Lipid droplets modulate proteostasis, SQST-1/SQSTM1 dynamics, and lifespan in C. elegans

Summary In several long-lived Caenorhabditis elegans strains, such as insulin/IGF-1 receptor daf-2 mutants, enhanced proteostatic mechanisms are accompanied by elevated intestinal lipid stores, but their role in longevity is unclear. Here, while determining the regulatory network of the selective autophagy receptor SQST-1/SQSTM1, we uncovered an important role for lipid droplets in proteostasis and longevity. Using genome-wide RNAi screening, we identified several SQST-1 modulators, including lipid droplets-associated and aggregation-prone proteins. Expansion of intestinal lipid droplets by silencing the conserved cytosolic triacylglycerol lipase gene atgl-1/ATGL enhanced autophagy, and extended lifespan. Notably, a substantial amount of ubiquitinated proteins were found on lipid droplets. Reducing lipid droplet levels exacerbated the proteostatic collapse when autophagy or proteasome function was compromised, and significantly reduced the lifespan of long-lived daf-2 animals. Altogether, our study uncovered a key role for lipid droplets in C. elegans as a proteostatic mediator that modulates ubiquitinated protein accumulation, facilitates autophagy, and promotes longevity.


INTRODUCTION
One of the major hallmarks of aging is the accumulation of damaged proteins that progressively compartmentalize in inclusions and aggregates. 1 In the nematode Caenorhabditis elegans, numerous proteins display impaired solubility with age. 2,3This phenomenon suggests that mechanisms that mediate proteostasis, such as chaperone-mediated folding and stabilization, actively contribute to somatic maintenance by preventing the collapse of the proteome. 4,5The enhanced proteasomal 6 and autophagic 7,8 capacities, the increased chaperone function, 9 and alterations in ribosomal biogenesis 10 and function 11,12 in long-lived nematodes support the notion that the balance between synthesis, folding, and efficient clearance of proteins is important for conferring organismal longevity.
Generally, proteins destined for degradation are tagged via poly-ubiquitination and recognized by ubiquitin binding domain-containing proteins that direct cargo toward proteasomal or autophagic degradation.The autophagy receptor, Sequestosome-1 (SQST-1/SQSTM1) is a well-established and conserved mediator of cargo recognition, which includes ubiquitinated targets. 13The SQST-1/SQSTM1-cargo complex can interact with the autophagosome proteins LGG-1/GABARAP and LGG-2/LC3 to enable cargo sequestration in the nascent autophagosome. 146][17] Additionally, selective autophagy of mitochondria has been linked to longevity, 18 suggesting that autophagic degradation of specific cargos and organelles is beneficial against organismal aging.Although SQST-1/SQSTM1 has been implicated in the selective clearance of aggregated proteins, 19 much remains to be understood about the modulation of this autophagy receptor, its cargoes, and the nature and extent of its contribution to lifespan.
In nematodes, intestinal cells have a unique role in autophagy-mediated longevity as they manage nutrient influx and signals from neurons 20 and the germline 21 to coordinate appropriate responses necessary for organismal survival.In particular, intestinal autophagy genes are required for lifespan extension 22 by maintaining intestinal tissue integrity and proteostasis.1][32][33] Accordingly, redistributing lipids intended for secreted lipoproteins to intestinal lipid droplets is sufficient guide for selective autophagy, we developed a tandem reporter system expressing SQST-1 fused to both GFP and RFP 7 (GFP signal is quenched in low pH environments).We confirmed that the conversion of tandem SQST-1 into an RFP-only signal is reduced in lgg-1or lgg-2-silenced animals (Figure S1E) and autophagy-deficient atg-18 mutants (Figure S1F).While relatively modest at 20 C (Figures 1H and  1I), increasing temperature up to 30 C for 24 h significantly enhanced the conversion to the RFP-only SQST-1 signal in the SQST-1 tandem reporter, suggesting that selective autophagy is induced with heat stress.Substantial temperature-dependent intestinal and neuronal accumulation of SQST-1 occurred during aging, and over-expressing SQST-1 at 25 C was also accompanied by higher levels of total polyubiquitinated proteins (Figures 1A-1F inset; S1G, and S1H).While over-expressing SQST-1 was detrimental at 25 C, so was the loss of sqst-1 (Figures S1I and S1J), suggesting that SQST-1 function has a temperature-specific role in the lifespan of wild-type animals.Expression and steady-state levels of SQST-1 likely need to be tightly coordinated with cargo targeting and more importantly, with the rate of protein degradation systems (i.e., autophagy and proteasome).Altogether, we found that solely increasing SQST-1 is detrimental for lifespan at 25 C and inconsequential at 20 C, indicating a temperature-dependent effect on SQST-1 dynamics in aging.

