Gergely Talaber1, Gabriella Miklossy1, Zachary Oaks2, Yuxin Liu1, Sharon A Tooze3, Dmitriy M Chudakov4, Katalin Banki5, Andras Perl6. 1. Departments of Medicine, State University of New York, Upstate Medical University, Syracuse, New York, United States of America. 2. Departments of Medicine, State University of New York, Upstate Medical University, Syracuse, New York, United States of America ; Biochemistry and Molecular Biology, State University of New York, Upstate Medical University, Syracuse, New York, United States of America. 3. Cancer Research UK London Research Institute, London, England, United Kingdom. 4. Shemiakin-Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia. 5. Department of Pathology, State University of New York, Upstate Medical University, Syracuse, New York, United States of America. 6. Departments of Medicine, State University of New York, Upstate Medical University, Syracuse, New York, United States of America ; Biochemistry and Molecular Biology, State University of New York, Upstate Medical University, Syracuse, New York, United States of America ; Microbiology and Immunology, State University of New York, Upstate Medical University, Syracuse, New York, United States of America.
Abstract
HRES-1/Rab4 is a small GTPase that regulates endocytic recycling. It has been colocalized to mitochondria and the mechanistic target of rapamycin (mTOR), a suppressor of autophagy. Since the autophagosomal membrane component microtubule-associated protein light chain 3 (LC3) is derived from mitochondria, we investigated the impact of HRES-1/Rab4 on the formation of LC3(+) autophagosomes, their colocalization with HRES-1/Rab4 and mitochondria, and the retention of mitochondria during autophagy induced by starvation and rapamycin. HRES-1/Rab4 exhibited minimal baseline colocalization with LC3, which was enhanced 22-fold upon starvation or 6-fold upon rapamycin treatment. Colocalization of HRES-1/Rab4 with mitochondria was increased >2-fold by starvation or rapamycin. HRES-1/Rab4 overexpression promoted the colocalization of mitochondria with LC3 upon starvation or rapamycin treatment. A dominant-negative mutant, HRES-1/Rab4(S27N) had reduced colocalization with LC3 and mitochondria upon starvation but not rapamycin treatment. A constitutively active mutant, HRES-1/Rab4(Q72L) showed diminished colocalization with LC3 but promoted the partitioning of mitochondria with LC3 upon starvation or rapamycin treatment. Phosphorylation-resistant mutant HRES-1/Rab4(S204Q) showed diminished colocalization with LC3 but increased partitioning to mitochondria. A newly discovered C-terminally truncated native isoform, HRES-1/Rab4(1-121), showed enhanced localization to LC3 and mitochondria without starvation or rapamycin treatment. HRES-1/Rab4(1-121) increased the formation of LC3(+) autophagosomes in resting cells, while other isoforms promoted autophagosome formation upon starvation. HRES-1/Rab4, HRES-1/Rab4(1-121), HRES-1/Rab4(Q72L) and HRES-1/Rab4(S204Q) promoted the accumulation of mitochondria during starvation. The specificity of HRES-1/Rab4-mediated mitochondrial accumulation is indicated by its abrogation by dominant-negative HRES-1/Rab4(S27N) mutation. The formation of interconnected mitochondrial tubular networks was markedly enhanced by HRES-1/Rab4(Q72L) upon starvation, which may contribute to the retention of mitochondria during autophagy. The present study thus indicates that HRES-1/Rab4 regulates autophagy through promoting the formation of LC3(+) autophagosomes and the preservation of mitochondria.
HRES-1/Rab4 is a small GTPase that regulates endocytic recycling. It has been colocalized to mitochondria and the mechanistic target of rapamycin (mTOR), a suppressor of autophagy. Since the autophagosomal membrane component microtubule-associated protein light chain 3 (LC3) is derived from mitochondria, we investigated the impact of HRES-1/Rab4 on the formation of LC3(+) autophagosomes, their colocalization with HRES-1/Rab4 and mitochondria, and the retention of mitochondria during autophagy induced by starvation and rapamycin. HRES-1/Rab4 exhibited minimal baseline colocalization with LC3, which was enhanced 22-fold upon starvation or 6-fold upon rapamycin treatment. Colocalization of HRES-1/Rab4 with mitochondria was increased >2-fold by starvation or rapamycin. HRES-1/Rab4 overexpression promoted the colocalization of mitochondria with LC3 upon starvation or rapamycin treatment. A dominant-negative mutant, HRES-1/Rab4(S27N) had reduced colocalization with LC3 and mitochondria upon starvation but not rapamycin treatment. A constitutively active mutant, HRES-1/Rab4(Q72L) showed diminished colocalization with LC3 but promoted the partitioning of mitochondria with LC3 upon starvation or rapamycin treatment. Phosphorylation-resistant mutant HRES-1/Rab4(S204Q) showed diminished colocalization with LC3 but increased partitioning to mitochondria. A newly discovered C-terminally truncated native isoform, HRES-1/Rab4(1-121), showed enhanced localization to LC3 and mitochondria without starvation or rapamycin treatment. HRES-1/Rab4(1-121) increased the formation of LC3(+) autophagosomes in resting cells, while other isoforms promoted autophagosome formation upon starvation. HRES-1/Rab4, HRES-1/Rab4(1-121), HRES-1/Rab4(Q72L) and HRES-1/Rab4(S204Q) promoted the accumulation of mitochondria during starvation. The specificity of HRES-1/Rab4-mediated mitochondrial accumulation is indicated by its abrogation by dominant-negative HRES-1/Rab4(S27N) mutation. The formation of interconnected mitochondrial tubular networks was markedly enhanced by HRES-1/Rab4(Q72L) upon starvation, which may contribute to the retention of mitochondria during autophagy. The present study thus indicates that HRES-1/Rab4 regulates autophagy through promoting the formation of LC3(+) autophagosomes and the preservation of mitochondria.
Autophagy is a complex stress-induced catabolic process that breaks down and recycles organelles and cytoplasmic materials [1]. While autophagy is a fundamental mechanism of cell survival, its dysregulation has been widely implicated in cancer, inflammatory, and neurodegenerative diseases [1], [2]. During autophagy, a new organelle, the autophagosome, is assembled from membrane components of mitochondria [3], plasma membrane [4] and endoplasmic reticulum [5]. Recycling endosomes have been suggested to contribute to the biogenesis of autophagosomes [6]. A signature component of the autophagosome membrane is microtubule-associated protein light chain 3 (LC3) [7], which has been widely identified as a binding partner of endosomal traffic regulator Rab GTPases [8], [9]. The early endosomal marker Rab11 has been implicated in autophagosome formation [6], while the late endosomal Rab7 contributes to endosome maturation [9] and progression [10].We have previously documented that the small GTPase HRES-1/Rab4 regulated the recycling and lysosomal degradation of surface receptors, GLUT4 on adipocytes [11] as well as CD4 [12] and CD3ζ in T lymphocytes [13]. Expression of HRES-1/Rab4 is redox-controlled: induced by H2O2 and inhibited by glutathione [13]. HRES-1/Rab4 was colocalized with the lysosomes and the mechanistic target of rapamycin (mTOR), which acts as a suppressor of autophagy [14], [15]. Blockade of mTOR with rapamycin, which is an effective treatment in patients with systemic lupus erythematosus [16], [17], inhibited the oxidative stress-induced expression of HRES-1/Rab4 and the lysosomal degradation of CD4 and CD3ζ [13]. Along these lines, mTOR has been localized to endosomes [18], including those carrying HRES-1/Rab4 [13]. Overexpression of HRES-1/Rab4 and activation of mTOR can be detected in T cells of patients with systemic lupus erythematosus (SLE) [13] and lupus-prone mice, where autophagy appears to be involved in disease pathogenesis [19]. While HRES-1/Rab4 promotes the lysosomal degradation of proteins by autophagy, it appears to inhibit the autophagy of mitochondria or mitophagy [19]. The biogenesis of LC3+ autophagosomes is dependent on the supply of membranes from mitochondria [3]. In order to test the hypothesis that HRES-1/Rab4 influences the formation of autophagosomes, we investigated the colocalization of its wild-type and functionally distinct mutant isoforms with LC3 and mitochondria during autophagy, which was induced by starvation or mTOR blockade with rapamycin in HeLa cells, owing to the limited size and paucity of organelles in primary human T lymphocytes. Here, we show that HRES-1/Rab4 colocalizes with the autophagosomal membrane component LC3 and promotes its partitioning to the mitochondria during autophagy. Dominant-negative HRES-1/Rab4S27N mutation [20] blocked colocalization with LC3 and partitioning to the mitochondria induced by starvation but not by rapamycin. Constitutively active HRES-1/Rab4Q72L
[21] showed diminished colocalization with LC3 but promoted partitioning of mitochondria with LC3 upon starvation or rapamycin treatment. A newly discovered C-terminally truncated natural isoform, HRES-1/Rab41–121, showed enhanced localization to LC3 and its partitioning to mitochondria without starvation or rapamycin treatment. CDC kinase phosphorylation-deficient HRES-1/Rab4S204Q
[22] showed diminished colocalization with LC3 but facilitated its partitioning to mitochondria without starvation or rapamycin treatment. Only HRES-1/Rab41–121 increased the formation of LC3+ autophagosomes in resting cells, while other isoforms promoted autophagosome formation upon starvation. HRES-1/Rab4, HRES-1/Rab41–121, HRES-1/Rab4Q72L and HRES-1/Rab4S204Q, but not HRES-1/Rab4S27N, enhanced the accumulation of mitochondria during starvation. The formation of interconnected mitochondrial tubular networks was enhanced by HRES-1/Rab4Q72L upon starvation, which may contribute to the preservation of mitochondria during autophagy. Overall, these results reveal a new role of HRES-1/Rab4 in autophagy and mitochondrial homeostasis.
Results
HRES-1/Rab4 colocalizes with the autophagosomal membrane component LC3
The colocalization of HRES-1/Rab4 and LC3 was investigated in HeLa cells transfected with LC3 fused to FP650 (FP650-LC3) and HRES-1/Rab4 isoforms, including wild-type HRES-1/Rab4, HRES-1/Rab4S27N, HRES-1/Rab4Q72L, HRES-1/Rab4S204Q, and C-terminally truncated HRES-1/Rab41–121, each tagged to the C-terminus of eGFP (Fig. 1). Among the mutant isoforms, HRES-1/Rab4S27N acts as a dominant negative mutation that prevents GTP binding [20]. HRES-1/Rab4Q72L is constitutively active due to elimination of GTPase activity [21]. HRES-1/Rab4S204Q cannot be phosphorylated by p34cdc2 kinase in mitotic cells and remains endosome-associated throughout the cell cycle [22]. HRES-1/Rab41–121 is encoded by a newly identified alternatively spliced mRNA that represents a 36-nucleotide out-of-frame deletion (GenBank submission number: 1591873). This results in a frame shift with an amino acid sequence corresponding to the 96 N-terminal residues of HRES-1/Rab4 continuing into 25 C-terminal residues unrelated to residues 97–218 of wild-type HRES-1/Rab4 (Fig. 1B).
Figure 1
Detection of LC3 fused to FP650 (FP650-LC3) and HRES-1/Rab4 isoforms, including wild-type HRES-1/Rab4, C-terminally truncated HRES-1/Rab41–121, dominant-negative/GTP binding-deficient HRES-1/Rab4S27N, constitutively active/GTPase-deficient HRES-1/Rab4Q72L and phosphorylation-resistant form HRES-1/Rab4S204Q, tagged with eGFP.
A, Functional domains of proteins encoded by the HRES-1/Rab4 cDNA at 1q42 [12] (Genbank accession number: AY585832). Amino acid changes previously shown to affect Rab4 activity are typed in red. HRES-1/Rab4S27N prevents GTP binding and acts as a dominant negative mutation [20]. HRES-1/Rab4Q72L is constitutively active due to elimination of GTPase activity [21]. HRES-1/Rab4S204Q will not be phosphorylated by p34cdc2 kinase in mitotic cells and remains endosome-associated throughout the cell cycle [22]. B, Amino acid sequence of HRES-1/Rab41–121, representing a 36-nucleotide out-of-frame deletion, is attributed to alternative splicing (GenBank submission number 1591873). This results in a frameshift with an amino acid sequence corresponding to the 96 N-terminal residues of HRES-1/Rab4 continuing into 25 C-terminal residues (typed in red characters), which are unrelated to the amino acid sequence of residues 97-218 in wild-type HRES-1/Rab4. C, Confocal microscopy of HeLa cells transfected with expression vectors producing FP650-LC3 (emitting red fluorescence) and HRES-1/Rab4 isoforms fused to eGFP (emitting green fluorescence) relative to control cells transfected with vectors expressing fluorescent proteins FP650 and eGFP alone. D, Western blot analysis of HeLa cells transduced with expression vectors producing eGFP-HRES-1/Rab4 and FP650-LC3 fusion proteins. HRES-1/Rab4 isoforms were detected with antibody SC312 directed to the C-terminus which is absent in HRES-1/Rab41–121. E, Western blot analysis of HRES-1/Rab41–121 expression in HeLa cells transfected with pAAV-HRES-1/Rab41–121-IRES-GFP vector (clone 8466), pAAV-hrGFP-HRES-1/Rab41–121 vector (clone 9214), and pAAV-HRES-1/Rab4-IRES-GFP vector (clone 8467). Cells were incubated without (control) or with 0.1% DMSO, bafilomycin A1 (200 nM), or leupeptin (10 μg/ml). HRES-1/Rab41–121 and HRES-1/Rab41–121-GFP fusion protein were detected with rabbit antibody G1432. HRES-1/Rab4 was detected with rabbit antibody 13407 [12].
Detection of LC3 fused to FP650 (FP650-LC3) and HRES-1/Rab4 isoforms, including wild-type HRES-1/Rab4, C-terminally truncated HRES-1/Rab41–121, dominant-negative/GTP binding-deficient HRES-1/Rab4S27N, constitutively active/GTPase-deficient HRES-1/Rab4Q72L and phosphorylation-resistant form HRES-1/Rab4S204Q, tagged with eGFP.
