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Spatiotemporal regulation of autophagy during Caenorhabditis elegans aging

  1. Jessica T Chang
  2. Caroline Kumsta
  3. Andrew B Hellman
  4. Linnea M Adams
  5. Malene Hansen Is a corresponding author
  1. Sanford Burnham Prebys Medical Discovery Institute, United States
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Cite as: eLife 2017;6:e18459 doi: 10.7554/eLife.18459

Abstract

Autophagy has been linked to longevity in many species, but the underlying mechanisms are unclear. Using a GFP-tagged and a new tandem-tagged Atg8/LGG-1 reporter, we quantified autophagic vesicles and performed autophagic flux assays in multiple tissues of wild-type Caenorhabditis elegans and long-lived daf-2/insulin/IGF-1 and glp-1/Notch mutants throughout adulthood. Our data are consistent with an age-related decline in autophagic activity in the intestine, body-wall muscle, pharynx, and neurons of wild-type animals. In contrast, daf-2 and glp-1 mutants displayed unique age- and tissue-specific changes in autophagic activity, indicating that the two longevity paradigms have distinct effects on autophagy during aging. Although autophagy appeared active in the intestine of both long-lived mutants, inhibition of intestinal autophagy significantly abrogated lifespan extension only in glp-1 mutants. Collectively, our data suggest that autophagic activity normally decreases with age in C. elegans, whereas daf-2 and glp-1 long-lived mutants regulate autophagy in distinct spatiotemporal-specific manners to extend lifespan.

https://doi.org/10.7554/eLife.18459.001

Introduction

Macroautophagy (hereafter referred to as autophagy) is a multistep cellular recycling process in which cytosolic components are encapsulated in membrane vesicles and ultimately degraded in the lysosome. Multiple autophagy (ATG) genes are involved in the initial formation of a crescent-shaped double-membrane vesicle called the phagophore or isolation membrane (IM), which elongates and engulfs cytosolic cargo, forming the autophagosome (AP). APs then fuse with lysosomes to form autolysosomes (AL), where degradation of cargo takes place (Mizushima, 2007).

As interest in this pathway and its pathophysiological roles has increased, it has become clear that measurement of autophagic vesicle levels at steady state, without monitoring the overall pathway flux, can lead to controversial results. Autophagy is commonly monitored by enumerating APs under steady-state conditions, also referred to as the AP pool size (Loos et al., 2014), using a GFP-tagged Atg8 marker. During AP formation, Atg8 is cleaved, conjugated to phosphatidylethanolamine, and inserted into the vesicle membrane, thus serving as a marker for IMs and APs (Klionsky et al., 2016). However, GFP-Atg8 only reports on the size of the IM and AP pools, not the rate by which IMs and APs are formed, or converted to ALs. For example, an increase in the number of GFP-Atg8 punctae could result from increased formation of APs or blockade of the downstream steps. A tandem-tagged mCherry-GFP-Atg8 reporter, which monitors both IMs/APs (yellow [green/red] punctae) and ALs (red punctae due to GFP fluorescence quenching in the acidic autolysosome environment) can help distinguish between these possibilities (Kimura et al., 2007). We acknowledge that APs can also fuse with acidic endosomes and give rise to amphisomes, which are similarly highlighted as red punctae, and which ultimately fuse with lysosomes to form ALs (Gordon and Seglen, 1988). Specifically, when used in combination with chemical inhibitors of autophagy, such as Bafilomycin A (BafA), tandem-tagged reporters can assess autophagic activity in so called autophagic flux assays (Klionsky et al., 2016). Although tandem-tagged Atg8 markers have been used extensively to monitor autophagy in mammalian cells (Klionsky et al., 2016), as well as in adult Drosophila melanogaster and in Caenorhabditis elegans embryos (Mauvezin et al., 2014; Manil-Ségalen et al., 2014), this reporter has not previously been used in adult C. elegans, and no comprehensive spatial or temporal analyses of autophagic activity have been reported in any animal thus far.

Autophagy plays important roles in numerous cellular processes and has been linked to normal physiological aging as well as the development of age-related diseases (Levine and Kroemer, 2008). Furthermore, accumulating evidence in long-lived species demonstrates that autophagy genes are required for extended longevity. In particular, autophagy is essential for lifespan extension by inhibition of the nutrient sensor mTOR in Saccharomyces cerevisiae, C. elegans, and D. melanogaster (Gelino and Hansen, 2012; Lapierre et al., 2015). In C. elegans, autophagy genes are also required for the long lifespan induced by other conserved longevity paradigms, such as reduced insulin/IGF-1 signaling, germline ablation, and reduced mitochondrial respiration, and all these longevity mutants have increased transcript levels of several autophagy genes (Lapierre et al., 2015). Conversely, neuronal overexpression of Atg1 or Atg8 in D. melanogaster (Bai et al., 2013; Simonsen et al., 2008; Ulgherait et al., 2014) or ubiquitous overexpression of ATG5 in mice (Pyo et al., 2013) is sufficient to extend lifespan. Consistent with these findings, all long-lived C. elegans mutants examined to date display elevated numbers of GFP::Atg8-positive punctae in their hypodermis during larval development and/or in their intestine during early adulthood (Hansen, 2016). Long-lived D. melanogaster overexpressing Atg1 in neurons also have more GFP::Atg8-positive punctae in neurons and in the intestine than do control animals (Ulgherait et al., 2014). Thus, it has been proposed, but not proven, that autophagic activity is elevated in long-lived animals and that this increase is critical for lifespan extension.

Considerable evidence suggests that autophagic activity changes with age, but it is unclear whether increases or decreases in autophagy are causally related to age-associated impairment of cellular function and organismal health. Aging rats have been shown to have reduced lysosomal proteolysis in liver lysates, and reduced autophagic activity in the liver as assessed by electron microscopy and flux assays (Del Roso et al., 2003, Donati et al., 2001). Moreover, aging is accompanied by a decrease in several autophagy gene transcripts in both D. melanogaster (Demontis and Perrimon, 2010; Ling and Salvaterra, 2009; Simonsen et al., 2008) and in rodent tissues (Cuervo and Dice, 2000; Vittorini et al., 1999; Ye et al., 2011; Kaushik et al., 2012), as well as with a decrease in lysosomal protease activity in C. elegans (Sarkis et al., 1988). In contrast, a recent study proposed that autophagic activity is increased in multiple tissues of C. elegans with age, as measured by a fluorescently labeled Atg8 reporter protein containing a lysosomal hydrolase-cleavable linker (Chapin et al., 2015). However, this steady-state assay did not conclusively evaluate autophagic activity, and the precise step in the autophagic process marked by the reporter was not identified. Taken together, these observations demonstrate a need for additional assays to evaluate and quantify tissue- and age-specific changes in autophagic activity.

To better understand how aging affects autophagy in C. elegans, we employed a GFP-tagged and a novel tandem-tagged (mCherry/GFP) form of LGG-1 (a C. elegans ortholog of Atg8) to investigate the spatial and temporal autophagy landscape in wild-type (WT) and long-lived daf-2 insulin/IGF-1 receptor mutants and germline-less glp-1 animals. To estimate IM/AP (hereafter referred to as AP) and AL pool sizes, we quantified these LGG-1/Atg8 reporters in the intestine, body-wall muscle, pharynx, and nerve-ring neurons between Day 1 and Day 10 of adulthood. We also performed flux assays (i.e. carried out analyses in BafA-injected animals) to assess autophagic activity in each tissue. Our data indicate that WT animals displayed an age-dependent increase in AP and AL numbers in all tissues, which flux assays suggest reflects a decrease in autophagic activity over time. In contrast, daf-2 and glp-1 mutants showed unique age- and tissue-specific differences consistent with select tissues displaying elevated, and in one case possibly reduced autophagic activity compared with WT animals. Moreover, tissue-specific inhibition of autophagy in the intestine significantly reduced the long lifespan of glp-1 mutants but not of daf-2 mutants, suggesting that autophagy in the intestine of daf-2 mutants may be dispensable for lifespan extension. Our study represents the first efforts to comprehensively analyze autophagic activity in a spatiotemporal manner of a live organism and provides evidence for an age-dependent decline in autophagic activity, and for a complex spatiotemporal regulation of autophagy in long-lived daf-2 and glp-1 mutants.

Results

Spatiotemporal analysis of the GFP::LGG-1 reporter in adult C. elegans

To perform an in-depth analysis of autophagy in adult C. elegans, we used previously published GFP::LGG-1 reporters (Meléndez et al., 2003; Kang et al., 2007; Gelino et al., 2016) to assess the isolation membrane (IM) and autophagosome (AP) pool size (for simplicity referred to as AP pool size, Figure 1A) in multiple somatic tissues of adult WT animals during aging, including the intestine (Figure 1B), body-wall muscle (hereafter referred to as muscle; Figure 1C), pharynx (the foregut of the animal; Figure 1D), as well as nerve-ring neurons (hereafter referred to as neurons; Figure 1E). Specifically, we used confocal microscopy and counted the number of GFP::LGG-1 positive punctae in each of these tissues from the first day of adulthood (i.e. when animals become reproductively active) until Day 10 of adulthood (Figure 1F–I), at which time GFP::LGG-1-positive punctae could still be identified in these major tissues (Figure 1B’–E’). Notably, all tissues examined showed an increase in GFP::LGG-1-positive punctae over time with the muscle showing the largest (~9-fold) increase (Figure 1F–I). We confirmed that the GFP::LGG-1-positive punctae likely represented APs and not GFP::LGG-1 aggregates, since WT C. elegans expressing a mutant form of GFP-tagged LGG-1/Atg8 protein (G116A) that is expected to be defective in lipidation and autophagosome-membrane targeting (Manil-Ségalen et al., 2014) showed no punctae formation in the intestine or muscle, and only a small increase in the pharynx over this 10-day time period (Figure 1—figure supplement 1, see figure legend for comment on neurons, which have yet to be evaluated in detail). Taken together, these experiments show that the GFP::LGG-1 reporter can be used to monitor autophagic events in multiple somatic tissues well into old age (i.e. at least until Day 10), and indicate that the AP pool size generally increases over time without forming lipidation-independent aggregates in WT animals. However, these observations are insufficient to evaluate whether the observed increase in GFP::LGG-1-positive punctae represents an induction in the formation, or a block in the turnover of APs.

Figure 1 with 1 supplement see all
The autophagosome pool size increases with age in C. elegans.

(A) Schematic representation of GFP::LGG-1 fluorescence states in the autophagy pathway. IM, isolation membrane; AP, autophagosome; AL, autolysosome. (B–E’) Adult transgenic WT animals expressing gfp::lgg-1, imaged at Day 1 (B–E) and Day 10 (B’–E’) of adulthood. APs (arrows) can be seen in the intestine (B,B’), body-wall muscle (C,C’), pharynx (D,D’), and nerve-ring neurons (E,E’). Dotted lines outline individual intestinal cells (B,B’) and pharyngeal bulbs (D–E’). AB, anterior pharyngeal bulb; TB, terminal pharyngeal bulb. Scale bars = 20 µm. (F–I) Quantification of autophagosomes (AP; GFP punctae) in the intestine (F), body-wall muscle (G), pharynx (H), and nerve-ring neurons (I) at Days 1, 3, 5, 7, or 10 of adulthood in WT animals. Day 7 was omitted for neurons due to a counting issue at this time point. Data are the mean ± SEM of ≥20 animals combined from three independent experiments per time point. ###p<0.00 and ##p<0.001 for WT control at Day 1, 3, 5, 7, or 10 vs. WT control at Day 1 by Poisson regression.

https://doi.org/10.7554/eLife.18459.002

Expression of an mCherry::GFP::LGG-1 reporter in adult C. elegans

To expand our spatiotemporal analysis, we constructed and expressed a dual-fluorescent mCherry::GFP::LGG-1 protein to monitor both APs as well as autolysosomes (ALs), as originally done in mammalian cells (Kimura et al., 2007). With this reporter, APs are visualized as punctae positive for both GFP and mCherry fluorescence, while ALs (and amphisomes, for simplicity referred to here as ALs) emit only the mCherry signal due to quenching of GFP in the acidic environment (Figure 2A). Expression of mcherry::gfp::lgg-1 from the endogenous lgg-1 promoter produced a full-length mCherry::GFP::LGG-1 protein (Figure 2—figure supplement 1A–C) that was functional since it was able to rescue an embryonic lethal lgg-1(tm3489) mutant (see Materials and methods, data not shown), as previously shown for GFP::LGG-1 expressed from the lgg-1 promoter (Manil-Ségalen et al., 2014). mCherry::GFP::LGG-1 was expressed in several major tissues of adult WT animals (Figure 2B–G, Figure 2—figure supplement 2A–E), consistent with the expression profile of GFP::LGG-1 (Figure 1B–E and [Meléndez et al., 2003]). As previously done for GFP::LGG-1 (Gelino et al., 2016), we also expressed mCherry::GFP::LGG-1 from a pan-neuronal rgef-1 promoter to specifically visualize neurons (Figure 2G, Figure 2—figure supplement 2E). mCherry/GFP double-labeled and mCherry single-labeled punctae were observed in multiple tissues, including hypodermal seam cells, the tissue most commonly characterized thus far when assessing autophagy in C. elegans (Hansen, 2016; Zhang et al., 2015), the intestine, muscle, pharynx, and neurons of Day 1 adult animals (Figure 2C–G, Figure 2—figure supplement 2A–E). Immunofluorescence analysis of the intestine of Day 1 mCherry::GFP::LGG-1-expressing animals showed that both GFP- and mCherry-positive punctae co-localized with structures stained by an LGG-1 antibody (Figure 2—figure supplement 1D–E), consistent with only full-length protein being present in double-labeled punctae. As observed with the GFP::LGG-1 reporter (Figure 1B’–E’), mCherry/GFP and mCherry-only punctae were observed in mCherry::gfp::lgg-1 transgenic animals at least until Day 10 (Figure 2D’–G’, Figure 2—figure supplement 2B’–E’). Hypodermal seam cells could not be visualized in older animals because they fuse and appear as a single line shortly after Day 1 of adulthood. Taken together, these data confirm that transgenic C. elegans express an mCherry::GFP::LGG-1 protein that localizes to dual- and single-fluorescent punctae in young and old adults.

Figure 2 with 4 supplements see all
Expression and validation of a novel mCherry::gfp::lgg-1 reporter in C. elegans.

(A) Schematic representation of mCherry::GFP::LGG-1 fluorescence states in the autophagy pathway. IM, isolation membrane; AP, autophagosome; AL, autolysosome. (B) Whole-body expression of mCherry::GFP::LGG-1 in a wild-type (WT) animal at Day 1 of adulthood. Scale bar = 100 µm. Note that the intensity of red fluorescence compared to green is stronger; thus, the red channel was purposely set lower (see Materials and methods). (C–G’) Adult transgenic WT animals expressing mCherry::gfp::lgg-1, imaged at Day 1 (C–G) or Day 10 (D’–G’) of adulthood. APs (mCherry/GFP; yellow arrowheads) and ALs (mCherry only; white arrowheads) can be seen in the hypodermal seam cells (C), intestine (D,D’), body-wall muscle (E,E’), pharynx (F,F’), and nerve-ring neurons (G,G’). Dotted lines outline individual intestinal cells (D,D’) and pharyngeal bulbs (F,G’). AB, anterior pharyngeal bulb; TB, terminal pharyngeal bulb. Scale bars = 20 µm (D–G’) and (C) = 10 μm. (H) Intestine of Day 1 WT transgenic animals expressing mCherry::gfp::lgg-1 (green, GFP; red, mCherry) and stained with LysoTracker (light blue). Scale bar = 5 µm. (I) Quantification of punctae containing mCherry alone or co-localized with GFP or LysoTracker in the intestine of WT transgenic animals. Data are representative of three independent experiments, each with ≥10 animals. (J) Schematic representation of basal AP and AL pool sizes and the effect of inhibiting autophagy by rab-7 RNAi or Bafilomycin (BafA) treatment. (K) Quantification of APs and ALs in the intestine of Day 1 WT transgenic animals fed from hatching with bacteria expressing empty vector (control) or dsRNA encoding rab-7. Data are the mean ± SEM of ≥40 animals combined from three experiments. ***p<0.0001 by Student’s t-test. (L) Quantification of APs and ALs in the hypodermal seam cells of Day 1 WT transgenic animals injected with BafA or DMSO (control). Data are the mean ± SEM of ≥30 animals combined from three experiments. ***p<0.0001 and **p<0.001 by Student’s t-test.

https://doi.org/10.7554/eLife.18459.004

mCherry/GFP and mCherry-only punctae represent autophagosomes and autolysosomes, respectively, in mCherry::GFP::LGG-1 transgenic animals

We sought to verify that the mCherry/GFP and mCherry-only punctae observed in mCherry::GFP::LGG-1-expressing animals represented bona fide autophagic vesicles. Irrespective of tissue type, the mCherry/GFP-positive punctae (Figure 2D–G, Figure 2—figure supplement 2B–E) were similar in morphology and number to the GFP-positive punctae observed in transgenic animals expressing GFP::LGG-1 (Figure 1B–E and [Meléndez et al., 2003]), supporting their identity as APs. To determine whether mCherry-only punctae represent ALs, we examined their co-localization with LysoTracker, a pH-sensitive dye that fluoresces in acidic compartments (Hersh et al., 2002). Indeed, ~75% of mCherry-positive punctae in the intestine of Day 1 adult WT animals co-localized with LysoTracker and ~10% co-localized with GFP (Figure 2H–I) consistent with labeling of ALs and APs, respectively. Of note, ~15% of mCherry-positive puncta co-localized with neither GFP nor LysoTracker (Figure 2H–I). These mCherry-only punctae may reflect inefficient labeling of Lysotracker in the intestine (which was highly variable between animals and experiments, data not shown), or they could represent mCherry aggregates rather than autophagic vesicles; however, we did not observe free mCherry on Western blots (Figure 2—figure supplement 1B). While we cannot rule out that this relatively low percentage of mCherry-positive punctae in the intestine could represent aggregates, our data are consistent with the mCherry::GFP::LGG-1 reporter being able to distinguish between APs and ALs in adult C. elegans, at least in the intestine of the animal. Hereafter, we will refer to mCherry/GFP and mCherry-only punctae as APs and ALs, respectively. Interestingly, ALs were more abundant than APs in all tissues of WT transgenic animals (Figure 2D–G, Figure 2—figure supplement 2A–E, Figure 3), suggesting that turnover of APs (i.e. formation of APs and conversion to ALs) is faster than turnover of ALs. If so, this would indicate that AL turnover may be rate limiting in C. elegans (further addressed in the Discussion; see also Figure 2—figure supplement 3A–C).

