young , healthy organisms strive to maintain their proteome in a functional state through the tight control of rates of protein synthesis , folding , and degradation .
extensive quality - control systems are set up throughout the cell to prevent and manage protein damage .
as the organism ages , these control mechanisms become less efficient , leading to a disruption in protein homeostasis ( balch et  al . , 2008 , david , 2012 ) .
aging is the main risk factor for a variety of neurodegenerative diseases where specific proteins accumulate as pathological aggregates .
recently , there has been considerable interest in investigating widespread protein aggregation in the absence of disease .
multiple studies have demonstrated that several hundred proteins become highly detergent - insoluble in aged animals ( ayyadevara et  al . , 2016 , david , 2012 , david et  al . , 2010 , demontis and perrimon , 2010 , reis - rodrigues et  al . , 2012 ,
, 2016 , walther et  al . , 2015 ) . computational analysis of the insoluble proteome indicates an overrepresentation of proteins with functional and structural similarities ( david et  al . , 2010 ) .
the examination of some of these proteins in  vivo reveals their assembly into large 
the discovery of endogenous age - dependent protein aggregation in model organisms gives us the unprecedented opportunity to dissect the intrinsic cellular machineries responsible for preventing protein aggregation without using ectopically expressed human disease - associated proteins . at this time , very little is known concerning the regulation of widespread protein insolubility with age and its consequences for the health of the organism .
interestingly , several studies show that protein insolubility is modified in long - lived animals with reduced insulin / insulin growth factor ( igf)-1 daf-2 signaling , but it remains unclear to which extent ( david et  al .
, 2010 , demontis and perrimon , 2010 , walther et  al . , 2015 ) .
a growing number of familial and sporadic forms of neurodegenerative diseases show pathological inclusions caused by  abnormal aggregation of rna - binding proteins ( rbps ) .
the first rbps identified in these inclusions were tar dna binding protein of 43  kda ( tdp-43 ) and fused in sarcoma ( fus ) , associated with amyotrophic lateral sclerosis ( als ) and frontotemporal lobar degeneration ( ftld ) ( arai et  al . , 2006 , neumann et  al . , 2009 , neumann et  al . , 2006 ) . since then additional rbps such  as taf15 , ewsr1 , hnrnpa2b1 , hnrnpa1 , and hnrnpa3
all of the known rbps associated with dementia contain a low - complexity ( lc )  prion - like  domain enriched in glycines and uncharged polar amino acids , and similar to the sequences driving yeast prion aggregation ( alberti et  al .
lc prion - like domains are also present in key rbps that mediate the assembly of rna granules by liquid - liquid phase separation ( lin et  al . , 2015 ,
significantly , a small proportion of liquid droplets made by rbps transform into solid aggregates over time in  vitro ( lin et  al . , 2015 ,
, 2015 , murakami et  al . , 2015 , patel et  al . , 2015 ) . for clarity
, we will use the term aggregation only when referring to the formation of non - dynamic rbp aggregates .
an important question is whether the special assembly properties of rbps puts them at risk of aggregating during aging in a multicellular organism and not just in the context of disease .
interestingly , several rbps with lc prion - like domains were identified in the insoluble proteome of aged animals ( david et  al . , 2010 ) .
overall , it is imperative to know the causes and consequences of wild - type rbp aggregation during aging in order to fully understand rbp aggregation in neurodegenerative diseases .
furthermore , it is likely that the organism has evolved specific mechanisms to control liquid droplet protein aggregation . in the current study , we chose to focus on key rbps responsible for stress granule formation .
stress granules are a specific type of rna granule that protect the cell by sequestering mrna from the translational machinery during periods of stress .
importantly , stress granule proteins are often found to co - localize with pathological inclusions of tdp-43 and fus ( bentmann et  al .
whether these stress granule proteins are innocent bystanders transiently interacting with tdp-43 and fus or whether they co - aggregate and accelerate disease - associated rbp aggregation remains intensely debated ( bentmann et  al . , 2013 , li et  al . , 2013 ) .
we show that key stress - granule - related rbps ( sgrbps ) accumulate in aberrant stress granule - like puncta and in large solid aggregates in aged c.  elegans .
proteomic analysis revealed that long - lived animals with reduced daf-2 signaling preferentially abrogate the insolubility of rna granule components .
importantly , sgrbp aggregates are associated with reduced animal size , motility , and lifespan .
we show that sgrbp aggregation is triggered at an earlier age by their co - aggregation with other misfolded proteins , a process that is prevented by daf-16 in daf-2 mutants .
in addition , the proteostasis network established by heat shock transcription factor 1 ( hsf-1 ) during development is required to maintain dynamic stress granule proteins throughout the animal s life .
to identify and quantify changes in aggregation - prone proteins in animals with reduced daf-2 signaling , we performed an in - depth proteomic analysis of the insoluble proteome from both control and long - lived animals ( figure  1a ; table s1 ) . because protein misfolding and aggregation is highly abundant in aged c.  elegans gonads and masks changes in other somatic tissues ( david et  al .
