Sang-Won Park1, Pureum Jeon2, Yong-Woo Jun1, Ju-Hui Park1, Seung-Hwan Lee1, Sangkyu Lee3, Jin-A Lee4, Deok-Jin Jang5. 1. Department of Ecological Science, College of Ecology and Environment, Kyungpook National University, 2559, Gyeongsang-daero, Sangju-si, Gyeongsangbuk-do, 37224, Republic of Korea. 2. Department of Biological Science and Biotechnology, College of Life Science and Nano Technology, Hannam University, 1646, Yuseong-daero, Yuseong-gu, Daejeon, 34054, Republic of Korea. 3. Center for Cognition and Sociality, Institute for Basic Science (IBS), Daejeon, 34126, Republic of Korea. 4. Department of Biological Science and Biotechnology, College of Life Science and Nano Technology, Hannam University, 1646, Yuseong-daero, Yuseong-gu, Daejeon, 34054, Republic of Korea. leeja@hnu.kr. 5. Department of Ecological Science, College of Ecology and Environment, Kyungpook National University, 2559, Gyeongsang-daero, Sangju-si, Gyeongsangbuk-do, 37224, Republic of Korea. jangdj@knu.ac.kr.
Abstract
Xenophagy is a selective lysosomal degradation pathway for invading pathogens in host cells. However, invading bacteria also develop survival mechanisms to inhibit host autophagy. RavZ is a protein secreted by Legionella that irreversibly delipidates mammalian autophagy-related protein 8 (mATG8) on autophagic membranes in host cells via efficient autophagic membrane targeting. In this study, we leveraged the autophagic membrane-targeting mechanism of RavZ and generated a new autophagosome probe by replacing the catalytic domain of RavZ with GFP. This probe is efficiently localized to mATG8-positive autophagic membranes via a synergistic combination between mATG8 protein-binding mediated by the LC3-interacting region (LIR) motifs and phosphoinositide-3-phosphate (PI3P) binding mediated by the membrane-targeting (MT) domain. Furthermore, the membrane association activity of this new probe with an MT domain was more efficient than that of probes with a hydrophobic domain that were previously used in LIR-based autophagosome sensors. Finally, by substituting the LIR motifs of RavZ with selective LIR motifs from Fyco1 or ULK2, we developed new probes for detecting LC3A/B- or GABARAP subfamily-positive autophagic membranes, respectively. We propose that these new RavZ-based sensors will be useful for monitoring and studying the function of mATG8-positive autophagic membranes in different cellular contexts for autophagy research.
Xenophagy is a selective lysosomal degradation pathway for invading pathogens in host cells. However, invading bacteria also develop survival mechanisms to inhibit host autophagy. RavZ is a protein secreted by Legionella that irreversibly delipidates mammalian autophagy-related protein 8 (mATG8) on autophagic membranes in host cells via efficient autophagic membrane targeting. In this study, we leveraged the autophagic membrane-targeting mechanism of RavZ and generated a new autophagosome probe by replacing the catalytic domain of RavZ with GFP. This probe is efficiently localized to mATG8-positive autophagic membranes via a synergistic combination between mATG8 protein-binding mediated by the LC3-interacting region (LIR) motifs and phosphoinositide-3-phosphate (PI3P) binding mediated by the membrane-targeting (MT) domain. Furthermore, the membrane association activity of this new probe with an MT domain was more efficient than that of probes with a hydrophobic domain that were previously used in LIR-based autophagosome sensors. Finally, by substituting the LIR motifs of RavZ with selective LIR motifs from Fyco1 or ULK2, we developed new probes for detecting LC3A/B- or GABARAP subfamily-positive autophagic membranes, respectively. We propose that these new RavZ-based sensors will be useful for monitoring and studying the function of mATG8-positive autophagic membranes in different cellular contexts for autophagy research.
Xenophagy is selective autophagy by which host cells degrade invading pathogens in lysosomes[1,2]. However, many bacteria have developed a survival mechanism to escape host autophagy by inhibiting the functions of host autophagic proteins[1,3,4]. One component that is essential to mammalian autophagy is mATG8, a mammalian homolog of yeast autophagy-related protein 8. mATG8 plays key roles in autophagosome formation, cargo recognition, and the recruitment of cargos into the autophagosomal membrane[5-9]. In mammals, there are two subgroups of ATG8-like proteins: microtubule-associated protein light chain 3 (LC3) proteins LC3A, LC3B, and LC3C and γ-aminobutyric acid receptor-associated proteins (GABARAPs) GABARAP, GABARAP-L1, and GABARAP-L2[10,11]. These proteins are lipidated by phosphatidylethanolamine (PE) conjugation to their C-terminal regions and are incorporated into membranes depending on different cellular contexts, leading to autophagosome formation and maturation[6,11-14]. However, few methods are available to date for monitoring the cellular localization of each endogenous LC3-, GABARAP-subfamily protein in live cells, and changes to cellular localization in certain physiological or pathogenic conditions[15,16].RavZ is a cysteine protease that is secreted from the intracellular pathogen Legionella pneumophila into the cytoplasm of host cells and irreversibly delipidates mATG8-PE proteins in autophagic membranes by hydrolyzing the amide bond between the C-terminal glycine residue and an adjacent aromatic residue, impairing autophagosome formation and ultimately inhibiting xenophagy in host cells[17]. To efficiently inhibit host autophagy, RavZ must be properly targeted to autophagosomes. RavZ has two LC3-interacting region (LIR) motifs at the N-terminal region (LIR1/2 motifs) and one LIR motif at the C-terminal region (LIR3 motif) that bind to mATG8 proteins in autophagosomes[18,19]. In addition to these LIR motifs, there is a catalytic domain and a phosphatidylinositol 3-phosphate (PI3P)-binding membrane-targeting (MT) domain in RavZ. Since PI3P is enriched in pre-autophagosomal and autophagosomal membranes, a PI3P-binding MT domain might lead to the targeting of RavZ into high-curvature autophagic vesicles[20].In addition to our interest in elucidating the functions of RavZ in host cells, we also became interested in understanding the targeting mechanism of RavZ into autophagosomal membrane since our group and others have recently developed LIR-based autophagosome sensors to detect endogenous autophagosomes[15,16,21]. Our group developed autophagosome sensors using LIR motifs and hydrophobic domains (HyD) with enhanced membrane association that efficiently detect endogenous mATG8-positive autophagosomes[11,15]. Other groups have identified selective mATG8-binding peptides by screening peptide libraries using phage display screening. Combining these peptides with the PB1 (Phox/Bem 1p) domain of p62, which helps self-oligomerization, they developed selective autophagosome sensors, including an LC3C-specific probe[16]. HyD and PB1 domains have been used for efficient targeting of LIR-based autophagosome sensors, but their assisting mechanisms are different. A HyD domain assists membrane association of the probe on the autophagosome, while a PB1 domain induces multimerization of LIR motifs, leading to the enhancement of autophagosome targeting via multiple mATG8 associations on the autophagic membrane. However, within the cells, there are many PB1 domain-containing proteins to interfere with the function of the PB1-containing probe, and multimers of LIR motifs also have increased non-specific binding with other proteins, including other LC3- or GABARAP-subfamily proteins. Therefore, using a membrane association domain instead of a dimerization/multimerization domain might have an advantage[22]. PI3P is involved in the formation and the regulation of autophagosome maturation, although it also exists in the endosomal membrane[23-28]. Therefore, PI3P binding motifs are good candidates for assisting the probes in associating with autophagic membranes if combined with an LIR motif. There are several PI3P-binding motifs, including conserved region 2 (C2), Fab1 YOTB Vac1 EEA1 (FYVE), phox homologue (PX), pleckstrin‐homology domain (PH), GRAM-Like Ubiquitin-binding in EAP45 (GLUE) and glucosyltransferase, Rab‐like GTPase activator, and myotubularin (GRAM) domains[29]. Among these PI3P motifs, an FYVE motif was used to enhance autophagosome detection in a previous study, but it was less efficient as a probe than a PB1 domain[16]. However, if strong PI3P binding domains are used for the probes, they are basally localized to early endosomes and sequester and alter PI3P dynamics in cells. Therefore, weak PI3P-binding domains that are not localized to, but help the localization of the proteins into early endosomes or autophagosomes, can minimize inhibition effects on PI3P function and are therefore useful for the generation of autophagosome-detecting probes.Interestingly, RavZ has a unique PI3P binding MT domain, which helps autophagosome targeting via membrane association[17,20]. Although MT domains and LIR motifs of RavZ can be involved in autophagosome targeting, their contributions remain elusive. Therefore, in this study, we tested the possibility of constructing new autophagosome probes using the PI3P-binding MT domain and LIR motifs from RavZ to enhance its autophagosome targeting. To do this, RavZ(ΔCA)-GFP was generated by replacing the RavZ enzyme activity domain with GFP. RavZ(ΔCA)-GFP was efficiently localized to autophagic membranes through mATG8 binding mediated by LIR motifs and PI3P binding mediated by an MT domain within the RavZ protein. An MT domain or LIR motif alone was insufficient or weak for autophagic membrane targeting. However, an MT domain combined with one or more LIR motifs leads to efficient targeting of the RavZ-based sensor to autophagic membranes. Interestingly, an MT domain was even more efficient than a HyD domain for facilitating autophagic membrane targeting. Furthermore, to increase selective targeting of RavZ-based sensors into LC3- or GABARAP-positive autophagic membranes, we replaced the LIR motifs of RavZ with selective LC3- or GABARAP subfamily-binding LIR motifs and developed additional RavZ-based probes that were selectively detecting for LC3- or GABARAP-positive autophagic membranes in cells. Thus, compared to HyD-LIR(x)-GFP, our newly developed RavZ-based fluorescent autophagosome probes are potentially useful for monitoring mATG8 family proteins in autophagy research with different types of cells under physiological or pathological conditions.
