Cell Cycle 9:15, 3112-3118; August 1, 2010; © 2010 Landes Bioscience

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*Correspondence to: Mette Prætorius Ibba; Email: ibba.2@osu.eduSubmitted: 03/05/10; Revised: 05/19/10; Accepted: 05/28/10Previously published online: www.landesbioscience.com/journals/cc/article/12525DOI: 10.4161/cc.9.15.12525

Introduction

Autophagy (macroautophagy) has recently gained tremendous attention due to its important role in initiation and propaga-tion of human diseases such as cardiovascular diseases, heart failure, neurodegeneration and cancer.1-6 Detailed understand-ing of how autophagy can be modulated is a prerequisite for the development of strategies to use this pathway as a therapeutic target.

Autophagy is a catabolic process that allows cells to degrade and recycle cytosolic constituents, such as long-lived proteins and organelles, by lysosomal degradation.7 The degradation products (nucleotides, amino acids, sugars and lipids) are shuttled from the lysosome to the cytosol, where they are recycled as building blocks for vital cellular processes that sustain cell growth, i.e., protein synthesis and ATP generation. Under favorable growth conditions, the autophagic pathway operates at a low constitu-tive (housekeeping) level to provide cytoplasmic quality control through elimination of damaged organelles and misfolded pro-tein aggregates, thereby ensuring cell homeostasis. During cel-lular stress such as nutrient deprivation, this pathway is strongly induced. Even though autophagy may provide a temporary sur-vival pathway for metabolically-stressed cells, it may also promote cell death if excessively upregulated.8-11

Cells respond to nutrient deprivation a variety of ways. In addition to global downregulation of cap-dependent protein synthesis mediated by the GCN2 and mtoRC1 signaling pathways, a catabolic process autophagy is upregulated to provide internal building blocks and energy needed to sustain viability. It has recently been shown that during nutrient deprivation tRNAs accumulate in the nucleus, but the functional role of this accumulation remains unknown. this study investigates whether subcellular localization of tRNAs plays a role in signaling nutritional stress and autophagy. We report that human fibroblasts that accumulate tRNA in the nucleus due to downregulation of their transportin, Xpo-t, show reduced mtoRC1 activity and upregulated autophagy. this suggests that subcellular localization of tRNAs may regulate an intracellular response to starvation independently of the cellular nutritional status.

Linking tRNA localization with activation of nutritional stress responses

Le-Nguyen Huynh,1 Muthusamy thangavel,1 timothy Chen,1 Ryan Cottrell,1 Joy Maria Mitchell1 and Mette prætorius-Ibba1,2,*

1Department of Molecular and Cellular Biochemistry; College of Medicine; Comprehensive Cancer Center; 2the ohio State University; Columbus, oH USA

Keywords: tRNA, Xpo-t, mTOR, autophagy

Abbreviations: mTOR, mammalian target of rapamycin; GCN2, general control non-derepressible-2; tRNA, transfer RNA; miRNA, microRNA; siRNA, small interfering RNA; Xpo-t, exportin-T; XPOT, gene encoding exportin-T; TNPO3, transportin 3; LC-3, MAPLC3B microtubule-associated protein 1 light chain 3B; GFP, green fluorescent protein; D. discoidium, Dictyostelium

discoideum; FITC, fluorescein isothiocyanate; DIG, digoxigenin; TEM, transmission electron microscopy; FISH, fluorescent in situ hybridization; Kar, karyopherin; mTORC1, mammalian target of rapamycin complex 1

Regulation of the autophagic pathway is complex and involves a number of different signaling cascades. Despite increasing knowledge about these pathways, many key aspects remain unknown. For example, whereas it is well-known that amino acid depletion downregulates the activity of the serine/threonine kinase mTOR (target of rapamycin) and upregulates autophagy (Fig. 1), little is known about upstream factors which transduce the starvation signal to mTOR. However, it was recently shown that the four RAG GTPases make amino acid dependent interac-tions with the mTOR kinase and thus play an important role in amino acids sensing by mTORC1.12-14