, 2010 , goudeau and aguilaniu , 2010 , zimmerman et  al . , 2015 ) , we used a gonad - less mutant to focus our analysis on protein insolubility in non - reproductive tissues .
we isolated large aggregates that are pelleted by low centrifugal forces ( 20,000   g ) and insoluble in 0.5% sds .
in three biological replicates , we identified 260 insoluble proteins , of which 186 were highly prone to aggregate with age in control animals ( figure  1b ) .
the strong correlations between the control replicates ( r  = 0.77 , r  = 0.85 , and r  = 0.67 ) and long - lived replicates ( r  = 0.87 , r  = 0.86 . and
r  = 0.82 ) attest to the quality of the quantification and experimental reproducibility ( figures s1a and s1b ) .
none of these insoluble proteins was more prone to aggregate with age in the long - lived animals as compared with controls .
these results are consistent with previous observations ( david et  al . , 2010 , demontis and perrimon , 2010 ) .
surprisingly , a recent proteomic study showed that endogenous protein insolubility is higher in daf-2 mutants than in wild - type animals ( walther et  al . , 2015 ) .
to account for procedural differences , we performed the extraction following the less stringent extraction protocol from walther et  al .
( 2015 ) . by omitting sds and using ultracentrifugation at 500,000   g , walther et  al . analyzed highly insoluble large aggregates ( as in this study ) , as well as smaller and more soluble aggregates .
however , we did not observe a general change in the action of daf-2 signaling on protein insolubility with age after using the less stringent extraction protocol ( figure  s1c ) .
next , we asked whether the inconsistencies between the studies could be related to protein aggregation in the gonad and indeed , we found that long - lived animals with gonads have proportionally more insoluble proteins compared with wild - type animals with gonads ( figure  s1d ) .
these results suggest that aggregation in the gonad masks the protective effect of reduced daf-2 signaling in somatic tissues .
importantly , we confirmed this protective action of reduced daf-2 signaling with several candidates ( see below ) . taken together , these data demonstrate that reduced daf-2 signaling promotes protein solubility in most tissues with age . to investigate specifically changes in insolubility regulated by reduced daf-2 signaling , we sorted proteins prone to aggregate with age in control animals ( > 2-fold ) by their fold change in insolubility with age in the long - lived conditions .
we restricted our analysis to 87 proteins that aggregated less with age ( < 0.83-fold ) and 52 proteins that continued to aggregate with age ( > 1.2-fold ) in the long - lived animals ( figure  1b ; table s1 ) .
previous bioinformatics analysis of aggregation - prone proteins revealed an enrichment in both -sheet propensity and aliphatic amino acids ( david et  al . , 2010 ) .
a similar enrichment in the whole insoluble proteome was identified in this study ( figures s1e and s1f ) .
intriguingly , when examining the two groups of insoluble proteins that were differentially regulated by reduced daf-2 signaling , we found a segregation of the two properties .
proteins with abrogated aggregation with the daf-2 rnai treatment were highly enriched in aliphatic amino acids , in particular alanine , glycine , and valines , but not in  sheets ( figures 1c , 1d , and s1 g ) .
conversely , proteins that still aggregate in the daf-2( ) condition had a significant propensity to form  sheets but were only modestly enriched in aliphatic amino acids ( figures 1c and 1d ) .
next , we searched for functional differences between the two  groups differentially regulated by reduced daf-2 signaling .
strikingly , ribosomal proteins and rna granule components , including stress granule and p - granule rbps , were highly overrepresented among the proteins that were prevented from aggregating with age in the long - lived animals ( tables s2a and s3 ) .