A, Functional domains of proteins encoded by the HRES-1/Rab4 cDNA at 1q42 [12] (Genbank accession number: AY585832). Amino acid changes previously shown to affect Rab4 activity are typed in red. HRES-1/Rab4S27N prevents GTP binding and acts as a dominant negative mutation [20]. HRES-1/Rab4Q72L is constitutively active due to elimination of GTPase activity [21]. HRES-1/Rab4S204Q will not be phosphorylated by p34cdc2 kinase in mitotic cells and remains endosome-associated throughout the cell cycle [22]. B, Amino acid sequence of HRES-1/Rab41–121, representing a 36-nucleotide out-of-frame deletion, is attributed to alternative splicing (GenBank submission number 1591873). This results in a frameshift with an amino acid sequence corresponding to the 96 N-terminal residues of HRES-1/Rab4 continuing into 25 C-terminal residues (typed in red characters), which are unrelated to the amino acid sequence of residues 97-218 in wild-type HRES-1/Rab4. C, Confocal microscopy of HeLa cells transfected with expression vectors producing FP650-LC3 (emitting red fluorescence) and HRES-1/Rab4 isoforms fused to eGFP (emitting green fluorescence) relative to control cells transfected with vectors expressing fluorescent proteins FP650 and eGFP alone. D, Western blot analysis of HeLa cells transduced with expression vectors producing eGFP-HRES-1/Rab4 and FP650-LC3 fusion proteins. HRES-1/Rab4 isoforms were detected with antibody SC312 directed to the C-terminus which is absent in HRES-1/Rab41–121. E, Western blot analysis of HRES-1/Rab41–121 expression in HeLa cells transfected with pAAV-HRES-1/Rab41–121-IRES-GFP vector (clone 8466), pAAV-hrGFP-HRES-1/Rab41–121 vector (clone 9214), and pAAV-HRES-1/Rab4-IRES-GFP vector (clone 8467). Cells were incubated without (control) or with 0.1% DMSO, bafilomycin A1 (200 nM), or leupeptin (10 μg/ml). HRES-1/Rab41–121 and HRES-1/Rab41–121-GFP fusion protein were detected with rabbit antibody G1432. HRES-1/Rab4 was detected with rabbit antibody 13407 [12].Expression of transduced eGFP-HRES-1/Rab4 and FP650-LC3 fusion proteins was monitored by fluorescence microscopy (Figs. 1C), flow cytometry (Figs. S1A and B), and western blot (Figs. 1D and E). The colocalization of HRES-1/Rab4 isoforms and LC3 was analyzed by confocal microscopy (Fig. 2) and quantified relative to both the total LC3 (Fig. 3A) and total HRES-1/Rab4 signal pools (Fig. 3B). HRES-1/Rab4 formed round-shaped structures around LC3+ vesicles. (Fig. 2). While minimal colocalization was observed between HRES-1/Rab4 and LC3 in cells cultured in complete medium (1.6±0.5%; Fig. 3), starvation initiated by withdrawal of serum and glutamine for 4 h enhanced their colocalization 22-fold (23.0±5.4%, p = 0.003; Figs. 3A). Colocalization was also induced, however, to a lesser extent, 6-fold, after exposure to rapamycin (Rapa) (9.8±3.5%; p = 0.020; Fig. 3A). Unlike starvation-induced colocalization, the effect of Rapa was not reduced by balifomycin A1 (Baf). HRES-1/Rab41–121 showed the most robust colocalization with LC3 but no responsiveness to starvation or Rapa. The disproportionately strong colocalization of HRES-1/Rab41–121 with LC3 may be related to an instability and susceptibility to lysosomal degradation of this truncated isoform (Fig. 1E). Colocalization of HRES-1/Rab4S27N with LC3 was induced 11.6-fold (p = 0.020) by Rapa but not starvation (Fig. 3A). Colocalization of HRES-1/Rab4Q72L with LC3 was robustly induced by Rapa (70-fold; p = 0.0005) and reversed by Baf (p = 0.04; Fig. 3A). HRES-1/Rab4S204Q was least prone to colocalize with LC3 upon starvation (Fig. 3A). The resistance of HRES-1/Rab4S27N, HRES-1/Rab4Q72L, and HRES-1/Rab4S204Q to starvation-induced colocalization with LC3 implies that this process is dependent on GTP binding, GTPase activity, and phosphorylation at S204.
Figure 2
Confocal microscopy of HeLa cells transfected with expression vector producing LC3 tagged with FP650 (emitting red fluorescence) and different isoforms of HRES-1/Rab4, including wild-type (HRES-1/Rab4), C-terminally truncated form (HRES-1/Rab41–121), dominant-negative form (Rab4S27N), constitutively active (HRES-1/Rab4Q72L) and phosphorylation-resistant form (HRES-1/Rab4S204Q) tagged with eGFP (emitting green fluorescence).
Cells were kept in complete medium (Control), starved in serum-free medium without glutamine (Star), or treated with autophagy-modifying agents, bafilomycin A1 (Baf) and rapamycin (Rapa) for 4 hours. Starved and rapamycin treated cells were also treated with Baf. Images are representative of 6–40 cells analyzed during three independent experiments. Image inserts bracketed by broken lines correspond to LC3 vesicles magnified from the original image. Individual and composite color channels are shown for each experimental condition.
Figure 3
Cumulative analysis of colocalization of HRES-1/Rab4 and LC3 induced by starvation and rapamycin.
Colocalization of HRES-1/Rab4 and LC3 was quantified relative to total LC3 (panel A) and total HRES-1/Rab4 content (panel B). The colocalization of HRES-1/Rab4 with LC3 was most profoundly skewed by HRES-1/Rab4S27N which blocked colocalization under starvation and promoted colocalization during mTOR blockade. In contrast to HRES-1/Rab4S27N, HRES-1/Rab4Q72L blocked colocalization with LC3 both under starvation and mTOR blockade. Data represent mean ± SEM of 6–29 cells acquired in 3 independent experiments. * indicates p values<0.05 reflecting comparison to control cells among cell cultures transfected with the same construct using paired two-tailed t-tests; brackets connecting bars within each construct also reflect comparison with paired two-tailed t-tests. Brackets connecting bars between constructs reflect p<0.05 using ANOVA followed by Bonferroni's post-test.
Confocal microscopy of HeLa cells transfected with expression vector producing LC3 tagged with FP650 (emitting red fluorescence) and different isoforms of HRES-1/Rab4, including wild-type (HRES-1/Rab4), C-terminally truncated form (HRES-1/Rab41–121), dominant-negative form (Rab4S27N), constitutively active (HRES-1/Rab4Q72L) and phosphorylation-resistant form (HRES-1/Rab4S204Q) tagged with eGFP (emitting green fluorescence).
Cells were kept in complete medium (Control), starved in serum-free medium without glutamine (Star), or treated with autophagy-modifying agents, bafilomycin A1 (Baf) and rapamycin (Rapa) for 4 hours. Starved and rapamycin treated cells were also treated with Baf. Images are representative of 6–40 cells analyzed during three independent experiments. Image inserts bracketed by broken lines correspond to LC3 vesicles magnified from the original image. Individual and composite color channels are shown for each experimental condition.
Cumulative analysis of colocalization of HRES-1/Rab4 and LC3 induced by starvation and rapamycin.
Colocalization of HRES-1/Rab4 and LC3 was quantified relative to total LC3 (panel A) and total HRES-1/Rab4 content (panel B). The colocalization of HRES-1/Rab4 with LC3 was most profoundly skewed by HRES-1/Rab4S27N which blocked colocalization under starvation and promoted colocalization during mTOR blockade. In contrast to HRES-1/Rab4S27N, HRES-1/Rab4Q72L blocked colocalization with LC3 both under starvation and mTOR blockade. Data represent mean ± SEM of 6–29 cells acquired in 3 independent experiments. * indicates p values<0.05 reflecting comparison to control cells among cell cultures transfected with the same construct using paired two-tailed t-tests; brackets connecting bars within each construct also reflect comparison with paired two-tailed t-tests. Brackets connecting bars between constructs reflect p<0.05 using ANOVA followed by Bonferroni's post-test.The fraction of LC3 colocalizing with the HRES-1/Rab4 was increased to 0.23±0.06 upon starvation from 0.015±0.006 (p = 0.0001) in complete medium (Fig. 3A). While Baf alone moderately increased this ratio (0.046±0.015, p = 0.02) relative to cells grown in complete medium, it failed to influence the impact of starvation. The ratio of LC3 bound to HRES-1/Rab4 was increased by Rapa to 0.098±0.03 (p = 0.001) and further augmented by the combination of Rapa and Baf to 0.24±0.06 (p = 0.00005). This pattern of changes was influenced by using mutated HRES-1/Rab4 isoforms. LC3 showed the greatest colocalization with C-terminally truncated HRES-1/Rab41–121 at baseline and the least responsiveness to starvation or treatment with Rapa or Baf, suggesting that this naturally occurring HRES-1/Rab4 isoform has limited functional capacity. LC3 failed to colocalize with HRES-1/Rab4S27N in complete medium or upon starvation. However, Rapa markedly increased the ratio of HRES-1/Rab4-positive LC3 vesicles to 0.41±0.16 from 0.035±0.018 at baseline (p = 0.02). Baf did not influence the colocalization of LC3 with HRES-1/Rab4S27N in the absence or presence of Rapa. The colocalization of LC3 with HRES-1/Rab4Q72L and LC3 was moderately responsive to Baf, starvation combined with Baf, and Rapa. The Rapa-induced colocalization of LC3 with HRES-1/Rab4Q72L was reduced at 0.037±0.01 relative to that with HRES-1/Rab4S27N at 0.41±0.16 (p = 0.029; Fig. 3A). Starvation did not influence to the colocalization of LC3 with HRES-1/Rab4Q72L. LC3 also showed increased colocalization with HRES-1/Rab4S204Q upon treatment with Rapa (p = 0.005). 2-way ANOVA and Bonferroni post-tests revealed resistance to starvation-induced colocalization of LC3 to mutated HRES-1/Rab4 isoforms and greatest colocalization to HRES-1/Rab4S27N in response to treatment with Rapa (Fig. 3A).We also quantified the ratio of HRES-1/Rab4 colocalizing with LC3 relative to total LC3 (Fig. 3A) or HRES-1/Rab4 (Fig. 3B). Starvation induced the fraction of HRES-1/Rab4 colocalizing with LC3 from 0.009±0.003 in complete medium to 0.17±0.04 upon starvation (p = 0.00007). The ratio of HRES-1/Rab4 colocalized with LC3 was also increased by Rapa at 0.05±0.02 (p = 0.006), but this was less pronounced than the impact of starvation (p = 0.015). HRES-1/Rab41–121 was most prominently associated with LC3 at baseline 0.22±0.06 relative to any other isoform tested (p<0.01), and that was further augmented by starvation and Baf to 0.64±0.18 (p = 0.03, Fig. 2B). HRES-1/Rab4S27N was resistant to starvation-induced colocalization with LC3 relative to wild-type HRES-1/Rab4 (ANOVA p<0.01; Fig. 3B). HRES-1/Rab4Q72L failed to colocalize with LC3 upon starvation, but it was effectively induced by Rapa to 0.096±0.0036 from a baseline of 0.0013±0.0006 (p = 0.0055) in complete medium (Fig. 3B). Colocalization of HRES-1/Rab4S204Q with LC3 was also induced by Rapa at 0.034±0.01 (p = 0.005, Fig. 3B). 2-way ANOVA and Bonferroni post-tests revealed an increased localization of HRES-1/Rab41–121 to LC3 over all other HRES-1/Rab4 isoforms. Starvation-induced colocalization of HRES-1/Rab4 with LC3 was blocked by C-terminal truncation in HRES-1/Rab41–121, inability to bind GTP, to exert GTPase activity, or to respond to phosphorylation at S204 (Fig. 3B).
Colocalization of LC3 with mitochondria is modulated by HRES-1/Rab4
After we found significant colocalization of HRES-1/Rab4 with LC3 upon starvation, we examined whether the colocalization between LC3 and MTDR, was affected by HRES-1/Rab4. As evident from representative images (Fig. 4) and cumulative analyses (Figs. 5), there was limited MTDR-LC3 colocalization at baseline, except with introduction of HRES-1/Rab41–121. Upon starvation, wild-type HRES-1/Rab4 increased the ratio of LC3+ mitochondria/total mitochondria to 0.226±0.049 from 0.015±0.005 at baseline (p = 4.8×10−5) and from 0.096±0.024 with starvation in the presence of transduced LC3 alone (p = 0.0229, Fig. 5A). In contrast, mutated HRES-1/Rab41–121, HRES-1/Rab4S27N, HRES-1/Rab4Q72L, and HRES-1/Rab4S204Q limited the formation of LC3+ mitochondria upon starvation (Figs. 4 and 5A). HRES-1/Rab4 also promoted the Rapa-induced formation of LC3+ mitochondria (0.087±0.027; p = 0.0008 relative to baseline), which was less pronounced with HRES-1/Rab4Q72L and HRES-1/Rab4S204Q. In the presence of Rapa and Baf, the latter being a lysosomal inhibitor, the fraction of LC3+ mitochondria was markedly enhanced to 0.259±0.111 relative to untreated controls (0.046±0.022; p = 0.0008, Fig. 5A). This phenomenon, i.e. Rapa-induced colocalization of mitochondria with LC3 in the presence of Baf, was obliterated by HRES-1/Rab4Q72L and HRES-1/Rab4S204Q (Fig. 5A).
Figure 4
Confocal microscopy of HRES-1/Rab4, mitochondria, and LC3+ autophagosomes in HeLa cells under starvation (Star) and treatment with rapamycin (Rapa) and bafilomycin A1 (Baf).
eGFP-tagged HRES-1/Rab4 isoforms were identified by green fluorescence. Mitochondria were stained with MTDR and visualized by blue fluorescence. LC3+ autophagosomes were visualized by red fluorescence of FP650-LC3. Individual and composite color channels are shown for each experimental condition. A, HeLa cells were transfected with FP650-LC3 alone. B, HeLa cells were transfected with FP650-LC3 and eGFP-tagged HRES-1/Rab4. C, HeLa cells were transfected with FP650-LC3 and eGFP-tagged HRES-1/Rab41–121. D, HeLa cells were transfected with FP650-LC3 and eGFP-tagged HRES-1/Rab4S27N. E, HeLa cells were transfected with FP650-LC3 and eGFP-tagged HRES-1/Rab4Q72L. F, HeLa cells were transfected with FP650-LC3 and eGFP-tagged HRES-1/Rab4S204Q. The areas showing the formation of mitochondrial tubular networks are delineated by white dotted rectangles in panels A and E.
Figure 5
Quantitative analysis of colocalization between LC3+ autophagosomes and mitochondria in HeLa cells transfected with eGFP-tagged HRES-1/Rab4 isoforms and FP650-LC3.