Proteins that bind lipid droplets modulate SQST-1 dynamics
In order to better understand how SQST-1 is regulated, we opted for an unbiased genome-wide RNAi screening approach in an integrated SQST-1 over-expressing strain (psqst-1::sqst-1::gfp) at 25 C during development.SQST-1 modulators were subsequently validated by gene silencing in a separate over-expressing strain (psqst-1::sqst-1::rfp) during development and adulthood.Interestingly, the vast majority of modifiers led to the intestinal accumulation of fluorescently tagged SQST-1 in both development and adulthood (Table S1).A substantial portion of genetic modifiers clustered in ribosomal-related proteins, including proteins coding for small and large subunits, suggesting that ribosomal assembly and function substantially impact SQST-1 dynamics (Figure 2A; Table S1).This is in line with studies in C. elegans 2 and killifish, 39 highlighting the age-related instability of several ribosomal proteins and consequent ribosomal mis-assembly.Loss of ribosome subunit RPL-43 was also found to increase SQST-1 accumulation during development. 40Since SQSTM1 was also recently reported to associate with lipid droplets in macrophages, 41 we wondered if SQST-1 might also associate with lipid droplets.We co-expressed SQST-1:RFP and the  S1  and S2.
lipid resident protein DHS-3 fused to GFP 42 in nematodes and we found that close to 50% of SQST-1:RFP localizes to lipid droplets when autophagy is inhibited (Figures 2B and S2A).Thus, we investigated whether SQST-1 modulators may also be related to lipid droplet metabolism.We compared our SQST-1 modulators to proteins that have been found to bind lipid droplets 43 and found significant overlap (p < 5.745e-60) (Figure 2C).Interestingly, several proteins prone to age-dependent aggregation 2 also modulated SQST-1 accumulation (p < 6.030e-10) (Figure 2C).Overall, 17 SQST-1 modulators have the propensity to both aggregate with age and bind lipid droplets (Figure 2C; Table S2).Several ribosomal subunits (rps and rpl genes) and mRNA-related proteins (inf-1 and pab-1) emerged, along with the HSP70 chaperone family member hsp-1/HSPA8, indicating that ribosomal assembly, translation initiation, and protein folding are important regulators of SQST-1 dynamics (Figure 2D).SQST-1:RFP accumulation upon silencing of these modifiers was mostly gonadal and intestinal (Figures 2D and  S2B).Interestingly, the expression of sqst-1 was differentially regulated by the 17 SQST-1 modulators whereas silencing large ribosomal proteins generally increased sqst-1 expression (Figure S2B).This observation highlights that sqst-1 expression is potentially sensitive to ribosomal protein stoichiometry.Here, our comparative analysis suggests that age-related SQST-1 accumulation may be partly attributed to altered interactions between lipid droplets and progressively unstable protein complexes like the ribosome.
As our lifespan analyses highlighted novel proteostatic and longevity roles for lipid droplets, we reasoned that long-lived animals with large lipid stores should accumulate less intestinal SQST-1.Strikingly, daf-2(e1370) expressing SQST-1:GFP (Figure 3I) or SQST-1:RFP (Figure S2I) accumulated negligible intestinal SQST-1 during aging (GFP signal in mid-section was entirely gonadal, see wild-type comparison in Figure 1B).In addition, SQST-1 over-expression did not significantly affect the long lifespan of daf-2 animals (Figure 3I).Loss of sqst-1 did not affect the lifespan of daf-2 animals (Figure S2J), as previously shown, 16 highlighting that SQST-1 function becomes less important at 25 C for the lifespan of organisms with relatively stable proteomes and high lipid stores.The extent of heat-induced increase in SQST-1-mediated selective autophagy was also attenuated in daf-2 animals (Figure S2K) compared to wild-type (Figure 1H), suggesting that elevated lipid droplets may buffer the need for SQST-1 function during heat stress.Silencing atgl-1 in daf-2 animals enhanced their intestinal lipid stores and further extended their lifespan (Figure 3J), indicating that elevated lipid droplet accumulation can also extend lifespan in animals with enhanced proteostasis.Altogether, our data present an important and previously unrecognized role for lipid droplets in SQST-1 dynamics and longevity.