conversely , chaperones and vitellogenin yolk proteins were overrepresented among proteins that were still prone to aggregate with age in daf-2( ) conditions ( table s2b ) . among the rbps , four
are predicted to have lc prion - like domains ( alberti et  al . , 2009 ) :
pab-1 , fib-1 , hrp-1 , and car-1 ( figure  s2a ) . by western blot
, we confirmed that reduced daf-2 signaling abrogated their aggregation with age ( figures 1e and  s2b ) .
in addition , we evaluated two proteins without rna - binding or lc prion - like domains , par-5 and daf-21 , quantified by mass spectrometry as more insoluble with age in both control  and long - lived animals .
we confirmed that par-5 and daf-21 continued to aggregate with age in long - lived animals ( by > 7-fold and 10-fold , respectively ) , albeit to a reduced extent compared with controls ( figure  s2c ) . of note
, changes in aggregation were not correlated with changes in total protein levels ( figure  s3 ) .
a previous study found that the chemical b - isox exclusively causes rna granules to precipitate out from whole - cell lysates by inducing their assembly into a hydrogel ( han et  al .
proteins precipitated by b - isox were also enriched in aliphatic amino acids ( in particular glycine and to a lesser extent alanine and valine ) and not in the propensity to form  sheets ( figures 1c , 1d , and s1 g ) .
we checked for proteins in common with our study and found a very significant overlap between proteins precipitated by b - isox and proteins no longer aggregating in the long - lived conditions ( figure  1f ; table s4 ) .
together , these results indicate that different types of rna granule components including key rbps responsible for their assembly become insoluble with age , and long - lived animals with reduced daf-2 signaling are highly successful in preventing their aggregation .
interestingly , higher levels of aliphatic amino acids in rna granule components could help their assembly and/or drive their age - dependent aggregation .
to investigate further the aggregation of key rbps with lc prion - like domains and to understand the mechanisms involved , we generated c.  elegans strains expressing pab-1 and tiar-2 in the pharyngeal muscles , fused to fluorescent tags .
pab-1 and tiar-2 are the c.  elegans homologs of human polyadenylate - binding protein 1 ( pabp-1 ) and t - cell - restricted intracellular antigen-1 ( tia-1 ) , two prominent rbps that localize to stress granules and are also minor components of pathological inclusions that occur in als and fltd ( bentmann et  al . , 2012 ) .
both of these rbps harbor lc prion - like domains ( figure  s2a ) ( alberti et  al . , 2009 ) .
exposing cells to stressors such as heat induces rbps with lc prion - like domains to form liquid droplets ( molliex et  al . , 2015 , patel et  al . , 2015 ) .
similarly , heat shock in c.  elegans caused pab-1 and tiar-2 to form stress granules ( figures 2a and 2c ; figure  s4a ) ( murakami et  al .
, 2012 , rousakis et  al . , 2014 ) . when co - expressed , pab-1 and tiar-2 localized to the same stress granules ( figure  s4b ) .
consistent with the dynamic nature of stress granules , these puncta were no longer observed 24  hr after the heat shock ( figures 2a and 2c ) . using antibodies
, we observed a similar change in pattern with endogenous pab-1 , indicating the effect is not merely due to overexpression or the fluorescent tag ( figure  s4c ) .
as an additional control , we showed that kinase kin-19 , which is not an rbp and lacks a lc prion - like domain , does not localize to stress granules upon heat shock ( figure  s4d ) . with age
, we observed a striking change in the distribution pattern of these key stress granule rbps . whereas the majority of pab-1 and tiar-2 proteins were diffusely localized in non - stressed young animals , we found that both stress granule components accumulated in distinct puncta in aged animals ( figures 2b and 2d ; figure  s4f )
this change was specific for pab-1 and tiar-2 as the fluorescent tags alone ( venus or tagrfp ) remained diffuse with age ( figure  s4e ) ( david et  al . , 2010 ) .
the results were not simply caused by overexpression because endogenous pab-1 formed similar puncta with age , in the different head regions where it is natively expressed ( figure  s4 g ) .
for both pab-1 and tiar-2 , we observed a significant increase with age in the number of worms with large puncta that are visible by low - magnification microscopy ( figures 2e and 2f ; figure  s4h ) .
interestingly , imaging at high magnification revealed that tiar-2 in aged animals was localized predominantly in small puncta highly reminiscent of stress granules assembled during heat shock , whereas pab-1 accumulated to a greater extent in large puncta that were not normally observed upon heat shock in young animals ( figures 2b , 2d , single - plane insets , 3a , 3c , and 3d ) .