We have observed that a yeast strain deleted for the tRNA transporting karyopherin LOS1 showed increased autophago-some-like punctates when expressing a GFP-tagged version of the autophagic marker ATG8 (Fig. S1). This led us to sug-gest that subcellular localization of tRNA may play a role in autophagy regulation, perhaps serving as a sensor of amino acid availability. To assess this hypothesis, and to extend our analysis to the mammalian cells, we analyzed autophagy regulation in human foreskin fibroblasts (HFF) that accu-mulate tRNAs in the nucleus as a result of reduced expres-sion of karyopherin Xpo-t. Our data demonstrate that Xpo-t knocked down fibroblasts indeed show elevated autophagy. Furthermore, the Xpo-t knocked down cells display reduced

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in the nucleus. The underlying reason for the latter is yet unknown.26-28

To determine whether tRNA retention in the nuclei of HFF would influence the autophagic process simi-larly to the deletion of yeast LOS1 (Fig. S1), we chose to knock-down the karyopherin Xpo-t by nucleofection with XPOT-specific siRNA. Using both real-time PCR and immunoblotting, we confirmed that the expression of the karyopherin was reduced by more than 80% as compared to cells nucleofected with a non-targeting siRNA (Fig. 2A and B).

We next tested whether siRNA-mediated knock-down of Xpo-t in fibroblasts would lead to tRNA accumulation in the nucleus. Fluorescent in situ hybridization (FISH) using a DIG-labeled oligonucle-otide designed to hybridize to tRNALys and fluorescent anti-DIG antibodies, demonstrated that in control cells, tRNALys was accumulated in the nuclei only fol-lowing starvation; this effect was reversed when the cells were refed. By contrast, the Xpo-t knocked down cells showed similar levels of increased nuclear accumu-lation of tRNALys under all conditions (Fig. 2C and D). Nuclear and cytosolic extractions followed by quanti-tative RT-PCR also confirmed an increase of tRNALys (1.8 fold) in the nucleus in Xpo-t knocked down cells as compared to control cells (Fig. 2E and F). These experiments confirm that reduction in Xpo-t levels in fibroblasts by siRNA knock-down impairs nucleocyto-plasmic transport of tRNAs and that this effect is inde-pendent of the cells nutritional status.

Knock-down of Xpo-t leads to changes of autophagic mark-ers and accumulation of autophagosomes. To test whether accumulation of tRNA caused by defects in nuclear export contributes to the onset of a starvation-like response, the Xpo-t knocked down fibroblasts were assessed for upregulation of autophagy. We first examined changes in the expression level and the modification state of the commonly used autopha-gic marker LC3 (MAPLC3B microtubule-associated protein 1 light chain 3B), a mammalian ortholog of yeast Atg8. LC3 is a key component required for autophagosome formation in mammalian cells: after LC3 is processed and conjugated to phosphatidylethanolamine (PE), the lipidated (LC3-II) form becomes tightly associated with the inner and outer autopha-gosomal membranes. LC3-II plays an additional role in protein targeting: it directly binds to, and selectively targets, p62 into autophagosomes.29,30

LC3-I and its lipidated form, LC3-II, were visualized by immunoblotting using protein extracts from HFF cells knocked down for Xpo-t or treated with a non-targeting (control) siRNA and siKar RNA directed towards TNPO3 karyopherin that has not been shown to participate in tRNA export from the nucleus (Fig. 3A). Extracts from HFF grown in nutrient replete media were compared to those from cells grown in nutrient deplete medium or medium containing chloroquine (CHL), which blocks the later steps in autophagic pathway, such as lysosomal degradation of LC3-II and p62.

activity of the mTORC1 pathway. These data support a role for tRNAs in starvation signaling through their subcellular localization.