Figure 3 with 3 supplements see all
The pool size of autophagic vesicles increases with age in all tissues of wild-type animals.

(A–D) Quantification of autophagosomes (AP) and autolysosomes (AL) in adult Days 1, 3, 5, 7, and 10 wild-type (WT) transgenic animals expressing mCherry::gfp::lgg-1 and injected with DMSO (control, black lines) or Bafilomycin A (BafA, gray lines). Tissues examined were the intestine (A), body-wall muscle (B), pharynx (C), and nerve-ring neurons (D). WT animals were raised at 25°C and incubated at 20°C from Day 1 of adulthood. Data are the mean ± SEM of ≥30 animals combined from three independent experiments per time point. ***p<0.0001, **p<0.001, and *p<0.01 for WT control vs WT + BafA on each day; ###p<0.0001 for WT control at Days 1, 3, 5, 7, or 10 vs. WT control at Day 1 by Poisson regression.

https://doi.org/10.7554/eLife.18459.009

We further validated the mCherry::GFP::LGG-1 reporter by investigating the effects of genetic (RNAi) or pharmacological inhibition of autophagy on AP and AL numbers. RNAi-mediated inhibition was achieved by feeding mCherry::gfp::lgg-1 transgenic animals with bacteria expressing dsRNA for rab-7, a small GTPase required for AP–lysosome fusion (Figure 2J and [Manil-Ségalen et al., 2014]). WT mcherry::gfp::lgg-1 transgenic animals subjected to rab-7 RNAi from hatching (whole-life RNAi) contained increased numbers of APs and reduced ALs in their intestines, as would be expected when autophagy is active and AP–lysosome fusion is inhibited (Figure 2K). We also pharmacologically inhibited autophagy by injecting WT mcherry::gfp::lgg-1 transgenic animals with Bafilomycin A (BafA), which blocks vacuolar type H+-ATPases thus inhibiting lysosomal acidification (Figure 2J and [Klionsky et al., 2008]). We confirmed this by showing that BafA abrogates LysoTracker staining in the intestine of Day 1 animals (Figure 2—figure supplement 4A–B). Consistent with a block in autophagy, we observed an increase in APs and a reduction in ALs in hypodermal seam cells of Day 1 transgenic WT animals injected with BafA compared with those injected with DMSO (Figure 2L). Lastly, we examined animals with a loss-of-function mutation in cst-1, the C. elegans ortholog of mammalian STK4; such mutants experience a block in autophagy (Wilkinson et al., 2015). While the number of APs in hypodermal seam cells of untreated mCherry::GFP::LGG-1-expressing cst-1(tm1900)  mutants was higher than that of WT transgenic animals, as previously reported (Wilkinson et al., 2015), we observed no change in AP or AL pool sizes following BafA treatment of these animals (Figure 2—figure supplement 4C), consistent with the pre-existing genetic block in autophagy. Collectively, these results support the notion that the new mCherry::GFP::LGG-1 reporter identifies both AP and AL compartments and can be used to monitor changes in these compartments upon modulation of autophagy in adult C. elegans.

Aging of WT C. elegans is associated with an increase in the number of autophagic vesicles, which corresponds to a decrease in autophagic activity

We next characterized tissue-specific changes in AP and AL pool sizes in the intestine, muscle, pharynx, and neurons of adult Days 1, 3, 5, 7, and 10 WT animals expressing mCherry::gfp::lgg-1. Similar to our analysis of WT animals expressing gfp::lgg-1 (Figure 1F–I), we observed that WT animals expressing mCherry::GFP::LGG-1 displayed an increase in AP numbers in the intestine, muscle, and pharynx during late adulthood (Figure 3A–C), with the largest (~15-fold) increase occurring in the muscle. Somewhat differently, the number of ALs in these tissues increased early in adulthood and then remained relatively constant until Day 10 (Figure 3A–C). In contrast, while neurons overall showed an increased number of APs when assessed with either the GFP::LGG-1 (Figure 1I), or the mCherry::GFP::LGG-1 reporter (Figure 3D), the number of ALs decreased with age (Figure 3D). Together, data with both fluorescent-tagged LGG-1 reporters indicate that the steady-state AP pool size increased with age in all the examined tissues of WT C. elegans, albeit with different trajectories, overall increasing confidence that APs are reliably monitored with the new tandem LGG-1 reporter. Likewise, the AL pool size appeared first to increase but then stagnate in the intestine, muscle, and pharynx of WT animals, whereas the number of ALs instead decreased over time in neurons.

An increase in APs and ALs could be due to an induction or a block in autophagy (Klionsky et al., 2016). To distinguish between these possibilities, we injected BafA into WT transgenic animals during adulthood and quantified APs and ALs in each tissue two hours later, when steady-state conditions were reached (Figure 3—figure supplement 1A–D). Since BafA inhibits lysosomal acidification (Klionsky et al., 2008), no change in AP and AL numbers following BafA treatment would be indicative of a block in autophagy, whereas BafA-induced changes in either AP or AL number, or both, would indicate active autophagy (Figure 3—figure supplement 2). Specifically, complete inhibition of lysosomal acidification by BafA would reduce AL numbers (mCherry-only punctae) while increasing AP numbers (mCherry/GFP punctae) since the GFP signal would no longer be quenched (Figure 3—figure supplement 2Ai,B). However, BafA may also prevent the later step of cargo degradation by incompletely inhibiting acidification of the lysosome resulting in reduced efficiency of lysosomal enzymes, which could lead to a concomitant increase in AL numbers (Figure 3—figure supplement 2Aii,B and Ni et al., 2011]). We found that BafA treatment increased AP numbers in all tissues and at all ages of mCherry::gfp::lgg-1 transgenic animals compared with controls, but the increases were generally dampened in older animals (Figure 3A–D). Similar observations were made in BafA-injected gfp::lgg-1 transgenic animals (Figure 3—figure supplement 3A–H; analyzed at Day 1 and Day 7). This dampening of the response was not due to BafA being less effective at older age, since BafA equally quenched Lysotracker in the intestine of Day 1 and Day 10 animals (Figure 2—figure supplement 4A–B, and data not shown). Interestingly, AL numbers were increased by BafA in the intestine and pharynx at all ages and in the muscle of young animals (Figure 3A–C), whereas the effect on neurons varied with time (Figure 3D). Collectively, the observed changes in AP and AL pool sizes following BafA treatment suggest that autophagy is active in all tissues of WT C. elegans throughout life, similar to conclusions from a recent study (Chapin et al., 2015). However, the observation that older animals generally showed reduced responses to BafA is consistent with an overall reduction in autophagic activity in all tissues with age. Although we cannot at this point explain the variable age-related effects of BafA on AL pool size in neurons, we note that the BafA response in both AP and AL compartments at Day 10 was larger in neurons than in other tissues, suggesting that autophagic capacity in neurons may decline less with age. This possibility remains to be directly tested.

Taken together, our analysis is consistent with the GFP::LGG-1 and mCherry::GFP::LGG-1 reporters similarly monitoring APs, that the AP and AL pool sizes generally increase with age, and that autophagy is active in all tissues throughout adulthood in WT C. elegans, consistent with (Chapin et al., 2015). Importantly, however, the increased pool sizes appear to reflect an age-dependent decline in autophagic activity in all tissues.

Autophagic activity in hypodermal seam cells is increased in daf-2 mutants, but can not be accurately assessed in glp-1 mutants

Mutations in the daf-2 insulin/IGF-1 receptor or the glp-1/Notch receptor extend the lifespan of C. elegans (Arantes-Oliveira et al., 2002; Kenyon et al., 1993), and reported evidence suggests that this is dependent on induction of autophagy. However, it is not known whether daf-2 and glp-1 mutants differentially regulate autophagy in a tissue- or age-specific manner to secure lifespan extension. To begin to investigate this, we assessed autophagy in a spatiotemporal manner in long-lived daf-2(e1370) and glp-1(e2141) mutants expressing the different fluorescently-tagged LGG-1 reporters.

We first analyzed hypodermal seam cells, the cell type that, thus far, has been the most frequently monitored using the GFP::LGG-1 reporter in C. elegans (reviewed in Zhang et al., 2015; Hansen, 2016). These cells display an increased number of GFP::LGG-1-positive punctae in both daf-2(e1370) and glp-1(e2141) larvae (Meléndez et al., 2003; Hansen et al., 2008; Lapierre et al., 2011). Consistently, we found that Day 1 daf-2(e1370) or glp-1(e2141) mutants expressing either GFP::LGG-1 or mCherry::GFP::LGG-1 displayed an increased AP pool size compared with WT, whereas AL numbers were similar between WT and the long-lived mutants (Figure 4—figure supplement 1A–G). However, expression of lipidation-deficient GFP::LGG-1(G116A) unexpectedly resulted in GFP-positive punctae in hypodermal seam cells of glp-1(e2141) mutants, whereas WT and daf-2(e1370) animals, as expected, showed diffuse GFP::LGG-1(G116A) localization (Figure 4—figure supplement 1D–E). Notably, both BafA (Figure 4—figure supplement 1A–C) and rab-7 RNAi (Figure 4—figure supplement 1F) treatments caused an increase in APs and a reduction in ALs in daf-2(e1370) mutants expressing mCherry::GFP::LGG-1, but had no effect on AP or AL numbers in glp-1(e2141) mutants (Figure 4—figure supplement 1A–C,G; we note that BafA was equally effective in quenching Lysotracker in the intestine of WT, daf-2, and glp-1 animals at Day 1 and Day 10 (Figure 2—figure supplement 4A–B,D, and data not shown). Taken together, these data are consistent with autophagy being induced in hypodermal seam cells of daf-2 mutants, as proposed previously (Meléndez et al., 2003). In contrast, hypodermal seam cells of glp-1 animals may be different and cause LGG-1 to aggregate, as observed in autophagy-deficient mammalian cells expressing lipidation-deficient Atg8/LC3 (Kuma et al., 2007). Consistently, we observed punctate structures in atg-3(bp412) and atg-18(gk378) autophagy mutants expressing GFP::LGG-1(G116A) (Figure 4—figure supplement 1H–I). The exact nature of such lipidation-deficient GFP::LGG-1(G116A) punctate structures in C. elegans remains to be determined.

daf-2 mutants generally display increased autophagic activity

Since the GFP::LGG-1(G116A) reporter remained largely diffuse over time in the other major tissues we analyzed in daf-2 and glp-1 mutants, similar to WT animals (Figure 1—figure supplement 1), we next analyzed the GFP::LGG-1 and mCherry::GFP::LGG-1 reporters in the intestine, muscle, pharynx, and in neurons over time. Using both reporters, we observed the number of APs in the intestine and muscle were higher in daf-2(e1370) mutants than in WT animals at Day 1 and increased modestly with age until Day 10, at which time the AP levels in daf-2(e1370) mutants were lower than in WT animals (Figure 4A–B; Figure 3—figure supplement 3A–B,E–F). In contrast, while AL numbers were also elevated in the intestine and muscle of Day 1 daf-2(e1370) mutants with age (Figure 4A–B), AL numbers in the muscle increased in early adulthood and remained at relatively constant levels thereafter, whereas in the intestine, they continued to increase for longer. In both tissues, AL numbers in daf-2(e1370) mutants were higher than in WT animals at older ages (Figure 4A–B). Notably, BafA treatment generally increased the AP pool size in the intestine and muscle of daf-2(e1370) mutants expressing mCherry::GFP::LGG-1 (Figure 4A–B), or GFP::LGG-1 (Figure 3—figure supplement 3E–F). Of particular note, Day 10 animals showed the largest induction following BafA treatment in the intestine and muscle of daf-2(e1370) mutants, and AL pool size was also generally increased (Figure 4A–B). Collectively, these results suggest that autophagic activity may be elevated in the intestine and muscle of daf-2 mutants compared with WT animals and may remain so throughout adulthood.

Figure 4 with 1 supplement see all
daf-2 insulin/IGF-1 receptor mutants display increased autophagic activity in most tissues.

(A–D) Quantification of autophagosomes (AP) and autolysosomes (AL) in adult Day 1, 3, 5, 7, and 10 daf-2(e1370) animals expressing mCherry::gfp::lgg-1 and injected with DMSO (control, dark green lines) or Bafilomycin A (BafA, light green lines). Tissues examined were the intestine (A), body-wall muscle (B), pharynx (C), and nerve-ring neurons (D). The black dashed lines in (A–C) show data from wild-type (WT) control animals from Figure 3 for comparison (animals were analyzed in parallel). The black dashed line in (D) shows data from WT animals incubated at 20°C for their entire lifespan. Data are the mean ± SEM of ≥25 animals combined from three independent experiments. , WT control vs. daf-2 control at Days 1, 3, 5, 7, and 10; *, daf-2 control vs. daf-2 + BafA at Days 1, 3, 5, 7, and 10; #, daf-2 control at Days 3, 5, 7, and 10 vs. daf-2 control at Day 1. ***/∧∧∧/###p<0.0001, **/∧∧/##p<0.001, *//#p<0.01 by Poisson regression. See also Figure 3—figure supplement 1A–H for quantification of APs in gfp::lgg-1 transgenic animals.

https://doi.org/10.7554/eLife.18459.013

In the pharynx, the AP pool size was similar in daf-2(e1370) and WT animals at most time points, whereas the AL pool size was initially larger in daf-2(e1370) mutants; however, in older daf-2(e1370) mutants, both AP and AL numbers were similar or lower than WT animals (Figure 4C, Figure 3—figure supplement 3C,G). Interestingly, BafA treatment did not cause any significant changes in AP numbers in the pharynx of daf-2(e1370) mutants, except at Day 10 of adulthood (Figure 4C, Figure 3—figure supplement 3C,G), whereas AL numbers were changed from Day 5 to Day 10 (Figure 4C). Collectively, these data indicate the interesting possibility that autophagy in the pharynx of daf-2 mutants may be blocked prior to Day 5 of adulthood. Moreover, while enumeration of APs alone would suggest a continuous block in autophagy in the pharynx of daf-2 mutants, the AL flux data is consistent with possible reactivation of autophagic activity that exceeds that of WT in late life. Additional experiments are needed to evaluate this possibility and to conclusively interpret the AL flux data, especially at older age.

The effect of age on the GFP::LGG-1 and mCherry::GFP::LGG-1 reporters in neurons was quite different to that in other tissues in daf-2 mutants, but similar to that observed in WT animals. APs and ALs were more and less abundant, respectively, in daf-2(e1370) animals than in WT animals in early adulthood, but both strains displayed an overall decrease in APs and an increase in ALs as they aged (Figure 4D, Figure 3—figure supplement 3D,H). BafA treatment altered both AP and AL numbers throughout early adulthood in daf-2(e1370) animals (Figure 4D, Figure 3—figure supplement 3H), while at Day 10, BafA altered APs but not ALs (Figure 4D). Collectively, these data are consistent with autophagic activity in the neurons of daf-2 mutants possibly being higher than in WT animals in early adulthood, whereas both strains display an age-related impairment of autophagic activity over time.