quantification confirmed that stress granules induced during heat shock and aberrant tiar-2 stress granule - like puncta formed during aging had similar sizes ( figure  3b ) .
remarkably , when co - expressed , pab-1 preferentially co - localized within tiar-2-positive age - dependent stress - granule - like puncta ( figure  2 g ) , suggesting that interactions between rbps change their aggregation patterns . a hallmark of protein aggregation associated with disease is the immobility of proteins within the aggregates .
to evaluate this aspect , we monitored fluorescence recovery after photobleaching ( frap ) in large pab-1 and tiar-2 puncta ( figures 3e3i ) .
all large tiar-2 puncta and half of the large pab-1 puncta in aged animals showed no fluorescence recovery , demonstrating that these are solid aggregates ( figures 3e3i ) .
because of the small size of the age - dependent stress - granule - like puncta , it was not possible to assess the mobility of tiar-2 or pab-1 in these structures .
consequently , we used the large puncta as a readout for sgrbp aggregation in subsequent experiments .
the consequences of age - dependent protein aggregation for the animal s health are poorly understood .
here , we evaluated the impact of the aggregation of a key stress granule component during aging . surprisingly , pab-1 overexpression was protective as animals , grown at 15c , 20c , or 25c all their life , lived longer than non - transgenic animals ( table s5 ) .
however , this effect is likely due to higher levels of functional pab-1 and not related to protein aggregation because pab-1 does not aggregate in animals at 15c ( figure  s5a ) . in order to distinguish effects specifically related to pab-1 aggregation , we separated pab-1 transgenics at day 7 into three groups depending on their aggregation levels .
we found that animals with pab-1 aggregation were significantly smaller in size compared with animals without pab-1 aggregation ( figure  4a ; figure  s5b ) .
in addition , smaller animals were visibly less motile ( movie s1 ) . importantly ,
mildly stressed animals with the highest levels of aggregation died earlier ( figure  4b ; table s5 ) .
our proteomic study revealed that reduced daf-2 signaling efficiently prevents the insolubility of stress granule components with age .
we confirmed that long - lived daf-2 mutants greatly delay the formation of both stress - granule - like structures and large aggregates of pab-1 and tiar-2 ( figures 5a and 5b ; figures s6a and s6b ) .
to gain insight into the mechanisms controlling protein aggregation , we investigated the role of the transcription factor hsf-1 activated by reduced daf-2 signaling ( hsu et  al . , 2003 ,
hsp40 modulate stress granule dynamics in saccharomyces cerevisiae and/or in cell culture ( cherkasov et  al .
because chaperone expression is controlled by hsf-1 in c.  elegans , we speculated that hsf-1 may regulate sgrbp aggregation .
indeed , impairing hsf-1 activity in both daf-2( ) and wild - type backgrounds caused severe pab-1 aggregation already in young adults as well as in aged individuals , an effect that was reversed by overexpressing hsf-1 ( figures 5c5e ; figures s6c and s6d ) .
interestingly , hsf-1 in daf-2 mutants did not control the aggregation of the kinase kin-19 , which was previously shown to misfold and form solid aggregates with age ( figure  5 g ) ( david et  al . , 2010 ) .
these results suggest that hsf-1 regulates different types of endogenous protein aggregation with age to different extents .
it was previously shown that to assure daf-2( ) longevity , hsf-1 is most highly expressed and acts mainly during development ( volovik et  al . , 2012 ) .
we observed that impairing hsf-1 activity by rnai during adulthood had no effect on pab-1 aggregation ( figure  5f ; figure  s6e ) .
conversely , reducing hsf-1 activity by rnai during development caused pab-1 aggregation in young adults , albeit mainly in the anterior bulb ( figure  5f ) .
the same chaperones discovered to regulate stress granule dynamics in other model systems could also play a role in preventing sgrbp aggregation . in yeast the hsp40 proteins sis1 and ydj1 were shown to co - localize with stress granules and to play a role in stress granule disassembly ( walters et  al . , 2015 ) .
we evaluated worm strains overexpressing yellow fluorescent protein ( yfp)-tagged dnj-13 and dnj-19 , the worm orthologs of sis1 and ydj1 , together with tagrfp::pab-1 .
we observed occasional co - localization of both chaperones with both heat - induced pab-1 stress granules and age - dependent large and stress - granule - like pab-1 puncta ( figures s6 g and s6h ) .
