Aging Cell (2013) 12, pp814–822
Doi: 10.1111/acel.12110
Germline stem cell arrest inhibits the collapse of somatic
proteostasis early in Caenorhabditis elegans adulthood
Netta Shemesh*, Nadav Shai* and Anat Ben-Zvi
Department of Life Sciences and The National Institute for Biotechnology in
the Negev, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel
Summary
All cells rely on highly conserved protein folding and clearance
pathways to detect and resolve protein damage and to maintain
protein homeostasis (proteostasis). Because age is associated
with an imbalance in proteostasis, there is a need to understand
how protein folding is regulated in a multicellular organism that
undergoes aging. We have observed that the ability of Caenorhabditis elegans to maintain proteostasis declines sharply following the onset of oocyte biomass production, suggesting that
a restricted protein folding capacity may be linked to the onset of
reproduction. To test this hypothesis, we monitored the effects
of different sterile mutations on the maintenance of proteostasis
in the soma of C. elegans. We found that germline stem cell (GSC)
arrest rescued protein quality control, resulting in maintenance of
robust proteostasis in different somatic tissues of adult animals.
We further demonstrated that GSC-dependent modulation of
proteostasis requires several different signaling pathways,
including hsf-1 and daf-16/kri-1/tcer-1, daf-12, daf-9, daf-36,
nhr-80, and pha-4 that differentially modulate somatic quality
control functions, such that each signaling pathway affects
different aspects of proteostasis and cannot functionally complement the other pathways. We propose that the effect of GSCs
on the collapse of proteostasis at the transition to adulthood is
due to a switch mechanism that links GSC status with maintenance of somatic proteostasis via regulation of the expression
and function of different quality control machineries and cellular
stress responses that progressively lead to a decline in the
maintenance of proteostasis in adulthood, thereby linking
reproduction to the maintenance of the soma.
Key words: aging; daf-16;
proteostasis; reproduction.
germline
stem
cells;
hsf-1;
Introduction
Aging Cell
The correct folding and assembly of proteins and protein complexes is
essential for cellular function. Accordingly, cells have evolved specialized
machines to maintain proper protein homeostasis (proteostasis). These
protein quality control networks are comprised of molecular chaperones
Correspondence
Dr. Anat Ben-Zvi, Department of Life Sciences, National Institute for Biotechnology
in the Negev, Ben-Gurion University of the Negev, 1 Ben Gurion Blvd.,
Beer Sheva 84105, Israel. Tel.: +972 8 647 9059; fax: +972 8 647 9041;
e-mail: anatbz@bgu.ac.il
*These authors contributed equally to this work.
Accepted for publication 22 May 2013
814
and degradation machineries, such as the proteasome and the
autophagy pathway, that ensure proper folding and the efficient
clearance of misfolded or damaged proteins, respectively (Finley, 2009;
Gidalevitz et al., 2010; Haynes & Ron, 2010; Tyedmers et al., 2010;
Arias & Cuervo, 2011; Walter & Ron, 2011).
When the proteostatic capacity of the cell becomes limiting, an
imbalance in the folding environment ensues, leading to the induction of
cytoprotective stress responses designed to suppress protein misfolding
and to enhance clearance, such as the heat-shock response (HSR;
Akerfelt et al., 2010; Haynes & Ron, 2010; Walter & Ron, 2011).
Activation of the heat-shock transcription factor (HSF-1) adjusts the
expression of chaperones and other cytoprotective genes to match the
elevated levels of damaged proteins, thereby enabling the cell to
rebalance proteostasis and increase survival and recovery from stress
(Akerfelt et al., 2010). Yet, despite these protective mechanisms, the
accumulation of damaged proteins is a well-established marker of aging,
with many age-dependent diseases being associated with protein
misfolding and aggregation (Gidalevitz et al., 2010; Taylor & Dillin,
2011).