Overall, these data indicate that autophagic activity in the intestine, muscle, and neurons may be higher in daf-2 mutants than in WT animals, at least in early adulthood. Notably, activity in the pharynx may possibly be blocked in younger daf-2 mutants. This observation constitutes the first report of a potential block in autophagy in a long-lived animal, and needs to be investigated in more detail.

glp-1 animals display a different autophagic activity profile than daf-2 mutants

We performed a similar spatiotemporal analysis of the autophagy reporters in the major tissues of long-lived, germline-less glp-1 mutants. We previously reported that GFP::LGG-1 punctae are more abundant in the intestine of glp-1 mutants than of WT animals at Day 1 of adulthood (Lapierre et al., 2011). Consistent with this, we observed elevated numbers of APs in the intestine of glp-1(e2141) animals compared with WT animals expressing either GFP::LGG-1 or mCherry::GFP::LGG-1 throughout adulthood except at old age (Figure 5A, Figure 3—figure supplement 3A,E’). In contrast, the intestinal AL pool size was lower in glp-1(e2141) mutants than in WT animals at Day 1 of adulthood and fluctuated thereafter (Figure 5A). BafA treatment had variable effects on AP numbers but increased AL numbers in the intestine of both young and old animals (Figure 5A, Figure 3—figure supplement 3E’). These results are consistent with autophagy being active in the intestine of glp-1 animals throughout life, although this dataset is insufficient to make a direct comparison of the relative autophagic activity in glp-1 mutants and WT animals.

In muscle, AP numbers were higher in glp-1(e2141) mutants than in WT animals in early adulthood but they increased similarly in both strains thereafter (Figure 5B, Figure 3—figure supplement 3B,F'). In contrast, AL numbers were generally lower in glp-1(e2141) mutants than in WT animals and remained relatively constant over time (Figure 5B). In this tissue, the BafA treatment, for unknown reasons, had variable effects on the two different reporter strains. Whereas glp-1(e2141) animals expressing GFP::LGG-1 showed induction of APs at both Day 1 and Day 7 (Figure 3—figure supplement 3F’), BafA had no significant effect on APs at any time point in glp-1(e2141) mutants expressing mCherry::GFP::LGG-1 (Figure 5B). ALs were similarly unaffected by BafA treatment in Days 1–5 glp-1(e2141) animals, yet altered in Day 7 and Day 10 glp-1(e2141) animals (Figure 5B). Thus, while additional experiments are needed to conclusively evaluate autophagy activity in the muscle of younger glp-1 mutants, our observations indicate that autophagy may be active in this tissue of older glp-1 mutants.

In the pharynx, glp-1(e2141) and WT animal’s AP numbers were generally similar throughout adulthood (Figure 5C, Figure 3—figure supplement 3C,G’), whereas the number of ALs increased somewhat with age but was consistently lower than in WT animals after Day 1 (Figure 5C). AP numbers in glp-1(e2141) mutants were generally unaffected by BafA treatment, whereas AL counts were altered in animals irrespective of age (Figure 5C, Figure 3—figure supplement 3C,G'). Thus, while the AL compartment needs to be fully investigated in older animals, it is possible that autophagy may remain active in the pharynx of glp-1 mutants throughout life. However, similar to the intestine, it is not possible to evaluate the relative autophagic activity in glp-1 mutants to WT animals based on this dataset.

In neurons, APs were more abundant in glp-1(e2141) mutants than in WT animals at Day 1, but were similar in older animals (Figure 5D, Figure 3—figure supplement 3D,H'). ALs were less abundant in the neurons of young Day 1 and Day 3 glp-1(e2141) mutants compared with WT animals, but ALs gradually decreased with age in WT animals while they remained relatively constant in glp-1(e2141) mutants (Figure 5D). Of note, BafA treatment of young glp-1(e2141) animals expressing either of the two LGG-1 reporters altered both AP and AL pool sizes (Figure 5D, Figure 3—figure supplement 3H’), caused no significant effect on AP or AL number on Day 7 (Figure 5D, Figure 3—figure supplement 3H’), but resulted in a significant increase in AP and ALs at Day 10 (Figure 5D). Taken together, these data indicate that autophagic activity may be increased in the neurons of young glp-1 animals compared to age-matched WT animals, whereas the autophagic activity may become more comparable to that in WT animals by mid-life.

Germline-less glp-1 mutants display a different autophagic activity profile than daf-2 mutants.

(A–D) Quantification of autophagosomes (AP) and autolysosomes (AL) in adult Days 1, 3, 5, 7, and 10 glp-1(e2141) animals expressing mCherry::gfp::lgg-1 and injected with DMSO (control, dark blue lines) or Bafliomycin A (BafA, light blue lines). Tissues examined were the intestine (A), body-wall muscle (B), pharynx (C), and nerve-ring neurons (D). The black dashed lines in (A–D) show data from wild-type (WT) control animals from Figure 3 for comparison (animals were analyzed in parallel). Data are the mean ± SEM of ≥25 animals combined from three independent experiments. , WT + control vs. glp-1 control at Days 1, 3, 5, 7, and 10; *, glp-1 control vs. glp-1 + BafA at Days 1, 3, 5, 7, and 10, #, glp-1 control at Days 3, 5, 7, and 10 vs. glp-1 control at Day 1. ***/∧∧∧/###p<0.0001, **/∧∧/##p<0.001, *//#p<0.01 by Poisson regression analysis.

https://doi.org/10.7554/eLife.18459.015

Collectively, these data suggest that autophagy remains active in the intestine, pharynx, and possibly also muscle of glp-1 mutants throughout adulthood. In neurons, autophagy appeared to be increased in young glp-1 animals compared to WT animals and remains active and potentially comparable to WT by mid-life. Overall, aging had different tissue-specific effects of autophagy in glp-1 mutants versus daf-2 mutants, highlighting the possibility, which needs to be further addressed by yet-to-be developed biochemical assays, that autophagy may be regulated differentially at the individual tissue level in long-lived mutants.

Autophagy gene expression in the intestine is required for lifespan extension of glp-1 mutants but not of daf-2 mutants

The intestine is a critical tissue in both daf-2 and glp-1 mutants, since the downstream effector DAF-16, a FOXO transcription factor functions in this tissue to ensure lifespan extension (Libina et al., 2003). Since our tissue analyses indicated that autophagy is active in the intestine of both mutants, we asked whether intestine-specific inhibition of autophagy prevents lifespan extension in daf-2 and glp-1 animals, as has been observed with whole-body inhibition of autophagy (Hars et al., 2007; Lapierre et al., 2011; Meléndez et al., 2003; Hansen et al., 2008). To this end, we used sid-1(qt9) mutants, which carry a mutation in the dsRNA transporter sid-1 rendering them systemically refractory to RNAi, but with reconstituted sid-1 expression specifically in the intestine using the vha-6 promoter (Melo and Ruvkun, 2012). This strain was crossed to daf-2(e1370) and glp-1(e2141) mutants and lifespan analyses were carried out. The lifespans of the RNAi-refractory strains daf-2(e1370); sid-1(qt9) and glp-1(e2141); sid-1(qt9) with or without intestinal-specific sid-1 re-expression were similar to the lifespans of daf-2(e1370) and glp-1(e2141) single mutants, respectively (Supplementary file 1A–B). Moreover, the lifespan of daf-2(e1370); sid-1(qt9) and glp-1(e2141); sid-1(qt9) double mutants were not affected by atg-18/Wipi RNAi (Supplementary file 1A–B), whereas the double mutants expressing vha-6p::sid-1 were RNAi competent in the intestine, but not in the other tissues examined (Figure 6—figure supplement 1A–E, and data not shown). Together, these results validate the use of these strains for tissue-specific lifespan analyses.

While whole-body and intestinal-specific RNAi of daf-16/Foxo potently shortened the lifespan of both glp-1(e2141) and daf-2(e1370) mutants (Figure 6A–B; Supplementary file 1A-B), consistent with daf-16 functioning in the intestine to extend the lifespan of these long-lived animals (Libina et al., 2003), we found striking differences in longevity of glp-1 and daf-2 animals subjected to intestinal atg-18 RNAi. Specifically, we observed that intestinal-specific inhibition of autophagy by atg-18/Wipi RNAi was sufficient to significantly reduce the lifespan of glp-1(e2141) animals, as previously observed with whole-body atg-18/Wipi RNAi (Figure 6C, Supplementary file 1A, and Lapierre et al., 2011). In contrast, knockdown of atg-18/Wipi or of lgg-1/Atg8 in the intestine of daf-2(e1370) animals did not have a significant effect on lifespan in a total of six out of six experiments, whereas whole-body atg-18/Wipi and lgg-1/Atg8 RNAi had significant lifespan-shortening effects (Figure 6D, Supplementary file 1B), similar to the effect of whole-body RNAi of other autophagy genes (Meléndez et al., 2003; Hansen et al., 2008; Hars et al., 2007). Notably, we confirmed that atg-18/Wipi RNAi reduced the expression of an mCherry::atg-18 transgene to the same extent in the intestine of WT and in daf-2(e1370) mutants (Figure 6—figure supplement 1F,G), indicating that knockdown of atg-18 by RNAi is not somehow compromised in the intestine of daf-2 mutants. Collectively, these data therefore indicate that intestinal autophagy is required for the lifespan extension of glp-1 mutants, but not daf-2 mutants.

Figure 6 with 1 supplement see all
Autophagy genes expressed in intestinal cells are required for lifespan extension of glp-1 mutants, but not of daf-2 mutants.

(A,C) Kaplan–Meier survival curves of glp-1(e2141) single mutants and glp-1(e2141); sid-1(qt9) double mutants expressing sid-1 cDNA in the intestine (vha-6 promoter). Animals were fed from Day 1 of adulthood with bacteria expressing empty vector (control), daf-16/Foxo dsRNA (A), or atg-18/Wipi dsRNA (C). Intestine-specific inhibition of daf-16/Foxo shortened the lifespan of glp-1(e2141) mutants in two out of two experiments ach with ≥100 animals. p<0.0001 for whole body control RNAi vs. whole body daf-16/Foxo RNAi; p<0.0001 for intestine-specific control RNAi vs. intestine-specific daf-16 RNAi by log-rank test. Intestine-specific inhibition of atg-18/Wipi shortened the lifespan of glp-1(e2141) mutants in five out of seven experiments, each with ≥100 animals. p<0.0001 for whole-body control RNAi vs. whole-body atg-18/Wipi RNAi; p=0.0008 for intestine-specific control RNAi vs. intestine-specific atg-18/Wipi RNAi by log-rank test. (B,D) Kaplan–Meier survival curves of daf-2(e1370) single mutants and daf-2(e1370); sid-1(qt9) double mutants expressing sid-1 cDNA in the intestine (vha-6 promoter). Animals were fed from Day 1 of adulthood with bacteria expressing empty vector (control), daf-16/Foxo dsRNA (B), or atg-18/Wipi dsRNA (D). Intestine-specific inhibition of daf-16/Foxo shortened the lifespan of daf-2(e1370) mutants in two out of two experiments, each with ≥100 animals. p<0.0001 for whole body control RNAi vs. whole body daf-16/Foxo RNAi; p<0.0001 for intestine-specific control RNAi vs. intestine-specific daf-16 RNAi by log-rank test. Intestine-specific inhibition of atg-18/Wipi had no significant effect on the lifespan of daf-2(e1370) mutants in all of six experiments, each with ≥100 animals. p<0.0001 for whole-body control RNAi vs. whole-body atg-18/Wipi RNAi; p=0.66 for intestine-specific control RNAi vs. intestine-specific atg-18/Wipi RNAi, by log-rank test. See Supplementary file 1 for details on lifespan analyses and additional repeats.

https://doi.org/10.7554/eLife.18459.016

This was a surprising result, because our autophagy flux assays indicated that daf-2 mutants displayed active autophagy in the intestine (Figure 4A, Figure 3—figure supplement 3A,E), similar to glp-1 mutants (Figure 5A, Figure 3—figure supplement 3A,E’). Autophagy in other tissues may instead play a role in the longevity of daf-2 mutants. In support of this, we found that muscle-specific knockdown of atg-18/Wipi decreased the lifespan of daf-2(e1370); sid-1(qt9) animals expressing sid-1 from the myo-3 promoter (Supplementary file 1B). Of note, we observed the same lifespan-shortening effect of muscle-specific atg-18/Wipi RNAi in glp-1(e2141) mutants; in this case, however, glp-1(e2141); sid-1(qt9) mutants with sid-1 re-expression in muscle were unexpectedly shorter-lived than glp-1(e2141); sid-1(qt9) mutants (Supplementary file 1A). Since the reason for this remains unclear, we refrain from drawing conclusions from this observation. Taken together, our lifespan analyses suggest that intestinal autophagy is required for lifespan extension in glp-1 mutants, whereas autophagy in the muscle, but not the intestine, may contribute to the longevity of daf-2 mutants.

Discussion

Changes in autophagy have been linked to aging in many species, but it is not yet clear how autophagy is modulated spatially and temporally during an animal’s lifespan. Our analysis of C. elegans using multiple Atg8 reporters and autophagy flux assays is the first effort to comprehensively estimate autophagic activity in a living animal during aging. We found that (i) aging of wild-type (WT) animals is accompanied by increased numbers of autophagic vesicles in all tissues examined that likely reflects a reduction in autophagy activity; (ii) long-lived daf-2 insulin/IGF-1 mutants and germline-less glp-1 mutants differentially regulate autophagy spatially and temporally compared to WT animals, and (iii) glp-1 mutants, but not daf-2 mutants, require autophagy genes in the intestine for lifespan extension, further emphasizing important differences in autophagy regulation in individual tissues between these conserved longevity paradigms.

For this study, we used previously published GFP::LGG-1 (Gelino et al., 2016; Kang et al., 2007; Meléndez et al., 2003) and new mCherry::GFP::LGG-1 reporters (which were expressed from endogenous or neuronal promoters) to quantify autophagic events in four major somatic tissues, that is, intestine, muscle, pharynx, and neurons during C. elegans adulthood. As an important control to help verify that GFP::LGG-1-positive punctae observed in WT, daf-2, and glp-1 animals likely represented APs and not unspecific aggregates, we also used a lipidation-deficient GFP::LGG-1(G116A) reporter expressed from the endogenous promoter (Manil-Ségalen et al., 2014). Similar point-mutated Atg8 proteins are assumed unable to attach to the autophagosomal membrane since they cannot be post-translationally modified to bind phosphatidylethanolamine (Kabeya et al., 2004). Consistent with this, GFP::LGG-1(G116A) appeared diffusely localized in essentially all the settings we investigated here, that is, in the intestine, muscle, and pharynx, with the notable exception of hypodermal seam cells in glp-1 mutants in which GFP::LGG-1(G116A) expression led to punctae formation, similar to glp-1 mutants expressing GFP::LGG-1 (Lapierre et al., 2011). GFP::LGG-1 punctae in hypodermal seam cells of glp-1 mutants may therefore have been incorrectly interpreted as APs. While GFP::LGG-1(G116A) punctate structures in C. elegans remains to be investigated in detail, they may be similar in nature to the intracellular protein aggregates or inclusion bodies observed in mammalian Atg5 knockout cells expressing GFP-tagged Atg8/LC3 (Kuma et al., 2007). Since Atg8 proteins are capable of oligomerization (Nymann-Andersen et al., 2002; Baisamy et al., 2009), GFP::LGG-1(G116A) punctate structures may alternatively still represent APs with mutant LGG-1(G116A) protein. However, methods to concretely determine whether GFP::LGG-1(G116A) punctae represent aggregates or APs (with WT and mutant LGG-1 protein) are not currently available in C. elegans. Collectively, these observations emphasize the use of analyzing a LGG-1(G116A) reporter in any given condition in which a fluorescent LGG-1 reporter is utilized to help assess autophagic vesicles, while the data from these reporters should be interpreted with caution. To this end, we note that a comprehensive analysis of such a reporter in neurons needs to be carried out since the GFP::LGG-1(G116A) reporter we used here limited us to only evaluate hypodermal seam cells, intestine, muscle, and pharynx in detail.

We used the GFP::LGG-1 and mCherry::GFP::LGG-1 reporters in combination with Bafilomycin (BafA) flux assays to quantify AP and AL numbers and estimate autophagic activity. These reporters equivalently monitored changes in the AP compartment in all tissues and at all ages, collectively increasing confidence that the AP compartment was accurately visualized with these reporters. Moreover, the mCherry::GFP::LGG-1 reporter rescued an embryonically lethal lgg-1/Atg8 mutant, indicating that the full-length protein is functional. We sought to use this new reporter to monitor the AL compartment for the first time in adult C. elegans after validating it in multiple ways Specifically, we found that co-staining with Lysotracker highlighted the majority of red-only punctae in the intestine of young WT animals, as expected for acidic autolysosomes. In addition, we observed that inhibition of autophagy by either BafA, which inhibits lysosomal acidification, RNAi of rab-7, a gene required for AP-lysosome fusion, or genetic inhibition of autophagy by mutation of the Hippo kinase cst-1, all modulated AP and AL numbers in young WT and long-lived mutants as expected. While these observations collectively support the use of mCherry::GFP::LGG-1 to reliably monitor the AP and AL compartments, we were unable to account for ~15% red-only punctae in the intestine of Day 1 WT animals expressing mCherry::GFP::LGG-1. A similar low percentage of red-only punctae was observed in the intestine of Day 1 daf-2 and glp-1 mutants (Figure 2—figure supplement 4D). These punctae could represent aggregates, or, alternatively, inefficient Lysotracker staining (as noted in results), however, at present we cannot distinguish between these possibilities and this estimate does not significantly offset any of our conclusions. While this estimate is very low, and has, to our knowledge, not previously been estimated for a tandem-tagged Atg8 reporter in any model system including mammalian cell culture, we emphasize that additional experiments are needed to fully verify the AL compartment in other C. elegans tissues and at later time points. To this end, it will be important to evaluate a lipidation-deficient mCherry::GFP::LGG-1(G116A) mutant. Moreover, it would be of interest to estimate autophagic activity via the Atg8 paralog LGG-2 in C. elegans (Alberti et al., 2010).