Autophagy was induced by starvation (Star) or treatment with rapamycin (Rapa) in the presence or absence of bafilomycin A1 (Baf). A, Colocalization of FP-650 LC3 and MTDR-stained mitochondria relative to the total mitochondrial pool. B, Colocalization of FP-650 LC3 and MTDR-stained mitochondria relative to the total LC3 pool. Data represent mean ± SEM of 6–30 cells acquired in 3 independent experiments. * indicates p values<0.05 reflecting comparison to control cells among cell cultures transfected with the same construct using paired two-tailed t-tests; brackets connecting bars within each construct also reflect comparison with paired two-tailed t-tests. Brackets connecting bars between constructs reflect p<0.05 using ANOVA followed by Bonferroni's post-test.
Confocal microscopy of HRES-1/Rab4, mitochondria, and LC3+ autophagosomes in HeLa cells under starvation (Star) and treatment with rapamycin (Rapa) and bafilomycin A1 (Baf).
eGFP-tagged HRES-1/Rab4 isoforms were identified by green fluorescence. Mitochondria were stained with MTDR and visualized by blue fluorescence. LC3+ autophagosomes were visualized by red fluorescence of FP650-LC3. Individual and composite color channels are shown for each experimental condition. A, HeLa cells were transfected with FP650-LC3 alone. B, HeLa cells were transfected with FP650-LC3 and eGFP-tagged HRES-1/Rab4. C, HeLa cells were transfected with FP650-LC3 and eGFP-tagged HRES-1/Rab41–121. D, HeLa cells were transfected with FP650-LC3 and eGFP-tagged HRES-1/Rab4S27N. E, HeLa cells were transfected with FP650-LC3 and eGFP-tagged HRES-1/Rab4Q72L. F, HeLa cells were transfected with FP650-LC3 and eGFP-tagged HRES-1/Rab4S204Q. The areas showing the formation of mitochondrial tubular networks are delineated by white dotted rectangles in panels A and E.
Quantitative analysis of colocalization between LC3+ autophagosomes and mitochondria in HeLa cells transfected with eGFP-tagged HRES-1/Rab4 isoforms and FP650-LC3.
Autophagy was induced by starvation (Star) or treatment with rapamycin (Rapa) in the presence or absence of bafilomycin A1 (Baf). A, Colocalization of FP-650 LC3 and MTDR-stained mitochondria relative to the total mitochondrial pool. B, Colocalization of FP-650 LC3 and MTDR-stained mitochondria relative to the total LC3 pool. Data represent mean ± SEM of 6–30 cells acquired in 3 independent experiments. * indicates p values<0.05 reflecting comparison to control cells among cell cultures transfected with the same construct using paired two-tailed t-tests; brackets connecting bars within each construct also reflect comparison with paired two-tailed t-tests. Brackets connecting bars between constructs reflect p<0.05 using ANOVA followed by Bonferroni's post-test.In contrast to the strong influence of HRES-1/Rab4 on partitioning of mitochondria to LC3 (Fig. 5A), the influence on partitioning of LC3 to mitochondria was modest (Fig. 5B). Interestingly, the partitioning of LC3 to mitochondria was reduced from 0.112±0.041 to 0.011±0.004 in the presence of HRES-1/Rab4 at baseline (p = 0.042; Fig. 5B). During starvation, HRES-1/Rab4 and HRES-1/Rab4S204Q promoted the partitioning of LC3 to mitochondria at 0.239±0.042 (p = 2.9×10−7) and 0.115±0.031 (p = 0.004), respectively (Fig. 5B). The greatest shift of LC3 to mitochondria was elicited by HRES-1/Rab4S27N in the presence of Rapa and Baf (0.586±0.263) relative to baseline (0.067±0.022; p = 0.010; Fig. 5B). The Rapa/Baf-induced partitioning of LC3 to mitochondria was diminished by wild-type HRES-1/Rab4 and HRES-1/Rab4Q72L (Fig. 5B).
Starvation and bafilomycin A1 (Baf) induce colocalization of HRES-1/Rab4 with mitochondria
During autophagy, proteins and organelles alike are carried to the lysosome as endosomal cargo [23].Since HRES-1/Rab4 influenced the colocalization of mitochondria with LC3, we investigated if mitochondria were associated with HRES-1/Rab4-carrying endosomes. As shown in Fig. 4B, HRES-1/Rab4 colocalized with mitochondria. Starvation strongly promoted the association of HRES-1/Rab4 with mitochondria (Fig. 6). HRES-1/Rab41–121 exhibited the greatest colocalization with mitochondria, approaching 65% in response to starvation and Baf (Fig. 6). HRES-1/Rab4Q72L also showed colocalization with mitochondria in response to starvation and Baf or Rapa and Baf (Fig. 6).Starvation did not induce the partitioning of HRES-1/Rab4S27N or HRES-1/Rab4S204Q to mitochondria. Rapa only induced the colocalization of HRES-1/Rab4S204Q to mitochondria, which was reversed by Baf (Fig. 6). Thus, Baf promoted the association of HRES-1/Rab4Q72L but inhibited the association of HRES-1/Rab4S204Q with mitochondria in the presence of Rapa (Fig. 6).
Figure 6
Quantitative analysis of colocalization between HRES-1/Rab4 and mitochondria in HeLa cells transfected with eGFP-tagged HRES-1/Rab4 isoforms and FP650-LC3.
Autophagy was induced by starvation (Star) or treatment with rapamycin (Rapa) in the presence or absence of bafilomycin A1 (Baf). A, Colocalization of HRES-1/Rab4 and MTDR-stained mitochondria relative to the total mitochondrial pool. B, Colocalization of HRES-1/Rab4 and MTDR-stained mitochondria relative to the total HRES-1/Rab4 pool. Bars represent mean ± SEM. Data represent mean ± SEM of 6–29 cells acquired in 3 independent experiments. * indicates p values<0.05 reflecting comparison to control cells among cell cultures transfected with the same construct using paired two-tailed t-tests; brackets connecting bars within each construct also reflect comparison with paired two-tailed t-tests. Brackets connecting bars between constructs reflect p<0.05 using ANOVA followed by Bonferroni's post-test.
Quantitative analysis of colocalization between HRES-1/Rab4 and mitochondria in HeLa cells transfected with eGFP-tagged HRES-1/Rab4 isoforms and FP650-LC3.
Autophagy was induced by starvation (Star) or treatment with rapamycin (Rapa) in the presence or absence of bafilomycin A1 (Baf). A, Colocalization of HRES-1/Rab4 and MTDR-stained mitochondria relative to the total mitochondrial pool. B, Colocalization of HRES-1/Rab4 and MTDR-stained mitochondria relative to the total HRES-1/Rab4 pool. Bars represent mean ± SEM. Data represent mean ± SEM of 6–29 cells acquired in 3 independent experiments. * indicates p values<0.05 reflecting comparison to control cells among cell cultures transfected with the same construct using paired two-tailed t-tests; brackets connecting bars within each construct also reflect comparison with paired two-tailed t-tests. Brackets connecting bars between constructs reflect p<0.05 using ANOVA followed by Bonferroni's post-test.
Effect of HRES-1/Rab4 on the formation of LC3+ autophagosomes
Among the HRES-1/Rab4 isoforms, only HRES-1/Rab41–121 increased the formation of LC3+ autophagosomes in the absence of starvation or Rapa treatment, using two-way ANOVA and Bonferroni's post-test comparison (Fig. 7A). Under starvation, autophagosome formation was enhanced by wild-type HRES-1/Rab4, HRES-1/Rab4S27N, HRES-1/Rab4Q72L, and HRES-1/Rab4S27N, using paired t-test (Fig. 7A). Relative to starvation, Rapa reduced the formation of LC3+ autophagosomes with overexpression of HRES-1/Rab4 and HRES-1/Rab4Q72L. This effect of Rapa was sustained in the presence of Baf (Fig. 7A).
Figure 7
Quantitative analyses of the effect by HRES-1/Rab4 on the accumulation of LC3+ autophagosomes (panel A) and MTDR-stained mitochondria (panel B).
HeLa cells were transfected with eGFP-tagged HRES-1/Rab4 isoforms and FP650-LC3. Autophagy was induced by starvation (Star) or treatment with rapamycin (Rapa) in the presence or absence of bafilomycin A1 (Baf). Data represent mean ± SEM of 6–30 cells acquired in 3 independent experiments. * indicates p values<0.05 reflecting comparison to cells transfected with LC3 alone using paired two-tailed t-tests; brackets connecting bars within each construct also reflect comparison with paired two-tailed t-tests. Brackets connecting bars between constructs reflect p<0.05 using ANOVA followed by Bonferroni's post-test.
Quantitative analyses of the effect by HRES-1/Rab4 on the accumulation of LC3+ autophagosomes (panel A) and MTDR-stained mitochondria (panel B).
HeLa cells were transfected with eGFP-tagged HRES-1/Rab4 isoforms and FP650-LC3. Autophagy was induced by starvation (Star) or treatment with rapamycin (Rapa) in the presence or absence of bafilomycin A1 (Baf). Data represent mean ± SEM of 6–30 cells acquired in 3 independent experiments. * indicates p values<0.05 reflecting comparison to cells transfected with LC3 alone using paired two-tailed t-tests; brackets connecting bars within each construct also reflect comparison with paired two-tailed t-tests. Brackets connecting bars between constructs reflect p<0.05 using ANOVA followed by Bonferroni's post-test.
HRES-1/Rab4, HRES-1/Rab41–121, HRES-1/Rab4Q72L and HRES-1/Rab4S204Q promote the accumulation of mitochondria during starvation
We investigated the impact of HRES-1/Rab4 on the mitochondrial mass of HeLa cells. As opposed to primary lymphocytes and Jurkat T cells [19], HRES-1/Rab4 did not influence the accumulation of mitochondria in resting HeLa cells. However, HRES-1/Rab4, HRES-1/Rab41–121, HRES-1/Rab4Q72L and HRES-1/Rab4S204Q promoted the accumulation of mitochondria during starvation (Fig. 7B). In contrast, dominant-negative HRES-1/Rab4S27N did not have such effect (Fig. 7B). Rapa only promoted the accumulation of mitochondria in the presence of HRES-1/Rab4S204Q, which was reversed by Baf (Fig. 7B). In contrast, Rapa promoted the accumulation of mitochondria in the presence of HRES-1/Rab4Q72L when Baf was also provided (Fig. 7B).
HRES-1/Rab4Q72L promotes the formation of mitochondrial tubular network upon starvation
Mitochondria fuse and form a highly interconnected tubular network upon starvation to resist degradation [24]. Upon starvation of cells transfected with LC3 only, 17±11% of mitochondria assumed tubular shape (Fig. 4A). The formation of highly interconnected tubular mitochondria was enhanced to 49±17% in cells transfected with HRES-1/Rab4Q72L (p = 0.02; Fig. 4E). No other HRES-1/Rab4 isoform increased the formation of mitochondrial tubular network (cumulative data not shown).
Discussion
The present study provides evidence that the endosomal recycling regulator small GTPase HRES-1/Rab4 colocalizes with the autophagosomal membrane component LC3 and mitochondria, and, moreover, it promotes the colocalization between LC3 and mitochondria. These findings are consistent with recent observations that 1) recycling endosomes contribute to the biogenesis of autophagosomal membrane [6], 2) mitochondria and endosomes are juxtaposed under cellular stress [25], and 3) autophagosomes originate from membrane components of mitochondria [3], which may be linked to the turnover of mitochondria, termed mitophagy [26]. Thus, the enhanced colocalization of LC3 and mitochondria by HRES-1/Rab4 may reflect increased formation of autophagosomes. All active isoforms of HRES-1/Rab4, HRES-1/Rab41–121, HRES-1/Rab4Q72L and HRES-1/Rab4S204Q, but not the dominant-negative HRES-1/Rab4S27N promoted the accumulation of mitochondria during starvation, suggesting that this small GTPase inhibits mitophagy, particularly under the conditions of cellular stress. Along these lines, blockade of mTOR by Rapa promoted the accumulation of mitochondria in the presence of HRES-1/Rab4S204Q, which was reversed by Baf. Since HRES-1/Rab4S204Q cannot be phosphorylated by p34cdc2 kinase and remains endosome-associated throughout the cell cycle [22], this indicates that mTOR blockade may promote the accumulation of mitochondria [16] through interacting with endosome-bound HRES-1/Rab4. This notion is supported by the finding that Rapa-induced colocalization of mitochondria with LC3 was obliterated by HRES-1/Rab4Q72L. Rapa also increased mitochondrial mass in the presence of HRES-1/Rab4Q72L when Baf was provided, suggesting that such accumulation of mitochondria may depend on lysosomal function. The marked enhancement of interconnected mitochondrial tubular networks by HRES-1/Rab4Q72L documents its impact on a key checkpoint of mitochondrial preservation during autophagy. Therefore, the colocalization of LC3 with mitochondria upon starvation and the enhancement of this colocalization by HRES-1/Rab4 may represent coordinated steps between autophagosome formation and retention of mitochondria through reduced mitophagy (Fig. 8).
Figure 8
Schematic diagram of the impact by HRES-1/Rab4 on autophagy.
HRES-1/Rab4 promotes the formation of LC3+ autophagosomes, the accumulation of mitochondria, and their colocalization during autophagy induced by starvation or treatment with rapamycin. LC3+ autophagosomes are encircled by HRES-1/Rab4+ endosomes. The formation of interconnected mitochondrial tubular networks is enhanced by HRES-1/Rab4Q72L upon starvation.
Schematic diagram of the impact by HRES-1/Rab4 on autophagy.