Results and Discussion
Generation and cellular localization of RavZ(ΔCA)-GFP into LC3 or GABARAP-positive autophagic membranes in an MT domain, LIR1/2 motif, or LIR3 motif-dependent manner
It has been reported that RavZ protein secreted from Legionella is targeted to autophagic membranes and delipidates mATG8-PE on autophagic membranes in cells[19,20]. Consistent with this, overexpression of 3xFlag-RavZ but not 3xFlag-RavZC258S, a catalytic mutant of RavZ, reduced LC3B-II in HEK293T Cells (Fig. 1A). These results indicate that RavZ protein is targeted to autophagic membranes and the catalytic domain of RavZ could delipidate mATG8-PE on autophagic membranes. Therefore, deletion of the catalytic domain of RavZ can be used as the target for new autophagic membrane probes. We first deleted the catalytic domain of RavZ and replaced it with GFP to generate RavZ(ΔCA)-GFP (Fig. 1B, upper and Supplemental Fig. 1A). As shown in Fig. 1B (lower), RavZ(ΔCA)-GFP was localized with mRFP-LC3B- or mRFP-GABARAP-positive autophagic membranes in rapamycin/NH4Cl-treated mouse embryonic fibroblast (MEF) cells. Quantitative analysis showed that RavZ(ΔCA)-GFP was co-localized with mRFP-LC3B- or mRFP-GABARAP-positive autophagic membranes at similar levels (Fig. 1C). In addition, RavZ(ΔCA)-GFP detected a vesicle structure in wild-type HeLa cells, but not in ATG5- or ATG7-knockout HeLa cells in an autophagy-dependent manner (Supplemental Fig. 2).
Figure 1
Efficient autophagosome targeting of RavZ(ΔCA)-GFP. (A) Representative Western blots of endogenous LC3B in cells expressing 3xFlag-RavZ protein or 3xFlag-RavZC258S catalytic mutant in HEK293T cells upon autophagy induction (100 nM rapamycin). Extended blot images including these data are presented in Supplementary Fig. 5A. (B) Schematic diagram of GFP-fused RavZ mutant protein (RavZ(ΔCA)-GFP) (upper) and confocal images depicting the cellular localization of RavZ(ΔCA)-GFP co-expressed with mRFP-LC3B or mRFP-GABARAP in MEF cells treated with 100 nM rapamycin (rapa) + 10 mM NH4Cl. Scale bar, 10 µm. (C) The bar graphs illustrate the percentages of mRFP-LC3B- or mRFP-GABARAP-positive RavZ(ΔCA)-GFP spots (n = 25 for each group). (D,E) Confocal images showing cellular localization of GFP, GFP-LC3B or RavZ(ΔCA)-GFP with Lysotracker into MEFs upon 100 nM rapa treatment. The bar graph illustrates the ratios of LysoTracker-positive RavZ(ΔCA)-GFP spots number per cell (n = 25 for each group). The data are presented as the mean ± SEM. ***P < 0.001, according to one-way ANOVA followed by Tukey’s post-hoc test. Scale bar, 10 µm. (F,G) Autophagic flux indicates differences in the levels of LC3-II of GABARAP-II in the presence and absence of chloroquine (CQ). The bar graphs illustrate the level of LC3-II or GABARAP-II. The levels of LC3-II and GABARAP-II in the GFP- or RavZ(ΔCA)-GFP-expressing cells were normalized to that of actin in HEK293T cells expressing GFP or RavZ(ΔCA)-GFP. The data are presented as the mean ± SEM of five independent experiments. Extended blot images including these data are presented in Supplementary Fig. 5B. RAP, GABARAP.
Efficient autophagosome targeting of RavZ(ΔCA)-GFP. (A) Representative Western blots of endogenous LC3B in cells expressing 3xFlag-RavZ protein or 3xFlag-RavZC258S catalytic mutant in HEK293T cells upon autophagy induction (100 nM rapamycin). Extended blot images including these data are presented in Supplementary Fig. 5A. (B) Schematic diagram of GFP-fused RavZ mutant protein (RavZ(ΔCA)-GFP) (upper) and confocal images depicting the cellular localization of RavZ(ΔCA)-GFP co-expressed with mRFP-LC3B or mRFP-GABARAP in MEF cells treated with 100 nM rapamycin (rapa) + 10 mM NH4Cl. Scale bar, 10 µm. (C) The bar graphs illustrate the percentages of mRFP-LC3B- or mRFP-GABARAP-positive RavZ(ΔCA)-GFP spots (n = 25 for each group). (D,E) Confocal images showing cellular localization of GFP, GFP-LC3B or RavZ(ΔCA)-GFP with Lysotracker into MEFs upon 100 nM rapa treatment. The bar graph illustrates the ratios of LysoTracker-positive RavZ(ΔCA)-GFP spots number per cell (n = 25 for each group). The data are presented as the mean ± SEM. ***P < 0.001, according to one-way ANOVA followed by Tukey’s post-hoc test. Scale bar, 10 µm. (F,G) Autophagic flux indicates differences in the levels of LC3-II of GABARAP-II in the presence and absence of chloroquine (CQ). The bar graphs illustrate the level of LC3-II or GABARAP-II. The levels of LC3-II and GABARAP-II in the GFP- or RavZ(ΔCA)-GFP-expressing cells were normalized to that of actin in HEK293T cells expressing GFP or RavZ(ΔCA)-GFP. The data are presented as the mean ± SEM of five independent experiments. Extended blot images including these data are presented in Supplementary Fig. 5B. RAP, GABARAP.Next, we examined whether RavZ(ΔCA)-GFP could detect autolysosomes by using LysoTracker. For this purpose, LysoTracker was added to detect acidic vesicles in MEFs expressing GFP, GFP-LC3B or RavZ(ΔCA)-GFP. As shown in Fig. 1D,E, RavZ(ΔCA)-GFP or GFP-LC3B was localized to LysoTracker-positive autolysosomes in rapamycin-treated MEF cells similarly, suggesting that RavZ(ΔCA)-GFP-positive autophagic membranes were also recruited to autolysosomes as similar with GFP-LC3B positive autophagic membranes.Because RavZ(ΔCA)-GFP binds to the mATG8 proteins, it may also affect autophagic flux via expression of the exogenous LIR motif. The latter process may stimulate sequestration of endogenous LC3B and/or modify its functions. To measure autophagic flux, the levels of autophagic substrates, such as LC3B-II or GABARAP-II, in HEK293T (rapamycin treatment) cells expressing GFP or RavZ(ΔCA)-GFP in the presence or absence of 50 μM CQ were quantified by western blotting[30,31]. As shown in Fig. 1F,G, the level of substrate proteins, which indicates the autophagic flux rate, in HEK293T cells expressing RavZ(ΔCA)-GFP was similar to that in control cells expressing GFP. This suggests that the expression of RavZ(ΔCA)-GFP sensors did not affect autophagic flux in the turnover assay of endogenous LC3B or GABARAP.It has been reported that in RavZ protein, LIR motifs bind directly to mATG8 proteins, whereas an MT domain specifically binds to PI3P on the cytoplasmic surface of the intracellular membrane[19,20]. To further evaluate the contributions of LIR motifs and MT domains within RavZ to the autophagic membrane targeting of RavZ(ΔCA)-GFP, we generated RavZ(ΔCA)mLIR1/2-3-GFP, an LIR1/2-3 mutant of RavZ(ΔCA)-GFP, and LIR(1/2-3)-GFP, an MT domain deletion mutant of RavZ(ΔCA)-GFP (Fig. 2A). Each construct was co-expressed with mRFP-LC3 or mRFP-GABARAP proteins in MEF cells. To quantify the autophagic membrane localization, the ratio of GFP fluorescence intensity in autophagic membranes to that of cytosol (the A/C ratio) was measured (Fig. 2B). As shown in Fig. 2A,B, RavZ(ΔCA)-GFP was strongly localized to mRFP-LC3B- or mRFP-GABARAP-positive autophagic membranes, while LIR(1/2-3)-GFP was more weakly localized with mRFP-LC3B- or mRFP-GABARAP-positive autophagic membranes than RavZ(ΔCA)-GFP (***P < 0.001, one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test). However, the LIR mutant RavZ(ΔCA)mLIR1/2-3-GFP was not localized to autophagic membranes even in rapamycin/NH4Cl-treated MEF cells. Taken together, these results indicate that an MT domain alone, which is known to bind to PI3P, is insufficient for early endosome and autophagic membrane targeting in cells. In addition, the LIR motif alone was weakly targeted to autophagic membranes. However, the combination of an LIR motif and an MT domain resulted in efficient LC3B or GABARAP-positive autophagic membrane targeting.
Figure 2
Roles of the LIR motifs of RavZ on mATG8-positive autophagic membrane targeting. (A,B) Contribution of the LIR motifs of GFP-fused LIR motifs of RavZ in autophagosome targeting. RavZ(ΔCA)mLIR1/2-3-GFP, an LIR1/2/3 mutant of RavZ(ΔCA)-GFP; LIR(1/2-3)-GFP, an MT domain deletion mutant of RavZ(ΔCA)-GFP. Confocal images (A) depicting the cellular localization of GFP-fused LIR motifs of RavZ in rapamycin (Rapa)/NH4Cl-treated MEF cells. Scale bar: 10 μm. The bar graphs (B) illustrate the GFP fluorescence intensities of the autophagic membranes and the cytosol (the A/C ratio) (n = 75 for each group). ***P < 0.001 (one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test). (C,D) mATG8 protein-binding properties of the GFP-fused LIR motifs of RavZ proteins using GST-pulldown assays and quantification analysis for the binding. Extended blot images including these data are presented in Supplementary Fig. 6A. The bar graphs (D) illustrate relative quantification of the level of bound GFP-constructs in GST-pulldown assay. The levels of bound GFP-constructs intensity were normalized to the intensity of the expressed GFP-constructs (input). The data are presented as the mean ± SEM of three independent experiments. RAP, RAP-L1, GABARAP-L1; RAP-L2, GABARAP-L2.