Results and Discussion

siRNA-mediated knock-down of karyopherin Xpo-t in human fibroblasts leads to nuclear accumulation of tRNA. Nucleocytoplasmic trafficking of macromolecules such as tRNAs is a tightly controlled process that relies on several fac-tors including (1) karyopherins, transport receptors that recog-nize, bind to, and assist in transport of, macromolecules across the nuclear envelope; (2) nucleoporins that are confined to the nuclear pore complex; and (3) RanGTPase that controls the rate at which macromolecules are translocated.15 Xpo-t and its yeast orthologue Los1 are tRNA-specific nuclear export recep-tors; they bind newly synthesized tRNAs in the nucleus and mediate their transport to the cytoplasm.16-18 Although los1 dele-tion strains show impaired tRNA splicing and accumulation of tRNAs in the nucleus, the LOS1 gene is not essential for growth, suggesting that other, partially redundant tRNA transport sys-tems exist.19,20

During amino acid deprivation, several cellular tRNA-related events take place. One such event is the activation of the nutri-ent-responsive kinase GCN2 by uncharged tRNAs present in the cytosol.21-23,25 Another is the accumulation of cytoplasmic tRNA

Figure 1. the main mechanisms sensing nutrient availability. A summary of the nutrient-responsive pathways and their role in autophagy regulation. our hypoth-esis that tRNA subcellular localization affects autophagy is also incorporated in the figure. the circled components are of main interest to this study.

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the observed change was not dramatic, we used an autophagic marker p62/SQSTM to substantiate our findings. The p62/SQSTM protein binds to and escorts proteins destined for lyso-somal degradation to the autophagic vesicles. In the course of this process, p62/SQSTM is itself sequestered and degraded in the autolysosomes.29-31 Immunoblot analysis showed that in cells grown in presence of nutrients the level of p62/SQSTM was

The lipidated form of LC3, LC3-II (which migrates as a 17 kD species) is easily distinguished from LC3-I (15 kD). As expected, the levels of LC3-II were markedly increased in CHL-treated samples (Fig. 3A). We observed a small yet reproducible increase in LC3-II levels in CHL-treated cells knocked down for Xpo-t (but not the cells treated with control and kar siR-NAs) during both normal growth and during starvation. Since

Figure 2. Knock-down levels of Xpo-t and cellular localization of tRNALys in HFF cells. HFF were transfected with a non-targeting control siRNA, with siRNA towards Xpo-t or without any siRNA (none, mock transfected). Cells were grown in non-starvation or starvation medium (6 h); to test the recovery from starvation, growth was continued in non-starvation media for 24 h following 6-h starvation. All individual siRNA transfections were tested for knocked down levels of Xpo-t. (A) the Xpo-t mRNA levels were determined by quantitative Rt-pCR and normalized to GApDH mRNA. error bars indicate s.d.; **p < 0.01; n = 6. (B) protein levels of Xpo-t measured by immunoblotting; actin was used as a control throughout this work. (C and D) Subcellular localization of tRNAs in HFF cells was visualized by FISH using DIG-labeled probes complementary to human tRNACUU

Lys and FItC-conju-gated anti-DIG antibodies. Nuclei were visualized by DApI staining. (e and F) Quantitative Rt-pCRs were performed with nuclear or cytoplasmic RNA isolated from luciferase or Xpo-t siRNA transfected HFFs. (e) 2.5% agarose gel loaded with the cDNA samples. (F) Relative levels of tRNALys cDNA in the cytoplasm or nucleus in the transfected cells were normalized to siRNA control levels.