HRES-1/Rab4 promotes the formation of LC3+ autophagosomes, the accumulation of mitochondria, and their colocalization during autophagy induced by starvation or treatment with rapamycin. LC3+ autophagosomes are encircled by HRES-1/Rab4+ endosomes. The formation of interconnected mitochondrial tubular networks is enhanced by HRES-1/Rab4Q72L upon starvation.The colocalization of HRES-1/Rab4 with LC3 was enhanced by starvation and Rapa, both of which induce autophagy [14], [15]. The induction of this colocalization was compromised by mutations at S27, Q72, and S204, indicating that this activity requires the complete functional integrity of HRES-1/Rab4. The association of HRES-1/Rab4 with LC3 and mitochondria was also enhanced by Baf. While Baf has been widely used as an inhibitor of autophagy [27], it can also lead to mitochondrial membrane depolarization and mitochondrial damage [28], [29], which is consistent with our findings.Here, we documented the existence of a novel splice variant of HRES-1/Rab4, encoding a 121 amino-acid-long N-terminal polypeptide, HRES-1/Rab41–121, which exhibited the most robust colocalization with both LC3 and mitochondria relative to all other HRES-1/Rab4 isoforms. The increased localization of HRES-1/Rab41–121 to LC3 and mitochondria was also the least regulated by starvation, Rapa, or Baf, possibly due to the absence of C-terminal prenylation site that allows association with membrane moieties. As HRES-1/Rab41–121 showed the greatest colocalization with LC3 without starvation or Rapa treatment, this truncated isoform may not act through promoting but rather retaining the association of mitochondria with LC3+ lysosomes. Alternatively, the strong association of HRES-1/Rab41–121 with LC3 may reflect its targeting for degradation through the autophagy-lysosome pathway [15], [30], which has been also documented for other proteins such as misfolded superoxide dismutase [31] and truncated Cln6 [32].These findings complement earlier observations about the involvement of early endosomal small GTPases in autophagy [8], such as Rab11 in autophagosome formation [6] and Rab7 in autophagosome progression [10] and maturation [9]. The role of HRES-1/Rab4 in mitophagy may be particularly relevant for the pathogenesis of mitochondrial dysfunction and oxidative stress in T cells of SLEpatients [33]. Redox-controlled overexpression of HRES-1/Rab4 in lupus T cells is partially reversed by Rapa [13]. Interestingly, the accumulation of mitochondria in lupus T cells is resistant to mTOR blockade by Rapa [16] or NAC [34]. As shown in this study, the accumulation of mitochondria was induced during starvation by wild-type HRES-1/Rab4, C-terminally truncated HRES-1/Rab41–121, constitutively active/GTPase-deficient HRES-1/Rab4Q72L and phosphorylation-resistant HRES-1/Rab4S204Q but not by dominant-negative/GTP binding-deficient HRES-1/Rab4S27N. Thus, the specificity of HRES-1/Rab4-mediated mitochondrial accumulation is indicated through its abrogation by the dominant-negative HRES-1/Rab4S27N mutation. These findings are consistent with recent observations that HRES-1/Rab4 depletes the mitophagy initiator Drp1 and thus facilitates the accumulation of mitochondria [19]. Along these lines, dominant-negative HRES-1/Rab4S27N or pharmacological blockade of HRES-1/Rab4 enhanced Drp1 levels and diminished mitochondrial mass in human and mouse T cells [19]. In accordance with these observations, pharmacological blockade of Drp1 also elicited the accumulation of mitochondria [35].The present study further substantiates the involvement of HRES-1/Rab4 in autophagy, indicating its potential roles in formation of autophagosomes and preservation of mitochondria by reduced mitophagy.
Materials and Methods
Vector constructs
HRES-1/Rab4 cDNA was inserted into the multiple cloning site (MCS) of the pAAV-MCS vector fused to the C-terminus of enhanced green fluorescent protein (eGFP; Stratagene, La Jolla, CA) or fluorescent protein 650 (FP650), as described previously [12], [36]. We also made bicistronic constructs having the internal ribosomal entry site positioned between the two open reading frames (pAAV-HRES-1/Rab4-IRES-GFP) [12]. Point mutations were created by site-directed mutagenesis using the PCR-based Quick Change method (Stratagene, La Jolla, CA) to produce wild-type HRES-1/Rab4, GDP-locked HRES-1/Rab4S27N (TCT→AAT), GTP-locked HRES-1/Rab4Q72L (CAA→CTA), HRES-1/Rab41–121 (C-terminally truncated) and cell cycle phosphorylation-defective HRES-1/Rab4S204Q mutants (TCA→CAA). The GDP-locked HRES-1/Rab4S27N prevents GTP binding and creates a dominant-negative mutation [20]. The GTP-locked HRES-1/Rab4Q72L mutant is constitutively active due to elimination of GTPase activity [21]. HRES-1/Rab4S204Q will not be phosphorylated by p34cdc2 kinase in mitotic cells and remains endosome-associated throughout the cell cycle [22]. Amino acid sequence of HRES-1/Rab41–121 represents a 36-nucleotide out-of-frame deletion, which was attributed to alternative splicing (GenBank submission number 1591873). This results in a frameshift with a new protein corresponding to the 96 N-terminal amino acid residues of HRES-1/Rab4 continuing into 25 C-terminal amino acid residues, which are unrelated to the C-terminal 97–218 peptide domain of wild-type HRES-1/Rab4. A schematic diagram of all investigated HRES-1/Rab4 isoforms is shown in Figs. 1A and B.
Transfections
HeLa cell were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 10 µg/mL amphotericin B. 24 h before transfection. 105 cells per well were seeded in glass bottom dishes Petri dishes (MatTek, Ashland, MA; Cat no. P35GC-1.5-10-C). Cells were washed in PBS and transfected with 1200 ng of DNA in serum-free DMEM for 6 hours using Lipofectamine 2000 reagent and protocol (Life Technologies, Grand Island, NY). After transfection, cells were washed with PBS, incubated in complete DMEM medium for 48 hours, followed by induction of autophagy, and subsequent analyses by microscopy, western blot, and flow cytometry (Fig. S1).
Autophagy induction
After transfection, cells were washed with PBS and starved with serum-free DMEM and compared to cells maintained in complete DMEM for 4 hours. Parallel cultures were treated for 4 hours with 200 nM bafilomycin A1 (Baf), a lysosomal or vacuolar type H+-ATPase inhibitor commonly used to prevent autophagosome-lysosome fusion [37], [38]. However, the effect of Baf on lysosomes may be non-selective, recent studies revealed its mitochondrial toxicity [29], [39]. Before flow cytometry and microscopy, cells were loaded with 100 nM Mito Tracker Deep Red (MTDR) for 30 minutes and washed with PBS twice. Indeed, we observed a 32±5% loss of MTDR fluorescence by flow cytometry after 4 h treatment with Baf (p = 0.001; data not shown). To inhibit mTOR [13], cells were treated by 100 nM rapamycin (Rapa) for 4 hours. Control cultures included 0.1% dimethylsulfoxide (DMSO) used as solvent for Baf and Rapa.
Flow cytometry
Transfection of expression vectors producing eGFP (excitation: 488 nm, emission: 507 nm; FL1 A channel) and FP650 (excitation: 592 nm, emission: 650 nm; YDC-A channel) and staining of mitochondria with 100 nM MitoTracker Deep Red (MTDR, excitation: 644 nm, emission: 665 nm; Red C-A channel) were monitored by flow cytometry. Samples were analyzed using a Becton Dickinson LSRII flow cytometer equipped with 20 mW solid-state Ng-YAG (emission at 355 nm), 20 mW argon (emission at 488 nm), 10 mW diode-pumped solid-state yellow-green (emission at 535 nm), and 16 mW helium-neon lasers (emission at 634 nm). Data were analyzed with Flow Jo 7.6 software (TreeStar Corporation, Ashland, OR). Dead cells and debris were excluded from the analysis by electronic gating of forward (FSC) and side scatter (SSC) measurements. Each measurement was carried out on ≥10,000 cells.
Western blot analysis
Cells were trypsinized, washed once with PBS, and resuspended in radio-immunoprecipitation assay buffer (150 mM NaCl, 2% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8.0, 1 mM PMSF, 1 μg/ml aproptinin, 1 μg/ml pepstatin, 1 μg/ml leupeptin, 1 mM NaF, 1 mM sodium orthovanadate, 0.1 mM sodium molybdate, 10 mM sodium pyrophosphate) at a density of 107 cells/ml on ice, followed by addition of equal volumes of Laemmli protein sample buffer (60 mM Tris-Cl pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.01% bromophenol blue) and heated to 95°C for 5 minutes prior to separation on SDS-PAGE gels and transfer to 0.45 μm nitrocellulose membranes. HRES-1/Rab4 was detected by primary rabbit antibodies directed to the C-terminus (Santa Cruz SC312) and Ab 13407 directed to full-length native protein [12]. Expression of HRES-1/Rab41–121 was detected by G1432 rabbit antibody directed to peptide residues 100–121 (Genemed, San Antonio, TX). LC3A/B (#4108), and LC3B (#2775) antibodies were obtained from Cell Signaling. Reactivities to primary antibodies were detected with horseradish peroxidase-conjugated secondary antibodies (Jackson, West Grove, PA) and visualized by enhanced chemiluminescence (Western Lightning Chemiluminescence Reagent Plus, GE Health Care/PerkinElmer Life Sciences, Inc., Boston, Massachusetts). Automated densitometry was used to quantify the relative levels of protein expression using a Kodak Image Station 440CF with Kodak 1D Image Analysis Software (Eastman Kodak Company, Rochester, NY).
Confocal microscopy
After autophagy induction, culture dishes with adherent cells were kept in PBS on ice until images were taken using a Zeiss LSM 510 confocal microscope and 63x oil objective. Visualization of the specimens on the slides was carried out under a Zeiss 510 LSM Meta confocal microscope using Zeiss LSM Image Browser software version 4.2 (Carl Zeiss Microimaging, Thornwood, NJ). Camera gain was calibrated on cells that have been untransfected and unstained. Sequential scanning was used to record eGFP (excitation: 488 nm, emission: 507 nm), FP650 (excitation: 592 nm, emission: 650 nm), and MTDR (excitation: 644 nm, emission: 665 nm); the RGB images were converted to 8-bit grayscales and pseudo-colored in green, red, and blue, respectively. The means for negative controls were set at 0 intensity. Fluorescence intensities were recorded in a range of 0–255. Fluorescence intensity was determined for each pixel using ImageJ(http://rsbweb.nih.gov/ij/). ImageJ was used to quantify colocalization between eGFP-tagged Rab4, FP650 tagged LC3 and Mitotracker Deep Red loaded mitochondria within cells. Cells were selected and the region of interest (ROI) was defined by an ROI analyzer plugin. Mean intensity values were taken in each channels for each pixel. For each experiment data were generated from analyses of 21.39±0.85 cells (range 6–40 cells). Colocalized signal between Rab4, LC3 and MTDR were quantified and divided by the mean value of the corresponding channel to determine colocalization ratio [40]. Tubular morphology of mitochondria was quantified as earlier described [24].
Statistical analysis
Data are presented as mean ± SEM, unless stated otherwise. Statistical analyses were done using the Graphpad Prism 5.0 software. Student's t-test was used to analyze the effect of various treatments within a mutation and 2-way ANOVA was done with Bonferroni post-tests to analyze the effect between mutations and treatments. Changes were considered significant at p value<0.05.Flow cytometry detection of HRES-1/Rab4 isoforms, including wild-type HRES-1/Rab4, C-terminally truncated HRES-1/Rab4 A) Flow cytometry of HeLa cells transfected with backbone vector pAAV-MCS and expression constructs producing HRES-1/Rab4-eGFP fusion proteins and eGFP alone. B, Flow cytometry of HeLa cells transfected with backbone vector pAAV-MCS, and expression vectors producing FP650-LC3 fusion protein and FP650 alone.(PDF)Click here for additional data file.