Silencing atgl-1 elicits limited changes in gene expression
Lipid droplets have been recently shown to coordinate transcriptional programs by sequestering factors that modulate transcription. 50Since HSF-1 was required for lifespan extension by atgl-1 silencing, we hypothesized that the lipid droplet increase associated with atgl-1 silencing might affect the expression of HSF-1-regulated targets.Using RNA sequencing (RNA-seq), we found that enhancing lipid stores by silencing atgl-1 in WT or daf-2 animals had limited effect on global transcription.Silencing atgl-1 resulted in 13 overlapping differentially expressed genes (DEG) in wild-type animals and daf-2 mutants of which 6 genes besides atgl-1 are down-regulated in both cases (Figures S3A and  S3B).The mRNA levels of sqst-1 remained unchanged by atgl-1 silencing (Figure S3C).Altering atgl-1 levels (silencing or over-expression) led to differential expression of 50 overlapping genes, with no discernable transcription factor signature (Figures S3D and S3E).In addition, expression of sqst-1 was unchanged in wild-type animals with low or high levels of atgl-1 (Figure S3E).Overall, we concluded that transcriptional regulation may not contribute significantly to the proteostatic-enhancing and lifespan-extending effects of lipid droplets.Therefore, lipid droplets may impact proteostasis via a more direct mechanism on the proteome itself.

Lipid droplets enhance proteostasis by modulating the accumulation of ubiquitinated proteins
The emerging connection between lipid droplet stores and proteome stability led us to investigate how lipid droplet loss affects proteostasis and SQST-1 abundance.First, we tested whether lipid droplet depletion affects lifespan using ATGL-1:GFP over-expressing animals. 51Opposite of the effect observed at 20 C (Table S3), 52 we found that over-expressing ATGL-1 was detrimental to lifespan at 25 C (Figure 4A) and led to a significant increase in SQST-1 intestinal accumulation accompanied by lower lipid stores (Figure 4B), suggesting that loss of lipid droplet stores can interfere with SQST-1 dynamics.When autophagy or proteasome function was reduced by silencing autophagosome protein lgg-1 or proteasome subunit rpn-6.1,respectively, ubiquitinated protein levels were higher in animals with lower lipid stores, suggesting that lipid droplets may buffer the proteome and facilitate the processing of unstable or misfolded proteins (Figures 4C and S4A).Strikingly, analyzing lipid droplets revealed substantial accumulation of ubiquitinated proteins in lipid droplet-enriched fractions (Figures 4D and S4B), indicating that lipid droplets contain a significant amount of proteins potentially bound for degradation.Lipid droplet-associated accumulation of ubiquitinated proteins was increased when autophagic or proteasomal degradation was reduced (Figure 4D), suggesting that lipid droplets have (H and I) (H) Levels of autophagosomes and autolysosomes were measured in animals expressing the tandem reporter mCherry:GFP::LGG-1 7 after feeding Day 1 animals with control bacteria or bacteria expressing dsRNA against atgl-1 for 2 days at 25 C. n = 10 per condition GSD t-test # p < 0.06 (I).Lifespan analysis of daf-2(e1370) and daf-2;SQST-1::GFP animals raised at 20 C and then grown at 25 C during adulthood on OP50 E. coli (with representative image of Day 5 animals, comparative image of wild-type animals in Figure 1B).(J) daf-2(e1370) animals were raised at 20 C on OP50 E. coli and then grown at 25 C during adulthood on control bacteria or bacteria expressing dsRNA against atgl-1 (n = 100).Corresponding images of Day 5 animals stained with ORO demonstrating intestinal lipid store accretion under atgl-1 RNAi (blue borders).Details about lifespan analyses and repeats are available in Tables S3 and S4, Mantel-Cox log rank.n.s.: not significant, **p < 0.01,****p < 0.001.
the capacity to harbor many degradation cargoes, which may become particularly relevant for proteostasis when autophagic and proteasomal systems are failing during aging.