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Figure 3. Levels of mtoR and autophagic markers in Xpo-t knocked down HFF cells. Levels of autophagic markers LC3 (A) and p62/SQStM (B) were determined by immunoblotting of extracts from HFF cells treated as described in Figure 2 legend. Where indicated, the cells were treated with chloroquine (CHL) for 3 h. the levels of LC3-II and p62 (normalized to actin) are indicated below each lane. (C) Activity of the mtoRC1 pathway was assessed by measuring levels of phosphorylated forms of mtoR and S6 (mtoRSer2448-P and S6Ser235/236-P, respectively) as compared to their total levels, as well as those of a phosphorylated form of 4e-Bp, 4e-Bpthr37/46-P, using immunoblotting. (D) transmission electron microscopy was carried out to visual-ize and quantify autophagosome structures (indicated by arrows) in cells nucleofected with a control siRNA or Xpo-t siRNA. (e) Autophagosomes were counted and compared to the number of autophagosomes in non-starved cells treated with control siRNA.

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We also probed the activity of another mTOR complex, TORC2, which phosphorylates and activates AKT1 at position Ser473 (AKTSer473-P). We found no indication of altered AKT1 phosphorylation in cells knocked down for Xpo-t relative to con-trol cells (Fig. S2). This suggests that the observed differences are specific for the mTORC1pathway.

Xpo-t knock down increases autophagosome formation. One of the most powerful ways to study upregulation of autophagy is the direct visualization of the double-membrane autophagosomes in the cytoplasm by transmission electron microscopy (TEM). We found that after nutrient starvation for 24 hours, the cells transfected with a control siRNA showed an ∼1.5 fold increase in the number of autophagosomes (Fig. 3D and E). We next visualized autophagosome-like structures in HFFs treated with the Xpo-t -specific siRNA. We observed a comparable increase (∼2-fold) of autophagosomes upon downregulation of Xpo-t in cells grown in rich or nutrient-depleted medium. These data demonstrate that the reduced expression of Xpo-t mimics the effect of nutrient limitation on the induction of autophagy.

Amino acid starvation is signaled through tRNA accumu-lation in the nucleus. We reported here that, similarly to yeast cells, human fibroblasts also accumulate tRNAs in the nucleus when tRNA transport is disrupted as a result of reduced expres-sion of the tRNA-specific karyopherin Xpo-t. Furthermore, our study revealed changes in activity of the mTORC1 nutrient-responsive signaling pathways in cells accumulating tRNAs in the nucleus. A possible role for tRNAs in triggering a nutrient starvation response through mTORC1 has been suggested pre-viously.34-36 Whereas these studies have assessed the effect of uncharged/mischarged tRNAs accumulating in the cell after blocking aminoacylation of tRNA, we examined changes in nutrient-related responses and autophagy upon accumulation of tRNAs in the nucleus. In agreement with some of these studies, our work supports a hypothesis that the subcellular distribution of tRNAs, in addition to their aminoacylation state, may be an important factor for regulation of nutrient-responsive signaling pathways in human fibroblasts. Thus, it is possible that the sub-cellular localization of tRNAs may signal the amino acid avail-ability through signaling to mTOR pathway. Thus, beside their essential role in protein synthesis to support cell growth and pro-liferation, tRNAs also seem to play important roles in improv-ing cellular homeostasis by modulating signaling pathways and processes that functions to protects cell from nutrient limitations. It is interesting to note that tRNA (cytosolic and mitochondrial) also recently has been shown to be involved in preventing apop-tosis by binding cytochrome c. Thus, tRNA may be involved in both apoptosis and autophagy, perhaps depending on their cel-lular localization.42

The role of tRNA in sensing stress and starvation. The use of tRNAs as a signal is ancient and variants thereof are found in all kingdoms. In eukaryotes the important roles of tRNAs in cell proliferation and stress response have been well demon-strated. During amino acid starvation uncharged tRNAs directly bind and activate the eIF2 kinase GCN2,21,22 which in turn ini-tiates general amino acid control pathway, wherein global pro-tein synthesis is reduced, allowing cells to adapt to the changing

reduced 2-fold in extracts from cells knocked down for Xpo-t, but not in those treated with siControl or siKar RNAs (Fig. 3B). As expected, all samples treated with CHL accumulated p62/SQSTM due to the blockage of lysosomal degradation of the auophagy cargo (Fig. 3B). During starvation, the level of p62/SQSTM in Xpo-t knockdown cells was also reduced as com-pared to control knocked down cells. These data strongly sup-port the hypothesis that nuclear accumulation of tRNAs caused by the reduced level of Xpo-t upregulates autophagy.