Authors: Michael R Mancuso; Rachel Davis; Scott M Norberg; Shaun O'Brien; Barbara Sennino; Tsutomu Nakahara; Virginia J Yao; Tetsuichiro Inai; Peter Brooks; Bruce Freimark; David R Shalinsky; Dana D Hu-Lowe; Donald M McDonald Journal: J Clin Invest Date: 2006-10 Impact factor: 14.808
Authors: Gyorgy Nagy; Jeffrey Ward; Dick D Mosser; Agnes Koncz; Peter Gergely; Christina Stancato; Yueming Qian; David Fernandez; Brian Niland; Craig E Grossman; Tiffany Telarico; Katalin Banki; Andras Perl Journal: J Biol Chem Date: 2006-08-24 Impact factor: 5.157
Authors: Vera V Teplova; Anton A Tonshin; Pavel A Grigoriev; Nils-Erik L Saris; Mirja S Salkinoja-Salonen Journal: J Bioenerg Biomembr Date: 2007-08 Impact factor: 2.945
Authors: Tiffany N Caza; David R Fernandez; Gergely Talaber; Zachary Oaks; Mark Haas; Michael P Madaio; Zhi-Wei Lai; Gabriella Miklossy; Ram R Singh; Dmitriy M Chudakov; Walter Malorni; Frank Middleton; Katalin Banki; Andras Perl Journal: Ann Rheum Dis Date: 2013-07-29 Impact factor: 19.103
Authors: Aparna Godavarthy; Ryan Kelly; John Jimah; Miguel Beckford; Tiffany Caza; David Fernandez; Nick Huang; Manuel Duarte; Joshua Lewis; Hind J Fadel; Eric M Poeschla; Katalin Banki; Andras Perl Journal: JCI Insight Date: 2020-01-16
Authors: Billur Akkaya; Alexander S Roesler; Pietro Miozzo; Brandon P Theall; Jafar Al Souz; Margery G Smelkinson; Juraj Kabat; Javier Traba; Michael N Sack; Joseph A Brzostowski; Mirna Pena; David W Dorward; Susan K Pierce; Munir Akkaya Journal: J Immunol Date: 2018-10-29 Impact factor: 5.422
Authors: Daniel J Klionsky; Kotb Abdelmohsen; Akihisa Abe; Md Joynal Abedin; Hagai Abeliovich; Abraham Acevedo Arozena; Hiroaki Adachi; Christopher M Adams; Peter D Adams; Khosrow Adeli; Peter J Adhihetty; Sharon G Adler; Galila Agam; Rajesh Agarwal; Manish K Aghi; Maria Agnello; Patrizia Agostinis; Patricia V Aguilar; Julio Aguirre-Ghiso; Edoardo M Airoldi; Slimane Ait-Si-Ali; Takahiko Akematsu; Emmanuel T Akporiaye; Mohamed Al-Rubeai; Guillermo M Albaiceta; Chris Albanese; Diego Albani; Matthew L Albert; Jesus Aldudo; Hana Algül; Mehrdad Alirezaei; Iraide Alloza; Alexandru Almasan; Maylin Almonte-Beceril; Emad S Alnemri; Covadonga Alonso; Nihal Altan-Bonnet; Dario C Altieri; Silvia Alvarez; Lydia Alvarez-Erviti; Sandro Alves; Giuseppina Amadoro; Atsuo Amano; Consuelo Amantini; Santiago Ambrosio; Ivano Amelio; Amal O Amer; Mohamed Amessou; Angelika Amon; Zhenyi An; Frank A Anania; Stig U Andersen; Usha P Andley; Catherine K Andreadi; Nathalie Andrieu-Abadie; Alberto Anel; David K Ann; Shailendra Anoopkumar-Dukie; Manuela Antonioli; Hiroshi Aoki; Nadezda Apostolova; Saveria Aquila; Katia Aquilano; Koichi Araki; Eli Arama; Agustin Aranda; Jun Araya; Alexandre Arcaro; Esperanza Arias; Hirokazu Arimoto; Aileen R Ariosa; Jane L Armstrong; Thierry Arnould; Ivica Arsov; Katsuhiko Asanuma; Valerie Askanas; Eric Asselin; Ryuichiro Atarashi; Sally S Atherton; Julie D Atkin; Laura D Attardi; Patrick Auberger; Georg Auburger; Laure Aurelian; Riccardo Autelli; Laura Avagliano; Maria Laura Avantaggiati; Limor Avrahami; Suresh Awale; Neelam Azad; Tiziana Bachetti; Jonathan M Backer; Dong-Hun Bae; Jae-Sung Bae; Ok-Nam Bae; Soo Han Bae; Eric H Baehrecke; Seung-Hoon Baek; Stephen Baghdiguian; Agnieszka Bagniewska-Zadworna; Hua Bai; Jie Bai; Xue-Yuan Bai; Yannick Bailly; Kithiganahalli Narayanaswamy Balaji; Walter Balduini; Andrea Ballabio; Rena Balzan; Rajkumar Banerjee; Gábor Bánhegyi; Haijun Bao; Benoit Barbeau; Maria D Barrachina; Esther Barreiro; Bonnie Bartel; Alberto Bartolomé; Diane C Bassham; Maria Teresa Bassi; Robert C Bast; Alakananda Basu; Maria Teresa Batista; Henri Batoko; Maurizio Battino; Kyle Bauckman; Bradley L Baumgarner; K Ulrich Bayer; Rupert Beale; Jean-François Beaulieu; George R Beck; Christoph Becker; J David Beckham; Pierre-André Bédard; Patrick J Bednarski; Thomas J Begley; Christian Behl; Christian Behrends; Georg Mn Behrens; Kevin E Behrns; Eloy Bejarano; Amine Belaid; Francesca Belleudi; Giovanni Bénard; Guy Berchem; Daniele Bergamaschi; Matteo Bergami; Ben Berkhout; Laura Berliocchi; Amélie Bernard; Monique Bernard; Francesca Bernassola; Anne Bertolotti; Amanda S Bess; Sébastien Besteiro; Saverio Bettuzzi; Savita Bhalla; Shalmoli Bhattacharyya; Sujit K Bhutia; Caroline Biagosch; Michele Wolfe Bianchi; Martine Biard-Piechaczyk; Viktor Billes; Claudia Bincoletto; Baris Bingol; Sara W Bird; Marc Bitoun; Ivana Bjedov; Craig Blackstone; Lionel Blanc; Guillermo A Blanco; Heidi Kiil Blomhoff; Emilio Boada-Romero; Stefan Böckler; Marianne Boes; Kathleen Boesze-Battaglia; Lawrence H Boise; Alessandra Bolino; Andrea Boman; Paolo Bonaldo; Matteo Bordi; Jürgen Bosch; Luis M Botana; Joelle Botti; German Bou; Marina Bouché; Marion Bouchecareilh; Marie-Josée Boucher; Michael E Boulton; Sebastien G Bouret; Patricia Boya; Michaël Boyer-Guittaut; Peter V Bozhkov; Nathan Brady; Vania Mm Braga; Claudio Brancolini; Gerhard H Braus; José M Bravo-San Pedro; Lisa A Brennan; Emery H Bresnick; Patrick Brest; Dave Bridges; Marie-Agnès Bringer; Marisa Brini; Glauber C Brito; Bertha Brodin; Paul S Brookes; Eric J Brown; Karen Brown; Hal E Broxmeyer; Alain Bruhat; Patricia Chakur Brum; John H Brumell; Nicola Brunetti-Pierri; Robert J Bryson-Richardson; Shilpa Buch; Alastair M Buchan; Hikmet Budak; Dmitry V Bulavin; Scott J Bultman; Geert Bultynck; Vladimir Bumbasirevic; Yan Burelle; Robert E Burke; Margit Burmeister; Peter Bütikofer; Laura Caberlotto; Ken Cadwell; Monika Cahova; Dongsheng Cai; Jingjing Cai; Qian Cai; Sara Calatayud; Nadine Camougrand; Michelangelo Campanella; Grant R Campbell; Matthew Campbell; Silvia Campello; Robin Candau; Isabella Caniggia; Lavinia Cantoni; Lizhi Cao; Allan B Caplan; Michele Caraglia; Claudio Cardinali; Sandra Morais Cardoso; Jennifer S Carew; Laura A Carleton; Cathleen R Carlin; Silvia Carloni; Sven R Carlsson; Didac Carmona-Gutierrez; Leticia Am Carneiro; Oliana Carnevali; Serena Carra; Alice Carrier; Bernadette Carroll; Caty Casas; Josefina Casas; Giuliana Cassinelli; Perrine Castets; Susana Castro-Obregon; Gabriella Cavallini; Isabella Ceccherini; Francesco Cecconi; Arthur I Cederbaum; Valentín Ceña; Simone Cenci; Claudia Cerella; Davide Cervia; Silvia Cetrullo; Hassan Chaachouay; Han-Jung Chae; Andrei S Chagin; Chee-Yin Chai; Gopal Chakrabarti; Georgios Chamilos; Edmond Yw Chan; Matthew Tv Chan; Dhyan Chandra; Pallavi Chandra; Chih-Peng Chang; Raymond Chuen-Chung Chang; Ta Yuan Chang; John C Chatham; Saurabh Chatterjee; Santosh Chauhan; Yongsheng Che; Michael E Cheetham; Rajkumar Cheluvappa; Chun-Jung Chen; Gang Chen; Guang-Chao Chen; Guoqiang Chen; Hongzhuan Chen; Jeff W Chen; Jian-Kang Chen; Min Chen; Mingzhou Chen; Peiwen Chen; Qi Chen; Quan Chen; Shang-Der Chen; Si Chen; Steve S-L Chen; Wei Chen; Wei-Jung Chen; Wen Qiang Chen; Wenli Chen; Xiangmei Chen; Yau-Hung Chen; Ye-Guang Chen; Yin Chen; Yingyu Chen; Yongshun Chen; Yu-Jen Chen; Yue-Qin Chen; Yujie Chen; Zhen Chen; Zhong Chen; Alan Cheng; Christopher Hk Cheng; Hua Cheng; Heesun Cheong; Sara Cherry; Jason Chesney; Chun Hei Antonio Cheung; Eric Chevet; Hsiang Cheng Chi; Sung-Gil Chi; Fulvio Chiacchiera; Hui-Ling Chiang; Roberto Chiarelli; Mario Chiariello; Marcello Chieppa; Lih-Shen Chin; Mario Chiong; Gigi Nc Chiu; Dong-Hyung Cho; Ssang-Goo Cho; William C Cho; Yong-Yeon Cho; Young-Seok Cho; Augustine Mk Choi; Eui-Ju Choi; Eun-Kyoung Choi; Jayoung Choi; Mary E Choi; Seung-Il Choi; Tsui-Fen Chou; Salem Chouaib; Divaker Choubey; Vinay Choubey; Kuan-Chih Chow; Kamal Chowdhury; Charleen T Chu; Tsung-Hsien Chuang; Taehoon Chun; Hyewon Chung; Taijoon Chung; Yuen-Li Chung; Yong-Joon Chwae; Valentina Cianfanelli; Roberto Ciarcia; Iwona A Ciechomska; Maria Rosa Ciriolo; Mara Cirone; Sofie Claerhout; Michael J Clague; Joan Clària; Peter Gh Clarke; Robert Clarke; Emilio Clementi; Cédric Cleyrat; Miriam Cnop; Eliana M Coccia; Tiziana Cocco; Patrice Codogno; Jörn Coers; Ezra Ew Cohen; David Colecchia; Luisa Coletto; Núria S Coll; Emma Colucci-Guyon; Sergio Comincini; Maria Condello; Katherine L Cook; Graham H Coombs; Cynthia D Cooper; J Mark Cooper; Isabelle Coppens; Maria Tiziana Corasaniti; Marco Corazzari; Ramon Corbalan; Elisabeth Corcelle-Termeau; Mario D Cordero; Cristina Corral-Ramos; Olga Corti; Andrea Cossarizza; Paola Costelli; Safia Costes; Susan L Cotman; Ana Coto-Montes; Sandra Cottet; Eduardo Couve; Lori R Covey; L Ashley Cowart; Jeffery S Cox; Fraser P Coxon; Carolyn B Coyne; Mark S Cragg; Rolf J Craven; Tiziana Crepaldi; Jose L Crespo; Alfredo Criollo; Valeria Crippa; Maria Teresa Cruz; Ana Maria Cuervo; Jose M Cuezva; Taixing Cui; Pedro R Cutillas; Mark J Czaja; Maria F Czyzyk-Krzeska; Ruben K Dagda; Uta Dahmen; Chunsun Dai; Wenjie Dai; Yun Dai; Kevin N Dalby; Luisa Dalla Valle; Guillaume Dalmasso; Marcello D'Amelio; Markus Damme; Arlette Darfeuille-Michaud; Catherine Dargemont; Victor M Darley-Usmar; Srinivasan Dasarathy; Biplab Dasgupta; Srikanta Dash; Crispin R Dass; Hazel Marie Davey; Lester M Davids; David Dávila; Roger J Davis; Ted M Dawson; Valina L Dawson; Paula Daza; Jackie de Belleroche; Paul de Figueiredo; Regina Celia Bressan Queiroz de Figueiredo; José de la Fuente; Luisa De Martino; Antonella De Matteis; Guido Ry De Meyer; Angelo De Milito; Mauro De Santi; Wanderley de Souza; Vincenzo De Tata; Daniela De Zio; Jayanta Debnath; Reinhard Dechant; Jean-Paul Decuypere; Shane Deegan; Benjamin Dehay; Barbara Del Bello; Dominic P Del Re; Régis Delage-Mourroux; Lea Md Delbridge; Louise Deldicque; Elizabeth Delorme-Axford; Yizhen Deng; Joern Dengjel; Melanie Denizot; Paul Dent; Channing J Der; Vojo Deretic; Benoît Derrien; Eric Deutsch; Timothy P Devarenne; Rodney J Devenish; Sabrina Di Bartolomeo; Nicola Di Daniele; Fabio Di Domenico; Alessia Di Nardo; Simone Di Paola; Antonio Di Pietro; Livia Di Renzo; Aaron DiAntonio; Guillermo Díaz-Araya; Ines Díaz-Laviada; Maria T Diaz-Meco; Javier Diaz-Nido; Chad A Dickey; Robert C Dickson; Marc Diederich; Paul Digard; Ivan Dikic; Savithrama P Dinesh-Kumar; Chan Ding; Wen-Xing Ding; Zufeng Ding; Luciana Dini; Jörg Hw Distler; Abhinav Diwan; Mojgan Djavaheri-Mergny; Kostyantyn Dmytruk; Renwick Cj Dobson; Volker Doetsch; Karol Dokladny; Svetlana Dokudovskaya; Massimo Donadelli; X Charlie Dong; Xiaonan Dong; Zheng Dong; Terrence M Donohue; Kelly S Doran; Gabriella D'Orazi; Gerald W Dorn; Victor Dosenko; Sami Dridi; Liat Drucker; Jie Du; Li-Lin Du; Lihuan Du; André du Toit; Priyamvada Dua; Lei Duan; Pu Duann; Vikash Kumar Dubey; Michael R Duchen; Michel A Duchosal; Helene