Our BafA flux assays indicated that although autophagy is active in the intestine, muscle, pharynx, and neurons of aging WT C. elegans, autophagic activity appeared to decrease with age. These results are consistent with flux assays and proteolysis experiments in rat liver (Del Roso et al., 2003, Donati et al., 2001), gene expression studies in D. melanogaster and rodents (Cuervo and Wong, 2014; Demontis and Perrimon, 2010; Kaushik et al., 2012; Ling and Salvaterra, 2009; Simonsen et al., 2008; Vittorini et al., 1999; Ye et al., 2011), and lysosomal protease studies in C. elegans (Sarkis et al., 1988). Our observations are also consistent with the findings of a recent study of tissue-specific autophagy in C. elegans, which showed that autophagy is active in all major somatic tissues during aging (Chapin et al., 2015). In that study, C. elegans expressing a lysosomal-cleavable dual-fluorescent LGG-1 protein showed an age-associated increase in mono-fluorescent LGG-1 protein levels, which the authors interpreted as an increase in autophagic activity (Chapin et al., 2015). However, the accumulation of this mono-fluorescent protein would also be consistent with an age-dependent decline in autophagic activity if degradation is inhibited, perhaps through reduced lysosomal pH or enzymatic activity, as has been observed in aging yeast (Hughes and Gottschling, 2012). How lysosomal pH and activity might change in individual C. elegans tissues during aging is an important question for the future and will be especially interesting to investigate in neurons, which appeared to behave differently to other major tissues with respect to autophagic activity. The apparent decline in autophagic activity observed here could have several explanations, including an age-associated failure to clear the autophagic machinery (as evidenced by the increases in AP and AL pool sizes) or to degrade otherwise detrimental cargo. Additional assays are required to confirm these interpretations, including biochemical approaches to directly address how autophagy might fail with age in the different tissues of C. elegans; such efforts may be facilitated by novel tissue-isolation methods (Kaletsky et al., 2016). It will also be interesting to investigate how an age-dependent decline of autophagy in specific tissues might contribute to loss of organ-specific functions during aging.

Our analysis suggested that autophagic activity might be increased in certain tissues of long-lived daf-2 and glp-1 mutants compared with WT animals, as we and others have previously proposed (Lapierre et al., 2011; Meléndez et al., 2003). However, our results indicate that these two longevity mutants differentially regulate autophagy in a tissue-specific manner during aging since daf-2 and glp-1 mutants showed distinct age-related changes in AP and AL pool sizes, as well as in autophagic flux. Of particular note, we found that autophagy appeared to be blocked in the pharynx of Day 1 adult daf-2 animals. This is an interesting scenario as it would constitute the first example of a possible autophagy block in a long-lived animal; however, we caution that steady-state conditions for the BafA treatment could vary between genetic background, tissue, as well as age. Irrespectively, it remains to be determined how daf-2 and glp-1 mutants may differentially regulate autophagy, and to this point, it is interesting to note that although both mutants require the transcription factor HLH-30/TFEB for longevity (Lapierre et al., 2013), glp-1 mutants upregulate more predicted HLH-30 target genes with roles in autophagy than daf-2 mutants (Lapierre et al., 2013). While the endpoint of our analysis was on Day 10 of adulthood, it will also be of interest to estimate autophagic activity in physiologically old long-lived mutants, including in their late state of decrepitude likely caused by bacterial colonization (Podshivalova et al., 2017).

We observed that intestine-specific inhibition of autophagy genes significantly shortened the lifespan of glp-1 animals, but not daf-2 animals, suggesting that the apparent increase in intestinal autophagic activity in daf-2 mutants may not be required for their long lifespan. Since systemic inhibition of autophagy reduces the lifespan of both mutants (reviewed in Hansen, 2016, and this study), autophagy in other tissues, such as muscle, may be more important for longevity in daf-2 mutants. These results further highlight important differences in autophagy regulation between daf-2 and glp-1 mutants and suggest that autophagy in different organs may have distinct biological roles; a possibility that is further emphasized by the observed differential requirement for autophagy in lifespan determination in the intestine of daf-2 versus glp-1 animals. To this end, it will be interesting to determine whether autophagy is regulated in a tissue-specific manner in other long-lived C. elegans mutants, or in other long-lived species.

In this study, we evaluated overall autophagic activity by quantifying steady-state levels of APs and ALs in combination with autophagic flux assays. However, autophagy is a multistep process and further insight can be gained into its regulation by taking into account that each step occurs at a distinct rate. Below, we reassess our dataset with this in mind. If we assume that autophagy can be represented by three steps: isolation membrane (IM) to AP, AP to AL, and degradation of ALs, the rates of each step under steady-state conditions can be defined as the concentration of IMs, APs, and ALs multiplied by the rate constants α, β, and γ, respectively (Figure 2—figure supplement 3A–B and [Loos et al., 2014]). Changes in the steady-state levels of APs and ALs can therefore provide information about the relative contribution of the change in each step to the overall autophagic flux. For example, in WT C. elegans under steady-state conditions, ALs were consistently more abundant than APs in all tissues examined, as has been observed in some mammalian cell types (Klionsky et al., 2016), and in D. melanogaster tissues (Mauvezin et al., 2014). Assuming α is constant, this observation suggests that β is greater than γ (i.e. the degradation step is slower than the AP to AL step), implying that turnover of ALs may be rate limiting in C. elegans (Figure 2—figure supplement 3B–C). We can similarly approximate how BafA treatment affects each step and rate constant since the ratio of [AP] in control-treated animals to [AP] in BafA-treated animals (and similarly for [AL]) is inversely correlated to the change in β (or γ). For example, an increase in both [AP] and [AL] following BafA treatment implies that BafA reduces both β and γ (Figure 2—figure supplement 3B,D). In fact, we observed that each tissue in WT, daf-2, and glp-1 animals responded to BafA in a different manner according to the genetic background and age of the animal, suggesting that tissue-, age-, and genotype-specific differences may exist in the autophagy-rate constants. The above analysis also clearly indicates that, while quantification of autophagic vesicles at steady state provides valuable information about autophagic activity, it at times is not sufficient to accurately determine the relative difference in autophagic activity between WT, daf-2, and glp-1 animals, or between the tissues examined. Moreover, a hypothetical modeling of our data shows that steady-state numbers of APs and ALs can be interpreted as increased, decreased, or unchanged autophagic flux when the rate constants differ (Figure 2—figure supplement 3E), again cautioning that steady-state measurements of AP or AL pool sizes may not reflect the overall autophagic flux. Therefore, additional methods that directly measure the rate of at least one step in autophagy or the overall rate of autophagic degradation will be crucial to accurately measure autophagic flux and fully understand its role in longevity.

In conclusion, our autophagic flux analysis using LGG-1/Atg8 reporters indicated an age-dependent decline in autophagy in several major tissues of C. elegans, and that the reduction occurs at a step after AP formation. In contrast, long-lived animals show differential regulation of autophagy in distinct tissues. Further experiments are needed to confirm these observations by directly measuring autophagic activity. Understanding the tissue- and age-specific regulation of autophagy in C. elegans is likely to shed light on the role of autophagy in aging and age-related diseases in mammals, including humans.

Materials and methods

C. elegans and bacterial strains

C. elegans strains were maintained and cultured under standard conditions at 20°C on the standard E. coli strain OP50 (see Supplementary file 2 for strain list). HT115 was used as the food source for feeding RNAi experiments (Brenner, 1974). For experiments with the temperature-sensitive glp-1(e2141) mutant, both glp-1(e2141) and wild-type (WT) N2 animals were allowed to hatch at 20°C for 24 hr and then moved to 25°C until Day 1 of adulthood. At Day 1 of adulthood, all strains were placed at 20°C for the rest of the analysis. All daf-2(e1370) strains were maintained at 20°C for their entire lifespan. WT animals were raised at 25°C and placed at 20°C at Day 1 of adulthood, with the exception of WT animals expressing the mCherry::GFP::LGG-1 transgene specifically in neurons (Figure 4D) or otherwise noted, which were maintained at 20°C for their entire lifespan. No significant difference was observed in AP and AL pool sizes in Day 1 or Day 3 adult animals raised at 20°C or 25°C (Figure 3—figure supplement 1E–H).

RNA interference (RNAi)

HT115 RNAi clones used were bli-3, bli-4, elt-2, lin-26, lgg-1, pept-1, rab-7, and unc-112 (all Ahringer library), atg-18 (Vidal library), as well as daf-16 and gfp (kind gifts from Dr. Andrew Dillin). All RNAi bacterial clones were verified by sequencing. The empty vector (L4440) for controls was provided by Dr. Andrew Fire. RNAi experiments were carried out as previously described (Gelino et al., 2016). Briefly, HT115 bacteria were grown in liquid LB medium containing 0.1 mg/ml carbenicillin (BioPioneer, San Diego, CA), 80 µl bacteria spotted onto 6 cm NGM plates supplemented with carbenicillin and grown for 1–2 days at room temperature, and 80 µl 0.1M IPTG (Promega, Sunnyvale, CA) added to bacterial lawn to induce dsRNA expression. Eggs (whole-life RNAi) or adults (adult-only RNAi) were transferred to plates. RNAi of bli-3, bli-4, elt-2, gfp, lin-26, pept-1, rab-7, and unc-112 was performed from hatching, and lgg-1, daf-16, and atg-18 RNAi used in lifespan analyses was performed from Day 1 of adulthood.

To confirm efficacy of atg-18 RNAi in daf-2 animals, transgenic WT or daf-2(e1370) animals expressing atg-18p::atg-18::mCherry from and extrachromosomal array were fed bacteria expressing atg-18 dsRNA from hatching and imaged using a Leica fluorescence dissecting microscope at 23x magnification. Images were acquired using a Leica DFC310 FX camera with 4 second exposure. Fluorescence intensity in the anterior intestine was determined using Image J.

Construction of transgenic strains

To construct the lgg-1p::mcherry::gfp::lgg-1 vector, mCherry was amplified by PCR from a Clontech pmCherry expression vector and the PCR fragment was inserted into TOPO TA vector. mCherry was verified by sequencing and then removed using Kpn1 and inserted into an lgg-1p::gfp:lgg-1 vector (Meléndez et al., 2003) upstream of gfp. The final product lgg-1p::mcherry::gfp::lgg-1 (pMH878) was verified by sequencing.

An expression plasmid for neuronal-specific rgef-1p::mCherry::gfp::lgg-1 was generated by Gateway cloning technology lambda (Thermo Fisher Scientific, Waltham, MA) (Hartley et al., 2000) using pDONR P4-P1R-rgef-1p (~1.6 kb, amplified from a vector provided by the Dillin lab), pDONR221-mCherry::gfp::lgg-1 open-reading frame (amplified from pMH878), and pDONR-P2RP3-unc-54 3′UTR (amplified from pGH8 [Frøkjaer-Jensen et al., 2008]). The final product rgef-1p::mcherry::gfp::lgg-1 (pMH1130) was verified by sequencing. An expression plasmid for atg-18p::atg-18::mCherry was similarly made using pDONR P4-P1R-atg-18p (540 bp, amplified from genomic DNA) and pDONR221-atg-18 open-reading frame (amplified from genomic DNA). The final product atg-18p::atg-18::mCherry (pMH791) was verified by sequencing.

Plasmid DNA was prepared using a Mini or Midi Prep kit (Invitrogen, Waltham, MA or Qiagen, Hilden, Germany). Transgenic animals expressing an extrachromosomal array were created by gonadal microinjection of pMH878/lgg-1p::mCherry::gfp::lgg-1, pMH1130/rgef-1p::mCherry::gfp::lgg-1, or pMH791/atg-18p::atg-18::mCherry plus pRF4/rol-6 co-injection marker into N2-Hansen animals. A list of strains made in this study is provided in Supplementary file 2. Integration of lgg-1p::mCherry::gfp::lgg-1 was subsequently performed by γ-irradiation followed by outcrossing four times to N2-Hansen WT animals.

To show that lgg-1p::mCherry::gfp::lgg-1 was functional and could rescue the lgg-1 mutant embryonic lethality, MAH215/lgg-1p::mCherry::gfp::lgg-1 was crossed to FX3489/lgg-1(tm3489) II/+, and the mCherry/GFP-positive F2 progeny were assayed for the homozygous lgg-1 mutation by PCR. Mutated lgg-1 is ~800 bp and WT lgg-1 is ~1000 bp. Viable and fertile animals that were positive for mCherry/GFP were identified with mutant lgg-1. Primer information is available in Supplementary file 3.

Quantification of autophagic vesicles

C. elegans were mounted live on a 2% agarose pad in M9 medium containing 0.1% NaN3 and imaged using an LSM Zeiss 710 scanning confocal microscope, Z-stack images were acquired at 0.6 µm slice intervals at 63x. GFP excitation/emission was set to 493/517 nm to eliminate background autofluorescence. GFP::LGG-1 (GFP) or mCherry::GFP::LGG-1 (mCherry/GFP or mCherry only)-positive punctae were counted in the hypodermal seam cells, intestine, body-wall muscle, pharynx, and nerve-ring neurons in one 0.6 µm slice. The Z-position was selected where the nucleus could be clearly seen (for hypodermal seam cells and intestine), where striation could be seen (body-wall muscle), and where the lumen of the pharyngeal bulbs was in focus (for the pharynx and nerve-ring neurons). Punctae were quantified from images as follows: for body-wall muscle, punctae in one 1000 µm2 area per 0.6 µm slice per animal, for the nerve-ring neurons total punctae between the pharyngeal bulbs in one 0.6 µm slice per animal, for the pharynx the number of punctae in the posterior pharyngeal bulb, and for the intestine and hypodermal seam cells, the number of punctae per cell per 0.6 µm slice. The number of mCherry-only punctae was calculated as (the total number of mCherry-positive punctae – the number of GFP-positive punctae). Statistical analysis of punctae was performed using Poisson regression calculated with the R Core Team (2015). The intensity of red fluorescence compared to green is stronger and in order to see mCherry-positive punctae clearly, the gain of the red channel was purposely set lower. If the intensity of red fluorescence was increased (which would overexpose the red punctae), cytoplasmic mCherry::GFP::LGG-1 appeared yellow (data not shown). For GFP::LGG-1(G116A) experiments, punctae in hypodermal seam cells were counted using a Zeiss Imager Z1, while at the microscope. The total number of punctae was counted in all visible hypodermal seam cells, whereas punctae quantification in the intestine, body-wall muscle, and pharynx was done as specified above. Imaging was performed using Zeiss Imager Z1 including apotome.2 at 100x magnification with a Hamamastsu orca flash 4LT camera and Zen 2.3 software. The average and SEM were calculated and data were analyzed using one-way analysis of variance (ANOVA) or two-way ANOVA as applicable (GraphPad Prism, La Jolla, CA).

Bafilomycin A and LysoTracker treatment

Bafilomycin A (BafA; BioViotica, Dransfeld, Germany) was injected into the body cavity of C. elegans as previously described (Wilkinson et al., 2015). Briefly, 50 µM of BafA in 0.2% DMSO (25 mM stock in DMSO) or 0.2% DMSO (control) was co-injected with 2.5 ug/ml Texas Red Dextran or Cascade Blue Dextran, 3,000 Daltons (re-suspended in water at a stock concentration of 25 ug/ml; Thermo Fisher Scientific, Walltham, MA) into the anterior body cavity, intestine, or intestinal lumen, and the animals were allowed to recover for two hours before they were imaged by confocal microscopy. Injection of BafA could result in variability of LGG-1 punctae counts; however, injection of the 3,000 Dalton Texas Red Dextran alone (i.e. significantly larger than BafA) into ~100 worms showed highly reproducible Texas Red signal in the head region where LGG-1 punctae were quantified (data not shown), and the standard error of the mean was similar in DMSO- and in BafA-treated animals in all experiments performed (as well as in published work, see [Wilkinson et al., 2015]). Thus, notable variability in number of punctae is likely due to variability per animal rather than variable injection of BafA.

C. elegans were grown from egg to adulthood on E. coli OP50-seeded NGM plates containing 25 μM LysoTracker Deep Red (Thermo Fisher Scientific, Waltham, MA, mixed into plate media during prep) or an equivalent volume of DMSO (control) and subsequently imaged using confocal microscopy.