Roles of the LIR motifs of RavZ on mATG8-positive autophagic membrane targeting. (A,B) Contribution of the LIR motifs of GFP-fused LIR motifs of RavZ in autophagosome targeting. RavZ(ΔCA)mLIR1/2-3-GFP, an LIR1/2/3 mutant of RavZ(ΔCA)-GFP; LIR(1/2-3)-GFP, an MT domain deletion mutant of RavZ(ΔCA)-GFP. Confocal images (A) depicting the cellular localization of GFP-fused LIR motifs of RavZ in rapamycin (Rapa)/NH4Cl-treated MEF cells. Scale bar: 10 μm. The bar graphs (B) illustrate the GFP fluorescence intensities of the autophagic membranes and the cytosol (the A/C ratio) (n = 75 for each group). ***P < 0.001 (one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test). (C,D) mATG8 protein-binding properties of the GFP-fused LIR motifs of RavZ proteins using GST-pulldown assays and quantification analysis for the binding. Extended blot images including these data are presented in Supplementary Fig. 6A. The bar graphs (D) illustrate relative quantification of the level of bound GFP-constructs in GST-pulldown assay. The levels of bound GFP-constructs intensity were normalized to the intensity of the expressed GFP-constructs (input). The data are presented as the mean ± SEM of three independent experiments. RAP, RAP-L1, GABARAP-L1; RAP-L2, GABARAP-L2.Next, we performed GST-pulldown assays to elucidate the binding properties of each mutant with mATG8 proteins. As shown in Fig. 2C,D, RavZ(ΔCA)-GFP and LIR(1/2-3)-GFP bound to GST-LC3A/B and GST-GABARAP/-L1/-L2 but not to GST-LC3C at a similar level, whereas RavZ(ΔCA)mLIR1/2-3-GFP did not bind to any of the GST-mATG8 proteins tested. Considering the cellular localization and mATG8 protein binding results, LIR motifs are primarily involved in mATG8 binding and an MT domain is additionally required for efficient autophagic membrane targeting via membrane association.
Characterization of LC3- and GABARAP-binding to the LIR1/2 and LIR3 motifs of RavZ(ΔCA)-GFP
To further evaluate the differential roles of N-terminal LIR1/2 motifs and the C-terminal LIR3 motif within RavZ, we generated an LIR1/2 motif mutant of RavZ(ΔCA)-GFP (RavZ(ΔCA) mLIR1/2-GFP) and an LIR3 motif mutant of RavZ(ΔCA)-GFP (RavZ(ΔCA)mLIR3-GFP). As shown in Fig. 3A,B, RavZ(ΔCA)mLIR1/2-GFP was localized with mRFP-LC3B- or mRFP-GABARAP-positive autophagic membranes, albeit less efficiently than RavZ(ΔCA)-GFP (***P < 0.001, one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test). On the other hand, RavZ(ΔCA)mLIR3-GFP was localized with mRFP-LC3B-positive autophagic membranes, but not with mRFP-GABARAP-positive autophagic membranes. These results suggest that both LIR1/2 motifs and LIR3 motif are involved in autophagic membrane targeting of RavZ(ΔCA)-GFP.
Figure 3
Elucidation of the roles of N- or C-terminal LIR motifs for autophagosome targeting of RavZ(ΔCA)-GFP. (A,B) Schematic diagram of GFP-fused RavZ mutant proteins and confocal images depicting the cellular localization of GFP-fused LIR motifs in RavZ proteins in MEF cells treated with 100 nM rapamycin (rapa) + 10 mM NH4Cl. The bar graphs (B) illustrate the GFP fluorescence intensities of the autophagic membranes and the cytosol (the A/C ratio) (n = 75 for each group). ***P < 0.001 (n = 75 for each group) one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test). Scale bar: 10 μm. (C,D) mATG8 protein-binding properties of the GFP-fused LIR motifs of RavZ proteins using GST-pulldown assays and quantification analysis for the binding. Extended blot images including these data are presented in Supplementary Fig. 6B. The bar graphs (D) illustrate relative quantification of the level of bound GFP-constructs in GST pull-down assay. The levels of bound GFP-constructs intensity were normalized to the intensity of the expressed GFP-constructs (input). The data are presented as the mean ± SEM of three independent experiments. RavZ(ΔCA)mLIR1/2-3-GFP, LIR1/2/3 mutant of RavZ(ΔCA)-GFP; RavZ(ΔCA)mLIR1/2-GFP, LIR1/2 motif mutant of RavZ(ΔCA)-GFP; RavZ(ΔCA)mLIR3-GFP, LIR3 motif mutant of RavZ(ΔCA)-GFP. RAP, GABARAP; RAP-L1, GABARAP-L1; RAP-L2, GABARAP-L2; N.S., not significant.
Elucidation of the roles of N- or C-terminal LIR motifs for autophagosome targeting of RavZ(ΔCA)-GFP. (A,B) Schematic diagram of GFP-fused RavZ mutant proteins and confocal images depicting the cellular localization of GFP-fused LIR motifs in RavZ proteins in MEF cells treated with 100 nM rapamycin (rapa) + 10 mM NH4Cl. The bar graphs (B) illustrate the GFP fluorescence intensities of the autophagic membranes and the cytosol (the A/C ratio) (n = 75 for each group). ***P < 0.001 (n = 75 for each group) one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test). Scale bar: 10 μm. (C,D) mATG8 protein-binding properties of the GFP-fused LIR motifs of RavZ proteins using GST-pulldown assays and quantification analysis for the binding. Extended blot images including these data are presented in Supplementary Fig. 6B. The bar graphs (D) illustrate relative quantification of the level of bound GFP-constructs in GST pull-down assay. The levels of bound GFP-constructs intensity were normalized to the intensity of the expressed GFP-constructs (input). The data are presented as the mean ± SEM of three independent experiments. RavZ(ΔCA)mLIR1/2-3-GFP, LIR1/2/3 mutant of RavZ(ΔCA)-GFP; RavZ(ΔCA)mLIR1/2-GFP, LIR1/2 motif mutant of RavZ(ΔCA)-GFP; RavZ(ΔCA)mLIR3-GFP, LIR3 motif mutant of RavZ(ΔCA)-GFP. RAP, GABARAP; RAP-L1, GABARAP-L1; RAP-L2, GABARAP-L2; N.S., not significant.Next, we performed GST-pulldown assays to elucidate the binding properties of each mutant with the mATG8 protein family. As shown in Fig. 3C,D, RavZ(ΔCA)mLIR1/2-GFP bound to GST-GABARAP/-L1 but not to GST-LC3A/B/C and GST-GABARAP-L2, while RavZ(ΔCA)mLIR3-GFP bound to GST-LC3B and weakly to GST-GABARAP-L1. As a control, RavZ(ΔCA)-GFP, but not RavZ(ΔCA)mLIR1/2-3-GFP bound to GST-LC3A/B and GST-GABARAP/-L1/-L2 but not to GST-LC3C. Quantification of GST binding showed that the binding of any mATG8 to RavZ(ΔCA)-GFP was higher than the binding to either RavZ(ΔCA)mLIR1/2-GFP or RavZ(ΔCA)mLIR3-GFP (Fig. 3D), indicating that both the LIR1/2 and LIR3 motifs contributed to the LC3 or GABARAP subfamily binding of RavZ protein. Overall, the results of both cellular localization and GST-pulldown assays suggest that both the LIR1/2 and LIR3 motifs are required for mATG8 proteins binding except for LC3C binding.Our cellular analysis of the modified RavZ(ΔCA)-GFP indicated that the protein is efficiently targeted to autophagosomes via a combination of LIR motifs and an MT domain. An MT domain, an LIR1/2 motif, or an LIR3 motif alone is negligible or weak for targeting RavZ to autophagic membranes. However, when the three domains are combined, as in the RavZ(ΔCA)-GFP protein, the protein is efficiently targeted to autophagic membranes. It has been reported that the catalytic domain of the RavZ protein also has an α3 helix, which is involved in the association of the membrane with enzyme activity (Supplemental Fig. 1A)[20]. Therefore, in wild-type RavZ, efficient autophagic membrane targeting of RavZ protein is mediated by the combination of multiple domains including two membrane association domains (an α3 helix in the catalytic domain and a PI3P-binding MT domain) and multiple LIR motifs (an LIR1/2 motifs and an LIR3 motif) for direct mATG8 protein binding.
Comparative analysis of the autophagosome targeting efficiency between HyD motifs and MT domains
We recently developed LIR-based autophagosome sensors using a HyD motif, which enhances the membrane localization[15]. In RavZ proteins, an MT domain, another type of membrane association domain, plays an assisting role in autophagosome targeting of RavZ(ΔCA)-GFP (Fig. 2). Therefore, to compare the relative efficiency of autophagosome targeting between a HyD motif and an MT domain, we generated several constructs, as shown in Fig. 4A: GFP fused to an MT domain (GFP-MT) and to an LIR3 motif from RavZ fused to GFP, GFP-MT, or HyD-GFP (GFP-LIR3, GFP-MT-LIR3, and HyD-LIR3-GFP, respectively). Each construct was co-expressed with either mRFP-LC3B or mRFP-GABARAP in MEF cells. As shown in Fig. 4A, GFP-LIR3, GFP-HyD, or GFP-MT alone was diffusely localized to the cytosol and nucleus and was barely localized to autophagic membranes. However, GFP-MT-LIR3 and HyD-LIR3-GFP were co-localized with mRFP-LC3B- or mRFP-GABARAP-positive autophagic membranes in rapamycin/NH4Cl-treated cells (Fig. 4A,B). Thus, when LIR motifs were combined with an MT domain or a HyD motif, the A/C ratio was significantly enhanced. More intriguingly, GFP-MT-LIR3 was more efficiently localized to autophagic membranes than HyD-LIR3-GFP (***P < 0.001, one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test). Therefore, our comparative analysis of the A/C ratios between MT- and HyD-LIR probes suggests that an MT domain facilitates autophagic membrane-targeting through PI3P-binding more efficiently than a HyD motif through non-selective membrane association via hydrophobic interactions on autophagic membranes.
Figure 4
Comparison of the autophagosome targeting efficiency of HyD and MT domains. (A) Schematic diagram of GFP-fused RavZ mutant proteins and confocal images showing cellular localization of mRFP-LC3B or mRFP-GABARAP co-expressed with GFP-LIR3 or its derivative forms in MEF cells upon autophagy induction (100 nM rapamycin (rapa) + 10 mM NH4Cl, 3 h). Scale bar: 10 μm. The bar graphs (B) illustrate the GFP fluorescent intensities of the autophagosomes and the cytosol (the A/C ratio) (n = 75 for each group). GFP-MT, GFP fused to an MT domain; GFP-LIR3, an LIR3 motif from RavZ fused to GFP; GFP-MT-LIR3, an LIR3 motif from RavZ fused to GFP-MT; HyD-LIR3-GFP, an LIR3 motif from RavZ fused to HyD-GFP. ***P < 0.001 (one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test). mRFP-RAP, mRFP-GABARAP.