Duez; Isabelle Dugail; Verónica I Dumit; Mara C Duncan; Elaine A Dunlop; William A Dunn; Nicolas Dupont; Luc Dupuis; Raúl V Durán; Thomas M Durcan; Stéphane Duvezin-Caubet; Umamaheswar Duvvuri; Vinay Eapen; Darius Ebrahimi-Fakhari; Arnaud Echard; Leopold Eckhart; Charles L Edelstein; Aimee L Edinger; Ludwig Eichinger; Tobias Eisenberg; Avital Eisenberg-Lerner; N Tony Eissa; Wafik S El-Deiry; Victoria El-Khoury; Zvulun Elazar; Hagit Eldar-Finkelman; Chris Jh Elliott; Enzo Emanuele; Urban Emmenegger; Nikolai Engedal; Anna-Mart Engelbrecht; Simone Engelender; Jorrit M Enserink; Ralf Erdmann; Jekaterina Erenpreisa; Rajaraman Eri; Jason L Eriksen; Andreja Erman; Ricardo Escalante; Eeva-Liisa Eskelinen; Lucile Espert; Lorena Esteban-Martínez; Thomas J Evans; Mario Fabri; Gemma Fabrias; Cinzia Fabrizi; Antonio Facchiano; Nils J Færgeman; Alberto Faggioni; W Douglas Fairlie; Chunhai Fan; Daping Fan; Jie Fan; Shengyun Fang; Manolis Fanto; Alessandro Fanzani; Thomas Farkas; Mathias Faure; Francois B Favier; Howard Fearnhead; Massimo Federici; Erkang Fei; Tania C Felizardo; Hua Feng; Yibin Feng; Yuchen Feng; Thomas A Ferguson; Álvaro F Fernández; Maite G Fernandez-Barrena; Jose C Fernandez-Checa; Arsenio Fernández-López; Martin E Fernandez-Zapico; Olivier Feron; Elisabetta Ferraro; Carmen Veríssima Ferreira-Halder; Laszlo Fesus; Ralph Feuer; Fabienne C Fiesel; Eduardo C Filippi-Chiela; Giuseppe Filomeni; Gian Maria Fimia; John H Fingert; Steven Finkbeiner; Toren Finkel; Filomena Fiorito; Paul B Fisher; Marc Flajolet; Flavio Flamigni; Oliver Florey; Salvatore Florio; R Andres Floto; Marco Folini; Carlo Follo; Edward A Fon; Francesco Fornai; Franco Fortunato; Alessandro Fraldi; Rodrigo Franco; Arnaud Francois; Aurélie François; Lisa B Frankel; Iain Dc Fraser; Norbert Frey; Damien G Freyssenet; Christian Frezza; Scott L Friedman; Daniel E Frigo; Dongxu Fu; José M Fuentes; Juan Fueyo; Yoshio Fujitani; Yuuki Fujiwara; Mikihiro Fujiya; Mitsunori Fukuda; Simone Fulda; Carmela Fusco; Bozena Gabryel; Matthias Gaestel; Philippe Gailly; Malgorzata Gajewska; Sehamuddin Galadari; Gad Galili; Inmaculada Galindo; Maria F Galindo; Giovanna Galliciotti; Lorenzo Galluzzi; Luca Galluzzi; Vincent Galy; Noor Gammoh; Sam Gandy; Anand K Ganesan; Swamynathan Ganesan; Ian G Ganley; Monique Gannagé; Fen-Biao Gao; Feng Gao; Jian-Xin Gao; Lorena García Nannig; Eleonora García Véscovi; Marina Garcia-Macía; Carmen Garcia-Ruiz; Abhishek D Garg; Pramod Kumar Garg; Ricardo Gargini; Nils Christian Gassen; Damián Gatica; Evelina Gatti; Julie Gavard; Evripidis Gavathiotis; Liang Ge; Pengfei Ge; Shengfang Ge; Po-Wu Gean; Vania Gelmetti; Armando A Genazzani; Jiefei Geng; Pascal Genschik; Lisa Gerner; Jason E Gestwicki; David A Gewirtz; Saeid Ghavami; Eric Ghigo; Debabrata Ghosh; Anna Maria Giammarioli; Francesca Giampieri; Claudia Giampietri; Alexandra Giatromanolaki; Derrick J Gibbings; Lara Gibellini; Spencer B Gibson; Vanessa Ginet; Antonio Giordano; Flaviano Giorgini; Elisa Giovannetti; Stephen E Girardin; Suzana Gispert; Sandy Giuliano; Candece L Gladson; Alvaro Glavic; Martin Gleave; Nelly Godefroy; Robert M Gogal; Kuppan Gokulan; Gustavo H Goldman; Delia Goletti; Michael S Goligorsky; Aldrin V Gomes; Ligia C Gomes; Hernando Gomez; Candelaria Gomez-Manzano; Rubén Gómez-Sánchez; Dawit Ap Gonçalves; Ebru Goncu; Qingqiu Gong; Céline Gongora; Carlos B Gonzalez; Pedro Gonzalez-Alegre; Pilar Gonzalez-Cabo; Rosa Ana González-Polo; Ing Swie Goping; Carlos Gorbea; Nikolai V Gorbunov; Daphne R Goring; Adrienne M Gorman; Sharon M Gorski; Sandro Goruppi; Shino Goto-Yamada; Cecilia Gotor; Roberta A Gottlieb; Illana Gozes; Devrim Gozuacik; Yacine Graba; Martin Graef; Giovanna E Granato; Gary Dean Grant; Steven Grant; Giovanni Luca Gravina; Douglas R Green; Alexander Greenhough; Michael T Greenwood; Benedetto Grimaldi; Frédéric Gros; Charles Grose; Jean-Francois Groulx; Florian Gruber; Paolo Grumati; Tilman Grune; Jun-Lin Guan; Kun-Liang Guan; Barbara Guerra; Carlos Guillen; Kailash Gulshan; Jan Gunst; Chuanyong Guo; Lei Guo; Ming Guo; Wenjie Guo; Xu-Guang Guo; Andrea A Gust; Åsa B Gustafsson; Elaine Gutierrez; Maximiliano G Gutierrez; Ho-Shin Gwak; Albert Haas; James E Haber; Shinji Hadano; Monica Hagedorn; David R Hahn; Andrew J Halayko; Anne Hamacher-Brady; Kozo Hamada; Ahmed Hamai; Andrea Hamann; Maho Hamasaki; Isabelle Hamer; Qutayba Hamid; Ester M Hammond; Feng Han; Weidong Han; James T Handa; John A Hanover; Malene Hansen; Masaru Harada; Ljubica Harhaji-Trajkovic; J Wade Harper; Abdel Halim Harrath; Adrian L Harris; James Harris; Udo Hasler; Peter Hasselblatt; Kazuhisa Hasui; Robert G Hawley; Teresa S Hawley; Congcong He; Cynthia Y He; Fengtian He; Gu He; Rong-Rong He; Xian-Hui He; You-Wen He; Yu-Ying He; Joan K Heath; Marie-Josée Hébert; Robert A Heinzen; Gudmundur Vignir Helgason; Michael Hensel; Elizabeth P Henske; Chengtao Her; Paul K Herman; Agustín Hernández; Carlos Hernandez; Sonia Hernández-Tiedra; Claudio Hetz; P Robin Hiesinger; Katsumi Higaki; Sabine Hilfiker; Bradford G Hill; Joseph A Hill; William D Hill; Keisuke Hino; Daniel Hofius; Paul Hofman; Günter U Höglinger; Jörg Höhfeld; Marina K Holz; Yonggeun Hong; David A Hood; Jeroen Jm Hoozemans; Thorsten Hoppe; Chin Hsu; Chin-Yuan Hsu; Li-Chung Hsu; Dong Hu; Guochang Hu; Hong-Ming Hu; Hongbo Hu; Ming Chang Hu; Yu-Chen Hu; Zhuo-Wei Hu; Fang Hua; Ya Hua; Canhua Huang; Huey-Lan Huang; Kuo-How Huang; Kuo-Yang Huang; Shile Huang; Shiqian Huang; Wei-Pang Huang; Yi-Ran Huang; Yong Huang; Yunfei Huang; Tobias B Huber; Patricia Huebbe; Won-Ki Huh; Juha J Hulmi; Gang Min Hur; James H Hurley; Zvenyslava Husak; Sabah Na Hussain; Salik Hussain; Jung Jin Hwang; Seungmin Hwang; Thomas Is Hwang; Atsuhiro Ichihara; Yuzuru Imai; Carol Imbriano; Megumi Inomata; Takeshi Into; Valentina Iovane; Juan L Iovanna; Renato V Iozzo; Nancy Y Ip; Javier E Irazoqui; Pablo Iribarren; Yoshitaka Isaka; Aleksandra J Isakovic; Harry Ischiropoulos; Jeffrey S Isenberg; Mohammad Ishaq; Hiroyuki Ishida; Isao Ishii; Jane E Ishmael; Ciro Isidoro; Ken-Ichi Isobe; Erika Isono; Shohreh Issazadeh-Navikas; Koji Itahana; Eisuke Itakura; Andrei I Ivanov; Anand Krishnan V Iyer; José M Izquierdo; Yotaro Izumi; Valentina Izzo; Marja Jäättelä; Nadia Jaber; Daniel John Jackson; William T Jackson; Tony George Jacob; Thomas S Jacques; Chinnaswamy Jagannath; Ashish Jain; Nihar Ranjan Jana; Byoung Kuk Jang; Alkesh Jani; Bassam Janji; Paulo Roberto Jannig; Patric J Jansson; Steve Jean; Marina Jendrach; Ju-Hong Jeon; Niels Jessen; Eui-Bae Jeung; Kailiang Jia; Lijun Jia; Hong Jiang; Hongchi Jiang; Liwen Jiang; Teng Jiang; Xiaoyan Jiang; Xuejun Jiang; Xuejun Jiang; Ying Jiang; Yongjun Jiang; Alberto Jiménez; Cheng Jin; Hongchuan Jin; Lei Jin; Meiyan Jin; Shengkan Jin; Umesh Kumar Jinwal; Eun-Kyeong Jo; Terje Johansen; Daniel E Johnson; Gail Vw Johnson; James D Johnson; Eric Jonasch; Chris Jones; Leo Ab Joosten; Joaquin Jordan; Anna-Maria Joseph; Bertrand Joseph; Annie M Joubert; Dianwen Ju; Jingfang Ju; Hsueh-Fen Juan; Katrin Juenemann; Gábor Juhász; Hye Seung Jung; Jae U Jung; Yong-Keun Jung; Heinz Jungbluth; Matthew J Justice; Barry Jutten; Nadeem O Kaakoush; Kai Kaarniranta; Allen Kaasik; Tomohiro Kabuta; Bertrand Kaeffer; Katarina Kågedal; Alon Kahana; Shingo Kajimura; Or Kakhlon; Manjula Kalia; Dhan V Kalvakolanu; Yoshiaki Kamada; Konstantinos Kambas; Vitaliy O Kaminskyy; Harm H Kampinga; Mustapha Kandouz; Chanhee Kang; Rui Kang; Tae-Cheon Kang; Tomotake Kanki; Thirumala-Devi Kanneganti; Haruo Kanno; Anumantha G Kanthasamy; Marc Kantorow; Maria Kaparakis-Liaskos; Orsolya Kapuy; Vassiliki Karantza; Md Razaul Karim; Parimal Karmakar; Arthur Kaser; Susmita Kaushik; Thomas Kawula; A Murat Kaynar; Po-Yuan Ke; Zun-Ji Ke; John H Kehrl; Kate E Keller; Jongsook Kim Kemper; Anne K Kenworthy; Oliver Kepp; Andreas Kern; Santosh Kesari; David Kessel; Robin Ketteler; Isis do Carmo Kettelhut; Bilon Khambu; Muzamil Majid Khan; Vinoth Km Khandelwal; Sangeeta Khare; Juliann G Kiang; Amy A Kiger; Akio Kihara; Arianna L Kim; Cheol Hyeon Kim; Deok Ryong Kim; Do-Hyung Kim; Eung Kweon Kim; Hye Young Kim; Hyung-Ryong Kim; Jae-Sung Kim; Jeong Hun Kim; Jin Cheon Kim; Jin Hyoung Kim; Kwang Woon Kim; Michael D Kim; Moon-Moo Kim; Peter K Kim; Seong Who Kim; Soo-Youl Kim; Yong-Sun Kim; Yonghyun Kim; Adi Kimchi; Alec C Kimmelman; Tomonori Kimura; Jason S King; Karla Kirkegaard; Vladimir Kirkin; Lorrie A Kirshenbaum; Shuji Kishi; Yasuo Kitajima; Katsuhiko Kitamoto; Yasushi Kitaoka; Kaio Kitazato; Rudolf A Kley; Walter T Klimecki; Michael Klinkenberg; Jochen Klucken; Helene Knævelsrud; Erwin Knecht; Laura Knuppertz; Jiunn-Liang Ko; Satoru Kobayashi; Jan C Koch; Christelle Koechlin-Ramonatxo; Ulrich Koenig; Young Ho Koh; Katja Köhler; Sepp D Kohlwein; Masato Koike; Masaaki Komatsu; Eiki Kominami; Dexin Kong; Hee Jeong Kong; Eumorphia G Konstantakou; Benjamin T Kopp; Tamas Korcsmaros; Laura Korhonen; Viktor I Korolchuk; Nadya V Koshkina; Yanjun Kou; Michael I Koukourakis; Constantinos Koumenis; Attila L Kovács; Tibor Kovács; Werner J Kovacs; Daisuke Koya; Claudine Kraft; Dimitri Krainc; Helmut Kramer; Tamara Kravic-Stevovic; Wilhelm Krek; Carole Kretz-Remy; Roswitha Krick; Malathi Krishnamurthy; Janos Kriston-Vizi; Guido Kroemer; Michael C Kruer; Rejko Kruger; Nicholas T Ktistakis; Kazuyuki Kuchitsu; Christian Kuhn; Addanki Pratap Kumar; Anuj Kumar; Ashok Kumar; Deepak Kumar; Dhiraj Kumar; Rakesh Kumar; Sharad Kumar; Mondira Kundu; Hsing-Jien Kung; Atsushi Kuno; Sheng-Han Kuo; Jeff Kuret; Tino Kurz; Terry Kwok; Taeg Kyu Kwon; Yong Tae Kwon; Irene Kyrmizi; Albert R La Spada; Frank Lafont; Tim Lahm; Aparna Lakkaraju; Truong Lam; Trond Lamark; Steve Lancel; Terry H Landowski; Darius J R Lane; Jon D Lane; Cinzia Lanzi; Pierre Lapaquette; Louis R Lapierre; Jocelyn Laporte; Johanna Laukkarinen; Gordon W Laurie; Sergio Lavandero; Lena Lavie; Matthew J LaVoie; Betty Yuen Kwan Law; Helen Ka-Wai Law; Kelsey B Law; Robert Layfield; Pedro A Lazo; Laurent Le Cam; Karine G Le Roch; Hervé Le Stunff; Vijittra Leardkamolkarn; Marc Lecuit; Byung-Hoon Lee; Che-Hsin Lee; Erinna F Lee; Gyun Min Lee; He-Jin Lee; Hsinyu Lee; Jae Keun Lee; Jongdae Lee; Ju-Hyun Lee; Jun Hee Lee; Michael Lee; Myung-Shik Lee; Patty J Lee; Sam W Lee; Seung-Jae Lee; Shiow-Ju Lee; Stella Y Lee; Sug Hyung Lee; Sung Sik Lee; Sung-Joon Lee; Sunhee Lee; Ying-Ray Lee; Yong J Lee; Young H Lee; Christiaan Leeuwenburgh; Sylvain Lefort; Renaud Legouis; Jinzhi Lei; Qun-Ying Lei; David A Leib; Gil Leibowitz; Istvan Lekli; Stéphane D Lemaire; John J Lemasters; Marius