Lifespan analysis

Lifespan was measured at 20°C as previously described (Hansen et al., 2005). Briefly, synchronized animals were transferred onto E. coli OP50-seeded plates and were raised at 20°C for WT/daf-2 lifespans while animals for WT/glp-1 lifespans were left at 20°C for 24 hr then moved to 25°C for 48 hr. At Day 1 of adulthood all animals were moved to 20°C for the rest of their lives. The animals were then transferred to plates seeded with HT115 RNAi clones (adult-only RNAi treatment) or bacteria contain empty vector (controls) and scored every 1–3 days, as previously described (Hansen et al., 2005). Animals were scored as dead if they failed to respond to gentle prodding with a platinum wire pick. Censoring occurred if animals desiccated on the edge of the plate, escaped, ruptured, or suffered from internal hatching. Kaplan–Meier survival curves were constructed and statistical analysis was performed using STATA software (StataCorp, College Station, TX). p-values were calculated using the log-rank (Mantel–Cox) method.

Analysis of the sid-1 transgene

Intestine-specific sid-1 transgene expression was assessed by PCR (see Supplementary file 3 for primer list). All daf-2 and glp-1 strains carrying sid-1 transgenes were tested and confirmed for RNAi efficiency using multiple tissue-specific RNAi clones for genes expressed in the intestine, body-wall muscle or hypodermis (Figure 6—figure supplement 1A–B, data not shown) as previously described for WT tissue-specific RNAi strains (Figure 6—figure supplement 1, and Kumsta and Hansen, 2012, and data not shown). Briefly, strains carrying sid-1 transgenes were subjected to whole-life tissue-specific RNAi and imaged at Day 3 of adulthood. Worms were imaged in M9 medium containing 0.1% NaN3 on a Leica fluorescence dissecting microscope at 8x magnification. Images were acquired using a Leica DFC310 FX camera with 2-second exposure.

Immunoblotting

Total protein was extracted from 50 handpicked Day 1 adult animals grown on OP50 bacteria at 20°C. Animals were washed thoroughly in M9 buffer and centrifuged, and the pellets were lysed in 10 μl of 6x SDS sample buffer. The entire extract was separated by 4–20% SDS-PAGE (Thermo Fisher Scientific, Waltham, MA) and transferred to a PVDF membrane (Millipore, Hayward, CA). Immunoblotting was performed using primary anti-GFP (diluted 1:1000; Santa Cruz Biotechnology, Dallas, TX), anti-mCherry (diluted 1:500; Clontech, Mountain View, CA), and anti-LGG-1 (diluted 1:1000; sample kindly obtained from Abgent, San Diego, CA) antibodies and secondary horseradish peroxidase-conjugated goat anti-mouse (diluted 1:2000; Santa Cruz Biotechnology, Dallas, TX) or goat anti-rabbit (1:2000; Cell Signaling Technology, Danvers, MA) secondary antibodies. Immunoblots were developed using enhanced chemiluminescent reagent (Thermo Fisher Scientific, Waltham, MA).

Immunofluorescence in C. elegans intestines

Day 1 adult transgenic animals expressing mcherry::gfp::lgg-1 were picked into a drop of M9 on a glass slide and dissected by pulling the head and tail apart with forceps, resulting in the intestine popping out of the animal. Dissected intestines were transferred to a 1.5-ml tube and fixed in 4% paraformaldehyde in 0.1% PBS-Tween20 for one hour at room temperature, and rinsed at least three times in 0.1% PBS-Tween20. Intestines were blocked in 5% FBS +0.1% PBS-Tween20 overnight at 4°C. Primary antibodies were mouse anti-GFP (diluted 1:100; Santa Cruz Biotechnology, Dallas, TX), mouse anti-mCherry (diluted 1:50; Clontech, Mountain View, CA), and rabbit anti-LGG-1 (diluted 1:100; sample kindly providd by Abgent, San Diego, CA); they were diluted in block and incubated overnight at 4°C. Secondary antibodies used were goat anti-mouse AlexaFluor 546 (1:500 Life Technologies, Carlsbad, CA) and goat anti-rabbit AlexaFluor 488 (1:500 Life Technologies, Carlsbad, CA) and incubated in block overnight at 4°C. Dissected intestines were flat mounted in glycerol directly onto glass slides and imaged using an LSM Zeiss 710 scanning confocal microscope. Secondary antibody-only treatment did not have any specific antibody staining in the intestine (data not shown). Fixation quenched mCherry/GFP fluorescence in the intestine, but not in the pharynx, body-wall muscle or hypodermis, as these tissues were not exposed as much as the intestine to the fixing solution and sometimes appeared to be fluorescent (both green and red) following secondary antibody-only treatment. N2/WT animals treated with secondary antibodies only did not show fluorescence staining in the body-wall muscle, pharynx, or hypodermis demonstrating that fluorescence in transgenic animals is due to the reporter that was not quenched during fixation (data not shown).