Comparison of the autophagosome targeting efficiency of HyD and MT domains. (A) Schematic diagram of GFP-fused RavZ mutant proteins and confocal images showing cellular localization of mRFP-LC3B or mRFP-GABARAP co-expressed with GFP-LIR3 or its derivative forms in MEF cells upon autophagy induction (100 nM rapamycin (rapa) + 10 mM NH4Cl, 3 h). Scale bar: 10 μm. The bar graphs (B) illustrate the GFP fluorescent intensities of the autophagosomes and the cytosol (the A/C ratio) (n = 75 for each group). GFP-MT, GFP fused to an MT domain; GFP-LIR3, an LIR3 motif from RavZ fused to GFP; GFP-MT-LIR3, an LIR3 motif from RavZ fused to GFP-MT; HyD-LIR3-GFP, an LIR3 motif from RavZ fused to HyD-GFP. ***P < 0.001 (one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test). mRFP-RAP, mRFP-GABARAP.
Monitoring LC3 or GABARAP subfamily-positive autophagic membranes using RavZ-based probes modified by replacement of LIR motifs with those selective for members of the LC3 or GABARAP subfamily
If RavZ(ΔCA)-GFP constructs are to be used for monitoring LC3- or GABARAP-positive autophagic membranes selectively, the LIR motifs of RavZ must be replaced with other LIR motifs that specifically and selectively bind to LC3 or GABARAP proteins. Based on previous studies that analyzed the preferential binding properties of LIR motifs for either LC3 or GABARAP, we chose candidate LIR motifs from Fyco1 as an LC3 subfamily-specific motif and from ULK2 as GABARAP subfamily-specific motifs (Fig. 5A). To generate selective LC3- or GABARAP-positive autophagic membrane-detecting RavZ-based probes, we replaced the LIR1/2 and LIR3 motifs within RavZ(ΔCA)-GFP with LIR motifs from Fyco1 or ULK2, generating RavZ(ΔCA)Fy-GFP and RavZ(ΔCA)ULK2-GFP, respectively. Each probe, which contains two LIR motifs and an MT domain, was co-expressed with each 3xFlag-mATG8 protein in HEK293T cells and Flag co-immunoprecipitation (Flag co-IP) assays were performed to investigate the binding properties of these new LIR motifs. As shown in Supplemental Fig. 3, RavZ(ΔCA)Fy-GFP bound selectively to 3xFlag-LC3A or 3xFlag-LC3B, but not to 3xFlag-LC3C or 3xFlag-GABARAP, -L1, or -L2. In contrast, RavZ(ΔCA)ULK2-GFP bound to 3xFlag-GABARAP, -L1, or -L2 but not to 3xFlag-LC3A, B, or C.
Figure 5
Selective LC3A/B or GABARAP subfamily-positive autophagosome targeting of RavZ(ΔCA)Fy-GFP or RavZ(ΔCA)ULK2-GFP, respectively. (A) Schematic diagram of GFP-fused RavZ(ΔCA)X-GFP and its binding preference. (B) Confocal images showing the cellular localization of RavZ(ΔCA)Fy-GFP or RavZ(ΔCA)ULK2-GFP in rapamycin/NH4Cl-treated MEF cells. Scale bar: 10 μm. The bar graphs (C) illustrate the GFP fluorescent intensities of the autophagosomes and the cytosol (the A/C ratio) (n = 75 for each group). (D) Percentage of co-localization of mRFP-mATG8-positive autophagic membrane with RavZ(ΔCA)Fy-GFP or RavZ(ΔCA)ULK2-GFP-positive autophagic membranes (n = 25 for each group). (E,F) Autophagic flux assay in HEK293T cells expressing GFP, RavZ(ΔCA)Fy-GFP, or RavZ(ΔCA)ULK2-GFP upon rapamycin (rapa) treatment (in the presence or absence of chloroquine (CQ) for 3 h). The bar graphs illustrate the level of LC3-II or GABARAP-II. The levels of LC3-II and GABARAP-II in the GFP, RavZ(ΔCA)Fy-GFP, or RavZ(ΔCA)ULK2-GFP-expressing cells were normalized to that of actin in HEK293T cells expressing GFP, RavZ(ΔCA)Fy-GFP, or RavZ(ΔCA)ULK2-GFP-expressing cells. The cell lysates were then subjected to western blot analyses (E) and quantification analysis (F) with an anti-GFP, anti-LC3, anti-GABARAP, or anti-β-actin antibody. The data are presented as the mean ± SEM of five independent experiments. Extended blot images including these data are presented in Supplementary Fig. 7. RAP, GABARAP; RAP-L1, GABARAP-L1; RAP-L2, GABARAP-L2; N.S., not significant.
Selective LC3A/B or GABARAP subfamily-positive autophagosome targeting of RavZ(ΔCA)Fy-GFP or RavZ(ΔCA)ULK2-GFP, respectively. (A) Schematic diagram of GFP-fused RavZ(ΔCA)X-GFP and its binding preference. (B) Confocal images showing the cellular localization of RavZ(ΔCA)Fy-GFP or RavZ(ΔCA)ULK2-GFP in rapamycin/NH4Cl-treated MEF cells. Scale bar: 10 μm. The bar graphs (C) illustrate the GFP fluorescent intensities of the autophagosomes and the cytosol (the A/C ratio) (n = 75 for each group). (D) Percentage of co-localization of mRFP-mATG8-positive autophagic membrane with RavZ(ΔCA)Fy-GFP or RavZ(ΔCA)ULK2-GFP-positive autophagic membranes (n = 25 for each group). (E,F) Autophagic flux assay in HEK293T cells expressing GFP, RavZ(ΔCA)Fy-GFP, or RavZ(ΔCA)ULK2-GFP upon rapamycin (rapa) treatment (in the presence or absence of chloroquine (CQ) for 3 h). The bar graphs illustrate the level of LC3-II or GABARAP-II. The levels of LC3-II and GABARAP-II in the GFP, RavZ(ΔCA)Fy-GFP, or RavZ(ΔCA)ULK2-GFP-expressing cells were normalized to that of actin in HEK293T cells expressing GFP, RavZ(ΔCA)Fy-GFP, or RavZ(ΔCA)ULK2-GFP-expressing cells. The cell lysates were then subjected to western blot analyses (E) and quantification analysis (F) with an anti-GFP, anti-LC3, anti-GABARAP, or anti-β-actin antibody. The data are presented as the mean ± SEM of five independent experiments. Extended blot images including these data are presented in Supplementary Fig. 7. RAP, GABARAP; RAP-L1, GABARAP-L1; RAP-L2, GABARAP-L2; N.S., not significant.Next, RavZ(ΔCA)Fy-GFP or RavZ(ΔCA)ULK2-GFP was co-expressed with each of the mRFP-mATG8 proteins in MEF cells. As shown in Fig. 5B,C, RavZ(ΔCA)Fy-GFP was efficiently localized to mRFP-LC3A/B-positive autophagosomes, whereas RavZ(ΔCA)ULK2-GFP was efficiently localized to mRFP-GABARAP/-L1/-L2-positive autophagosomes in MEF cells (***P < 0.001, one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test). Similarly, RavZ(ΔCA)Fy-GFP-positive spots were more co-localized with mRFP-LC3 subfamily-positive spots than mRFP-GABARAP subfamily-positive spots (***P < 0.001, one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test) (Fig. 5D). On the other hand, RavZ(ΔCA)ULK2-GFP-positive spots were more co-localized with mRFP-GABARAP subfamily-positive spots than mRFP-LC3 subfamily-positive spots (***P < 0.001, one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test) (Fig. 5D). In addition, RavZ(ΔCA)Fy-GFP and RavZ(ΔCA)ULK2-GFP detected vesicle structures in wild-type HeLa cells, but not in ATG5- or ATG7-knockout HeLa cells in an autophagy-dependent manner (Supplemental Fig. 2).As shown in Fig. 5E,F, the expression of RavZ(ΔCA)Fy-GFP or RavZ(ΔCA)ULK2-GFP did not affect autophagic flux in the turnover assay of endogenous LC3B or GABARAP. These results suggest that RavZ(ΔCA)Fy-GFP and RavZ(ΔCA)ULK2-GFP can detect endogenous autophagic membranes and preferentially detect LC3A/B-positive and GABARAP-positive autophagic membranes, respectively.Next, we examined the dynamics of RavZ(ΔCA)X-GFP-positive autophagic membranes in MEF cells using live-cell imaging. Kymograph analysis of GFP-LC3B, GFP-GABARAP, RavZ(ΔCA)-GFP RavZ(ΔCA)Fy-GFP, and RavZ(ΔCA)ULK2-GFP mobility showed no difference between the groups (Supplemental Fig. 4).We summarized the A/C ratio of the constructs used in the experiments in Supplemental Table 1. The A/C ratio clearly showed that RavZ(ΔCA)-GFP efficiently detected both LCB and GABARAP-positive autophagic membranes. Meanwhile, RavZ(ΔCA)Fy-GFP selectively detected LC3A/B-positive autophagic membranes, whereas RavZ(ΔCA)ULK2-GFP efficiently detected GABARAP subfamily-positive autophagic membranes. RavZ(ΔCA)mLIR3-GFP also selectively detected LC3B-positive autophagic membranes but to a much weaker degree than RavZ(ΔCA)Fy-GFP. RavZ(ΔCA)mLIR1/2-GFP detected both LC3B- and GABARAP-positive autophagic membranes to a much weaker degree than RavZ(ΔCA)-GFP. Thus, RavZ(ΔCA)-GFP, RavZ(ΔCA)Fy-GFP, and RavZ(ΔCA)ULK2-GFP are useful for detecting all types of mATG8-positive, LC3A/B-positive, and GABARAP subfamily-positive autophagic membranes, respectively.Many mATG8-interacting proteins contain a canonical LIR motif with a core consensus sequence, (W/F/Y)-X-X-(L/I/V), which binds to LIR docking sites (LDS) in two hydrophobic pockets, HP1 and HP2, conserved in mATG8s using W/F/Y and L/I/V, respectively[9,32-35]. As shown in Supplemental Fig. 1B, canonical LIR motifs from RavZ, Fyco1, and ULK2 have “F” in a core LIR motifs commonly. Recently, the GABARAP-selective motif (GIM) was proposed to have a core consensus sequence ((W/F)-(I/V)-X-V)[36]. The LIR motif from ULK2 has “FVLV,” which follows the GIM consensus sequence. The LIR3 motif of RavZ has “FVTI,” which is similar to the GIM sequence ((W/F)-(I/V)-X-V) except for the presence of “I” instead of “V”. This might be the reason why the LIR3 motif of RavZ has GABARAP-preferential binding.In a previous study, we used a general membrane association motif, a HyD motif, to monitor endogenous mATG8 family proteins in autophagosomes in live cells without overexpression[15,21]. HyD motifs have mild hydrophobicity and, by themselves, have no organelle membrane targeting; instead, these motifs help enhance membrane association mediated by LIR motifs[15]. Similarly, we found that the MT domain of RavZ alone is localized to the cytosol, but not to the early endosome, where it fails to enrich PI3P probably due to weak PI3P binding, but helps to enhance membrane association mediated by LIR motifs (Figs 2A,B and 4). Therefore, using an MT domain of RavZ can minimize the sequestering and altering of PI3P dynamics in cells. In a previous study, we duplicated LIR motifs to enhance the efficiency of autophagic membranes further and generated GABARAP subfamily-positive autophagic membrane-targeting probes (HyD-2xLIR(ULK2)-GFP and HyD-2xLIR(Stbd1)-GFP)[21]. Interestingly, the wild-type RavZ protein has multiple LIR motifs that enhance autophagic membrane targeting through the synergistic binding avidity of the N- and C-terminal LIR motifs. Thus, RavZ has a useful structure for sensing autophagic membranes. We leveraged this advantage in our study to generate probes that detect LC3A/B- or GABARAP-positive autophagic membranes in cells. However, if LIR-based sensors are expressed at a higher level, they could potentially function as dominant-negative probes that sequester endogenous LIR-containing proteins or PI3P. Therefore, to be used as probes to detect LC3- or GABARAP-positive autophagic membranes, stable cell lines that express RavZ(ΔCA)X-GFP at a lower level or promoters that mediate lower expression levels need to be considered. Despite some limitations, we propose that RavZ(ΔCA)X-GFP constructs are an advanced version of LIR-based LC3- or GABARAP-positive autophagic membrane-detecting probes for autophagy research.
