HRES-1/Rab4 Promotes the Formation of LC3⁺ Autophagosomes and the Accumulation of Mitochondria during Autophagy

<div><p>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.</p></div

Detection of LC3 fused to FP650 (FP650-LC3) and HRES-1/Rab4 isoforms, including wild-type HRES-1/Rab4, C-terminally truncated HRES-1/Rab4^1–121, dominant-negative/GTP binding-deficient HRES-1/Rab4^S27N, constitutively active/GTPase-deficient HRES-1/Rab4^Q72L and phosphorylation-resistant form HRES-1/Rab4^S204Q, tagged with eGFP.

<p>A, Functional domains of proteins encoded by the HRES-1/Rab4 cDNA at 1q42 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084392#pone.0084392-Nagy1" target="_blank">[12]</a> (Genbank accession number: AY585832). Amino acid changes previously shown to affect Rab4 activity are typed in red. HRES-1/Rab4^S27N prevents GTP binding and acts as a dominant negative mutation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084392#pone.0084392-Lazzarino1" target="_blank">[20]</a>. HRES-1/Rab4^Q72L is constitutively active due to elimination of GTPase activity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084392#pone.0084392-Cormont1" target="_blank">[21]</a>. HRES-1/Rab4^S204Q will not be phosphorylated by p34cdc2 kinase in mitotic cells and remains endosome-associated throughout the cell cycle <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084392#pone.0084392-vanderSluis1" target="_blank">[22]</a>. B, Amino acid sequence of HRES-1/Rab4^1–121, 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/Rab4^1–121. E, Western blot analysis of HRES-1/Rab4^1–121 expression in HeLa cells transfected with pAAV-HRES-1/Rab4^1–121-IRES-GFP vector (clone 8466), pAAV-hrGFP-HRES-1/Rab4^1–121 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/Rab4^1–121 and HRES-1/Rab4^1–121-GFP fusion protein were detected with rabbit antibody G1432. HRES-1/Rab4 was detected with rabbit antibody 13407 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084392#pone.0084392-Nagy1" target="_blank">[12]</a>.</p

MAGERI: Computational pipeline for molecular-barcoded targeted resequencing

<div><p>Unique molecular identifiers (UMIs) show outstanding performance in targeted high-throughput resequencing, being the most promising approach for the accurate identification of rare variants in complex DNA samples. This approach has application in multiple areas, including cancer diagnostics, thus demanding dedicated software and algorithms. Here we introduce MAGERI, a computational pipeline that efficiently handles all caveats of UMI-based analysis to obtain high-fidelity mutation profiles and call ultra-rare variants. Using an extensive set of benchmark datasets including gold-standard biological samples with known variant frequencies, cell-free DNA from tumor patient blood samples and publicly available UMI-encoded datasets we demonstrate that our method is both robust and efficient in calling rare variants. The versatility of our software is supported by accurate results obtained for both tumor DNA and viral RNA samples in datasets prepared using three different UMI-based protocols.</p></div

Quantitative analysis of colocalization between LC3⁺ autophagosomes and mitochondria in HeLa cells transfected with eGFP-tagged HRES-1/Rab4 isoforms and FP650-LC3.

<p>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.</p

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).

<p>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/Rab4^1–121. D, HeLa cells were transfected with FP650-LC3 and eGFP-tagged HRES-1/Rab4^S27N. E, HeLa cells were transfected with FP650-LC3 and eGFP-tagged HRES-1/Rab4^Q72L. F, HeLa cells were transfected with FP650-LC3 and eGFP-tagged HRES-1/Rab4^S204Q. The areas showing the formation of mitochondrial tubular networks are delineated by white dotted rectangles in panels A and E.</p

Quantitative analyses of the effect by HRES-1/Rab4 on the accumulation of LC3⁺ autophagosomes (panel A) and MTDR-stained mitochondria (panel B).

<p>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.</p

Schematic diagram of the impact by HRES-1/Rab4 on autophagy.

<p>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/Rab4^Q72L upon starvation.</p

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/Rab4^1–121), dominant-negative form (Rab4^S27N), constitutively active (HRES-1/Rab4^Q72L) and phosphorylation-resistant form (HRES-1/Rab4^S204Q) tagged with eGFP (emitting green fluorescence).

<p>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.</p

Cumulative analysis of colocalization of HRES-1/Rab4 and LC3 induced by starvation and rapamycin.

<p>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/Rab4^S27N which blocked colocalization under starvation and promoted colocalization during mTOR blockade. In contrast to HRES-1/Rab4^S27N, HRES-1/Rab4^Q72L 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.</p