References

  1. 1
  2. 2
  3. 3
  4. 4
  5. 5
    The genetics of Caenorhabditis elegans
    1. S Brenner
    (1974)
    Genetics 77:71–94.
  6. 6
  7. 7
  8. 8
  9. 9
  10. 10
  11. 11
  12. 12
  13. 13
  14. 14
  15. 15
  16. 16
  17. 17
  18. 18
    Autophagy and Ageing
    1. M Hansen
    (2016)
    Ageing: Lessons from C. elegans, Autophagy and Ageing, Springer Internatinal Publishing.
  19. 19
  20. 20
  21. 21
  22. 22
  23. 23
  24. 24
  25. 25
  26. 26
  27. 27
  28. 28
  29. 29
  30. 30
    Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)
    1. DJ Klionsky
    2. K Abdelmohsen
    3. A Abe
    4. MJ Abedin
    5. H Abeliovich
    6. A Acevedo Arozena
    7. H Adachi
    8. CM Adams
    9. PD Adams
    10. K Adeli
    11. PJ Adhihetty
    12. SG Adler
    13. G Agam
    14. R Agarwal
    15. MK Aghi
    16. M Agnello
    17. P Agostinis
    18. PV Aguilar
    19. J Aguirre-Ghiso
    20. EM Airoldi
    21. S Ait-Si-Ali
    22. T Akematsu
    23. ET Akporiaye
    24. M Al-Rubeai
    25. GM Albaiceta
    26. C Albanese
    27. D Albani
    28. ML Albert
    29. J Aldudo
    30. H Algül
    31. M Alirezaei
    32. I Alloza
    33. A Almasan
    34. M Almonte-Beceril
    35. ES Alnemri
    36. C Alonso
    37. N Altan-Bonnet
    38. DC Altieri
    39. S Alvarez
    40. L Alvarez-Erviti
    41. S Alves
    42. G Amadoro
    43. A Amano
    44. C Amantini
    45. S Ambrosio
    46. I Amelio
    47. AO Amer
    48. M Amessou
    49. A Amon
    50. Z An
    51. FA Anania
    52. SU Andersen
    53. UP Andley
    54. CK Andreadi
    55. N Andrieu-Abadie
    56. A Anel
    57. DK Ann
    58. S Anoopkumar-Dukie
    59. M Antonioli
    60. H Aoki
    61. N Apostolova
    62. S Aquila
    63. K Aquilano
    64. K Araki
    65. E Arama
    66. A Aranda
    67. J Araya
    68. A Arcaro
    69. E Arias
    70. H Arimoto
    71. AR Ariosa
    72. JL Armstrong
    73. T Arnould
    74. I Arsov
    75. K Asanuma
    76. V Askanas
    77. E Asselin
    78. R Atarashi
    79. SS Atherton
    80. JD Atkin
    81. LD Attardi
    82. P Auberger
    83. G Auburger
    84. L Aurelian
    85. R Autelli
    86. L Avagliano
    87. ML Avantaggiati
    88. L Avrahami
    89. S Awale
    90. N Azad
    91. T Bachetti
    92. JM Backer
    93. DH Bae
    94. JS Bae
    95. ON Bae
    96. SH Bae
    97. EH Baehrecke
    98. SH Baek
    99. S Baghdiguian
    100. A Bagniewska-Zadworna
    101. H Bai
    102. J Bai
    103. XY Bai
    104. Y Bailly
    105. KN Balaji
    106. W Balduini
    107. A Ballabio
    108. R Balzan
    109. R Banerjee
    110. G Bánhegyi
    111. H Bao
    112. B Barbeau
    113. MD Barrachina
    114. E Barreiro
    115. B Bartel
    116. A Bartolomé
    117. DC Bassham
    118. MT Bassi
    119. RC Bast
    120. A Basu
    121. MT Batista
    122. H Batoko
    123. M Battino
    124. K Bauckman
    125. BL Baumgarner
    126. KU Bayer
    127. R Beale
    128. JF Beaulieu
    129. GR Beck
    130. C Becker
    131. JD Beckham
    132. PA Bédard
    133. PJ Bednarski
    134. TJ Begley
    135. C Behl
    136. C Behrends
    137. GM Behrens
    138. KE Behrns
    139. E Bejarano
    140. A Belaid
    141. F Belleudi
    142. G Bénard
    143. G Berchem
    144. D Bergamaschi
    145. M Bergami
    146. B Berkhout
    147. L Berliocchi
    148. A Bernard
    149. M Bernard
    150. F Bernassola
    151. A Bertolotti
    152. AS Bess
    153. S Besteiro
    154. S Bettuzzi
    155. S Bhalla
    156. S Bhattacharyya
    157. SK Bhutia
    158. C Biagosch
    159. MW Bianchi
    160. M Biard-Piechaczyk
    161. V Billes
    162. C Bincoletto
    163. B Bingol
    164. SW Bird
    165. M Bitoun
    166. I Bjedov
    167. C Blackstone
    168. L Blanc
    169. GA Blanco
    170. HK Blomhoff
    171. E Boada-Romero
    172. S Böckler
    173. M Boes
    174. K Boesze-Battaglia
    175. LH Boise
    176. A Bolino
    177. A Boman
    178. P Bonaldo
    179. M Bordi
    180. J Bosch
    181. LM Botana
    182. J Botti
    183. G Bou
    184. M Bouché
    185. M Bouchecareilh
    186. MJ Boucher
    187. ME Boulton
    188. SG Bouret
    189. P Boya
    190. M Boyer-Guittaut
    191. PV Bozhkov
    192. N Brady
    193. VM Braga
    194. C Brancolini
    195. GH Braus
    196. JM Bravo-San Pedro
    197. LA Brennan
    198. EH Bresnick
    199. P Brest
    200. D Bridges
    201. MA Bringer
    202. M Brini
    203. GC Brito
    204. B Brodin
    205. PS Brookes
    206. EJ Brown
    207. K Brown
    208. HE Broxmeyer
    209. A Bruhat
    210. PC Brum
    211. JH Brumell
    212. N Brunetti-Pierri
    213. RJ Bryson-Richardson
    214. S Buch
    215. AM Buchan
    216. H Budak
    217. DV Bulavin
    218. SJ Bultman
    219. G Bultynck
    220. V Bumbasirevic
    221. Y Burelle
    222. RE Burke
    223. M Burmeister
    224. P Bütikofer
    225. L Caberlotto
    226. K Cadwell
    227. M Cahova
    228. D Cai
    229. J Cai
    230. Q Cai
    231. S Calatayud
    232. N Camougrand
    233. M Campanella
    234. GR Campbell
    235. M Campbell
    236. S Campello
    237. R Candau
    238. I Caniggia
    239. L Cantoni
    240. L Cao
    241. AB Caplan
    242. M Caraglia
    243. C Cardinali
    244. SM Cardoso
    245. JS Carew
    246. LA Carleton
    247. CR Carlin
    248. S Carloni
    249. SR Carlsson
    250. D Carmona-Gutierrez
    251. LA Carneiro
    252. O Carnevali
    253. S Carra
    254. A Carrier
    255. B Carroll
    256. C Casas
    257. J Casas
    258. G Cassinelli
    259. P Castets
    260. S Castro-Obregon
    261. G Cavallini
    262. I Ceccherini
    263. F Cecconi
    264. AI Cederbaum
    265. V Ceña
    266. S Cenci
    267. C Cerella
    268. D Cervia
    269. S Cetrullo
    270. H Chaachouay
    271. HJ Chae
    272. AS Chagin
    273. CY Chai
    274. G Chakrabarti
    275. G Chamilos
    276. EY Chan
    277. MT Chan
    278. D Chandra
    279. P Chandra
    280. CP Chang
    281. RC Chang
    282. TY Chang
    283. JC Chatham
    284. S Chatterjee
    285. S Chauhan
    286. Y Che
    287. ME Cheetham
    288. R Cheluvappa
    289. CJ Chen
    290. G Chen
    291. GC Chen
    292. G Chen
    293. H Chen
    294. JW Chen
    295. JK Chen
    296. M Chen
    297. M Chen
    298. P Chen
    299. Q Chen
    300. Q Chen
    301. SD Chen
    302. S Chen
    303. SS Chen
    304. W Chen
    305. WJ Chen
    306. WQ Chen
    307. W Chen
    308. X Chen
    309. YH Chen
    310. YG Chen
    311. Y Chen
    312. Y Chen
    313. Y Chen
    314. YJ Chen
    315. YQ Chen
    316. Y Chen
    317. Z Chen
    318. Z Chen
    319. A Cheng
    320. CH Cheng
    321. H Cheng
    322. H Cheong
    323. S Cherry
    324. J Chesney
    325. CH Cheung
    326. E Chevet
    327. HC Chi
    328. SG Chi
    329. F Chiacchiera
    330. HL Chiang
    331. R Chiarelli
    332. M Chiariello
    333. M Chieppa
    334. LS Chin
    335. M Chiong
    336. GN Chiu
    337. DH Cho
    338. SG Cho
    339. WC Cho
    340. YY Cho
    341. YS Cho
    342. AM Choi
    343. EJ Choi
    344. EK Choi
    345. J Choi
    346. ME Choi
    347. SI Choi
    348. TF Chou
    349. S Chouaib
    350. D Choubey
    351. V Choubey
    352. KC Chow
    353. K Chowdhury
    354. CT Chu
    355. TH Chuang
    356. T Chun
    357. H Chung
    358. T Chung
    359. YL Chung
    360. YJ Chwae
    361. V Cianfanelli
    362. R Ciarcia
    363. IA Ciechomska
    364. MR Ciriolo
    365. M Cirone
    366. S Claerhout
    367. MJ Clague
    368. J Clària
    369. PG Clarke
    370. R Clarke
    371. E Clementi
    372. C Cleyrat
    373. M Cnop
    374. EM Coccia
    375. T Cocco
    376. P Codogno
    377. J Coers
    378. EE Cohen
    379. D Colecchia
    380. L Coletto
    381. NS Coll
    382. E Colucci-Guyon
    383. S Comincini
    384. M Condello
    385. KL Cook
    386. GH Coombs
    387. CD Cooper
    388. JM Cooper
    389. I Coppens
    390. MT Corasaniti
    391. M Corazzari
    392. R Corbalan
    393. E Corcelle-Termeau
    394. MD Cordero
    395. C Corral-Ramos
    396. O Corti
    397. A Cossarizza
    398. P Costelli
    399. S Costes
    400. SL Cotman
    401. A Coto-Montes
    402. S Cottet
    403. E Couve
    404. LR Covey
    405. LA Cowart
    406. JS Cox
    407. FP Coxon
    408. CB Coyne
    409. MS Cragg
    410. RJ Craven
    411. T Crepaldi
    412. JL Crespo
    413. A Criollo
    414. V Crippa
    415. MT Cruz
    416. AM Cuervo
    417. JM Cuezva
    418. T Cui
    419. PR Cutillas
    420. MJ Czaja
    421. MF Czyzyk-Krzeska
    422. RK Dagda
    423. U Dahmen
    424. C Dai
    425. W Dai
    426. Y Dai
    427. KN Dalby
    428. L Dalla Valle
    429. G Dalmasso
    430. M D'Amelio
    431. M Damme
    432. A Darfeuille-Michaud
    433. C Dargemont
    434. VM Darley-Usmar
    435. S Dasarathy
    436. B Dasgupta
    437. S Dash
    438. CR Dass
    439. HM Davey
    440. LM Davids
    441. D Dávila
    442. RJ Davis
    443. TM Dawson
    444. VL Dawson
    445. P Daza
    446. J de Belleroche
    447. P de Figueiredo
    448. RC de Figueiredo
    449. J de la Fuente
    450. L De Martino
    451. A De Matteis
    452. GR De Meyer
    453. A De Milito
    454. M De Santi
    455. W de Souza
    456. V De Tata
    457. D De Zio
    458. J Debnath
    459. R Dechant
    460. JP Decuypere
    461. S Deegan
    462. B Dehay
    463. B Del Bello
    464. DP Del Re
    465. R Delage-Mourroux
    466. LM Delbridge
    467. L Deldicque
    468. E Delorme-Axford
    469. Y Deng
    470. J Dengjel
    471. M Denizot
    472. P Dent
    473. CJ Der
    474. V Deretic
    475. B Derrien
    476. E Deutsch
    477. TP Devarenne
    478. RJ Devenish
    479. S Di Bartolomeo
    480. N Di Daniele
    481. F Di Domenico
    482. A Di Nardo
    483. S Di Paola
    484. A Di Pietro
    485. L Di Renzo
    486. A DiAntonio
    487. G Díaz-Araya
    488. I Díaz-Laviada
    489. MT Diaz-Meco
    490. J Diaz-Nido
    491. CA Dickey
    492. RC Dickson
    493. M Diederich
    494. P Digard
    495. I Dikic
    496. SP Dinesh-Kumar
    497. C Ding
    498. WX Ding
    499. Z Ding
    500. L Dini
    501. JH Distler
    502. A Diwan
    503. M Djavaheri-Mergny
    504. K Dmytruk
    505. RC Dobson
    506. V Doetsch
    507. K Dokladny
    508. S Dokudovskaya
    509. M Donadelli
    510. XC Dong
    511. X Dong
    512. Z Dong
    513. TM Donohue
    514. KS Doran
    515. G D'Orazi
    516. GW Dorn
    517. V Dosenko
    518. S Dridi
    519. L Drucker
    520. J Du
    521. LL Du
    522. L Du
    523. A du Toit
    524. P Dua
    525. L Duan
    526. P Duann
    527. VK Dubey
    528. MR Duchen
    529. MA Duchosal
    530. H Duez
    531. I Dugail
    532. VI Dumit
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    534. EA Dunlop
    535. WA Dunn
    536. N Dupont
    537. L Dupuis
    538. RV Durán
    539. TM Durcan
    540. S Duvezin-Caubet
    541. U Duvvuri
    542. V Eapen
    543. D Ebrahimi-Fakhari
    544. A Echard
    545. L Eckhart
    546. CL Edelstein
    547. AL Edinger
    548. L Eichinger
    549. T Eisenberg
    550. A Eisenberg-Lerner
    551. NT Eissa
    552. WS El-Deiry
    553. V El-Khoury
    554. Z Elazar
    555. H Eldar-Finkelman
    556. CJ Elliott
    557. E Emanuele
    558. U Emmenegger
    559. N Engedal
    560. AM Engelbrecht
    561. S Engelender
    562. JM Enserink
    563. R Erdmann
    564. J Erenpreisa
    565. R Eri
    566. JL Eriksen
    567. A Erman
    568. R Escalante
    569. EL Eskelinen
    570. L Espert
    571. L Esteban-Martínez
    572. TJ Evans
    573. M Fabri
    574. G Fabrias
    575. C Fabrizi
    576. A Facchiano
    577. NJ Færgeman
    578. A Faggioni
    579. WD Fairlie
    580. C Fan
    581. D Fan
    582. J Fan
    583. S Fang
    584. M Fanto
    585. A Fanzani
    586. T Farkas
    587. M Faure
    588. FB Favier
    589. H Fearnhead
    590. M Federici
    591. E Fei
    592. TC Felizardo
    593. H Feng
    594. Y Feng
    595. Y Feng
    596. TA Ferguson
    597. ÁF Fernández
    598. MG Fernandez-Barrena
    599. JC Fernandez-Checa
    600. A Fernández-López
    601. ME Fernandez-Zapico
    602. O Feron
    603. E Ferraro
    604. CV Ferreira-Halder
    605. L Fesus
    606. R Feuer
    607. FC Fiesel
    608. EC Filippi-Chiela
    609. G Filomeni
    610. GM Fimia
    611. JH Fingert
    612. S Finkbeiner
    613. T Finkel
    614. F Fiorito
    615. PB Fisher
    616. M Flajolet
    617. F Flamigni
    618. O Florey
    619. S Florio
    620. RA Floto
    621. M Folini
    622. C Follo
    623. EA Fon
    624. F Fornai
    625. F Fortunato
    626. A Fraldi
    627. R Franco
    628. A Francois
    629. A François
    630. LB Frankel
    631. ID Fraser
    632. N Frey
    633. DG Freyssenet
    634. C Frezza
    635. SL Friedman
    636. DE Frigo
    637. D Fu
    638. JM Fuentes
    639. J Fueyo
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    641. Y Fujiwara
    642. M Fujiya
    643. M Fukuda
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    645. C Fusco
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    647. M Gaestel
    648. P Gailly
    649. M Gajewska
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    651. G Galili
    652. I Galindo
    653. MF Galindo
    654. G Galliciotti
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    656. L Galluzzi
    657. V Galy
    658. N Gammoh
    659. S Gandy
    660. AK Ganesan
    661. S Ganesan
    662. IG Ganley
    663. M Gannagé
    664. FB Gao
    665. F Gao
    666. JX Gao
    667. L García Nannig
    668. E García Véscovi
    669. M Garcia-Macía
    670. C Garcia-Ruiz
    671. AD Garg
    672. PK Garg
    673. R Gargini
    674. NC Gassen
    675. D Gatica
    676. E Gatti
    677. J Gavard
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    679. L Ge
    680. P Ge
    681. S Ge
    682. PW Gean
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    684. AA Genazzani
    685. J Geng
    686. P Genschik
    687. L Gerner
    688. JE Gestwicki
    689. DA Gewirtz
    690. S Ghavami
    691. E Ghigo
    692. D Ghosh
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    694. F Giampieri
    695. C Giampietri
    696. A Giatromanolaki
    697. DJ Gibbings
    698. L Gibellini
    699. SB Gibson
    700. V Ginet
    701. A Giordano
    702. F Giorgini
    703. E Giovannetti
    704. SE Girardin
    705. S Gispert
    706. S Giuliano
    707. CL Gladson
    708. A Glavic
    709. M Gleave
    710. N Godefroy
    711. RM Gogal
    712. K Gokulan
    713. GH Goldman
    714. D Goletti
    715. MS Goligorsky
    716. AV Gomes
    717. LC Gomes
    718. H Gomez
    719. C Gomez-Manzano
    720. R Gómez-Sánchez
    721. DA Gonçalves
    722. E Goncu
    723. Q Gong
    724. C Gongora
    725. CB Gonzalez
    726. P Gonzalez-Alegre
    727. P Gonzalez-Cabo
    728. RA González-Polo
    729. IS Goping
    730. C Gorbea
    731. NV Gorbunov
    732. DR Goring
    733. AM Gorman
    734. SM Gorski
    735. S Goruppi
    736. S Goto-Yamada
    737. C Gotor
    738. RA Gottlieb
    739. I Gozes
    740. D Gozuacik
    741. Y Graba
    742. M Graef
    743. GE Granato
    744. GD Grant
    745. S Grant
    746. GL Gravina
    747. DR Green
    748. A Greenhough
    749. MT Greenwood
    750. B Grimaldi
    751. F Gros
    752. C Grose
    753. JF Groulx
    754. F Gruber
    755. P Grumati
    756. T Grune
    757. JL Guan
    758. KL Guan
    759. B Guerra
    760. C Guillen
    761. K Gulshan
    762. J Gunst
    763. C Guo
    764. L Guo
    765. M Guo
    766. W Guo
    767. XG Guo
    768. AA Gust
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    771. MG Gutierrez
    772. HS Gwak
    773. A Haas
    774. JE Haber
    775. S Hadano
    776. M Hagedorn
    777. DR Hahn
    778. AJ Halayko
    779. A Hamacher-Brady
    780. K Hamada
    781. A Hamai
    782. A Hamann
    783. M Hamasaki
    784. I Hamer
    785. Q Hamid
    786. EM Hammond
    787. F Han
    788. W Han
    789. JT Handa
    790. JA Hanover
    791. M Hansen
    792. M Harada
    793. L Harhaji-Trajkovic
    794. JW Harper
    795. AH Harrath
    796. AL Harris
    797. J Harris
    798. U Hasler
    799. P Hasselblatt
    800. K Hasui
    801. RG Hawley
    802. TS Hawley
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    804. CY He