Methods
DNA constructs
All primers are listed in Supplemental Table 2. The regions encoding individual RavZLIR1/2 or LIR3 motifs and MT domains were generated by PCR amplification of pcDNA3.1(−)-Flag-RavZ vectors and inserted into the N3-EGFP vector using restriction enzymes. The pcDNA3.1(−)-Flag-RavZ vectors were kindly provided by Dr. Song (Department of Life Sciences, Korea University, Korea)[19]. The Aplysia PDE4 short-form (N20) (SN20, HyD)-GFP was generated by PCR amplification of the full-length Aplysia PDE4 short-form gene and inserted into the pcDNA3.1-EGFP and N3-EGFP vectors. Mutations of the RavZ LIR motif were amplified by PCR using RavZLIR1/2 or 3 mutant primers (Supplemental Table 2) and inserted into N3-RavZ-EGFP vectors using restriction enzymes. Additionally, other LIR motifs, including FUNDC1, Fyco1, Stbd1, and ULK2, were amplified by PCR using primers (Supplemental Table 2) and inserted into N3-RavZ-GFP vectors to replace the RavZ LIR with another LIR. GST-LC3A, GST-LC3B, GST-LC3C, GST-GABARAP, GST-GABARAP-L1, and GST-GABARAP-L2 were obtained from Addgene (Cambridge, MA, USA). We also used previously described DNA constructs mRFP-LC3A, mRFP-LC3B, mRFP-LC3C, mRFP-GABARAP, mRFP-GABARAP-L1, and mRFP-GABARAP-L2[15] in this study.
Cell culture, transfection, confocal microscopy, and drug treatment
This method has been previously described[37]. Briefly, HEK293T, MEF, and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum and penicillin/streptomycin in a humidified atmosphere of 5% (v/v) CO2 at 37 °C. Cells were seeded in a Sticky-Slide 8-well system (Catalog #: 80828; Ibidi, Martinsried, Germany) to obtain 40–60% confluent cells on the day of imaging. Cells were transfected with DNA constructs using calcium phosphate or Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA) 20–24 h before imaging. The relative amount of each construct was empirically determined based on the relative expression of each construct combination.Cells were visualized with an inverted Zeiss LSM-700 scanning laser confocal microscope operated by ZEN software (Carl Zeiss, Oberkochen, Germany). The laser lines for excitation and the spectral detection windows for the fluorochromes were 488 with 508–543 nm for GFP and 561 with 578–649 nm for mRFP. Appropriate GFP (500–550 nm) and mRFP (575–625 nm) emission filters were used during the sequential imaging of each fluorescent protein. Most images were taken with live cells. Rapamycin was obtained from Sigma-Aldrich (Catalog #: R8781; St. Louis, MO, USA). All treatments and assays were performed at 37 °C unless otherwise indicated.
Quantitative analysis of A/C ratio
To calculate the ratio of autophagosome/cytosol (A/C) fluorescent intensities, the average value of the autophagosome or cytosol fluorescent intensity was obtained from at least five randomly selected points on autophagosomes or in the cytosol of a single cell using ZEN software. In the same manner, the quantitative A/C ratio of at least 25 randomly selected cells per experiment was obtained from three independent experiments. All statistical data were calculated and graphed using GraphPad Prism5 (GraphPad, Inc., La Jolla, CA, USA).
Co-localized spot number analysis
To determine the percent of co-localized spots of LC3/GABARAP-positive autophagosomes in autophagy-induced cells, the number of co-localized spots over a certain size in a single cell was counted using Image-J software. First, the cell image was changed to an 8-bit image and then inverted. Next, the background was removed so that only the spot was visible, and finally, the number of co-localized spots was counted using the “Analyze particles” function in the Image-J program. In the same manner, at least 25 randomly selected cells were quantified. All statistical data were calculated and graphed using GraphPad Prism5.
GST-pulldown assay
For GST-pulldown assays using HEK293T cell lysates, cells were transfected with the GFP construct-containing DNA using calcium phosphate (Takara Bio) transfection. After 24 h, cells were harvested, washed with PBS, and lysed in immunoprecipitation lysis buffer solution (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 2 mM EDTA; 1% Triton X ‐100; and protease and phosphatase inhibitors), and the supernatants were isolated after centrifugation. Cell lysates were incubated with purified GST-mATG8 protein and glutathione-agarose beads overnight at 4 °C. The next day, they were washed 3–5 times with immunoprecipitation lysis buffer solution at 4 °C. Proteins were separated by SDS–PAGE and analyzed by Western blot and Coomassie blue staining.
Immunoprecipitation
This method has been previously described[38]. Briefly, for transient transfections, HEK293T cells were plated at a density of 5–7 × 105 cells/well in six-well plates and cultured for 24 h. The cells were transfected with DNA constructs using calcium phosphate (Clontech) and incubated for 24 h. For Flag immunoprecipitation, the transfected HEK293T cells were washed twice with PBS and lysed with a buffer containing 1% Triton X-100, 50 mM Tris-HCl (pH 7.5), 150 mM sodium chloride (NaCl), 2 mM ethylenediaminetetraacetic acid (EDTA), and a protease inhibitor cocktail (Roche). The cell lysate was incubated with 50 μL (bead volume) of mouse anti-Flag M2 antibody-conjugated beads (Sigma) at 4 °C overnight. The beads were subsequently washed three times with lysis buffer. The immunoprecipitate was eluted by adding 2 μg/mL of 3xFlag peptides and analyzed by Western blot.
Western blot, antibodies and band quantitation
Protein samples from the GST-pulldown, immunoprecipitation assays, and flux assays were separated by SDS-PAGE, transferred to PVDF membranes, and incubated with primary antibodies overnight at 4 °C. After three washes, membranes were incubated with secondary antibodies and conjugated with horseradish peroxidase for an hour. Signals were visualized with ECL using Advansta WesternBright ECL (K-12045-D50). The antibodies using in the experiment were used: Flag (Sigma, F1804, 1:10000), GFP (Santa Cruz Biotechnology, sc-9996, 1:10000), LC3B (Cell Signaling Technology, #2775, 1:1000), GABARAP (Cell Signaling Technology, #13733, 1:1000), donkey anti-rabbit HRP (Santa Cruz Biotechnology, sc-2313, 1:10000) and goat anti-mouse HRP (Santa Cruz Biotechnology, sc-2005, 1:10000). In order to quantify the intensity of the western blot band, the area of each band was quantified using the ImageJ program. In the same manner, the Band Quantitation was obtained from three independent experiments. All statistical data were calculated and graphed using GraphPad Prism5 (GraphPad, Inc., La Jolla, CA, USA).
Live cell imaging and autophagosome dynamics analysis
MEFs were transfected using Lipofectamine 2000 and expressed for 24 h on 96-well glass-bottom plates (Ibidi, #89626). Before analysis, the cells were incubated with rapamycin (100 nM, 4 h) to induce autophagy. Images of autophagosome dynamics were acquired on an A1R confocal microscope (Nikon, Japan) with a Nikon CFI Plan Apochromat VC object (60x/1.40 numerical aperture) in a temperature-controlled chamber at 37 °C. A 525-nm laser was used for excitation. The images were captured every 5 s for 5 min for GFP-LC3, GFP-GABARAP, RavZ(ΔCat)-GFP, RavZ(ΔCat, Fyco1)-GFP, and RavZ(ΔCat, ULK2)-GFP. Kymograph images and movies were generated using ImageJ (NIH) software to compare the dynamics of autophagosomes. Autophagosome dynamics was analyzed using NIS-elements AR analysis program (Nikon).