The association and function of ATGL-1 with lipid droplets was recently found to be antagonized by the AAA-ATPase CDC-48/VCP. 53CDC-48/VCP is best known for its role in ER-associated degradation machinery, 54 but it has been associated with other functions including endocytosis 55 and more recently autophagy itself. 56CDC-48/VCP helps unfold unstable, ubiquitinated, and proteasomal degradationbound proteins in concert with heat shock protein HSP-70, a key regulated target of HSF-1. 57Thus, we assayed the effect of the loss of CDC-48 on SQST-1 and ATGL-1 levels using fluorescent reporters.Silencing cdc-48.2increased the levels of ATGL-1:GFP (Figure S4C) and led to the accumulation of SQST-1:RFP (Figure S4D).In mammalian cells, loss of proteasome function drives SQSTM1 to divert cargo to selective autophagy, 58 but stimulating proteasomal processing by over-expressing of VCP can reduce SQSTM1 accumulation. 59Here, we that CDC-48 functions related to processing ubiquitinated targets and autophagy may underlie the ability of nematodes with elevated lipid droplets to attenuate SQST-1 accumulation and modulate lifespan.Accordingly, silencing atgl-1 in cdc-48.1 or cdc-48.2mutants failed to significantly extend lifespan (Figure S4E; Table S4), indicating that lipid droplet-mediated lifespan extension requires functional CDC-48.
As lipid droplet size increases, its surface also increases, and it is possible that the capacity of this organelle to bind and stabilize proteins may also increase as well.Enhancing lipid droplet stores by silencing atgl-1 reduced the overall accumulation of ubiquitinated proteins, in particular in the lower solubility (5% SDS soluble) fraction (Figure 4E), suggesting that typically insoluble proteins are less likely to be  S3 and S4, Mantel-Cox log rank.n.s.: not significant, *p < 0.05,****p < 0.001.ubiquitinated when lipid droplets abound.Notably, the types of proteins that aggregate with age differ between wild-type and long-lived daf-2 mutants, as the latter tends to accumulate aggregating proteins that are less hydrophobic than those aggregating in wild-type animals. 4educing lipid droplet stores genetically by silencing lipogenic genes sbp-1(SREBP2), lpin-1(LIPN1) or fasn-1(FASN) enhanced SQST-1 accumulation in wild-type animals (Figure S4F).Similarly, loss of lipid droplet stores by lpin-1 in daf-2 resulted in increased SQST-1 accumulation and overall protein ubiquitination (Figures 4F, 4G, and S4G).Ultimately, in wild-type and more significantly in daf-2 animals, silencing of lpin-1 reduced their lifespan (Figure 4H).As proteostasis failure is a feature of neurodegeneration, we tested the effect of increasing lipid droplet levels in proteotoxic contexts.Animals intestinally expressing an aggregating poly-glutamine protein fused to YFP (Q44:YFP) 60 had reduced numbers of aggregates when atgl-1 was silenced (Figure S4H).Reducing the expression of atgl-1 in a muscle-expressing proteotoxic amyloid protein Ab-42 resulted in a marked protection against aggregation-associated paralysis 61 (Figure S4I).Altogether, our data demonstrate that lipid droplets are important for proteostasis and contribute to lifespan by facilitating autophagy and stabilizing the proteome.

DISCUSSION
Decades of aging research has uncovered key longevity-regulating pathways in C. elegans, 48 yet the role of lipid droplets in the lifespan extension of several established long-lived nematodes has remained unresolved.Here, while studying the regulation of the selective autophagy receptor SQST-1, we unexpectedly uncovered a direct role for lipid droplets in proteostasis and lifespan.Several SQST-1 modulators associate with lipid droplets and aggregate with age, including ribosomal, translation-related, and folding-related proteins. 43Ribosomal assembly dysfunction due to loss in subunit stoichiometry may burden the autophagy machinery, and contribute to age-related proteotoxicity. 39Importantly, lipid droplet accumulation, by silencing cytosolic triacylglycerol lipase atgl-1/ATGL, mitigates the progressive age-related SQST-1 accumulation, a benefit recapitulated in long-lived, lipid droplet-rich daf-2 mutant animals.Lipid droplets are cytoprotective as they prevent aberrant SQST-1 accumulation, in part via the functioning of AAA-ATPase CDC-48/VCP which processes ubiquitinated proteins for proteasomal degradation. 62Strikingly, we found that a substantial portion of soluble ubiquitinated proteins in nematodes was associated with lipid droplets, suggesting that unstable proteins bound for degradation may be stabilized by the surface of lipid droplets.Our findings also provide support to the importance of the emerging lipid-droplet-mediated protein degradation process 63,64 in longevity and aging.Altogether, our study strengthens the emerging concept that lipid droplets serve as a buffer for proteostasis 47 by stabilizing proteomes and coordinating protein degradation machineries.