Nuclear accumulation of tRNAs caused by Xpo-t deple-tion alters activity of the nutrient-responsive mTOR path-way. When nutrients are abundant, the serine/threonine kinase mTOR is found in its active, phosphorylated at Ser2448, form (mTORSer2448-P). As a part of the larger complex, mTORC1, the active mTOR kinase modulates cell size and growth through tight control of protein synthesis in response to nutrient availabil-ity. The mTORC1 pathway stimulates nutrient uptake and pro-motes ribosome biogenesis and protein synthesis; among mTOR kinase targets are S6K protein kinase, which phosphorylates ribosomal protein S6, and the translational inhibitor 4E-BP1 that acts through the initiation factor eIF4E.13 Under amino acid replete conditions, mTORC1 also inhibits autophagy;32 it has also been shown that TOR in Drosophila melanogaster directly phosphorylates Atg1, thereby reducing its activity and subse-quent autophagy induction.33 During amino acid starvation, phosphorylation of Ser2448 decreases. This results in reduced mTOR kinase activity, in turn leading to a decrease in phospho-(Thr389)-S6K and phospho- (Ser235/236) S6, and subsequent downregulation of the translational machinery. Conversely, Atg1 undergoes dephosphorylation, becomes activated, and induces the autophagy cascade. Besides the interaction of the RAG pro-teins with mTORC1, little is known about how mTORC1 senses the intracellular levels of amino acids.12,13,32,33

Since amino acid starvation leads to nuclear accumulation of tRNA, we intended to investigate whether subcellular localiza-tion of tRNAs may play a role in signaling amino acid avail-ability. We measured the levels of active mTORSer2448-P in protein extracts from fibroblasts knocked down for Xpo-t or the control karyopherin siKar, as well as cells treated with non-targeting control siRNA, by immunoblotting (Fig. 3C). Whereas no sig-nificant changes in mTORSer2448-P were detected in cells trans-fected with control or Kar siRNA, a significant reduction was observed in Xpo-t knocked down cells (Fig. 3C). This strongly indicates that tRNA accumulation caused by a decrease in the Xpo-t levels leads to downregulation of the mTORC1 pathway. To evaluate this indication, we examined the phosphorylation states of the downstream components of the mTORC1 path-way S6 (S6Ser235/236-P) and 4E-BP(4E-BPThr37/46-P). We observed a comparable reduction in the levels of phosphorylated forms of S6 and 4E-BP upon the Xpo-t knock down, whereas the levels of the unmodified proteins remained the same (Fig. 3C). The reduced phosphorylation of mTOR, S6 and 4E-BP observed in cells specifically knocked down for Xpo-t strongly suggests that the activity of the mTORC1 is downregulated at low levels of Xpo-t, a condition which leads to tRNA accumulation in the nucleus.

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environment by altering their transcription program. Recent global analysis of yeast tRNA charging status demonstrated that the charging pattern changes in response to both nutrient starvation and osmotic stress.37 Even in the case of amino acid starvation, the response appears to be global as starvation for a given amino acid favors deacylation of non-cognate tRNAs. In HeLa cells, immune response and oxidative stress lead to a ten-fold increase in misacylation of many tRNA species with Met, followed by incorporation of Met into growing peptide chains;38 interestingly, misacylation was exclusively cytosolic, highlighting the importance of subcellular tRNA localization. tRNA modi-fications, such as thiolation by a ubiquitin-like Urm1p protein, may be critical to regulate cellular responses to nutrient starvation and oxidative stress conditions.39 Furthermore, tRNAs can also be endonucleolytically cleaved by cytoplasmic nucleases (such as yeast Rny1) during stress, inducing a variety of responses that may range from GCN2 activation to general translational repres-sion to induction of siRNA and miRNA pathways.40

In this work, we report that tRNA retention in the nucleus trig-gers a starvation-like response even under nutrient-replete condi-tions. This effect could be mediated by nucleus-specific changes in the retained tRNAs. Modification and cleavage enzymes are expected to be specifically localized, thus the pattern of tRNA modifications and the regulatory signals induced by these modi-fications would also be compartmentalized.

