNA potently lowered TIAR expression in both the nucleus and the cytoplasm, although SGs still formed in response to UVC irradiation, as detected by immunofluorescence using a TIA-1-reactive antibody (Fig. 5B; see Fig. S3 in the supplemental material).
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Since changes in both nascent translation and proteolysis could account for the observed differences in steady-state protein levels (Fig. 6B), we directly monitored protein translation by assessing nascent protein synthesis. To this end, cells were subjected to a brief (20-min-long) incubation in the presence of 35S-labeled amino acids; immediately after labeling, cells were collected for IP, as previously described (see references 31 and 32), using antibodies that specifically recognized c-Myc, eIF4E, and eEF1B. While this labeling procedure is inefficient (lasting only 20 min) and hence yields relatively weak signals, it uniquely allows the measurement of true nascent translation, since any contributions from proteolysis are generally negligible during the short incubation period. As shown in Fig. 6C, the incorporation of radiolabeled amino acids was significantly lower for the three proteins after UVC irradiation in cells expressing normal TIAR levels ("control siRNA" group) but remained elevated in UVC-treated cells expressing reduced TIAR ("TIAR siRNA" group). It is important that the reduction in translation seen at 6 h (Fig. 6C) was not yet reflected in changes in steady-state protein levels on Western blots (6 h following UVC) (Fig. 6B), as the preexisting pool of c-Myc, eIF4E, and eEF1B had not yet been depleted.
The translational status of mRNAs encoding of MYC, EIF4A1, EIF4E2, EEF1B2, and GAPDH was also monitored by carrying out sucrose gradient fractionations (see Materials and Methods) (see Fig. 8A) of control and TIAR-silenced cells that were either left untreated or UVC irradiated. After pooling fractions 1 to 3, representing the mRNA that was not associated with the translation machinery ("nontranslating"), and fractions 5 to 11, representing the mRNA that was bound to the translation machinery ("translating"), qPCR was used to measure the abundance of specific mRNAs in each translation group (Fig. 7A) and verified when possible by Northern blotting (Fig. 7B). When the distribution of these mRNAs in the siRNA transfection groups was compared, MYC, EIF4A1, EIF4E2, and EEF1B2 mRNAs were significantly more abundant in the translating than in the nontranslating fractions, particularly after UVC irradiation; control GAPDH mRNA levels were virtually unchanged (Fig. 7A). To further document the changes in the relative distribution of these mRNAs along sucrose gradients, the MYC and EEF1B2 mRNAs in each gradient fraction of each transfection and treatment group were monitored by Northern blotting (Fig. 7B). Despite the intrinsic variability in the analysis of RNA data derived from polysome gradients (particularly coming from transfected and stress-treated populations), the inhibitory influence of TIAR upon its target mRNAs was also readily observed by Northern blotting (Fig. 7B). Collectively, these findings indicate that TIAR contributed to the translational repression of target mRNAs encoding c-Myc, eIF4E, and eEF1B.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, MMP13, Cox-2, and ß2-adrenergic receptor) (12, 18, 20, 45), whereas following stress, it transiently suppresses the translation of general mRNA pools (1). The latter set of events is triggered when stress-activated kinases phosphorylate eIF2 and thereby reduce the eIF2-GTP-tRNAMet that is required to load tRNAiMet onto the 40S ribosomal subunit to initiate protein synthesis. Under such conditions, TIA-1 and TIAR have been proposed to promote the assembly of translationally inactive preinitiation complexes (containing the small ribosomal subunit but lacking eIF2 and eIF5) and aggregate in SGs.
Our findings presented here provide experimental support for these two proposed functions of TIAR and expand its role to one of extension of general translational repression following stress, well after the disappearance of SGs. These functions are supported by evidence provided in this study that (i) SGs were readily visible by 3 h after UVC irradiation but disappeared almost completely by 6 h after UVC irradiation (Fig. 3A); (ii) global translation was repressed following UVC irradiation, while eIF2 phosphorylation, although readily detectable in both untreated and UVC-irradiated cells, was not noticeably elevated by UVC treatment and hence did not appear to significantly repress translation (Fig. 1C); (iii) silencing of TIAR caused translation to remain elevated, both in untreated and in UVC-irradiated cells (Fig. 1 and 7); and (iv) TIAR associated with mRNAs encoding several translation factors, components of the translation machinery, and other proteins that regulate translation (including eIF4A, eIF4E, eEF1B, and c-Myc) in a UVC-dependent manner and repressed their translation (Fig. 2, 4, and 6). Based on these observations, we postulate that in response to cellular stress, TIAR contributes to a global repression of translation via two sequential sets of events. First, TIAR would promote the general repression of translation through the transient assembly of SGs; these effects might occur in tandem with eIF2 phosphorylation, which reportedly increase after UVC phosphorylation (14, 44) and need not be strongly elevated in order to cause translational repression (33). Afterwards, despite the disappearance of SGs and the unchanged levels of eIF2 phosphorylation, the increased association of TIAR with target mRNAs encoding c-Myc, which regulates many translation control proteins, as well as translation factors (including eIF4E, eEF1B, and likely also eIF4A and other factors) (Fig. 2), would specifically block the translation of these proteins, causing their levels to drop. In turn, the reduced levels of translational initiation and elongation factors would contribute to the extension of a translationally repressed state until the cell has repaired the damage brought upon by the stress agent (Fig. 8B).