Statistical analysis
Kolmogorov-Smirnov (KS) tests were used to examine the distribution of the data. The data were normally distributed, and then one-way ANOVA, in conjunction with Tukey’s multiple comparison test for post-hoc analysis (group number > = 3) was used for statistical analysis.Supplementary information
Authors: Alexandra Stolz; Mateusz Putyrski; Ivana Kutle; Jessica Huber; Chunxin Wang; Viktória Major; Sachdev S Sidhu; Richard J Youle; Vladimir V Rogov; Volker Dötsch; Andreas Ernst; Ivan Dikic Journal: EMBO J Date: 2016-12-27 Impact factor: 11.598
Authors: Daniel J Klionsky; Fabio C Abdalla; Hagai Abeliovich; Robert T Abraham; Abraham Acevedo-Arozena; Khosrow Adeli; Lotta Agholme; Maria Agnello; Patrizia Agostinis; Julio A Aguirre-Ghiso; Hyung Jun Ahn; Ouardia Ait-Mohamed; Slimane Ait-Si-Ali; Takahiko Akematsu; Shizuo Akira; Hesham M Al-Younes; Munir A Al-Zeer; Matthew L Albert; Roger L Albin; Javier Alegre-Abarrategui; Maria Francesca Aleo; Mehrdad Alirezaei; Alexandru Almasan; Maylin Almonte-Becerril; Atsuo Amano; Ravi Amaravadi; Shoba Amarnath; Amal O Amer; Nathalie Andrieu-Abadie; Vellareddy Anantharam; David K Ann; Shailendra Anoopkumar-Dukie; Hiroshi Aoki; Nadezda Apostolova; Giuseppe Arancia; John P Aris; Katsuhiko Asanuma; Nana Y O Asare; Hisashi Ashida; Valerie Askanas; David S Askew; Patrick Auberger; Misuzu Baba; Steven K Backues; Eric H Baehrecke; Ben A Bahr; Xue-Yuan Bai; Yannick Bailly; Robert Baiocchi; Giulia Baldini; Walter Balduini; Andrea Ballabio; Bruce A Bamber; Edward T W Bampton; Gábor Bánhegyi; Clinton R Bartholomew; Diane C Bassham; Robert C Bast; Henri Batoko; Boon-Huat Bay; Isabelle Beau; Daniel M Béchet; Thomas J Begley; Christian Behl; Christian Behrends; Soumeya Bekri; Bryan Bellaire; Linda J Bendall; Luca Benetti; Laura Berliocchi; Henri Bernardi; Francesca Bernassola; Sébastien Besteiro; Ingrid Bhatia-Kissova; Xiaoning Bi; Martine Biard-Piechaczyk; Janice S Blum; Lawrence H Boise; Paolo Bonaldo; David L Boone; Beat C Bornhauser; Karina R Bortoluci; Ioannis Bossis; Frédéric Bost; Jean-Pierre Bourquin; Patricia Boya; Michaël Boyer-Guittaut; Peter V Bozhkov; Nathan R Brady; Claudio Brancolini; Andreas Brech; Jay E Brenman; Ana Brennand; Emery H Bresnick; Patrick Brest; Dave Bridges; Molly L Bristol; Paul S Brookes; Eric J Brown; John H Brumell; Nicola Brunetti-Pierri; Ulf T Brunk; Dennis E Bulman; Scott J Bultman; Geert Bultynck; Lena F Burbulla; Wilfried Bursch; Jonathan P Butchar; Wanda Buzgariu; Sergio P Bydlowski; Ken Cadwell; Monika Cahová; Dongsheng Cai; Jiyang Cai; Qian Cai; Bruno Calabretta; Javier Calvo-Garrido; Nadine Camougrand; Michelangelo Campanella; Jenny Campos-Salinas; Eleonora Candi; Lizhi Cao; Allan B Caplan; Simon R Carding; Sandra M Cardoso; Jennifer S Carew; Cathleen R Carlin; Virginie Carmignac; Leticia A M Carneiro; Serena Carra; Rosario A Caruso; Giorgio Casari; Caty Casas; Roberta Castino; Eduardo Cebollero; Francesco Cecconi; Jean Celli; Hassan Chaachouay; Han-Jung Chae; Chee-Yin Chai; David C Chan; Edmond Y Chan; Raymond Chuen-Chung Chang; Chi-Ming Che; Ching-Chow Chen; Guang-Chao Chen; Guo-Qiang Chen; Min Chen; Quan Chen; Steve S-L Chen; WenLi Chen; Xi Chen; Xiangmei Chen; Xiequn Chen; Ye-Guang Chen; Yingyu Chen; Yongqiang Chen; Yu-Jen Chen; Zhixiang Chen; Alan Cheng; Christopher H K Cheng; Yan Cheng; Heesun Cheong; Jae-Ho Cheong; Sara Cherry; Russ Chess-Williams; Zelda H Cheung; Eric Chevet; Hui-Ling Chiang; Roberto Chiarelli; Tomoki Chiba; Lih-Shen Chin; Shih-Hwa Chiou; Francis V Chisari; Chi Hin Cho; Dong-Hyung Cho; Augustine M K Choi; DooSeok Choi; Kyeong Sook Choi; Mary E Choi; Salem Chouaib; Divaker Choubey; Vinay Choubey; Charleen T Chu; Tsung-Hsien Chuang; Sheau-Huei Chueh; Taehoon Chun; Yong-Joon Chwae; Mee-Len Chye; Roberto Ciarcia; Maria R Ciriolo; Michael J Clague; Robert S B Clark; Peter G H Clarke; Robert Clarke; Patrice Codogno; Hilary A Coller; María I Colombo; Sergio Comincini; Maria Condello; Fabrizio Condorelli; Mark R Cookson; Graham H Coombs; Isabelle Coppens; Ramon Corbalan; Pascale Cossart; Paola Costelli; Safia Costes; Ana Coto-Montes; Eduardo Couve; Fraser P Coxon; James M Cregg; José L Crespo; Marianne J Cronjé; Ana Maria Cuervo; Joseph J Cullen; Mark J Czaja; Marcello D'Amelio; Arlette Darfeuille-Michaud; Lester M Davids; Faith E Davies; Massimo De Felici; John F de Groot; Cornelis A M de Haan; Luisa De Martino; Angelo De Milito; Vincenzo De Tata; Jayanta Debnath; Alexei Degterev; Benjamin Dehay; Lea M D Delbridge; Francesca Demarchi; Yi Zhen Deng; Jörn Dengjel; Paul Dent; Donna Denton; Vojo Deretic; Shyamal D Desai; Rodney J Devenish; Mario Di Gioacchino; Gilbert Di Paolo; Chiara Di Pietro; Guillermo Díaz-Araya; Inés Díaz-Laviada; Maria T Diaz-Meco; Javier Diaz-Nido; Ivan Dikic; Savithramma P Dinesh-Kumar; Wen-Xing Ding; Clark W Distelhorst; Abhinav Diwan; Mojgan Djavaheri-Mergny; Svetlana Dokudovskaya; Zheng Dong; Frank C Dorsey; Victor Dosenko; James J Dowling; Stephen Doxsey; Marlène Dreux; Mark E Drew; Qiuhong Duan; Michel A Duchosal; Karen Duff; Isabelle Dugail; Madeleine Durbeej; Michael Duszenko; Charles L Edelstein; Aimee L Edinger; Gustavo Egea; Ludwig Eichinger; N Tony Eissa; Suhendan Ekmekcioglu; Wafik S El-Deiry; Zvulun Elazar; Mohamed Elgendy; Lisa M Ellerby; Kai Er Eng; Anna-Mart Engelbrecht; Simone Engelender; Jekaterina Erenpreisa; Ricardo Escalante; Audrey Esclatine; Eeva-Liisa Eskelinen; Lucile Espert; Virginia Espina; Huizhou Fan; Jia Fan; Qi-Wen Fan; Zhen Fan; Shengyun Fang; Yongqi Fang; Manolis Fanto; Alessandro Fanzani; Thomas Farkas; Jean-Claude Farré; Mathias Faure; Marcus Fechheimer; Carl G Feng; Jian Feng; Qili Feng; Youji Feng; László Fésüs; Ralph Feuer; Maria E Figueiredo-Pereira; Gian Maria Fimia; Diane C Fingar; Steven Finkbeiner; Toren Finkel; Kim D Finley; Filomena Fiorito; Edward A Fisher; Paul B Fisher; Marc Flajolet; Maria L Florez-McClure; Salvatore Florio; Edward A Fon; Francesco Fornai; Franco Fortunato; Rati Fotedar; Daniel H Fowler; Howard S Fox; Rodrigo Franco; Lisa B Frankel; Marc Fransen; José M Fuentes; Juan Fueyo; Jun Fujii; Kozo Fujisaki; Eriko Fujita; Mitsunori Fukuda; Ruth H Furukawa; Matthias Gaestel; Philippe Gailly; Malgorzata Gajewska; Brigitte Galliot; Vincent Galy; Subramaniam Ganesh; Barry Ganetzky; Ian G Ganley; Fen-Biao Gao; George F Gao; Jinming Gao; Lorena Garcia; Guillermo Garcia-Manero; Mikel Garcia-Marcos; Marjan Garmyn; Andrei L Gartel; Evelina Gatti; Mathias Gautel; Thomas R Gawriluk; Matthew E Gegg; Jiefei Geng; Marc Germain; Jason E Gestwicki; David A Gewirtz; Saeid Ghavami; Pradipta Ghosh; Anna M Giammarioli; Alexandra N Giatromanolaki; Spencer B Gibson; Robert W Gilkerson; Michael L Ginger; Henry N Ginsberg; Jakub Golab; Michael S Goligorsky; Pierre Golstein; Candelaria Gomez-Manzano; Ebru Goncu; Céline Gongora; Claudio D Gonzalez; Ramon Gonzalez; Cristina González-Estévez; Rosa Ana González-Polo; Elena Gonzalez-Rey; Nikolai V Gorbunov; Sharon Gorski; Sandro Goruppi; Roberta A Gottlieb; Devrim Gozuacik; Giovanna Elvira Granato; Gary D Grant; Kim N Green; Aleš Gregorc; Frédéric Gros; Charles Grose; Thomas W Grunt; Philippe Gual; Jun-Lin Guan; Kun-Liang Guan; Sylvie M Guichard; Anna S