these findings were confirmed in single tagrfp::pab-1 transgenics using antibodies against dnj-13 and dnj-19 ( data not shown ) .
however , overexpression of dnj-19 and dnj-13 did not significantly reduce pab-1 aggregation with age ( figures s6f and s6i ) , indicating that these chaperones may not modulate sgrbp aggregation .
however , we observed no co - staining with either pab-1 stress granules induced by heat shock or pab-1 puncta formed during aging .
therefore , it is possible that hsp110 does not modulate stress granule dynamics and sgrbp aggregation in c.  elegans . finally , we investigated the role of hsp70 on sgrbp aggregation and found that inhibition of hsp70 by rnai did not alter pab-1 aggregation ( table s6 ) . of note , the later results could be caused by redundancy between chaperone functions .
collectively , these results reveal that hsf-1 is an important regulator of sgrbp aggregation throughout life , and it contributes to maintaining dynamic stress granule proteins in long - lived daf-2 mutants .
next , we examined the role of the transcription factor daf-16 activated by reduced daf-2 signaling ( lin et  al .
1997 ) . in daf-2 mutants , daf-16 protected against pab-1 aggregation in aged animals ( figures 5c and 5d )
importantly , daf-2 mutants use daf-16 to control different types of age - dependent protein aggregation because delayed aggregation of the kinase kin-19 was also dependent on daf-16 ( figure  5 g ) .
recent work performed in s.  cerevisiae and drosophila reveals that stress granules dynamically interact with misfolded proteins ( cherkasov et  al . , 2013 , kroschwald et  al . , 2015 ) .
we speculated that widespread protein misfolding and aggregation occurring with age could promote sgrbp aggregation . in support of this hypothesis
, we observed the co - localization of pab-1 and kin-19 in large immobile aggregates in double transgenics ( figures 6a and 6b ) .
next , we evaluated the rate of pab-1 and kin-19 aggregation in the double transgenics relative to their aggregation rates in single transgenics ( figures 6c and 6d ) . in the single transgenics ,
importantly , when both pab-1 and kin-19 were co - expressed , the presence of misfolded kin-19 triggered more abundant pab-1 aggregation at an earlier age ( figure  6c ) .
conversely , kin-19 aggregation was slightly impeded by pab-1 overexpression ( figure  6d ) , indicating that sgrbp aggregation does not cross - seed kin-19 aggregation .
notably , pab-1 aggregation was not accelerated by the co - expression of a fluorescent tag alone ( figure  6e ) .
furthermore , we did not observe a general induction of pab-1 stress granules in young double - transgenic animals expressing both kin-19 and pab-1 ( figure  s6j ) .
these data strongly suggest that pab-1 aggregation is not simply the consequence of generalized cellular stress induced by kin-19 overexpression .
rather , the co - localization of kin-19 and pab-1 in the same aggregates as well as the earlier and accelerated aggregation of pab-1 is consistent with a seeding mechanism related to kin-19 aggregation . taken together , we interpret these findings to imply that the accumulation of misfolded proteins with age acts as a seed for sgrbp aggregation . therefore , the overall reduction in widespread protein aggregation in long - lived daf-2( ) conditions as evidenced by our proteomic analysis , at least in part through increased daf-16 activity , could be an effective strategy to prevent sgrbp aggregation .
several experimental manipulations have been shown to extend the lifespan of c.  elegans and protect against proteotoxicity ( kenyon , 2010 , taylor and dillin , 2011 ) .
we found that both dietary restriction mimicked in eat-2 mutant animals as well as inhibition of mitochondrial function achieved by targeting cyc-1 with rnai strongly limited pab-1 aggregation with age ( figures 7a and 7b ) .
we show that a wide variety of rna granule components become highly insoluble with age in c.  elegans . together with previous in  vitro and cell culture results ,
our findings demonstrate that the capacity of rbps to cycle between assembled and disassembled states can become a liability in aging organisms . already in young animals , maintaining sgrbp dynamics necessitates an active control system established by hsf-1 .
the accumulation of other misfolded proteins during age acts as a seed for the aggregation of key sgrbps .
significantly , one of the main outcomes of the longevity program initiated by reduced daf-2 signaling while responding to widespread protein aggregation is the preservation of rbp solubility with age . in this study , we have examined in detail the aggregation pattern of pab-1 and tiar-2 , two key rbps with lc prion - like domains that are important for the formation of stress granules . during the aging process , both proteins spontaneously assembled into small puncta similar to liquid droplets induced during stress and into larger aggregates .