    805. F He
    806. G He
    807. RR He
    808. XH He
    809. YW He
    810. YY He
    811. JK Heath
    812. MJ Hébert
    813. RA Heinzen
    814. GV Helgason
    815. M Hensel
    816. EP Henske
    817. C Her
    818. PK Herman
    819. A Hernández
    820. C Hernandez
    821. S Hernández-Tiedra
    822. C Hetz
    823. PR Hiesinger
    824. K Higaki
    825. S Hilfiker
    826. BG Hill
    827. JA Hill
    828. WD Hill
    829. K Hino
    830. D Hofius
    831. P Hofman
    832. GU Höglinger
    833. J Höhfeld
    834. MK Holz
    835. Y Hong
    836. DA Hood
    837. JJ Hoozemans
    838. T Hoppe
    839. C Hsu
    840. CY Hsu
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    843. G Hu
    844. HM Hu
    845. H Hu
    846. MC Hu
    847. YC Hu
    848. ZW Hu
    849. F Hua
    850. Y Hua
    851. C Huang
    852. HL Huang
    853. KH Huang
    854. KY Huang
    855. S Huang
    856. S Huang
    857. WP Huang
    858. YR Huang
    859. Y Huang
    860. Y Huang
    861. TB Huber
    862. P Huebbe
    863. WK Huh
    864. JJ Hulmi
    865. GM Hur
    866. JH Hurley
    867. Z Husak
    868. SN Hussain
    869. S Hussain
    870. JJ Hwang
    871. S Hwang
    872. TI Hwang
    873. A Ichihara
    874. Y Imai
    875. C Imbriano
    876. M Inomata
    877. T Into
    878. V Iovane
    879. JL Iovanna
    880. RV Iozzo
    881. NY Ip
    882. JE Irazoqui
    883. P Iribarren
    884. Y Isaka
    885. AJ Isakovic
    886. H Ischiropoulos
    887. JS Isenberg
    888. M Ishaq
    889. H Ishida
    890. I Ishii
    891. JE Ishmael
    892. C Isidoro
    893. K Isobe
    894. E Isono
    895. S Issazadeh-Navikas
    896. K Itahana
    897. E Itakura
    898. AI Ivanov
    899. AK Iyer
    900. JM Izquierdo
    901. Y Izumi
    902. V Izzo
    903. M Jäättelä
    904. N Jaber
    905. DJ Jackson
    906. WT Jackson
    907. TG Jacob
    908. TS Jacques
    909. C Jagannath
    910. A Jain
    911. NR Jana
    912. BK Jang
    913. A Jani
    914. B Janji
    915. PR Jannig
    916. PJ Jansson
    917. S Jean
    918. M Jendrach
    919. JH Jeon
    920. N Jessen
    921. EB Jeung
    922. K Jia
    923. L Jia
    924. H Jiang
    925. H Jiang
    926. L Jiang
    927. T Jiang
    928. X Jiang
    929. X Jiang
    930. X Jiang
    931. Y Jiang
    932. Y Jiang
    933. A Jiménez
    934. C Jin
    935. H Jin
    936. L Jin
    937. M Jin
    938. S Jin
    939. UK Jinwal
    940. EK Jo
    941. T Johansen
    942. DE Johnson
    943. GV Johnson
    944. JD Johnson
    945. E Jonasch
    946. C Jones
    947. LA Joosten
    948. J Jordan
    949. AM Joseph
    950. B Joseph
    951. AM Joubert
    952. D Ju
    953. J Ju
    954. HF Juan
    955. K Juenemann
    956. G Juhász
    957. HS Jung
    958. JU Jung
    959. YK Jung
    960. H Jungbluth
    961. MJ Justice
    962. B Jutten
    963. NO Kaakoush
    964. K Kaarniranta
    965. A Kaasik
    966. T Kabuta
    967. B Kaeffer
    968. K Kågedal
    969. A Kahana
    970. S Kajimura
    971. O Kakhlon
    972. M Kalia
    973. DV Kalvakolanu
    974. Y Kamada
    975. K Kambas
    976. VO Kaminskyy
    977. HH Kampinga
    978. M Kandouz
    979. C Kang
    980. R Kang
    981. TC Kang
    982. T Kanki
    983. TD Kanneganti
    984. H Kanno
    985. AG Kanthasamy
    986. M Kantorow
    987. M Kaparakis-Liaskos
    988. O Kapuy
    989. V Karantza
    990. MR Karim
    991. P Karmakar
    992. A Kaser
    993. S Kaushik
    994. T Kawula
    995. AM Kaynar
    996. PY Ke
    997. ZJ Ke
    998. JH Kehrl
    999. KE Keller
    1000. JK Kemper
    1001. AK Kenworthy
    1002. O Kepp
    1003. A Kern
    1004. S Kesari
    1005. D Kessel
    1006. R Ketteler
    1007. IC Kettelhut
    1008. B Khambu
    1009. MM Khan
    1010. VK Khandelwal
    1011. S Khare
    1012. JG Kiang
    1013. AA Kiger
    1014. A Kihara
    1015. AL Kim
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    1017. DR Kim
    1018. DH Kim
    1019. EK Kim
    1020. HY Kim
    1021. HR Kim
    1022. JS Kim
    1023. JH Kim
    1024. JC Kim
    1025. JH Kim
    1026. KW Kim
    1027. MD Kim
    1028. MM Kim
    1029. PK Kim
    1030. SW Kim
    1031. SY Kim
    1032. YS Kim
    1033. Y Kim
    1034. A Kimchi
    1035. AC Kimmelman
    1036. T Kimura
    1037. JS King
    1038. K Kirkegaard
    1039. V Kirkin
    1040. LA Kirshenbaum
    1041. S Kishi
    1042. Y Kitajima
    1043. K Kitamoto
    1044. Y Kitaoka
    1045. K Kitazato
    1046. RA Kley
    1047. WT Klimecki
    1048. M Klinkenberg
    1049. J Klucken
    1050. H Knævelsrud
    1051. E Knecht
    1052. L Knuppertz
    1053. JL Ko
    1054. S Kobayashi
    1055. JC Koch
    1056. C Koechlin-Ramonatxo
    1057. U Koenig
    1058. YH Koh
    1059. K Köhler
    1060. SD Kohlwein
    1061. M Koike
    1062. M Komatsu
    1063. E Kominami
    1064. D Kong
    1065. HJ Kong
    1066. EG Konstantakou
    1067. BT Kopp
    1068. T Korcsmaros
    1069. L Korhonen
    1070. VI Korolchuk
    1071. NV Koshkina
    1072. Y Kou
    1073. MI Koukourakis
    1074. C Koumenis
    1075. AL Kovács
    1076. T Kovács
    1077. WJ Kovacs
    1078. D Koya
    1079. C Kraft
    1080. D Krainc
    1081. H Kramer
    1082. T Kravic-Stevovic
    1083. W Krek
    1084. C Kretz-Remy
    1085. R Krick
    1086. M Krishnamurthy
    1087. J Kriston-Vizi
    1088. G Kroemer
    1089. MC Kruer
    1090. R Kruger
    1091. NT Ktistakis
    1092. K Kuchitsu
    1093. C Kuhn
    1094. AP Kumar
    1095. A Kumar
    1096. A Kumar
    1097. D Kumar
    1098. D Kumar
    1099. R Kumar
    1100. S Kumar
    1101. M Kundu
    1102. HJ Kung
    1103. A Kuno
    1104. SH Kuo
    1105. J Kuret
    1106. T Kurz
    1107. T Kwok
    1108. TK Kwon
    1109. YT Kwon
    1110. I Kyrmizi
    1111. AR La Spada
    1112. F Lafont
    1113. T Lahm
    1114. A Lakkaraju
    1115. T Lam
    1116. T Lamark
    1117. S Lancel
    1118. TH Landowski
    1119. DJ Lane
    1120. JD Lane
    1121. C Lanzi
    1122. P Lapaquette
    1123. LR Lapierre
    1124. J Laporte
    1125. J Laukkarinen
    1126. GW Laurie
    1127. S Lavandero
    1128. L Lavie
    1129. MJ LaVoie
    1130. BY Law
    1131. HK Law
    1132. KB Law
    1133. R Layfield
    1134. PA Lazo
    1135. L Le Cam
    1136. KG Le Roch
    1137. H Le Stunff
    1138. V Leardkamolkarn
    1139. M Lecuit
    1140. BH Lee
    1141. CH Lee
    1142. EF Lee
    1143. GM Lee
    1144. HJ Lee
    1145. H Lee
    1146. JK Lee
    1147. J Lee
    1148. JH Lee
    1149. JH Lee
    1150. M Lee
    1151. MS Lee
    1152. PJ Lee
    1153. SW Lee
    1154. SJ Lee
    1155. SJ Lee
    1156. SY Lee
    1157. SH Lee
    1158. SS Lee
    1159. SJ Lee
    1160. S Lee
    1161. YR Lee
    1162. YJ Lee
    1163. YH Lee
    1164. C Leeuwenburgh
    1165. S Lefort
    1166. R Legouis
    1167. J Lei
    1168. QY Lei
    1169. DA Leib
    1170. G Leibowitz
    1171. I Lekli
    1172. SD Lemaire
    1173. JJ Lemasters
    1174. MK Lemberg
    1175. A Lemoine
    1176. S Leng
    1177. G Lenz
    1178. P Lenzi
    1179. LO Lerman
    1180. D Lettieri Barbato
    1181. JI Leu
    1182. HY Leung
    1183. B Levine
    1184. PA Lewis
    1185. F Lezoualc'h
    1186. C Li
    1187. F Li
    1188. FJ Li
    1189. J Li
    1190. K Li
    1191. L Li
    1192. M Li
    1193. M Li
    1194. Q Li
    1195. R Li
    1196. S Li
    1197. W Li
    1198. W Li
    1199. X Li
    1200. Y Li
    1201. J Lian
    1202. C Liang
    1203. Q Liang
    1204. Y Liao
    1205. J Liberal
    1206. PP Liberski
    1207. P Lie
    1208. AP Lieberman
    1209. HJ Lim
    1210. KL Lim
    1211. K Lim
    1212. RT Lima
    1213. CS Lin
    1214. CF Lin
    1215. F Lin
    1216. F Lin
    1217. FC Lin
    1218. K Lin
    1219. KH Lin
    1220. PH Lin
    1221. T Lin
    1222. WW Lin
    1223. YS Lin
    1224. Y Lin
    1225. R Linden
    1226. D Lindholm
    1227. LM Lindqvist
    1228. P Lingor
    1229. A Linkermann
    1230. LA Liotta
    1231. MM Lipinski
    1232. VA Lira
    1233. MP Lisanti
    1234. PB Liton
    1235. B Liu
    1236. C Liu
    1237. CF Liu
    1238. F Liu
    1239. HJ Liu
    1240. J Liu
    1241. JJ Liu
    1242. JL Liu
    1243. K Liu
    1244. L Liu
    1245. L Liu
    1246. Q Liu
    1247. RY Liu
    1248. S Liu
    1249. S Liu
    1250. W Liu
    1251. XD Liu
    1252. X Liu
    1253. XH Liu
    1254. X Liu
    1255. X Liu
    1256. X Liu
    1257. Y Liu
    1258. Y Liu
    1259. Z Liu
    1260. Z Liu
    1261. JP Liuzzi
    1262. G Lizard
    1263. M Ljujic
    1264. IJ Lodhi
    1265. SE Logue
    1266. BL Lokeshwar
    1267. YC Long
    1268. S Lonial
    1269. B Loos
    1270. C López-Otín
    1271. C López-Vicario
    1272. M Lorente
    1273. PL Lorenzi
    1274. P Lõrincz
    1275. M Los
    1276. MT Lotze
    1277. PE Lovat
    1278. B Lu
    1279. B Lu
    1280. J Lu
    1281. Q Lu
    1282. SM Lu
    1283. S Lu
    1284. Y Lu
    1285. F Luciano
    1286. S Luckhart
    1287. JM Lucocq
    1288. P Ludovico
    1289. A Lugea
    1290. NW Lukacs
    1291. JJ Lum
    1292. AH Lund
    1293. H Luo
    1294. J Luo
    1295. S Luo
    1296. C Luparello
    1297. T Lyons
    1298. J Ma
    1299. Y Ma
    1300. Y Ma
    1301. Z Ma
    1302. J Machado
    1303. GM Machado-Santelli
    1304. F Macian
    1305. GC MacIntosh
    1306. JP MacKeigan
    1307. KF Macleod
    1308. JD MacMicking
    1309. LA MacMillan-Crow
    1310. F Madeo
    1311. M Madesh
    1312. J Madrigal-Matute
    1313. A Maeda
    1314. T Maeda
    1315. G Maegawa
    1316. E Maellaro
    1317. H Maes
    1318. M Magariños
    1319. K Maiese
    1320. TK Maiti
    1321. L Maiuri
    1322. MC Maiuri
    1323. CG Maki
    1324. R Malli
    1325. W Malorni
    1326. A Maloyan
    1327. F Mami-Chouaib
    1328. N Man
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    1330. EM Mandelkow
    1331. MA Mandell
    1332. AA Manfredi
    1333. SN Manié
    1334. C Manzoni
    1335. K Mao
    1336. Z Mao
    1337. ZW Mao
    1338. P Marambaud
    1339. AM Marconi
    1340. Z Marelja
    1341. G Marfe
    1342. M Margeta
    1343. E Margittai
    1344. M Mari
    1345. FV Mariani
    1346. C Marin
    1347. S Marinelli
    1348. G Mariño
    1349. I Markovic
    1350. R Marquez
    1351. AM Martelli
    1352. S Martens
    1353. KR Martin
    1354. SJ Martin
    1355. S Martin
    1356. MA Martin-Acebes
    1357. P Martín-Sanz
    1358. C Martinand-Mari
    1359. W Martinet
    1360. J Martinez
    1361. N Martinez-Lopez
    1362. U Martinez-Outschoorn
    1363. M Martínez-Velázquez
    1364. M Martinez-Vicente
    1365. WK Martins
    1366. H Mashima
    1367. JA Mastrianni
    1368. G Matarese
    1369. P Matarrese
    1370. R Mateo
    1371. S Matoba
    1372. N Matsumoto
    1373. T Matsushita
    1374. A Matsuura
    1375. T Matsuzawa
    1376. MP Mattson
    1377. S Matus
    1378. N Maugeri
    1379. C Mauvezin
    1380. A Mayer
    1381. D Maysinger
    1382. GD Mazzolini
    1383. MK McBrayer
    1384. K McCall
    1385. C McCormick
    1386. GM McInerney
    1387. SC McIver
    1388. S McKenna
    1389. JJ McMahon
    1390. IA McNeish
    1391. F Mechta-Grigoriou
    1392. JP Medema
    1393. DL Medina
    1394. K Megyeri
    1395. M Mehrpour
    1396. JL Mehta
    1397. Y Mei
    1398. UC Meier
    1399. AJ Meijer
    1400. A Meléndez
    1401. G Melino
    1402. S Melino
    1403. EJ de Melo
    1404. MA Mena
    1405. MD Meneghini
    1406. JA Menendez
    1407. R Menezes
    1408. L Meng
    1409. LH Meng
    1410. S Meng
    1411. R Menghini
    1412. AS Menko
    1413. RF Menna-Barreto
    1414. MB Menon
    1415. MA Meraz-Ríos
    1416. G Merla
    1417. L Merlini
    1418. AM Merlot
    1419. A Meryk
    1420. S Meschini
    1421. JN Meyer
    1422. MT Mi
    1423. CY Miao
    1424. L Micale
    1425. S Michaeli
    1426. C Michiels
    1427. AR Migliaccio
    1428. AS Mihailidou
    1429. D Mijaljica
    1430. K Mikoshiba
    1431. E Milan
    1432. L Miller-Fleming
    1433. GB Mills
    1434. IG Mills
    1435. G Minakaki
    1436. BA Minassian
    1437. XF Ming
    1438. F Minibayeva
    1439. EA Minina
    1440. JD Mintern
    1441. S Minucci
    1442. A Miranda-Vizuete
    1443. CH Mitchell
    1444. S Miyamoto
    1445. K Miyazawa
    1446. N Mizushima
    1447. K Mnich
    1448. B Mograbi
    1449. S Mohseni
    1450. LF Moita
    1451. M Molinari
    1452. M Molinari
    1453. AB Møller
    1454. B Mollereau
    1455. F Mollinedo
    1456. M Mongillo
    1457. MM Monick
    1458. S Montagnaro
    1459. C Montell
    1460. DJ Moore
    1461. MN Moore
    1462. R Mora-Rodriguez
    1463. PI Moreira
    1464. E Morel
    1465. MB Morelli
    1466. S Moreno
    1467. MJ Morgan
    1468. A Moris
    1469. Y Moriyasu
    1470. JL Morrison
    1471. LA Morrison
    1472. E Morselli
    1473. J Moscat
    1474. PL Moseley
    1475. S Mostowy
    1476. E Motori
    1477. D Mottet
    1478. JC Mottram
    1479. CE Moussa
    1480. VE Mpakou
    1481. H Mukhtar
    1482. JM Mulcahy Levy
    1483. S Muller
    1484. R Muñoz-Moreno
    1485. C Muñoz-Pinedo
    1486. C Münz
    1487. ME Murphy
    1488. JT Murray
    1489. A Murthy
    1490. IU Mysorekar
    1491. IR Nabi
    1492. M Nabissi
    1493. GA Nader
    1494. Y Nagahara
    1495. Y Nagai
    1496. K Nagata
    1497. A Nagelkerke
    1498. P Nagy
    1499. SR Naidu
    1500. S Nair
    1501. H Nakano
    1502. H Nakatogawa
    1503. M Nanjundan
    1504. G Napolitano
    1505. NI Naqvi
    1506. R Nardacci
    1507. DP Narendra
    1508. M Narita
    1509. AC Nascimbeni
    1510. R Natarajan
    1511. LC Navegantes
    1512. ST Nawrocki
    1513. TY Nazarko
    1514. VY Nazarko
    1515. T Neill
    1516. LM Neri
    1517. MG Netea
    1518. RT Netea-Maier
    1519. BM Neves
    1520. PA Ney
    1521. IP Nezis
    1522. HT Nguyen
    1523. HP Nguyen
    1524. AS Nicot
    1525. H Nilsen
    1526. P Nilsson
    1527. M Nishimura
    1528. I Nishino
    1529. M Niso-Santano
    1530. H Niu
    1531. RA Nixon
    1532. VC Njar
    1533. T Noda
    1534. AA Noegel
    1535. EM Nolte
    1536. E Norberg
    1537. KK Norga
    1538. SK Noureini
    1539. S Notomi
    1540. L Notterpek
    1541. K Nowikovsky
    1542. N Nukina
    1543. T Nürnberger
    1544. VB O'Donnell
    1545. T O'Donovan
    1546. PJ O'Dwyer
    1547. I Oehme
    1548. CL Oeste
    1549. M Ogawa
    1550. B Ogretmen
    1551. Y Ogura
    1552. YJ Oh
    1553. M Ohmuraya
    1554. T Ohshima
    1555. R Ojha
    1556. K Okamoto
    1557. T Okazaki
    1558. FJ Oliver
    1559. K Ollinger
    1560. S Olsson
    1561. DP Orban
    1562. P Ordonez
    1563. I Orhon
    1564. L Orosz
    1565. EJ O'Rourke
    1566. H Orozco
    1567. AL Ortega
    1568. E Ortona
    1569. LD Osellame
    1570. J Oshima
    1571. S Oshima
    1572. HD Osiewacz
    1573. T Otomo
    1574. K Otsu
    1575. JH Ou
    1576. TF Outeiro
    1577. DY Ouyang
    1578. H Ouyang
    1579. M Overholtzer
    1580. MA Ozbun
    1581. PH Ozdinler
    1582. B Ozpolat
    1583. C Pacelli
    1584. P Paganetti
    1585. G Page
    1586. G Pages
    1587. U Pagnini
    1588. B Pajak
    1589. SC Pak
    1590. K Pakos-Zebrucka
    1591. N Pakpour
    1592. Z Palková
    1593. F Palladino
    1594. K Pallauf
    1595. N Pallet
    1596. M Palmieri
    1597. SR Paludan
    1598. C Palumbo
    1599. S Palumbo
    1600. O Pampliega
    1601. H Pan
    1602. W Pan
    1603. T Panaretakis
    1604. A Pandey
    1605. A Pantazopoulou
    1606. Z Papackova
    1607. DL Papademetrio
    1608. I Papassideri
    1609. A Papini
    1610. N Parajuli
    1611. J Pardo
    1612. VV Parekh
    1613. G Parenti
    1614. JI Park
    1615. J Park
    1616. OK Park
    1617. R Parker
    1618. R Parlato
    1619. JB Parys
    1620. KR Parzych
    1621. JM Pasquet
    1622. B Pasquier
    1623. KB Pasumarthi
    1624. D Patschan
    1625. C Patterson
    1626. S Pattingre
    1627. S Pattison
    1628. A Pause
    1629. H Pavenstädt
    1630. F Pavone
    1631. Z Pedrozo
    1632. FJ Peña
    1633. MA Peñalva
    1634. M Pende
    1635. J Peng
    1636. F Penna
    1637. JM Penninger
    1638. A Pensalfini
    1639. S Pepe
    1640. GJ Pereira
    1641. PC Pereira
    1642. V Pérez-de la Cruz
    1643. ME Pérez-Pérez
    1644. D Pérez-Rodríguez