Understanding the regulation of SQST-1/SQSTM1 during aging and in different disease contexts is important in order to determine the validity of stimulating SQSTM1-mediated selective autophagy as a therapeutic strategy to improve proteostasis in age-related diseases. 65Here, we find that over-expressing SQST-1 had no effect on the lifespan of wild-type animals at 20 C 16 whereas elevated SQST-1 level at 25 C was detrimental.While lab-to-lab variations in lifespan analyses can occur, we tested a variety of over-expressing strains for SQST-1 with different tags in order to more broadly assess the impact of high levels of SQST-1 on longevity.In hindsight, one could have predicted that over-expressing a ubiquitin-binding protein with a propensity for oligomerization with ubiquitinated cargoes 66 might not be necessarily advantageous.This is particularly relevant in C. elegans which is characterized by a proteome nearing its solubility limit 2,3 and displaying overall proteome instability and aggregated protein accumulation during aging. 5SQST-1 over-expression was primarily visible in neuronal, gonadal and intestinal cells and accumulated in a temperature-dependent manner and progressively during aging.The inability of stressed and aging animals to process ubiquitinated cargos bound for proteasomal or autophagic degradation may lead to a gradual build-up of SQST-1.Accordingly, SQST-1 over-expression may over-sensitize the animals to unstable, ubiquitination-prone proteins, posing an additional challenge to the autophagic machinery.This is exemplified by recent evidence that reducing SQST-1 specifically in the neurons of a nematode model of ALS is protective, 67 possibly since mutant FUsed in Sarcoma (FUS) expression promotes SQST-1 accumulation, which exacerbates proteotoxicity and neurodegeneration.Notably, SQSTM1 can phase separate when interacting with ubiquitinated cargoes, 68 but the impact of the formation of these condensates on overall proteostasis is unclear.
Lipid droplet accumulation is a prominent and under-explored phenotype of several long-lived nematodes, including well-established models such as insulin/IGF-1 receptor daf-2 mutants, germline-less glp-1 animals and protein synthesis rsks-1 mutants, 69 as well as in long-lived animals with reduced nuclear protein export. 70In addition, lifespan extension by inhibiting intestinal lipid secretion mediated by vitellogenins results in lipid redistribution to intestinal lipid droplet stores, 29 a process believed to generate precursors for lysosomallyderived lipid signals. 28While enhanced lysosomal lipolysis and lipophagy may stimulate longevity-associated lipid signaling, we found that overactive cytosolic lipolysis decreases the ability of cells to maintain a stable proteome, which may burden the selective autophagy machinery.Our findings support that lipid droplets are much more than just lipid storage organelles 47 and can serve as interfaces to enable protein stabilization and processing.