Experimental Procedures

Cells and growth media. Primary human foreskin fibroblast cells (HFF) were grown in DMEM (Dulbecco’s Modified Eagle’s Medium, Sigma-Aldrich) supplemented with 10% heat-inacti-vated fetal bovine serum (FBS, Invitrogen) and 5% Penicillin and Streptomycin (Sigma-Aldrich). Cells were grown at 37°C with 5% CO

2 until 75 to 90% confluent. HFF cells were starved (6 h)

by incubation in Earle’s Balanced Salt Solution (Sigma-Aldrich). Chloroquine 40 µM was added 3 h prior to harvesting the fibro-blasts, cells were washed twice with PBS (Phosphate Buffered Saline, pH 7.4) and detached by incubating with Trypsin (0.25% Trypsin-EDTA solution, Sigma Aldrich) for 1–3 minutes at 37°C. Cells were collected by centrifugation at 1,250 rpm for 8 minutes.

siRNA-mediated knock-down. Cells were harvested and concentrated to 2 x 105/ml prior to nucleofection, which were done according to manufacturer’s nucleofection protocol (Amaxa Nucleofector, Lonza) using 1.5 µg siRNA duplex specific to TNPO3 (sense 5'-CUG AAU UAC UGC CGU AUU U and antisense 5'-AAA UAC GGC AGU AAU UCA G), Xpo-t (oligo 1: sense 5'-GAU AGU UAG UUG GAG UAA A and antisense 5'-UUU ACU CCA ACU AAC UAU C and oligo 2: sense 5'-CCU ACU UCA UGA UCA UGA A and antisense 5'-UUC AUG AUC AUG AAG UAG G). The latter was used to repeat experimemts to verify that results obtained with Xpo-t oligo 1 are specific for knock-down of the karyoperin [Data not shown], non-targeting scrambled siRNA (5'-GAU CAU ACG UGC GAU CAG ATT and Antisense 5'-UCU GAU CGC ACG UAU GAU CTT) or siRNA towards luciferase (Sense 5'-AAA CAU GCA GAA AAU GCU G and Antisense 5'-CAG CAU UUU CUG CAU GUU U)

as controls (Sigma Aldrich). 2 x 106 cells were used per nucleo-fection. Cells were incubated 24 h (experiments that include 24 hours starvation) or 48 hours before they were used in the described experiments.

Isolation of nuclear and cytoplasmic RNA. RNA was iso-lated using a Cytoplasmic and Nuclear RNA Purification Kit (Norgen, Canada) using the manufacturer’s protocol. The RNA was used for quantitative RT-PCR as described below.

Quantitative RT-PCR. Cells were harvested in Trizol Reagent (Invitrogen), snap frozen to inhibit activity of endogenous RNases where after RNA was extracted according to the manufacturer’s protocol (Invitrogen). One µg of total RNA was used to reverse transcribe mRNA into cDNA using random hexamer primers and M-MLV Reverse Transcriptase (Invitrogen). qPCR (SYBR® Green I dye detection, Applied Biosystems, WA) was performed in triplicate on a sequence detection system (Prism 7300; Applied Biosystems) using SABiosciences primers to human XPOT (NM_007235.3 position 2551–2571); GADPH was used as an internal control (sense, 5'-CCC CTT CAT TGA CCT CAA CTA CAT-3' and antisense, 5'-CGC TCC TGG AAG ATG GTG A-3'). Oligos towards tRNALys were sense 5'-AGC TCA GTC GGT AGA GCA TGA-3' and antisense 5'-AAC CCA CG ACC CTG AGA TTA A. XPOT and tRNALys expressions were normalized to GADPH.