K Lemberg; Antoinette Lemoine; Shuilong Leng; Guido Lenz; Paola Lenzi; Lilach O Lerman; Daniele Lettieri Barbato; Julia I-Ju Leu; Hing Y Leung; Beth Levine; Patrick A Lewis; Frank Lezoualc'h; Chi Li; Faqiang Li; Feng-Jun Li; Jun Li; Ke Li; Lian Li; Min Li; Min Li; Qiang Li; Rui Li; Sheng Li; Wei Li; Wei Li; Xiaotao Li; Yumin Li; Jiqin Lian; Chengyu Liang; Qiangrong Liang; Yulin Liao; Joana Liberal; Pawel P Liberski; Pearl Lie; Andrew P Lieberman; Hyunjung Jade Lim; Kah-Leong Lim; Kyu Lim; Raquel T Lima; Chang-Shen Lin; Chiou-Feng Lin; Fang Lin; Fangming Lin; Fu-Cheng Lin; Kui Lin; Kwang-Huei Lin; Pei-Hui Lin; Tianwei Lin; Wan-Wan Lin; Yee-Shin Lin; Yong Lin; Rafael Linden; Dan Lindholm; Lisa M Lindqvist; Paul Lingor; Andreas Linkermann; Lance A Liotta; Marta M Lipinski; Vitor A Lira; Michael P Lisanti; Paloma B Liton; Bo Liu; Chong Liu; Chun-Feng Liu; Fei Liu; Hung-Jen Liu; Jianxun Liu; Jing-Jing Liu; Jing-Lan Liu; Ke Liu; Leyuan Liu; Liang Liu; Quentin Liu; Rong-Yu Liu; Shiming Liu; Shuwen Liu; Wei Liu; Xian-De Liu; Xiangguo Liu; Xiao-Hong Liu; Xinfeng Liu; Xu Liu; Xueqin Liu; Yang Liu; Yule Liu; Zexian Liu; Zhe Liu; Juan P Liuzzi; Gérard Lizard; Mila Ljujic; Irfan J Lodhi; Susan E Logue; Bal L Lokeshwar; Yun Chau Long; Sagar Lonial; Benjamin Loos; Carlos López-Otín; Cristina López-Vicario; Mar Lorente; Philip L Lorenzi; Péter Lõrincz; Marek Los; Michael T Lotze; Penny E Lovat; Binfeng Lu; Bo Lu; Jiahong Lu; Qing Lu; She-Min Lu; Shuyan Lu; Yingying Lu; Frédéric Luciano; Shirley Luckhart; John Milton Lucocq; Paula Ludovico; Aurelia Lugea; Nicholas W Lukacs; Julian J Lum; Anders H Lund; Honglin Luo; Jia Luo; Shouqing Luo; Claudio Luparello; Timothy Lyons; Jianjie Ma; Yi Ma; Yong Ma; Zhenyi Ma; Juliano Machado; Glaucia M Machado-Santelli; Fernando Macian; Gustavo C MacIntosh; Jeffrey P MacKeigan; Kay F Macleod; John D MacMicking; Lee Ann MacMillan-Crow; Frank Madeo; Muniswamy Madesh; Julio Madrigal-Matute; Akiko Maeda; Tatsuya Maeda; Gustavo Maegawa; Emilia Maellaro; Hannelore Maes; Marta Magariños; Kenneth Maiese; Tapas K Maiti; Luigi Maiuri; Maria Chiara Maiuri; Carl G Maki; Roland Malli; Walter Malorni; Alina Maloyan; Fathia Mami-Chouaib; Na Man; Joseph D Mancias; Eva-Maria Mandelkow; Michael A Mandell; Angelo A Manfredi; Serge N Manié; Claudia Manzoni; Kai Mao; Zixu Mao; Zong-Wan Mao; Philippe Marambaud; Anna Maria Marconi; Zvonimir Marelja; Gabriella Marfe; Marta Margeta; Eva Margittai; Muriel Mari; Francesca V Mariani; Concepcio Marin; Sara Marinelli; Guillermo Mariño; Ivanka Markovic; Rebecca Marquez; Alberto M Martelli; Sascha Martens; Katie R Martin; Seamus J Martin; Shaun Martin; Miguel A Martin-Acebes; Paloma Martín-Sanz; Camille Martinand-Mari; Wim Martinet; Jennifer Martinez; Nuria Martinez-Lopez; Ubaldo Martinez-Outschoorn; Moisés Martínez-Velázquez; Marta Martinez-Vicente; Waleska Kerllen Martins; Hirosato Mashima; James A Mastrianni; Giuseppe Matarese; Paola Matarrese; Roberto Mateo; Satoaki Matoba; Naomichi Matsumoto; Takehiko Matsushita; Akira Matsuura; Takeshi Matsuzawa; Mark P Mattson; Soledad Matus; Norma Maugeri; Caroline Mauvezin; Andreas Mayer; Dusica Maysinger; Guillermo D Mazzolini; Mary Kate McBrayer; Kimberly McCall; Craig McCormick; Gerald M McInerney; Skye C McIver; Sharon McKenna; John J McMahon; Iain A McNeish; Fatima Mechta-Grigoriou; Jan Paul Medema; Diego L Medina; Klara Megyeri; Maryam Mehrpour; Jawahar L Mehta; Yide Mei; Ute-Christiane Meier; Alfred J Meijer; Alicia Meléndez; Gerry Melino; Sonia Melino; Edesio Jose Tenorio de Melo; Maria A Mena; Marc D Meneghini; Javier A Menendez; Regina Menezes; Liesu Meng; Ling-Hua Meng; Songshu Meng; Rossella Menghini; A Sue Menko; Rubem Fs Menna-Barreto; Manoj B Menon; Marco A Meraz-Ríos; Giuseppe Merla; Luciano Merlini; Angelica M Merlot; Andreas Meryk; Stefania Meschini; Joel N Meyer; Man-Tian Mi; Chao-Yu Miao; Lucia Micale; Simon Michaeli; Carine Michiels; Anna Rita Migliaccio; Anastasia Susie Mihailidou; Dalibor Mijaljica; Katsuhiko Mikoshiba; Enrico Milan; Leonor Miller-Fleming; Gordon B Mills; Ian G Mills; Georgia Minakaki; Berge A Minassian; Xiu-Fen Ming; Farida Minibayeva; Elena A Minina; Justine D Mintern; Saverio Minucci; Antonio Miranda-Vizuete; Claire H Mitchell; Shigeki Miyamoto; Keisuke Miyazawa; Noboru Mizushima; Katarzyna Mnich; Baharia Mograbi; Simin Mohseni; Luis Ferreira Moita; Marco Molinari; Maurizio Molinari; Andreas Buch Møller; Bertrand Mollereau; Faustino Mollinedo; Marco Mongillo; Martha M Monick; Serena Montagnaro; Craig Montell; Darren J Moore; Michael N Moore; Rodrigo Mora-Rodriguez; Paula I Moreira; Etienne Morel; Maria Beatrice Morelli; Sandra Moreno; Michael J Morgan; Arnaud Moris; Yuji Moriyasu; Janna L Morrison; Lynda A Morrison; Eugenia Morselli; Jorge Moscat; Pope L Moseley; Serge Mostowy; Elisa Motori; Denis Mottet; Jeremy C Mottram; Charbel E-H Moussa; Vassiliki E Mpakou; Hasan Mukhtar; Jean M Mulcahy Levy; Sylviane Muller; Raquel Muñoz-Moreno; Cristina Muñoz-Pinedo; Christian Münz; Maureen E Murphy; James T Murray; Aditya Murthy; Indira U Mysorekar; Ivan R Nabi; Massimo Nabissi; Gustavo A Nader; Yukitoshi Nagahara; Yoshitaka Nagai; Kazuhiro Nagata; Anika Nagelkerke; Péter Nagy; Samisubbu R Naidu; Sreejayan Nair; Hiroyasu Nakano; Hitoshi Nakatogawa; Meera Nanjundan; Gennaro Napolitano; Naweed I Naqvi; Roberta Nardacci; Derek P Narendra; Masashi Narita; Anna Chiara Nascimbeni; Ramesh Natarajan; Luiz C Navegantes; Steffan T Nawrocki; Taras Y Nazarko; Volodymyr Y Nazarko; Thomas Neill; Luca M Neri; Mihai G Netea; Romana T Netea-Maier; Bruno M Neves; Paul A Ney; Ioannis P Nezis; Hang Tt Nguyen; Huu Phuc Nguyen; Anne-Sophie Nicot; Hilde Nilsen; Per Nilsson; Mikio Nishimura; Ichizo Nishino; Mireia Niso-Santano; Hua Niu; Ralph A Nixon; Vincent Co Njar; Takeshi Noda; Angelika A Noegel; Elsie Magdalena Nolte; Erik Norberg; Koenraad K Norga; Sakineh Kazemi Noureini; Shoji Notomi; Lucia Notterpek; Karin Nowikovsky; Nobuyuki Nukina; Thorsten Nürnberger; Valerie B O'Donnell; Tracey O'Donovan; Peter J O'Dwyer; Ina Oehme; Clara L Oeste; Michinaga Ogawa; Besim Ogretmen; Yuji Ogura; Young J Oh; Masaki Ohmuraya; Takayuki Ohshima; Rani Ojha; Koji Okamoto; Toshiro Okazaki; F Javier Oliver; Karin Ollinger; Stefan Olsson; Daniel P Orban; Paulina Ordonez; Idil Orhon; Laszlo Orosz; Eyleen J O'Rourke; Helena Orozco; Angel L Ortega; Elena Ortona; Laura D Osellame; Junko Oshima; Shigeru Oshima; Heinz D Osiewacz; Takanobu Otomo; Kinya Otsu; Jing-Hsiung James Ou; Tiago F Outeiro; Dong-Yun Ouyang; Hongjiao Ouyang; Michael Overholtzer; Michelle A Ozbun; P Hande Ozdinler; Bulent Ozpolat; Consiglia Pacelli; Paolo Paganetti; Guylène Page; Gilles Pages; Ugo Pagnini; Beata Pajak; Stephen C Pak; Karolina Pakos-Zebrucka; Nazzy Pakpour; Zdena Palková; Francesca Palladino; Kathrin Pallauf; Nicolas Pallet; Marta Palmieri; Søren R Paludan; Camilla Palumbo; Silvia Palumbo; Olatz Pampliega; Hongming Pan; Wei Pan; Theocharis Panaretakis; Aseem Pandey; Areti Pantazopoulou; Zuzana Papackova; Daniela L Papademetrio; Issidora Papassideri; Alessio Papini; Nirmala Parajuli; Julian Pardo; Vrajesh V Parekh; Giancarlo Parenti; Jong-In Park; Junsoo Park; Ohkmae K Park; Roy Parker; Rosanna Parlato; Jan B Parys; Katherine R Parzych; Jean-Max Pasquet; Benoit Pasquier; Kishore Bs Pasumarthi; Daniel Patschan; Cam Patterson; Sophie Pattingre; Scott Pattison; Arnim Pause; Hermann Pavenstädt; Flaminia Pavone; Zully Pedrozo; Fernando J Peña; Miguel A Peñalva; Mario Pende; Jianxin Peng; Fabio Penna; Josef M Penninger; Anna Pensalfini; Salvatore Pepe; Gustavo Js Pereira; Paulo C Pereira; Verónica Pérez-de la Cruz; María Esther Pérez-Pérez; Diego Pérez-Rodríguez; Dolores Pérez-Sala; Celine Perier; Andras Perl; David H Perlmutter; Ida Perrotta; Shazib Pervaiz; Maija Pesonen; Jeffrey E Pessin; Godefridus J Peters; Morten Petersen; Irina Petrache; Basil J Petrof; Goran Petrovski; James M Phang; Mauro Piacentini; Marina Pierdominici; Philippe Pierre; Valérie Pierrefite-Carle; Federico Pietrocola; Felipe X Pimentel-Muiños; Mario Pinar; Benjamin Pineda; Ronit Pinkas-Kramarski; Marcello Pinti; Paolo Pinton; Bilal Piperdi; James M Piret; Leonidas C Platanias; Harald W Platta; Edward D Plowey; Stefanie Pöggeler; Marc Poirot; Peter Polčic; Angelo Poletti; Audrey H Poon; Hana Popelka; Blagovesta Popova; Izabela Poprawa; Shibu M Poulose; Joanna Poulton; Scott K Powers; Ted Powers; Mercedes Pozuelo-Rubio; Krisna Prak; Reinhild Prange; Mark Prescott; Muriel Priault; Sharon Prince; Richard L Proia; Tassula Proikas-Cezanne; Holger Prokisch; Vasilis J Promponas; Karin Przyklenk; Rosa Puertollano; Subbiah Pugazhenthi; Luigi Puglielli; Aurora Pujol; Julien Puyal; Dohun Pyeon; Xin Qi; Wen-Bin Qian; Zheng-Hong Qin; Yu Qiu; Ziwei Qu; Joe Quadrilatero; Frederick Quinn; Nina Raben; Hannah Rabinowich; Flavia Radogna; Michael J Ragusa; Mohamed Rahmani; Komal Raina; Sasanka Ramanadham; Rajagopal Ramesh; Abdelhaq Rami; Sarron Randall-Demllo; Felix Randow; Hai Rao; V Ashutosh Rao; Blake B Rasmussen; Tobias M Rasse; Edward A Ratovitski; Pierre-Emmanuel Rautou; Swapan K Ray; Babak Razani; Bruce H Reed; Fulvio Reggiori; Markus Rehm; Andreas S Reichert; Theo Rein; David J Reiner; Eric Reits; Jun Ren; Xingcong Ren; Maurizio Renna; Jane Eb Reusch; Jose L Revuelta; Leticia Reyes; Alireza R Rezaie; Robert I Richards; Des R Richardson; Clémence Richetta; Michael A Riehle; Bertrand H Rihn; Yasuko Rikihisa; Brigit E Riley; Gerald Rimbach; Maria Rita Rippo; Konstantinos Ritis; Federica Rizzi; Elizete Rizzo; Peter J Roach; Jeffrey Robbins; Michel Roberge; Gabriela Roca; Maria Carmela Roccheri; Sonia Rocha; Cecilia Mp Rodrigues; Clara I Rodríguez; Santiago Rodriguez de Cordoba; Natalia Rodriguez-Muela; Jeroen Roelofs; Vladimir V Rogov; Troy T Rohn; Bärbel Rohrer; Davide Romanelli; Luigina Romani; Patricia Silvia Romano; M Isabel G Roncero; Jose Luis Rosa; Alicia Rosello; Kirill V Rosen; Philip Rosenstiel; Magdalena Rost-Roszkowska; Kevin A Roth; Gael Roué; Mustapha Rouis; Kasper M Rouschop; Daniel T Ruan; Diego Ruano; David C Rubinsztein; Edmund B Rucker; Assaf Rudich; Emil Rudolf; Ruediger Rudolf; Markus A Ruegg; Carmen Ruiz-Roldan; Avnika Ashok Ruparelia; Paola Rusmini; David W Russ; Gian Luigi Russo; Giuseppe Russo; Rossella Russo; Tor Erik Rusten; Victoria Ryabovol; Kevin M Ryan; Stefan W Ryter; David M Sabatini; Michael Sacher; Carsten Sachse; Michael N Sack; Junichi Sadoshima; Paul Saftig; Ronit Sagi-Eisenberg; Sumit Sahni; Pothana Saikumar; Tsunenori Saito; Tatsuya Saitoh; Koichi Sakakura; Machiko