significantly , upon co - expression , both pab-1 and tiar-2 co - localized in these age - related stress - granule - like structures . because our proteomic analysis revealed a number of stress granule components in the insoluble proteome , it is likely that secondary stress granule proteins are also incorporated .
therefore , an attractive hypothesis is that these small puncta represent stress granules formed as a response to stress related to aging .
the inherent aggregation propensity of sgrbps would induce at least some of these droplets to undergo the irreversible transition into a solid state . these stabilized stress granules
could then grow into large aggregates as we observed with pab-1 , or simply accumulate with age as seen with tiar-2 .
an important question remains how the inherent propensity of rna granule components to aggregate with age could influence pathogenesis in neurodegenerative diseases .
one possibility is that inherent rbp aggregation impacts cellular health and thereby indirectly accelerates pathology .
we observed reduced lifespan and a striking decrease in size and mobility of animals with higher levels of pab-1 aggregation .
as yet , it remains unclear whether reduced fitness is a cause or consequence of pab-1 aggregation . in support of a gain of function related to sgrbp aggregation ,
two rare diseases are caused by mutated pabpn1 and tia-1 , the human homologs of pab-1 and tiar-2 , which accumulate in pathological aggregates ( brais et  al .
our proteomic analysis of aging c.  elegans highlighted three other rbps with lc prion - like domains that are highly prone to aggregate with age : hrp-1 , fib-1 , and car-1 .
the aggregation of the human homologs of hrp-1 , hnrnp - a1 , and hnrnp - a3 was recently discovered to cause multisystem proteinopathy and als / ftld ( kim et  al .
indeed , a loss in nucleolar protein fib-1 function caused by its aggregation with age could impair ribosomal biogenesis ( tollervey et  al .
the mammalian lsm14b and lsm14a homologs of car-1 localize to p bodies ( eulalio et  al . , 2007 ) , and aggregation of key p - body components could impair non - sense - mediated decay . therefore , it will be important to investigate the consequences of fib-1 and car-1 aggregation on cellular health . apart from accelerating pathology indirectly by reducing cellular health ,
the presence of stress granule proteins in pathological protein aggregates is emerging as a common denominator in different types of neurodegenerative diseases including als , ftld , alzheimer s disease , and huntington s disease ( aulas and vande velde , 2015 , bentmann et  al .
the inherent aggregation propensity of stress granule proteins demonstrates that they are unlikely to be transient interacting partners in pathological aggregates .
it remains to be determined whether age - related sgrbp aggregation acts as a seed for disease - associated protein aggregation .
overall , the role of stress granules in neurodegenerative diseases is clearly highly complex because there is evidence supporting the recruitment of disease - associated proteins to stress granules and vice versa .
interestingly , recent cell culture data show that the assembly of stress granules caused by disease - associated protein aggregation in turn promotes pathological aggregation ( vanderweyde et  al . , 2016 ) .
the age - dependent aggregation of sgrbps and prevalence of stress granule components in neurodegenerative diseases underline their relevance as therapeutic targets .
one successful strategy would be to prevent the initial assembly of stress granules ( kim et  al . , 2014 ) .
, it will be important to understand how longevity pathways relying on dietary restriction or defective mitochondrial respiration efficiently prevent sgrbp aggregation . in the case of reduced daf-2 signaling , sgrbp aggregation is suppressed by at least two mechanisms : daf-16 activation prevents cross - seeding by delaying the accumulation of misfolded aggregation - prone proteins , and increased activity of hsf-1 during development assures enhanced sgrbp proteostasis throughout adulthood .
to date , cross - seeding of rbp aggregation has been observed only with the huntingtin protein containing an expanded polyglutamine repeat region ( furukawa et  al . , 2009 ) .
it will be important to investigate by which means endogenous aggregation - prone proteins lacking motifs similar to the lc prion - like domain would cross - seed rbps aggregation .
overall , both of these strategies used to restore the dynamic nature of stress granule proteins could also directly prevent disease - associated rbp aggregation .
a list of strains , strain maintenance , rnai treatment , and lifespan assays is described in the supplemental experimental procedures .
worms were either fixed for imaging analysis directly after heat shock or allowed to recover on normal growth ( ng ) plates kept at 20c before fixation .
images were taken of synchronized live c.  elegans using a leica fluorescence microscope m165 fc with a planapo 2.0 objective and leica dfc310 fx camera .