Immunoblotting. Cell extracts were prepared in 1X RIPA Buffer (Thermo Scientific) supplemented with Halt Phosphatase Inhibitor Cocktail and Protease Inhibitor Cocktail EDTA-free (Thermo Scientific). Proteins from cell extracts were resolved by SDS PAGE (6, 10 and 13%) and transferred onto 0.45 µm Pure Nitrocellulose membrane (Bio-Rad). The protein blots were incubated with indicated antibodies in PBS-T buffer (8 g NaCl, 0.2 g KCL, 1.44 g Sodium Phosphate, 0.24 g Potassium Phosphate per 1 L and 0.1% Tween 20 (Sigma-Aldrich) and 3% BSA (Albumin from bovine serum, Sigma-Aldrich). Monoclonal anti-Actin, anti-LC3B and anti-Xpo-t antibodies were purchased from Sigma-Aldrich, anti-p62/SQSTM from MBL and anti-mTOR, phospho-mTOR (Ser2448), phospho-S6 (Ser235/236), AKT1, phosphor-AKT1 (Ser473), and 4E-BPThr37/46 were pur-chased from Cell Signaling Technology. Goat anti-rabbit and goat anti-mouse Infrared IRDye®-labeled secondary antibod-ies (LI-COR) were used. Proteins recognized by the antibodies were detected using the Detection LI-COR/Odyssey Infrared Imaging System.

Fluorescent in-situ hybridization. HFF cells transfected with non-targeting siRNA or siRNA towards Xpo-t were grown on cover slips in non-starvation media or starvation media. Cells were then fixed and processed as previously described.41 Dig-labeled hybridization probes used were complementary to human tRNA

CUULys (CCA ACG TGG GGC TCG AAC CCA

CGA CCC TGA GAT TAA GAG TCT CAT GCT CTA CCG ACT) and as negative control to D. discoidium tRNAGlu (CCA GTG TTA GAG ACT AGA GTG TAC CGA CTA CAC CAA TGA) and were previously described.28 The oligonucleotides were from Sigma-Aldrich and FITC-conjugated anti-DIG anti-bodies were from Roche. Fluorescent images were observed by using an Olympus FV1000-Filter Confocal Microscope (Japan)

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and FITC intensity in the cytoplasm and nucleus quantified from 3 images per condition using NIS-Elements BR software.

Transmission electron microscopy. Monolayer cells were grown in chamber slides in non-starvation or starvation media. Prior to processing the cells were fixed for 30 min in 2.5% glu-taraldehyde in 0.1 M phosphate buffer pH 7.4. Further process-ing was performed by OSU Campus Microscopy and Imaging Facility. Images were obtained with a FEI Technai G

2 Spirit

Transmission Electron Microscope.

Acknowledgements

We would like to thank Irina Artsimovitch for her remarkable assistance and valuable comments and suggestions in writing this manuscript. We greatly appreciate Michael Ibba and Jerneja Tomsic for suggestions and critically reading of the manuscript. Daniel Klionsky is acknowledged for providing the plasmid

expressing GFP-Atg8 and Anita Hopper for yeast strains, Gustavo Leone for the HFF cell line and Rebecca Hurto for advice con-cerning the fluorescent in situ hybridization experiment. OSU Campus Microscopy and Imaging Facility is acknowledged for sample preparation and assistance with transmission electron microscopy, confocal and fluorescent microscopy and Randy J. Giedt and Rita Alevriadou for quantification of fluorescence intensities. The work is support by seed grants from OSU Comprehensive Cancer Center, an OSU Critical Difference for Women Award and a Beginning Grant-In-Aid from American Heart Association #09BGIA2230347.

Note

Supplementry materials can be found at:www.landesbioscience.com/supplement/HuynhCC9-15-sup.pdf

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