Rationing of intestinal lipid stores was originally evoked as a potential mechanism to provide long-term energy for dauer larvae that arrest feeding. 35Here, we propose a role for lipid stores during adulthood that focuses on their capacity to buffer proteostasis during aging in concert with HSF-1 and heat shock chaperones, thereby unburdening protein degradation systems.ATGL-1 over-expression leads to lipid store depletion, 52 but, more importantly, it also removes the protection conferred by lipid droplets and renders animals vulnerable to proteotoxic stress associated with reduced proteasomal function, heat and aging.Thus, regulating the activity of ATGL-1 via transcription, 71 post-translational modification, 72 or alterations in CDC48/VCP activity 53 provide a mechanism by which cells modify protein homeostasis.Overall, our data show that the lipid droplet-mediated decrease in protein ubiquitination and the enhancement in lifespan require the function of the AAA-ATPase CDC-48/VCP, a ubiquitin protein processing enzyme and autophagy modulator. 56ipid droplet expansion and accumulation may belong to an arsenal of proteostatic measures that lipid-storing cells and possibly neurons can employ to prevent protein misfolding and aggregation.While lipid droplet accumulation in immune cells such as macrophages 73 and microglia 74 may be inflammatory and contribute to aging, lipid droplets may confer some post-mitotic, terminally differentiated cells with added protection against protein aggregation 49 and mitochondrial lipid 75 For example, lipid droplet accumulation in Alzheimer's disease pathogenesis 76 may highlight an effort from neuronal cells to improve proteome stability and resilience against progressive proteotoxicity.Notably, drastic inhibition of triacylglycerol lipolysis becomes detrimental to neurons 77 as larger lipid droplets are less easily broken down. 51However, a recent study showed that lipid droplet accumulation protected hyper-activated neurons from cell death, 44 a phenomenon potentially relevant in Alzheimer's disease. 78Thus, moderate accumulation of lipid droplets may be more desirable to provide protection against age-related proteotoxicity.In agreement, our data show marked protection against paralysis in a proteostatic Alzheimer's disease model by attenuating cytosolic lipolysis.
Enhancing proteostasis by stimulating the autophagy process has emerged as an attractive strategy to mitigate several age-related diseases with pathological proteostatic decline such as neurodegenerative diseases. 79However, our study indicates that stimulating selective autophagy by specifically increasing the expression of one selective autophagy receptor, SQST-1/SQSTM1, is not sufficient to improve proteostasis and may exacerbate age-related proteostatic collapse.Overall, our study suggests that therapeutic improvement in proteostasis may benefit from a combinatorial approach in which the whole autophagy/lysosomal machinery is stimulated concomitantly with a modest reduction in lipid droplet breakdown.Such an approach may prevent pathogenic burdening of the autophagy/lysosome pathway with cargoes that could have otherwise been stabilized by lipid droplets or routed to the proteasome.

Limitations of the study
Our study uncovered novel regulators of SQST-1/SQSTM1 dynamics and place lipid droplets as an important organelle in SQST-1/SQSTM1 dynamics and ubiquitinated protein metabolism.Here, lifespan studies in SQST-1 transgenic animals were repeated with integrated and nonintegrated stains, and with different tags, which improved confidence in our interpretations.Our findings highlight challenges that can arise with nematode lifespan studies, including variations across laboratories and the role that temperature can play in the study of proteins in transgenic strains that respond to temperature at the transgene expression level and dynamically at the protein level.Notably, modulating lipid droplet levels using gene silencing (atgl-1, hosl-1 or lid-1 RNAi) and long-lived daf-2 mutants led to a substantial reduction in SQST-1 accumulation.Whether other nematode longevity models accumulate less SQST-1 with age could be tested in strains with high lipid droplet content, such as the germline-less glp-1 animals and the protein synthesis rsks-1 mutants.Also, we found interesting new roles of ribosomal subunits in SQST-1 accumulation which could be further clarified by studying the direct impact of ribosomal assembly and function on selective autophagy.Overall, while the mechanism by which lipid droplets modulate SQST-1/SQSTM1 dynamics and polyubiquitinated protein levels is not fully elucidated, our work lays the foundation to further study the role of lipid droplets in proteostasis and aging at the cellular, tissular, and organismal levels.

Figure 1 .
Figure 1.SQST-1 dynamics and lifespan modulation is temperature-dependent in C. elegans (A-F) Lifespan analysis of wild-type animals and animals over-expressing SQST-1 fused to RFP, GFP or GFP::RFP fed OP50 E. coli, developmentally raised at 20 C and then grown at 25 C (A-C) or 20 C (D-F) during adulthood (n = 100).Insets include corresponding representative images of transgenic animals at Day 5 of adulthood, and quantified fluorescence, GSD t-test ****p < 0.001.(G and H) (G) sqst-1 mRNA levels in wild-type and transgenic animals were quantified by qPCR.Biological triplicates GSD t-test *p < 0.05, **p < 0.01, ***p < 0.001 (H).Levels of GFP and RFP were measured in transgenic tandem SQST-1:GFP::RFP animals (leveraging the pH sensitivity of GFP to visualize SQST-1 accumulation in autolysosome) after incubating Day 1 animals at 20 C, 25 C or 30 C for 24 h.Average of 10 worms per image.n = 5 images per condition GSD ANOVA *p < 0.05, **p < 0.01, ***p < 0.001.Difference in signal between GFP and RFP is due to the low pH sensitivity of GFP.Thus, RFP-only signal represents SQST-1 protein in autolysosomes.(I) Representative images of transgenic animals (quantified in h.) incubated at 20 C, 25 C or 30 C for 24 h.Details about lifespan analyses and repeats are available in TableS4, Mantel-Cox log rank.n.s.: not significant, **p < 0.01,****p < 0.001.