body length was determined for each worm using fiji software ( schindelin et  al . , 2012 ) .
significance was evaluated by unpaired , two - tailed t test using graphpad prism 6 .
worms were examined with a leica sp8 confocal microscope . fixation and immunostaining protocol including imaging parameters
representative confocal images are displayed as maximum z stack projection unless mentioned otherwise . in a population of
synchronized live c.  elegans , aggregation levels were determined using a leica fluorescence microscope m165 fc with a planapo 2.0 objective .
animals overexpressing pmyo-2::venus::tiar-2 were divided into two categories : animals with up to 10 ( low aggregation ) or more than 10 venus::tiar-2 puncta ( intermediate aggregation ) in the anterior and posterior  pharyngeal bulb .
animals overexpressing pmyo-2:tagrfp : pab-1 were divided into three categories : animals with up to 10 ( low aggregation ) or more than 10 ( intermediate aggregation ) tagrfp::pab-1 puncta in the posterior bulb , or more than 10 ( high aggregation ) tagrfp : pab-1 puncta in the anterior bulb .
animals expressing pkin-19::kin-19::meos or pkin-19::kin-19::venus were divided into less than 10 puncta ( low aggregation ) , between 10 and 100 puncta ( intermediate aggregation ) , and more than 100 puncta in the anterior bulb ( high aggregation ) . to compare aggregation levels between kin-19::venus and tagrfp::pab-1
, we evaluated tagrfp::pab-1 puncta formation only in the anterior bulb and with the same counting scheme as for kin-19::venus .
when comparing different strains , counting was done in a blind fashion . for statistics , two - tailed fisher s exact test
significance of high  + intermediate against low aggregation levels was calculated unless indicated otherwise .
frap analysis was performed as previously described ( david et  al . , 2010 ) using the leica sp8 confocal microscope with the harmonic compound , plan , apochromatic ( hc pl apo ) cs2 63 1.30 glycerol objective and photomultiplier tube ( pmt ) detector .
further experimental details are described in the supplemental experimental procedures . to obtain large synchronized populations of aged animals and quantify protein aggregation only in the somatic tissues
, we used temperature - induced sterile gon-2 mutants as previously described ( david et  al . , 2010 ) .
to induce longevity , we subjected animals to daf-2 rnai and control animals to gfp rnai from the last larval stage l4 onward .
animals aged at 25c were collected at day 3 of adulthood ( young ) and when half the control animals had died between days 14 and 18 ( aged ) .
isolation of large sds insoluble aggregates for immunoblot and mass spectrometry analysis were performed as previously described ( david et  al . , 2010 ) .
the mass spectrometry proteomics data have been deposited to the proteomexchange consortium ( http://proteomecentral.proteomexchange.org ) via the pride partner repository ( vizcano et  al . , 2014 ) with the dataset identifier pxd003451 .
aliphatic amino acid residues were defined as a , g , i , l , and v. secondary structure content was predicted using psipred v2.6 ( jones , 1999 ) .
the p  values were calculated using the unequal variance t test compared with the background set of all proteins detected by mass spectrometry ( n  =  5,637 ) . additional details and functional analysis are described in the supplemental experimental procedures .
m.c.l . , n.g . , j.c.t . , and d.c.d . designed and performed experiments .
 summarylow - complexity  prion - like  domains in key rna - binding proteins ( rbps ) mediate the reversible assembly of rna granules . 
 individual rbps harboring these domains have been linked to specific neurodegenerative diseases . 
 although their aggregation in neurodegeneration has been extensively characterized , it remains unknown how the process of aging disturbs rbp dynamics . 
 we show that a wide variety of rna granule components , including stress granule proteins , become highly insoluble with age in c.  elegans and that reduced insulin / insulin - like growth factor 1 ( igf-1 ) daf-2 receptor signaling efficiently prevents their aggregation . 
 importantly , stress - granule - related rbp aggregates are associated with reduced fitness . 
 we show that heat shock transcription factor 1 ( hsf-1 ) is a main regulator of stress - granule - related rbp aggregation in both young and aged animals . during aging , increasing daf-16 activity 
 restores dynamic stress - granule - related rbps , partly by decreasing the buildup of other misfolded proteins that seed rbp aggregation . 
 longevity - associated mechanisms found to maintain dynamic rbps during aging could be relevant for neurodegenerative diseases . 
Introduction
Results
Discussion
Experimental Procedures
Author Contributions