Gukovskaya; Ilya Gukovsky; Jan Gunst; Asa B Gustafsson; Andrew J Halayko; Amber N Hale; Sandra K Halonen; Maho Hamasaki; Feng Han; Ting Han; Michael K Hancock; Malene Hansen; Hisashi Harada; Masaru Harada; Stefan E Hardt; J Wade Harper; Adrian L Harris; James Harris; Steven D Harris; Makoto Hashimoto; Jeffrey A Haspel; Shin-ichiro Hayashi; Lori A Hazelhurst; Congcong He; You-Wen He; Marie-Joseé Hébert; Kim A Heidenreich; Miep H Helfrich; Gudmundur V Helgason; Elizabeth P Henske; Brian Herman; Paul K Herman; Claudio Hetz; Sabine Hilfiker; Joseph A Hill; Lynne J Hocking; Paul Hofman; Thomas G Hofmann; Jörg Höhfeld; Tessa L Holyoake; Ming-Huang Hong; David A Hood; Gökhan S Hotamisligil; Ewout J Houwerzijl; Maria Høyer-Hansen; Bingren Hu; Chien-An A Hu; Hong-Ming Hu; Ya Hua; Canhua Huang; Ju Huang; Shengbing Huang; Wei-Pang Huang; Tobias B Huber; Won-Ki Huh; Tai-Ho Hung; Ted R Hupp; Gang Min Hur; James B Hurley; Sabah N A Hussain; Patrick J Hussey; Jung Jin Hwang; Seungmin Hwang; Atsuhiro Ichihara; Shirin Ilkhanizadeh; Ken Inoki; Takeshi Into; Valentina Iovane; Juan L Iovanna; Nancy Y Ip; Yoshitaka Isaka; Hiroyuki Ishida; Ciro Isidoro; Ken-ichi Isobe; Akiko Iwasaki; Marta Izquierdo; Yotaro Izumi; Panu M Jaakkola; Marja Jäättelä; George R Jackson; William T Jackson; Bassam Janji; Marina Jendrach; Ju-Hong Jeon; Eui-Bae Jeung; Hong Jiang; Hongchi Jiang; Jean X Jiang; Ming Jiang; Qing Jiang; Xuejun Jiang; Xuejun Jiang; Alberto Jiménez; Meiyan Jin; Shengkan Jin; Cheol O Joe; Terje Johansen; Daniel E Johnson; Gail V W Johnson; Nicola L Jones; Bertrand Joseph; Suresh K Joseph; Annie M Joubert; Gábor Juhász; Lucienne Juillerat-Jeanneret; Chang Hwa Jung; Yong-Keun Jung; Kai Kaarniranta; Allen Kaasik; Tomohiro Kabuta; Motoni Kadowaki; Katarina Kagedal; Yoshiaki Kamada; Vitaliy O Kaminskyy; Harm H Kampinga; Hiromitsu Kanamori; Chanhee Kang; Khong Bee Kang; Kwang Il Kang; Rui Kang; Yoon-A Kang; Tomotake Kanki; Thirumala-Devi Kanneganti; Haruo Kanno; Anumantha G Kanthasamy; Arthi Kanthasamy; Vassiliki Karantza; Gur P Kaushal; Susmita Kaushik; Yoshinori Kawazoe; Po-Yuan Ke; John H Kehrl; Ameeta Kelekar; Claus Kerkhoff; David H Kessel; Hany Khalil; Jan A K W Kiel; Amy A Kiger; Akio Kihara; Deok Ryong Kim; Do-Hyung Kim; Dong-Hou Kim; Eun-Kyoung Kim; Hyung-Ryong Kim; Jae-Sung Kim; Jeong Hun Kim; Jin Cheon Kim; John K Kim; Peter K Kim; Seong Who Kim; Yong-Sun Kim; Yonghyun Kim; Adi Kimchi; Alec C Kimmelman; Jason S King; Timothy J Kinsella; Vladimir Kirkin; Lorrie A Kirshenbaum; Katsuhiko Kitamoto; Kaio Kitazato; Ludger Klein; Walter T Klimecki; Jochen Klucken; Erwin Knecht; Ben C B Ko; Jan C Koch; Hiroshi Koga; Jae-Young Koh; Young Ho Koh; Masato Koike; Masaaki Komatsu; Eiki Kominami; Hee Jeong Kong; Wei-Jia Kong; Viktor I Korolchuk; Yaichiro Kotake; Michael I Koukourakis; Juan B Kouri Flores; Attila L Kovács; Claudine Kraft; Dimitri Krainc; Helmut Krämer; Carole Kretz-Remy; Anna M Krichevsky; Guido Kroemer; Rejko Krüger; Oleg Krut; Nicholas T Ktistakis; Chia-Yi Kuan; Roza Kucharczyk; Ashok Kumar; Raj Kumar; Sharad Kumar; Mondira Kundu; Hsing-Jien Kung; Tino Kurz; Ho Jeong Kwon; Albert R La Spada; Frank Lafont; Trond Lamark; Jacques Landry; Jon D Lane; Pierre Lapaquette; Jocelyn F Laporte; Lajos László; Sergio Lavandero; Josée N Lavoie; Robert Layfield; Pedro A Lazo; Weidong Le; Laurent Le Cam; Daniel J Ledbetter; Alvin J X Lee; Byung-Wan Lee; Gyun Min Lee; Jongdae Lee; Ju-Hyun Lee; Michael Lee; Myung-Shik Lee; Sug Hyung Lee; Christiaan Leeuwenburgh; Patrick Legembre; Renaud Legouis; Michael Lehmann; Huan-Yao Lei; Qun-Ying Lei; David A Leib; José Leiro; John J Lemasters; Antoinette Lemoine; Maciej S Lesniak; Dina Lev; Victor V Levenson; Beth Levine; Efrat Levy; Faqiang Li; Jun-Lin Li; Lian Li; Sheng Li; Weijie Li; Xue-Jun Li; Yan-bo Li; Yi-Ping Li; Chengyu Liang; Qiangrong Liang; Yung-Feng Liao; Pawel P Liberski; Andrew Lieberman; Hyunjung J Lim; Kah-Leong Lim; Kyu Lim; Chiou-Feng Lin; Fu-Cheng Lin; Jian Lin; Jiandie D Lin; Kui Lin; Wan-Wan Lin; Weei-Chin Lin; Yi-Ling Lin; Rafael Linden; Paul Lingor; Jennifer Lippincott-Schwartz; Michael P Lisanti; Paloma B Liton; Bo Liu; Chun-Feng Liu; Kaiyu Liu; Leyuan Liu; Qiong A Liu; Wei Liu; Young-Chau Liu; Yule Liu; Richard A Lockshin; Chun-Nam Lok; Sagar Lonial; Benjamin Loos; Gabriel Lopez-Berestein; Carlos López-Otín; Laura Lossi; Michael T Lotze; Peter Lőw; Binfeng Lu; Bingwei Lu; Bo Lu; Zhen Lu; Frédéric Luciano; Nicholas W Lukacs; Anders H Lund; Melinda A Lynch-Day; Yong Ma; Fernando Macian; Jeff P MacKeigan; Kay F Macleod; Frank Madeo; Luigi Maiuri; Maria Chiara Maiuri; Davide Malagoli; May Christine V Malicdan; Walter Malorni; Na Man; Eva-Maria Mandelkow; Stéphen Manon; Irena Manov; Kai Mao; Xiang Mao; Zixu Mao; Philippe Marambaud; Daniela Marazziti; Yves L Marcel; Katie Marchbank; Piero Marchetti; Stefan J Marciniak; Mateus Marcondes; Mohsen Mardi; Gabriella Marfe; Guillermo Mariño; Maria Markaki; Mark R Marten; Seamus J Martin; Camille Martinand-Mari; Wim Martinet; Marta Martinez-Vicente; Matilde Masini; Paola Matarrese; Saburo Matsuo; Raffaele Matteoni; Andreas Mayer; Nathalie M Mazure; David J McConkey; Melanie J McConnell; Catherine McDermott; Christine McDonald; Gerald M McInerney; Sharon L McKenna; BethAnn McLaughlin; Pamela J McLean; Christopher R McMaster; G Angus McQuibban; Alfred J Meijer; Miriam H Meisler; Alicia Meléndez; Thomas J Melia; Gerry Melino; Maria A Mena; Javier A Menendez; Rubem F S Menna-Barreto; Manoj B Menon; Fiona M Menzies; Carol A Mercer; Adalberto Merighi; Diane E Merry; Stefania Meschini; Christian G Meyer; Thomas F Meyer; Chao-Yu Miao; Jun-Ying Miao; Paul A M Michels; Carine Michiels; Dalibor Mijaljica; Ana Milojkovic; Saverio Minucci; Clelia Miracco; Cindy K Miranti; Ioannis Mitroulis; Keisuke Miyazawa; Noboru Mizushima; Baharia Mograbi; Simin Mohseni; Xavier Molero; Bertrand Mollereau; Faustino Mollinedo; Takashi Momoi; Iryna Monastyrska; Martha M Monick; Mervyn J Monteiro; Michael N Moore; Rodrigo Mora; Kevin Moreau; Paula I Moreira; Yuji Moriyasu; Jorge Moscat; Serge Mostowy; Jeremy C Mottram; Tomasz Motyl; Charbel E-H Moussa; Sylke Müller; Sylviane Muller; Karl Münger; Christian Münz; Leon O Murphy; Maureen E Murphy; Antonio Musarò; Indira Mysorekar; Eiichiro Nagata; Kazuhiro Nagata; Aimable Nahimana; Usha Nair; Toshiyuki Nakagawa; Kiichi Nakahira; Hiroyasu Nakano; Hitoshi Nakatogawa; Meera Nanjundan; Naweed I Naqvi; Derek P Narendra; Masashi Narita; Miguel Navarro; Steffan T Nawrocki; Taras Y Nazarko; Andriy Nemchenko; Mihai G Netea; Thomas P Neufeld; Paul A Ney; Ioannis P Nezis; Huu Phuc Nguyen; Daotai Nie; Ichizo Nishino; Corey Nislow; Ralph A Nixon; Takeshi Noda; Angelika A Noegel; Anna Nogalska; Satoru Noguchi; Lucia Notterpek; Ivana Novak; Tomoyoshi Nozaki; Nobuyuki Nukina; Thorsten Nürnberger; Beat Nyfeler; Keisuke Obara; Terry D Oberley; Salvatore Oddo; Michinaga Ogawa; Toya Ohashi; Koji Okamoto; Nancy L Oleinick; F Javier Oliver; Laura J Olsen; Stefan Olsson; Onya Opota; Timothy F Osborne; Gary K Ostrander; Kinya Otsu; Jing-hsiung James Ou; Mireille Ouimet; Michael Overholtzer; Bulent Ozpolat; Paolo Paganetti; Ugo Pagnini; Nicolas Pallet; Glen E Palmer; Camilla Palumbo; Tianhong