Figure 2 .
Figure 2. SQST-1 regulators include proteins that bind lipid droplets and that aggregate with age (A) Pathway enrichment of genes that regulate SQST-1 levels using WormCat. 44(B) SQST-1:RFP accumulates after autophagy is inhibited by lgg-1 silencing for 3 days during adulthood at 25 C. Representative confocal microscopy image of the distal intestine of animals expressing both SQST-1:RFP and the lipid droplet-resident protein DHS-3 fused to GFP, subjected to lgg-1 silencing for 3 days during adulthood at 25 C.The co-localization between DHS-3:GFP and SQST-1:GFP is quantified.(C) Overlap of SQST-1 regulators with age-dependent aggregating proteins 2 and lipid droplet-associated proteins. 40Bolded genes were selected as representative.(D) Representative images of gene silencing of a selected set of overlapping SQST-1 regulators during adulthood for 3 days at 25 C in animals over-expressing SQST-1:RFP.Fluorescence and tissue distribution quantified GSD ANOVA *p < 0.05,****p < 0.001.Details about SQST-1 regulators are available in TablesS1 and S2.

Figure 4 .
Figure 4. Lipid droplets modulate ubiquitinated protein accumulation, SQST-1 levels, and lifespan (A) Lifespan analysis of wild-type and transgenic animals over-expressing ATGL-1:GFP developmentally raised at 20 C and then grown at 25 C during adulthood on OP50 E. coli (n = 100).(B) Levels of RFP signal and lipid droplets from Day 3 animals over-expressing SQST-1:RFP in wild-type or transgenic ATGL-1:GFP over-expressing background.(C) Wild-type and ATGL-1:GFP over-expressing animals were raised at 20 C and then grown at 25 C during adulthood on control bacteria (Ctl) or bacteria expressing dsRNA against autophagy gene lgg-1 or proteasome subunit gene rpn-6.1 for 2 days.Levels of ubiquitinated proteins and actin were visualized by immunoblotting.Actin was used as loading control.Biological replicates shown.(D) Animals expressing lipid droplet-resident protein DHS-3 fused to GFP were raised at 20 C and then grown at 25 C during adulthood on control bacteria or bacteria expressing RNAi against lgg-1 or rpn-6 for 4 days.Levels of ubiquitinated proteins and DHS-3:GFP were visualized by immunoblotting from total input (I), cytosol (C) and lipid droplet (LD) fractions (comparative % loaded between fractions, i.e., 10%).(E) Day 1 wild-type animals were fed control bacteria or bacteria expressing dsRNA against rpn-6 or atgl-1 for 3 days and ubiquitination levels were detected by immunoblotting.(F) Levels of RFP signal and lipid droplets in Day 7 daf-2 animals over-expressing SQST-1:RFP and fed control bacteria or bacteria expressing dsRNA against lpin-1 during adulthood.(G) Day 1 daf-2 animals were fed control bacteria or bacteria expressing dsRNA against atgl-1, rpn-6 or lpin-1 for 5 days at 25 C and ubiquitination levels were detected by immunoblotting.Actin was used as loading control.Biological replicates shown.(H) Lifespan analysis of wild-type and daf-2(e1370) raised at 20 C on OP50 E. coli and then grown at 25 C during adulthood on control bacteria or bacteria expressing dsRNA against lpin-1 (n = 100).Details about lifespan analyses and repeats are available in TablesS3 and S4, Mantel-Cox log rank.n.s.: not significant, *p < 0.05,****p < 0.001.