    1645. D Pérez-Sala
    1646. C Perier
    1647. A Perl
    1648. DH Perlmutter
    1649. I Perrotta
    1650. S Pervaiz
    1651. M Pesonen
    1652. JE Pessin
    1653. GJ Peters
    1654. M Petersen
    1655. I Petrache
    1656. BJ Petrof
    1657. G Petrovski
    1658. JM Phang
    1659. M Piacentini
    1660. M Pierdominici
    1661. P Pierre
    1662. V Pierrefite-Carle
    1663. F Pietrocola
    1664. FX Pimentel-Muiños
    1665. M Pinar
    1666. B Pineda
    1667. R Pinkas-Kramarski
    1668. M Pinti
    1669. P Pinton
    1670. B Piperdi
    1671. JM Piret
    1672. LC Platanias
    1673. HW Platta
    1674. ED Plowey
    1675. S Pöggeler
    1676. M Poirot
    1677. P Polčic
    1678. A Poletti
    1679. AH Poon
    1680. H Popelka
    1681. B Popova
    1682. I Poprawa
    1683. SM Poulose
    1684. J Poulton
    1685. SK Powers
    1686. T Powers
    1687. M Pozuelo-Rubio
    1688. K Prak
    1689. R Prange
    1690. M Prescott
    1691. M Priault
    1692. S Prince
    1693. RL Proia
    1694. T Proikas-Cezanne
    1695. H Prokisch
    1696. VJ Promponas
    1697. K Przyklenk
    1698. R Puertollano
    1699. S Pugazhenthi
    1700. L Puglielli
    1701. A Pujol
    1702. J Puyal
    1703. D Pyeon
    1704. X Qi
    1705. WB Qian
    1706. ZH Qin
    1707. Y Qiu
    1708. Z Qu
    1709. J Quadrilatero
    1710. F Quinn
    1711. N Raben
    1712. H Rabinowich
    1713. F Radogna
    1714. MJ Ragusa
    1715. M Rahmani
    1716. K Raina
    1717. S Ramanadham
    1718. R Ramesh
    1719. A Rami
    1720. S Randall-Demllo
    1721. F Randow
    1722. H Rao
    1723. VA Rao
    1724. BB Rasmussen
    1725. TM Rasse
    1726. EA Ratovitski
    1727. PE Rautou
    1728. SK Ray
    1729. B Razani
    1730. BH Reed
    1731. F Reggiori
    1732. M Rehm
    1733. AS Reichert
    1734. T Rein
    1735. DJ Reiner
    1736. E Reits
    1737. J Ren
    1738. X Ren
    1739. M Renna
    1740. JE Reusch
    1741. JL Revuelta
    1742. L Reyes
    1743. AR Rezaie
    1744. RI Richards
    1745. DR Richardson
    1746. C Richetta
    1747. MA Riehle
    1748. BH Rihn
    1749. Y Rikihisa
    1750. BE Riley
    1751. G Rimbach
    1752. MR Rippo
    1753. K Ritis
    1754. F Rizzi
    1755. E Rizzo
    1756. PJ Roach
    1757. J Robbins
    1758. M Roberge
    1759. G Roca
    1760. MC Roccheri
    1761. S Rocha
    1762. CM Rodrigues
    1763. CI Rodríguez
    1764. SR de Cordoba
    1765. N Rodriguez-Muela
    1766. J Roelofs
    1767. VV Rogov
    1768. TT Rohn
    1769. B Rohrer
    1770. D Romanelli
    1771. L Romani
    1772. PS Romano
    1773. MI Roncero
    1774. JL Rosa
    1775. A Rosello
    1776. KV Rosen
    1777. P Rosenstiel
    1778. M Rost-Roszkowska
    1779. KA Roth
    1780. G Roué
    1781. M Rouis
    1782. KM Rouschop
    1783. DT Ruan
    1784. D Ruano
    1785. DC Rubinsztein
    1786. EB Rucker
    1787. A Rudich
    1788. E Rudolf
    1789. R Rudolf
    1790. MA Ruegg
    1791. C Ruiz-Roldan
    1792. AA Ruparelia
    1793. P Rusmini
    1794. DW Russ
    1795. GL Russo
    1796. G Russo
    1797. R Russo
    1798. TE Rusten
    1799. V Ryabovol
    1800. KM Ryan
    1801. SW Ryter
    1802. DM Sabatini
    1803. M Sacher
    1804. C Sachse
    1805. MN Sack
    1806. J Sadoshima
    1807. P Saftig
    1808. R Sagi-Eisenberg
    1809. S Sahni
    1810. P Saikumar
    1811. T Saito
    1812. T Saitoh
    1813. K Sakakura
    1814. M Sakoh-Nakatogawa
    1815. Y Sakuraba
    1816. M Salazar-Roa
    1817. P Salomoni
    1818. AK Saluja
    1819. PM Salvaterra
    1820. R Salvioli
    1821. A Samali
    1822. AM Sanchez
    1823. JA Sánchez-Alcázar
    1824. R Sanchez-Prieto
    1825. M Sandri
    1826. MA Sanjuan
    1827. S Santaguida
    1828. L Santambrogio
    1829. G Santoni
    1830. CN Dos Santos
    1831. S Saran
    1832. M Sardiello
    1833. G Sargent
    1834. P Sarkar
    1835. S Sarkar
    1836. MR Sarrias
    1837. MM Sarwal
    1838. C Sasakawa
    1839. M Sasaki
    1840. M Sass
    1841. K Sato
    1842. M Sato
    1843. J Satriano
    1844. N Savaraj
    1845. S Saveljeva
    1846. L Schaefer
    1847. UE Schaible
    1848. M Scharl
    1849. HM Schatzl
    1850. R Schekman
    1851. W Scheper
    1852. A Schiavi
    1853. HM Schipper
    1854. H Schmeisser
    1855. J Schmidt
    1856. I Schmitz
    1857. BE Schneider
    1858. EM Schneider
    1859. JL Schneider
    1860. EA Schon
    1861. MJ Schönenberger
    1862. AH Schönthal
    1863. DF Schorderet
    1864. B Schröder
    1865. S Schuck
    1866. RJ Schulze
    1867. M Schwarten
    1868. TL Schwarz
    1869. S Sciarretta
    1870. K Scotto
    1871. AI Scovassi
    1872. RA Screaton
    1873. M Screen
    1874. H Seca
    1875. S Sedej
    1876. L Segatori
    1877. N Segev
    1878. PO Seglen
    1879. JM Seguí-Simarro
    1880. J Segura-Aguilar
    1881. E Seki
    1882. C Sell
    1883. I Seiliez
    1884. CF Semenkovich
    1885. GL Semenza
    1886. U Sen
    1887. AL Serra
    1888. A Serrano-Puebla
    1889. H Sesaki
    1890. T Setoguchi
    1891. C Settembre
    1892. JJ Shacka
    1893. AN Shajahan-Haq
    1894. IM Shapiro
    1895. S Sharma
    1896. H She
    1897. CK Shen
    1898. CC Shen
    1899. HM Shen
    1900. S Shen
    1901. W Shen
    1902. R Sheng
    1903. X Sheng
    1904. ZH Sheng
    1905. TG Shepherd
    1906. J Shi
    1907. Q Shi
    1908. Q Shi
    1909. Y Shi
    1910. S Shibutani
    1911. K Shibuya
    1912. Y Shidoji
    1913. JJ Shieh
    1914. CM Shih
    1915. Y Shimada
    1916. S Shimizu
    1917. DW Shin
    1918. ML Shinohara
    1919. M Shintani
    1920. T Shintani
    1921. T Shioi
    1922. K Shirabe
    1923. R Shiri-Sverdlov
    1924. O Shirihai
    1925. GC Shore
    1926. CW Shu
    1927. D Shukla
    1928. AA Sibirny
    1929. V Sica
    1930. CJ Sigurdson
    1931. EM Sigurdsson
    1932. PS Sijwali
    1933. B Sikorska
    1934. WA Silveira
    1935. S Silvente-Poirot
    1936. GA Silverman
    1937. J Simak
    1938. T Simmet
    1939. AK Simon
    1940. HU Simon
    1941. C Simone
    1942. M Simons
    1943. A Simonsen
    1944. R Singh
    1945. SV Singh
    1946. SK Singh
    1947. D Sinha
    1948. S Sinha
    1949. FA Sinicrope
    1950. A Sirko
    1951. K Sirohi
    1952. BJ Sishi
    1953. A Sittler
    1954. PM Siu
    1955. E Sivridis
    1956. A Skwarska
    1957. R Slack
    1958. I Slaninová
    1959. N Slavov
    1960. SS Smaili
    1961. KS Smalley
    1962. DR Smith
    1963. SJ Soenen
    1964. SA Soleimanpour
    1965. A Solhaug
    1966. K Somasundaram
    1967. JH Son
    1968. A Sonawane
    1969. C Song
    1970. F Song
    1971. HK Song
    1972. JX Song
    1973. W Song
    1974. KY Soo
    1975. AK Sood
    1976. TW Soong
    1977. V Soontornniyomkij
    1978. M Sorice
    1979. F Sotgia
    1980. DR Soto-Pantoja
    1981. A Sotthibundhu
    1982. MJ Sousa
    1983. HP Spaink
    1984. PN Span
    1985. A Spang
    1986. JD Sparks
    1987. PG Speck
    1988. SA Spector
    1989. CD Spies
    1990. W Springer
    1991. DS Clair
    1992. A Stacchiotti
    1993. B Staels
    1994. MT Stang
    1995. DT Starczynowski
    1996. P Starokadomskyy
    1997. C Steegborn
    1998. JW Steele
    1999. L Stefanis
    2000. J Steffan
    2001. CM Stellrecht
    2002. H Stenmark
    2003. TM Stepkowski
    2004. ST Stern
    2005. C Stevens
    2006. BR Stockwell
    2007. V Stoka
    2008. Z Storchova
    2009. B Stork
    2010. V Stratoulias
    2011. DJ Stravopodis
    2012. P Strnad
    2013. AM Strohecker
    2014. AL Ström
    2015. P Stromhaug
    2016. J Stulik
    2017. YX Su
    2018. Z Su
    2019. CS Subauste
    2020. S Subramaniam
    2021. CM Sue
    2022. SW Suh
    2023. X Sui
    2024. S Sukseree
    2025. D Sulzer
    2026. FL Sun
    2027. J Sun
    2028. J Sun
    2029. SY Sun
    2030. Y Sun
    2031. Y Sun
    2032. Y Sun
    2033. V Sundaramoorthy
    2034. J Sung
    2035. H Suzuki
    2036. K Suzuki
    2037. N Suzuki
    2038. T Suzuki
    2039. YJ Suzuki
    2040. MS Swanson
    2041. C Swanton
    2042. K Swärd
    2043. G Swarup
    2044. ST Sweeney
    2045. PW Sylvester
    2046. Z Szatmari
    2047. E Szegezdi
    2048. PW Szlosarek
    2049. H Taegtmeyer
    2050. M Tafani
    2051. E Taillebourg
    2052. SW Tait
    2053. K Takacs-Vellai
    2054. Y Takahashi
    2055. S Takáts
    2056. G Takemura
    2057. N Takigawa
    2058. NJ Talbot
    2059. E Tamagno
    2060. J Tamburini
    2061. CP Tan
    2062. L Tan
    2063. ML Tan
    2064. M Tan
    2065. YJ Tan
    2066. K Tanaka
    2067. M Tanaka
    2068. D Tang
    2069. D Tang
    2070. G Tang
    2071. I Tanida
    2072. K Tanji
    2073. BA Tannous
    2074. JA Tapia
    2075. I Tasset-Cuevas
    2076. M Tatar
    2077. I Tavassoly
    2078. N Tavernarakis
    2079. A Taylor
    2080. GS Taylor
    2081. GA Taylor
    2082. JP Taylor
    2083. MJ Taylor
    2084. EV Tchetina
    2085. AR Tee
    2086. F Teixeira-Clerc
    2087. S Telang
    2088. T Tencomnao
    2089. BB Teng
    2090. RJ Teng
    2091. F Terro
    2092. G Tettamanti
    2093. AL Theiss
    2094. AE Theron
    2095. KJ Thomas
    2096. MP Thomé
    2097. PG Thomes
    2098. A Thorburn
    2099. J Thorner
    2100. T Thum
    2101. M Thumm
    2102. TL Thurston
    2103. L Tian
    2104. A Till
    2105. JP Ting
    2106. VI Titorenko
    2107. L Toker
    2108. S Toldo
    2109. SA Tooze
    2110. I Topisirovic
    2111. ML Torgersen
    2112. L Torosantucci
    2113. A Torriglia
    2114. MR Torrisi
    2115. C Tournier
    2116. R Towns
    2117. V Trajkovic
    2118. LH Travassos
    2119. G Triola
    2120. DN Tripathi
    2121. D Trisciuoglio
    2122. R Troncoso
    2123. IP Trougakos
    2124. AC Truttmann
    2125. KJ Tsai
    2126. MP Tschan
    2127. YH Tseng
    2128. T Tsukuba
    2129. A Tsung
    2130. AS Tsvetkov
    2131. S Tu
    2132. HY Tuan
    2133. M Tucci
    2134. DA Tumbarello
    2135. B Turk
    2136. V Turk
    2137. RF Turner
    2138. AA Tveita
    2139. SC Tyagi
    2140. M Ubukata
    2141. Y Uchiyama
    2142. A Udelnow
    2143. T Ueno
    2144. M Umekawa
    2145. R Umemiya-Shirafuji
    2146. BR Underwood
    2147. C Ungermann
    2148. RP Ureshino
    2149. R Ushioda
    2150. VN Uversky
    2151. NL Uzcátegui
    2152. T Vaccari
    2153. MI Vaccaro
    2154. L Váchová
    2155. H Vakifahmetoglu-Norberg
    2156. R Valdor
    2157. EM Valente
    2158. F Vallette
    2159. AM Valverde
    2160. G Van den Berghe
    2161. L Van Den Bosch
    2162. GR van den Brink
    2163. FG van der Goot
    2164. IJ van der Klei
    2165. LJ van der Laan
    2166. WG van Doorn
    2167. M van Egmond
    2168. KL van Golen
    2169. L Van Kaer
    2170. M van Lookeren Campagne
    2171. P Vandenabeele
    2172. W Vandenberghe
    2173. I Vanhorebeek
    2174. I Varela-Nieto
    2175. MH Vasconcelos
    2176. R Vasko
    2177. DG Vavvas
    2178. I Vega-Naredo
    2179. G Velasco
    2180. AD Velentzas
    2181. PD Velentzas
    2182. T Vellai
    2183. E Vellenga
    2184. MH Vendelbo
    2185. K Venkatachalam
    2186. N Ventura
    2187. S Ventura
    2188. PS Veras
    2189. M Verdier
    2190. BG Vertessy
    2191. A Viale
    2192. M Vidal
    2193. HL Vieira
    2194. RD Vierstra
    2195. N Vigneswaran
    2196. N Vij
    2197. M Vila
    2198. M Villar
    2199. VH Villar
    2200. J Villarroya
    2201. C Vindis
    2202. G Viola
    2203. MT Viscomi
    2204. G Vitale
    2205. DT Vogl
    2206. OV Voitsekhovskaja
    2207. C von Haefen
    2208. K von Schwarzenberg
    2209. DE Voth
    2210. V Vouret-Craviari
    2211. K Vuori
    2212. JM Vyas
    2213. C Waeber
    2214. CL Walker
    2215. MJ Walker
    2216. J Walter
    2217. L Wan
    2218. X Wan
    2219. B Wang
    2220. C Wang
    2221. CY Wang
    2222. C Wang
    2223. C Wang
    2224. C Wang
    2225. D Wang
    2226. F Wang
    2227. F Wang
    2228. G Wang
    2229. HJ Wang
    2230. H Wang
    2231. HG Wang
    2232. H Wang
    2233. HD Wang
    2234. J Wang
    2235. J Wang
    2236. M Wang
    2237. MQ Wang
    2238. PY Wang
    2239. P Wang
    2240. RC Wang
    2241. S Wang
    2242. TF Wang
    2243. X Wang
    2244. XJ Wang
    2245. XW Wang
    2246. X Wang
    2247. X Wang
    2248. Y Wang
    2249. Y Wang
    2250. Y Wang
    2251. YJ Wang
    2252. Y Wang
    2253. Y Wang
    2254. YT Wang
    2255. Y Wang
    2256. ZN Wang
    2257. P Wappner
    2258. C Ward
    2259. DM Ward
    2260. G Warnes
    2261. H Watada
    2262. Y Watanabe
    2263. K Watase
    2264. TE Weaver
    2265. CD Weekes
    2266. J Wei
    2267. T Weide
    2268. CC Weihl
    2269. G Weindl
    2270. SN Weis
    2271. L Wen
    2272. X Wen
    2273. Y Wen
    2274. B Westermann
    2275. CM Weyand
    2276. AR White
    2277. E White
    2278. JL Whitton
    2279. AJ Whitworth
    2280. J Wiels
    2281. F Wild
    2282. ME Wildenberg
    2283. T Wileman
    2284. DS Wilkinson
    2285. S Wilkinson
    2286. D Willbold
    2287. C Williams
    2288. K Williams
    2289. PR Williamson
    2290. KF Winklhofer
    2291. SS Witkin
    2292. SE Wohlgemuth
    2293. T Wollert
    2294. EJ Wolvetang
    2295. E Wong
    2296. GW Wong
    2297. RW Wong
    2298. VK Wong
    2299. EA Woodcock
    2300. KL Wright
    2301. C Wu
    2302. D Wu
    2303. GS Wu
    2304. J Wu
    2305. J Wu
    2306. M Wu
    2307. M Wu
    2308. S Wu
    2309. WK Wu
    2310. Y Wu
    2311. Z Wu
    2312. CP Xavier
    2313. RJ Xavier
    2314. GX Xia
    2315. T Xia
    2316. W Xia
    2317. Y Xia
    2318. H Xiao
    2319. J Xiao
    2320. S Xiao
    2321. W Xiao
    2322. CM Xie
    2323. Z Xie
    2324. Z Xie
    2325. M Xilouri
    2326. Y Xiong
    2327. C Xu
    2328. C Xu
    2329. F Xu
    2330. H Xu
    2331. H Xu
    2332. J Xu
    2333. J Xu
    2334. J Xu
    2335. L Xu
    2336. X Xu
    2337. Y Xu
    2338. Y Xu
    2339. ZX Xu
    2340. Z Xu
    2341. Y Xue
    2342. T Yamada
    2343. A Yamamoto
    2344. K Yamanaka
    2345. S Yamashina
    2346. S Yamashiro
    2347. B Yan
    2348. B Yan
    2349. X Yan
    2350. Z Yan
    2351. Y Yanagi
    2352. DS Yang
    2353. JM Yang
    2354. L Yang
    2355. M Yang
    2356. PM Yang
    2357. P Yang
    2358. Q Yang
    2359. W Yang
    2360. WY Yang
    2361. X Yang
    2362. Y Yang
    2363. Y Yang
    2364. Z Yang
    2365. Z Yang
    2366. MC Yao
    2367. PJ Yao
    2368. X Yao
    2369. Z Yao
    2370. Z Yao
    2371. LS Yasui
    2372. M Ye
    2373. B Yedvobnick
    2374. B Yeganeh
    2375. ES Yeh
    2376. PL Yeyati
    2377. F Yi
    2378. L Yi
    2379. XM Yin
    2380. CK Yip
    2381. YM Yoo
    2382. YH Yoo
    2383. SY Yoon
    2384. K Yoshida
    2385. T Yoshimori
    2386. KH Young
    2387. H Yu
    2388. JJ Yu
    2389. JT Yu
    2390. J Yu
    2391. L Yu
    2392. WH Yu
    2393. XF Yu
    2394. Z Yu
    2395. J Yuan
    2396. ZM Yuan
    2397. BY Yue
    2398. J Yue
    2399. Z Yue
    2400. DN Zacks
    2401. E Zacksenhaus
    2402. N Zaffaroni
    2403. T Zaglia
    2404. Z Zakeri
    2405. V Zecchini
    2406. J Zeng
    2407. M Zeng
    2408. Q Zeng
    2409. AS Zervos
    2410. DD Zhang
    2411. F Zhang
    2412. G Zhang
    2413. GC Zhang
    2414. H Zhang
    2415. H Zhang
    2416. H Zhang
    2417. H Zhang
    2418. J Zhang
    2419. J Zhang
    2420. J Zhang
    2421. J Zhang
    2422. JP Zhang
    2423. L Zhang
    2424. L Zhang
    2425. L Zhang
    2426. L Zhang
    2427. MY Zhang
    2428. X Zhang
    2429. XD Zhang
    2430. Y Zhang
    2431. Y Zhang
    2432. Y Zhang
    2433. Y Zhang
    2434. Y Zhang
    2435. M Zhao
    2436. WL Zhao
    2437. X Zhao
    2438. YG Zhao
    2439. Y Zhao
    2440. Y Zhao
    2441. YX Zhao
    2442. Z Zhao
    2443. ZJ Zhao
    2444. D Zheng
    2445. XL Zheng
    2446. X Zheng
    2447. B Zhivotovsky
    2448. Q Zhong
    2449. GZ Zhou
    2450. G Zhou
    2451. H Zhou
    2452. SF Zhou
    2453. XJ Zhou
    2454. H Zhu
    2455. H Zhu
    2456. WG Zhu
    2457. W Zhu
    2458. XF Zhu
    2459. Y Zhu
    2460. SM Zhuang
    2461. X Zhuang
    2462. E Ziparo
    2463. CE Zois
    2464. T Zoladek
    2465. WX Zong
    2466. A Zorzano
    2467. SM Zughaier
    (2016)
    Autophagy 12:1–222.
    https://doi.org/10.1080/15548627.2015.1100356