In the aforementioned study by Wu et al. (44), UVC irradiation was reported to inhibit global translation in various human and rodent cell lines, an effect that was shown to be specifically dependent on the phosphorylation of eIF2 by the endoplasmic reticulum-localized kinase PERK. However, the PERK-elicited inhibition was incomplete, since expression of a dominant negative variant (PERK
C) only partially restored protein synthesis; accordingly, 24 h after UVC irradiation, translation was suppressed by 18% in the presence of wild-type PERK, but it was relieved to 50% of translation in cells expressing PERKC (44). While those investigators employed different cell types and higher doses of UVC than we used, it is plausible that TIAR-dependent events (i.e., suppressed expression of eIF4E, eEF1B, eIF4A, and c-Myc) contribute, at least in part, to the fraction of translational inhibition that was PERK independent, particularly at late time points following UVC treatment. A formal analysis of these possibilities awaits further study.
Much remains to be learned about SGs, including their genesis, function, and disassembly. It is formally possible that the aggregated preinitiation complexes that give rise to SGs in response to cell damage (peaking in size by about 3 h after UVC irradiation, as determined by immunofluorescence) (Fig. 3A) subsequently disassemble but remain as noncanonical, translationally silent 48S associations that are undetectable by fluorescence microscopy. Nonetheless, the finding that lowering of the TIAR levels effectively increased global translation in both untreated and UVC-stimulated cells (Fig. 1 and 7) suggests that TIAR is involved in translational repression both constitutively and in response to cellular injury. The inhibitory influence of TIAR on the expression of the translation factors identified here (eIF4E and eEF1B) as well as c-Myc (Fig. 4 and 6) likely contributes to the global translational repression of TIAR. The expression levels of proteins encoded by additional TIAR target mRNAs (Fig. 2 and additional transcripts identified on the microarrays [http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE1433]) and their possible involvement in the TIAR-mediated translational repression are also under investigation.
The precise mechanisms whereby TIAR binds to and represses the translation of specific target ARE-containing mRNAs also remain to be fully elucidated. The findings that TIAR abundance (whole-cell and cytoplasmic levels) (Fig. 3) remained unchanged after UVC irradiation while binding of cytoplasmic TIAR to target transcripts increased dramatically following stress (Fig. 4) suggest that TIAR may function in a manner different from that of HuR, another ARE-RBP that also modulates the translation of target mRNAs. In the case of HuR, which is almost exclusively nuclear in unstimulated cells, both its cytoplasmic abundance and its association with many target transcripts increase in response to stress agents (41). These findings have prompted researchers in the field to propose a model whereby HuR binds target mRNAs in the nucleus and contributes to their export to the cytoplasm, where it modulates their stability and translation (8, 25). Although no changes in cytoplasmic TIAR levels were seen here following UVC irradiation, a possible dynamic shuttle of TIAR between the nucleus and cytoplasm could effectively prevent a net increase of TIAR in either compartment. Thus, it has yet to be determined whether TIAR associates with target mRNAs destined for translational suppression while the mRNA is still in the nucleus, possibly during splicing (a process in which TIAR and TIA-1 actively participate) (15, 27, 37) of a pre-mRNA precursor, and whether it plays a further role in the export of target mRNAs. The alternative possibility that TIAR associates with target mRNAs only in the cytoplasm also awaits further study. Given that the TIAR target ARE-mRNAs previously described (such as cyclooxygenase 2, MMP13, and TNF-) are also targets of other ARE-RBPs, it will be particularly interesting to examine whether TIAR competes or cooperates with other ARE-RBPs associating with common target mRNAs and whether stress stimuli influence these interactions. Finally, the analysis of possible posttranslational modifications of TIAR that modulate its association with target mRNAs is likely to be a fruitful area of future pursuit.
It is important that in populations in which TIAR was knocked down, SGs were still detected when an anti-TIA-1 antibody was used, although there appeared to be a slight delay in SG formation (see Fig. S4 in the supplemental material), suggesting that both proteins are required for optimal assembly of SGs. Whereas TIA-1 and TIAR appear to have somewhat interchangeable roles in SG formation, their relative affinity for target mRNAs has not been studied systematically. En masse analyses to identify TIAR and TIA-1 target mRNAs under way in our laboratory suggest the existence of both shared and specific target ARE-containing transcripts (30; data not shown), in keeping with the notion that TIAR and TIA-1 are functionally distinct (13, 42). Further efforts to elucidate the composition and function of RNP complexes involving TIAR, TIA-1, and other ARE-RBPs will decisively advance our understanding of the translational control of gene expression triggered by cell damage.
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ACKNOWLEDGMENTS |
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This research was supported by the Intramural Research Program of the NIA, NIH.
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FOOTNOTES |
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Supplemental material for this article may be found at http://mcb.asm.org/.
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