Pan; Theocharis Panaretakis; Udai Bhan Pandey; Zuzana Papackova; Issidora Papassideri; Irmgard Paris; Junsoo Park; Ohkmae K Park; Jan B Parys; Katherine R Parzych; Susann Patschan; Cam Patterson; Sophie Pattingre; John M Pawelek; Jianxin Peng; David H Perlmutter; Ida Perrotta; George Perry; Shazib Pervaiz; Matthias Peter; Godefridus J Peters; Morten Petersen; Goran Petrovski; James M Phang; Mauro Piacentini; Philippe Pierre; Valérie Pierrefite-Carle; Gérard Pierron; Ronit Pinkas-Kramarski; Antonio Piras; Natik Piri; Leonidas C Platanias; Stefanie Pöggeler; Marc Poirot; Angelo Poletti; Christian Poüs; Mercedes Pozuelo-Rubio; Mette Prætorius-Ibba; Anil Prasad; Mark Prescott; Muriel Priault; Nathalie Produit-Zengaffinen; Ann Progulske-Fox; Tassula Proikas-Cezanne; Serge Przedborski; Karin Przyklenk; Rosa Puertollano; Julien Puyal; Shu-Bing Qian; Liang Qin; Zheng-Hong Qin; Susan E Quaggin; Nina Raben; Hannah Rabinowich; Simon W Rabkin; Irfan Rahman; Abdelhaq Rami; Georg Ramm; Glenn Randall; Felix Randow; V Ashutosh Rao; Jeffrey C Rathmell; Brinda Ravikumar; Swapan K Ray; Bruce H Reed; John C Reed; Fulvio Reggiori; Anne Régnier-Vigouroux; Andreas S Reichert; John J Reiners; Russel J Reiter; Jun Ren; José L Revuelta; Christopher J Rhodes; Konstantinos Ritis; Elizete Rizzo; Jeffrey Robbins; Michel Roberge; Hernan Roca; Maria C Roccheri; Stephane Rocchi; H Peter Rodemann; Santiago Rodríguez de Córdoba; Bärbel Rohrer; Igor B Roninson; Kirill Rosen; Magdalena M Rost-Roszkowska; Mustapha Rouis; Kasper M A Rouschop; Francesca Rovetta; Brian P Rubin; David C Rubinsztein; Klaus Ruckdeschel; Edmund B Rucker; Assaf Rudich; Emil Rudolf; Nelson Ruiz-Opazo; Rossella Russo; Tor Erik Rusten; Kevin M Ryan; Stefan W Ryter; David M Sabatini; Junichi Sadoshima; Tapas Saha; Tatsuya Saitoh; Hiroshi Sakagami; Yasuyoshi Sakai; Ghasem Hoseini Salekdeh; Paolo Salomoni; Paul M Salvaterra; Guy Salvesen; Rosa Salvioli; Anthony M J Sanchez; José A Sánchez-Alcázar; Ricardo Sánchez-Prieto; Marco Sandri; Uma Sankar; Poonam Sansanwal; Laura Santambrogio; Shweta Saran; Sovan Sarkar; Minnie Sarwal; Chihiro Sasakawa; Ausra Sasnauskiene; Miklós Sass; Ken Sato; Miyuki Sato; Anthony H V Schapira; Michael Scharl; Hermann M Schätzl; Wiep Scheper; Stefano Schiaffino; Claudio Schneider; Marion E Schneider; Regine Schneider-Stock; Patricia V Schoenlein; Daniel F Schorderet; Christoph Schüller; Gary K Schwartz; Luca Scorrano; Linda Sealy; Per O Seglen; Juan Segura-Aguilar; Iban Seiliez; Oleksandr Seleverstov; Christian Sell; Jong Bok Seo; Duska Separovic; Vijayasaradhi Setaluri; Takao Setoguchi; Carmine Settembre; John J Shacka; Mala Shanmugam; Irving M Shapiro; Eitan Shaulian; Reuben J Shaw; James H Shelhamer; Han-Ming Shen; Wei-Chiang Shen; Zu-Hang Sheng; Yang Shi; Kenichi Shibuya; Yoshihiro Shidoji; Jeng-Jer Shieh; Chwen-Ming Shih; Yohta Shimada; Shigeomi Shimizu; Takahiro Shintani; Orian S Shirihai; Gordon C Shore; Andriy A Sibirny; Stan B Sidhu; Beata Sikorska; Elaine C M Silva-Zacarin; Alison Simmons; Anna Katharina Simon; Hans-Uwe Simon; Cristiano Simone; Anne Simonsen; David A Sinclair; Rajat Singh; Debasish Sinha; Frank A Sinicrope; Agnieszka Sirko; Parco M Siu; Efthimios Sivridis; Vojtech Skop; Vladimir P Skulachev; Ruth S Slack; Soraya S Smaili; Duncan R Smith; Maria S Soengas; Thierry Soldati; Xueqin Song; Anil K Sood; Tuck Wah Soong; Federica Sotgia; Stephen A Spector; Claudia D Spies; Wolfdieter Springer; Srinivasa M Srinivasula; Leonidas Stefanis; Joan S Steffan; Ruediger Stendel; Harald Stenmark; Anastasis Stephanou; Stephan T Stern; Cinthya Sternberg; Björn Stork; Peter Strålfors; Carlos S Subauste; Xinbing Sui; David Sulzer; Jiaren Sun; Shi-Yong Sun; Zhi-Jun Sun; Joseph J Y Sung; Kuninori Suzuki; Toshihiko Suzuki; Michele S Swanson; Charles Swanton; Sean T Sweeney; Lai-King Sy; Gyorgy Szabadkai; Ira Tabas; Heinrich Taegtmeyer; Marco Tafani; Krisztina Takács-Vellai; Yoshitaka Takano; Kaoru Takegawa; Genzou Takemura; Fumihiko Takeshita; Nicholas J Talbot; Kevin S W Tan; Keiji Tanaka; Kozo Tanaka; Daolin Tang; Dingzhong Tang; Isei Tanida; Bakhos A Tannous; Nektarios Tavernarakis; Graham S Taylor; Gregory A Taylor; J Paul Taylor; Lance S Terada; Alexei Terman; Gianluca Tettamanti; Karin Thevissen; Craig B Thompson; Andrew Thorburn; Michael Thumm; FengFeng Tian; Yuan Tian; Glauco Tocchini-Valentini; Aviva M Tolkovsky; Yasuhiko Tomino; Lars Tönges; Sharon A Tooze; Cathy Tournier; John Tower; Roberto Towns; Vladimir Trajkovic; Leonardo H Travassos; Ting-Fen Tsai; Mario P Tschan; Takeshi Tsubata; Allan Tsung; Boris Turk; Lorianne S Turner; Suresh C Tyagi; Yasuo Uchiyama; Takashi Ueno; Midori Umekawa; Rika Umemiya-Shirafuji; Vivek K Unni; Maria I Vaccaro; Enza Maria Valente; Greet Van den Berghe; Ida J van der Klei; Wouter van Doorn; Linda F van Dyk; Marjolein van Egmond; Leo A van Grunsven; Peter Vandenabeele; Wim P Vandenberghe; Ilse Vanhorebeek; Eva C Vaquero; Guillermo Velasco; Tibor Vellai; Jose Miguel Vicencio; Richard D Vierstra; Miquel Vila; Cécile Vindis; Giampietro Viola; Maria Teresa Viscomi; Olga V Voitsekhovskaja; Clarissa von Haefen; Marcela Votruba; Keiji Wada; Richard Wade-Martins; Cheryl L Walker; Craig M Walsh; Jochen Walter; Xiang-Bo Wan; Aimin Wang; Chenguang Wang; Dawei Wang; Fan Wang; Fen Wang; Guanghui Wang; Haichao Wang; Hong-Gang Wang; Horng-Dar Wang; Jin Wang; Ke Wang; Mei Wang; Richard C Wang; Xinglong Wang; Xuejun Wang; Ying-Jan Wang; Yipeng Wang; Zhen Wang; Zhigang Charles Wang; Zhinong Wang; Derick G Wansink; Diane M Ward; Hirotaka Watada; Sarah L Waters; Paul Webster; Lixin Wei; Conrad C Weihl; William A Weiss; Scott M Welford; Long-Ping Wen; Caroline A Whitehouse; J Lindsay Whitton; Alexander J Whitworth; Tom Wileman; John W Wiley; Simon Wilkinson; Dieter Willbold; Roger L Williams; Peter R Williamson; Bradly G Wouters; Chenghan Wu; Dao-Cheng Wu; William K K Wu; Andreas Wyttenbach; Ramnik J Xavier; Zhijun Xi; Pu Xia; Gengfu Xiao; Zhiping Xie; Zhonglin Xie; Da-zhi Xu; Jianzhen Xu; Liang Xu; Xiaolei Xu; Ai Yamamoto; Akitsugu Yamamoto; Shunhei Yamashina; Michiaki Yamashita; Xianghua Yan; Mitsuhiro Yanagida; Dun-Sheng Yang; Elizabeth Yang; Jin-Ming Yang; Shi Yu Yang; Wannian Yang; Wei Yuan Yang; Zhifen Yang; Meng-Chao Yao; Tso-Pang Yao; Behzad Yeganeh; Wei-Lien Yen; Jia-jing Yin; Xiao-Ming Yin; Ook-Joon Yoo; Gyesoon Yoon; Seung-Yong Yoon; Tomohiro Yorimitsu; Yuko Yoshikawa; Tamotsu Yoshimori; Kohki Yoshimoto; Ho Jin You; Richard J Youle; Anas Younes; Li Yu; Long Yu; Seong-Woon Yu; Wai Haung Yu; Zhi-Min Yuan; Zhenyu Yue; Cheol-Heui Yun; Michisuke Yuzaki; Olga Zabirnyk; Elaine Silva-Zacarin; David Zacks; Eldad Zacksenhaus; Nadia Zaffaroni; Zahra Zakeri; Herbert J Zeh; Scott O Zeitlin; Hong Zhang; Hui-Ling Zhang; Jianhua Zhang; Jing-Pu Zhang; Lin Zhang; Long Zhang; Ming-Yong Zhang; Xu Dong Zhang; Mantong Zhao; Yi-Fang Zhao; Ying Zhao; Zhizhuang J Zhao; Xiaoxiang Zheng; Boris Zhivotovsky; Qing Zhong; Cong-Zhao Zhou; Changlian Zhu; Wei-Guo Zhu; Xiao-Feng Zhu; Xiongwei Zhu; Yuangang Zhu; Teresa Zoladek; Wei-Xing Zong; Antonio Zorzano; Jürgen Zschocke; Brian Zuckerbraun Journal: Autophagy Date: 2012-04 Impact factor: 16.016
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