Sakoh-Nakatogawa; Yasuhito Sakuraba; María Salazar-Roa; Paolo Salomoni; Ashok K Saluja; Paul M Salvaterra; Rosa Salvioli; Afshin Samali; Anthony Mj Sanchez; José A Sánchez-Alcázar; Ricardo Sanchez-Prieto; Marco Sandri; Miguel A Sanjuan; Stefano Santaguida; Laura Santambrogio; Giorgio Santoni; Claudia Nunes Dos Santos; Shweta Saran; Marco Sardiello; Graeme Sargent; Pallabi Sarkar; Sovan Sarkar; Maria Rosa Sarrias; Minnie M Sarwal; Chihiro Sasakawa; Motoko Sasaki; Miklos Sass; Ken Sato; Miyuki Sato; Joseph Satriano; Niramol Savaraj; Svetlana Saveljeva; Liliana Schaefer; Ulrich E Schaible; Michael Scharl; Hermann M Schatzl; Randy Schekman; Wiep Scheper; Alfonso Schiavi; Hyman M Schipper; Hana Schmeisser; Jens Schmidt; Ingo Schmitz; Bianca E Schneider; E Marion Schneider; Jaime L Schneider; Eric A Schon; Miriam J Schönenberger; Axel H Schönthal; Daniel F Schorderet; Bernd Schröder; Sebastian Schuck; Ryan J Schulze; Melanie Schwarten; Thomas L Schwarz; Sebastiano Sciarretta; Kathleen Scotto; A Ivana Scovassi; Robert A Screaton; Mark Screen; Hugo Seca; Simon Sedej; Laura Segatori; Nava Segev; Per O Seglen; Jose M Seguí-Simarro; Juan Segura-Aguilar; Ekihiro Seki; Christian Sell; Iban Seiliez; Clay F Semenkovich; Gregg L Semenza; Utpal Sen; Andreas L Serra; Ana Serrano-Puebla; Hiromi Sesaki; Takao Setoguchi; Carmine Settembre; John J Shacka; Ayesha N Shajahan-Haq; Irving M Shapiro; Shweta Sharma; Hua She; C-K James Shen; Chiung-Chyi Shen; Han-Ming Shen; Sanbing Shen; Weili Shen; Rui Sheng; Xianyong Sheng; Zu-Hang Sheng; Trevor G Shepherd; Junyan Shi; Qiang Shi; Qinghua Shi; Yuguang Shi; Shusaku Shibutani; Kenichi Shibuya; Yoshihiro Shidoji; Jeng-Jer Shieh; Chwen-Ming Shih; Yohta Shimada; Shigeomi Shimizu; Dong Wook Shin; Mari L Shinohara; Michiko Shintani; Takahiro Shintani; Tetsuo Shioi; Ken Shirabe; Ronit Shiri-Sverdlov; Orian Shirihai; Gordon C Shore; Chih-Wen Shu; Deepak Shukla; Andriy A Sibirny; Valentina Sica; Christina J Sigurdson; Einar M Sigurdsson; Puran Singh Sijwali; Beata Sikorska; Wilian A Silveira; Sandrine Silvente-Poirot; Gary A Silverman; Jan Simak; Thomas Simmet; Anna Katharina Simon; Hans-Uwe Simon; Cristiano Simone; Matias Simons; Anne Simonsen; Rajat Singh; Shivendra V Singh; Shrawan K Singh; Debasish Sinha; Sangita Sinha; Frank A Sinicrope; Agnieszka Sirko; Kapil Sirohi; Balindiwe Jn Sishi; Annie Sittler; Parco M Siu; Efthimios Sivridis; Anna Skwarska; Ruth Slack; Iva Slaninová; Nikolai Slavov; Soraya S Smaili; Keiran Sm Smalley; Duncan R Smith; Stefaan J Soenen; Scott A Soleimanpour; Anita Solhaug; Kumaravel Somasundaram; Jin H Son; Avinash Sonawane; Chunjuan Song; Fuyong Song; Hyun Kyu Song; Ju-Xian Song; Wei Song; Kai Y Soo; Anil K Sood; Tuck Wah Soong; Virawudh Soontornniyomkij; Maurizio Sorice; Federica Sotgia; David R Soto-Pantoja; Areechun Sotthibundhu; Maria João Sousa; Herman P Spaink; Paul N Span; Anne Spang; Janet D Sparks; Peter G Speck; Stephen A Spector; Claudia D Spies; Wolfdieter Springer; Daret St Clair; Alessandra Stacchiotti; Bart Staels; Michael T Stang; Daniel T Starczynowski; Petro Starokadomskyy; Clemens Steegborn; John W Steele; Leonidas Stefanis; Joan Steffan; Christine M Stellrecht; Harald Stenmark; Tomasz M Stepkowski; Stęphan T Stern; Craig Stevens; Brent R Stockwell; Veronika Stoka; Zuzana Storchova; Björn Stork; Vassilis Stratoulias; Dimitrios J Stravopodis; Pavel Strnad; Anne Marie Strohecker; Anna-Lena Ström; Per Stromhaug; Jiri Stulik; Yu-Xiong Su; Zhaoliang Su; Carlos S Subauste; Srinivasa Subramaniam; Carolyn M Sue; Sang Won Suh; Xinbing Sui; Supawadee Sukseree; David Sulzer; Fang-Lin Sun; Jiaren Sun; Jun Sun; Shi-Yong Sun; Yang Sun; Yi Sun; Yingjie Sun; Vinod Sundaramoorthy; Joseph Sung; Hidekazu Suzuki; Kuninori Suzuki; Naoki Suzuki; Tadashi Suzuki; Yuichiro J Suzuki; Michele S Swanson; Charles Swanton; Karl Swärd; Ghanshyam Swarup; Sean T Sweeney; Paul W Sylvester; Zsuzsanna Szatmari; Eva Szegezdi; Peter W Szlosarek; Heinrich Taegtmeyer; Marco Tafani; Emmanuel Taillebourg; Stephen Wg Tait; Krisztina Takacs-Vellai; Yoshinori Takahashi; Szabolcs Takáts; Genzou Takemura; Nagio Takigawa; Nicholas J Talbot; Elena Tamagno; Jerome Tamburini; Cai-Ping Tan; Lan Tan; Mei Lan Tan; Ming Tan; Yee-Joo Tan; Keiji Tanaka; Masaki Tanaka; Daolin Tang; Dingzhong Tang; Guomei Tang; Isei Tanida; Kunikazu Tanji; Bakhos A Tannous; Jose A Tapia; Inmaculada Tasset-Cuevas; Marc Tatar; Iman Tavassoly; Nektarios Tavernarakis; Allen Taylor; Graham S Taylor; Gregory A Taylor; J Paul Taylor; Mark J Taylor; Elena V Tchetina; Andrew R Tee; Fatima Teixeira-Clerc; Sucheta Telang; Tewin Tencomnao; Ba-Bie Teng; Ru-Jeng Teng; Faraj Terro; Gianluca Tettamanti; Arianne L Theiss; Anne E Theron; Kelly Jean Thomas; Marcos P Thomé; Paul G Thomes; Andrew Thorburn; Jeremy Thorner; Thomas Thum; Michael Thumm; Teresa Lm Thurston; Ling Tian; Andreas Till; Jenny Pan-Yun Ting; Vladimir I Titorenko; Lilach Toker; Stefano Toldo; Sharon A Tooze; Ivan Topisirovic; Maria Lyngaas Torgersen; Liliana Torosantucci; Alicia Torriglia; Maria Rosaria Torrisi; Cathy Tournier; Roberto Towns; Vladimir Trajkovic; Leonardo H Travassos; Gemma Triola; Durga Nand Tripathi; Daniela Trisciuoglio; Rodrigo Troncoso; Ioannis P Trougakos; Anita C Truttmann; Kuen-Jer Tsai; Mario P Tschan; Yi-Hsin Tseng; Takayuki Tsukuba; Allan Tsung; Andrey S Tsvetkov; Shuiping Tu; Hsing-Yu Tuan; Marco Tucci; David A Tumbarello; Boris Turk; Vito Turk; Robin Fb Turner; Anders A Tveita; Suresh C Tyagi; Makoto Ubukata; Yasuo Uchiyama; Andrej Udelnow; Takashi Ueno; Midori Umekawa; Rika Umemiya-Shirafuji; Benjamin R Underwood; Christian Ungermann; Rodrigo P Ureshino; Ryo Ushioda; Vladimir N Uversky; Néstor L Uzcátegui; Thomas Vaccari; Maria I Vaccaro; Libuše Váchová; Helin Vakifahmetoglu-Norberg; Rut Valdor; Enza Maria Valente; Francois Vallette; Angela M Valverde; Greet Van den Berghe; Ludo Van Den Bosch; Gijs R van den Brink; F Gisou van der Goot; Ida J van der Klei; Luc Jw van der Laan; Wouter G van Doorn; Marjolein van Egmond; Kenneth L van Golen; Luc Van Kaer; Menno van Lookeren Campagne; Peter Vandenabeele; Wim Vandenberghe; Ilse Vanhorebeek; Isabel Varela-Nieto; M Helena Vasconcelos; Radovan Vasko; Demetrios G Vavvas; Ignacio Vega-Naredo; Guillermo Velasco; Athanassios D Velentzas; Panagiotis D Velentzas; Tibor Vellai; Edo Vellenga; Mikkel Holm Vendelbo; Kartik Venkatachalam; Natascia Ventura; Salvador Ventura; Patrícia St Veras; Mireille Verdier; Beata G Vertessy; Andrea Viale; Michel Vidal; Helena L A Vieira; Richard D Vierstra; Nadarajah Vigneswaran; Neeraj Vij; Miquel Vila; Margarita Villar; Victor H Villar; Joan Villarroya; Cécile Vindis; Giampietro Viola; Maria Teresa Viscomi; Giovanni Vitale; Dan T Vogl; Olga V Voitsekhovskaja; Clarissa von Haefen; Karin von Schwarzenberg; Daniel E Voth; Valérie Vouret-Craviari; Kristina Vuori; Jatin M Vyas; Christian Waeber; Cheryl Lyn Walker; Mark J Walker; Jochen Walter; Lei Wan; Xiangbo Wan; Bo Wang; Caihong Wang; Chao-Yung Wang; Chengshu Wang; Chenran Wang; Chuangui Wang; Dong Wang; Fen Wang; Fuxin Wang; Guanghui Wang; Hai-Jie Wang; Haichao Wang; Hong-Gang Wang; Hongmin Wang; Horng-Dar Wang; Jing Wang; Junjun Wang; Mei Wang; Mei-Qing Wang; Pei-Yu Wang; Peng Wang; Richard C Wang; Shuo Wang; Ting-Fang Wang; Xian Wang; Xiao-Jia Wang; Xiao-Wei Wang; Xin Wang; Xuejun Wang; Yan Wang; Yanming Wang; Ying Wang; Ying-Jan Wang; Yipeng Wang; Yu Wang; Yu Tian Wang; Yuqing Wang; Zhi-Nong Wang; Pablo Wappner; Carl Ward; Diane McVey Ward; Gary Warnes; Hirotaka Watada; Yoshihisa Watanabe; Kei Watase; Timothy E Weaver; Colin D Weekes; Jiwu Wei; Thomas Weide; Conrad C Weihl; Günther Weindl; Simone Nardin Weis; Longping Wen; Xin Wen; Yunfei Wen; Benedikt Westermann; Cornelia M Weyand; Anthony R White; Eileen White; J Lindsay Whitton; Alexander J Whitworth; Joëlle Wiels; Franziska Wild; Manon E Wildenberg; Tom Wileman; Deepti Srinivas Wilkinson; Simon Wilkinson; Dieter Willbold; Chris Williams; Katherine Williams; Peter R Williamson; Konstanze F Winklhofer; Steven S Witkin; Stephanie E Wohlgemuth; Thomas Wollert; Ernst J Wolvetang; Esther Wong; G William Wong; Richard W Wong; Vincent Kam Wai Wong; Elizabeth A Woodcock; Karen L Wright; Chunlai Wu; Defeng Wu; Gen Sheng Wu; Jian Wu; Junfang Wu; Mian Wu; Min Wu; Shengzhou Wu; William Kk Wu; Yaohua Wu; Zhenlong Wu; Cristina Pr Xavier; Ramnik J Xavier; Gui-Xian Xia; Tian Xia; Weiliang Xia; Yong Xia; Hengyi Xiao; Jian Xiao; Shi Xiao; Wuhan Xiao; Chuan-Ming Xie; Zhiping Xie; Zhonglin Xie; Maria Xilouri; Yuyan Xiong; Chuanshan Xu; Congfeng Xu; Feng Xu; Haoxing Xu; Hongwei Xu; Jian Xu; Jianzhen Xu; Jinxian Xu; Liang Xu; Xiaolei Xu; Yangqing Xu; Ye Xu; Zhi-Xiang Xu; Ziheng Xu; Yu Xue; Takahiro Yamada; Ai Yamamoto; Koji Yamanaka; Shunhei Yamashina; Shigeko Yamashiro; Bing Yan; Bo Yan; Xianghua Yan; Zhen Yan; Yasuo Yanagi; Dun-Sheng Yang; Jin-Ming Yang; Liu Yang; Minghua Yang; Pei-Ming Yang; Peixin Yang; Qian Yang; Wannian Yang; Wei Yuan Yang; Xuesong Yang; Yi Yang; Ying Yang; Zhifen Yang; Zhihong Yang; Meng-Chao Yao; Pamela J Yao; Xiaofeng Yao; Zhenyu Yao; Zhiyuan Yao; Linda S Yasui; Mingxiang Ye; Barry Yedvobnick; Behzad Yeganeh; Elizabeth S Yeh; Patricia L Yeyati; Fan Yi; Long Yi; Xiao-Ming Yin; Calvin K Yip; Yeong-Min Yoo; Young Hyun Yoo; Seung-Yong Yoon; Ken-Ichi Yoshida; Tamotsu Yoshimori; Ken H Young; Huixin Yu; Jane J Yu; Jin-Tai Yu; Jun Yu; Li Yu; W Haung Yu; Xiao-Fang Yu; Zhengping Yu; Junying Yuan; Zhi-Min Yuan; Beatrice Yjt Yue; Jianbo Yue; Zhenyu Yue; David N Zacks; Eldad Zacksenhaus; Nadia Zaffaroni; Tania Zaglia; Zahra Zakeri; Vincent Zecchini; Jinsheng Zeng; Min Zeng; Qi Zeng; Antonis S Zervos; Donna D Zhang; Fan Zhang; Guo Zhang; Guo-Chang Zhang; Hao Zhang; Hong Zhang; Hong Zhang; Hongbing Zhang; Jian Zhang; Jian Zhang; Jiangwei Zhang; Jianhua Zhang; Jing-Pu Zhang; Li Zhang; Lin Zhang; Lin Zhang; Long Zhang; Ming-Yong Zhang; Xiangnan Zhang; Xu Dong Zhang; Yan Zhang; Yang Zhang; Yanjin Zhang; Yingmei Zhang; Yunjiao Zhang; Mei Zhao; Wei-Li Zhao; Xiaonan Zhao; Yan G Zhao; Ying Zhao; Yongchao Zhao; Yu-Xia Zhao; Zhendong Zhao; Zhizhuang J Zhao; Dexian Zheng; Xi-Long Zheng; Xiaoxiang Zheng; Boris Zhivotovsky; Qing Zhong; Guang-Zhou Zhou; Guofei Zhou; Huiping Zhou; Shu-Feng Zhou; Xu-Jie Zhou; Hongxin Zhu; Hua Zhu; Wei-Guo Zhu; Wenhua Zhu; Xiao-Feng Zhu; Yuhua Zhu; Shi-Mei Zhuang; Xiaohong Zhuang; Elio Ziparo; Christos E Zois; Teresa Zoladek; Wei-Xing Zong; Antonio Zorzano; Susu M Zughaier Journal: Autophagy Date: 2016 Impact factor: 16.016
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8