Two possible mechanisms can explain the failure of protein quality
control networks to adjust to the cellular folding demands of aged
animals. Protein damage and misfolding in such individuals could result
from a limited efficiency of the cellular quality control networks in
repairing or removing misfolded proteins, leading to a gradual accumulation of damaged proteins over time. Alternatively, the ability of the
cellular proteostasis network to rebalance itself may be differentially
regulated during the lifespan of the organism. Given that the composition
of the cellular proteostasis machinery in Caenorhabditis elegans is
modulated during early adulthood (Liu et al., 2011; Taylor & Dillin, 2011;
Twumasi-Boateng et al., 2012; Volovik et al., 2012), we hypothesized
that the capacity of cellular protein quality control networks may be
differentially regulated upon transition to adulthood. This idea is
supported by the observed decline in the ability of C. elegans to keep
meta-stable proteins folded and mount effective stress responses early in
adulthood (Ben-Zvi et al., 2009; David et al., 2010; Taylor & Dillin, 2011).
Because this change in proteostasis composition and capacity coincides
with the onset of oocyte biomass production, we asked whether
disrupting the function of the reproductive system would affect the
observed decline in proteostasis maintenance seen during adulthood.
In C. elegans, reproduction is specifically linked to aging by endocrine signaling from germline stem cells (GSCs), a process that alters fat
metabolism and extends lifespan (Antebi, 2012). Several pathways have
been suggested to respond to life-extending signals sent from the GSC
to the soma. One pathway is a lipophilic hormone-signaling pathway
that is dependent on daf-12, a nuclear hormone receptor, and on the
enzymes that synthesize or modify the daf-12 ligands daf-9/cytochrome
P450 and the daf-36/Rieske oxygenase (Hsin & Kenyon, 1999; Gerisch
et al., 2007). A second responsive pathway activates daf-16 via a
regulatory mechanism that is distinct from that employed in insulin-like
signaling pathway (ILS) and which requires kri-1, encoding an intestinal
ankyrin-repeat protein, and tcer-1, encoding a putative transcription
elongation factor (Berman & Kenyon, 2006; Ghazi et al., 2009). Such
regulation alters the expression of genes encoding proteasome
components (Vilchez et al., 2012) and can, furthermore, modulate
ª 2013 The Anatomical Society and John Wiley & Sons Ltd
Germline stem cells regulate somatic proteostasis, N. Shemesh et al. 815
stress survival (Libina et al., 2003). GSC signaling also requires hsf-1
(Hansen et al., 2005), although it is not known whether such
regulation occurs independently of the ILS. Finally, GSC signaling
modulates fat metabolism in a manner that depends on the daf-12 and
daf-16 pathways, as well as on the nuclear receptor nhr-80 (Wang
et al., 2008; Goudeau et al., 2011; McCormick et al., 2012) and on the
activation of TOR signaling, which regulates autophagy through pha-4
and independently of daf-16 (Lapierre et al., 2011). Thus, GSC
proliferation coordinates a regulatory switch that alters endocrine and
metabolic signaling to modulate fatty acid metabolism, proteasomal
function and autophagy, as well as lifespan (Antebi, 2012). We
therefore asked whether reproduction, and specifically, signaling from
GSCs, regulates the decline in protein quality control seen early in
adulthood.
To determine whether the onset of reproduction is linked to the
decline in cellular proteostasis maintenance that occurs in early
adulthood, we considered C. elegans strains presenting diverse mutations in the reproductive system to assess whether interfering with
different aspects of reproductive function could rescue proteostasis
capacity. We found that inhibiting GSC proliferation resulted in robust
proteostasis in different somatic tissues of adult animals. Of the known
pathways linking signals from GSCs with lifespan, signaling via hsf-1
and daf-16/kri-1/tcer-1 modulated heat-shock (HS) activation in adulthood, while other signaling pathways were required to rescue
polyglutamine-dependent toxicity and temperature-sensitive (ts) metastable protein-associated dysfunction. We propose that GSC signaling
regulates a set of quality control machineries and cellular stress
responses, thereby promoting a regulated switch between the limited
and robust states of proteostatic capacity in the soma upon transition
to adulthood.
(A)
Results
Inhibition of GSC proliferation rescues the decline in thermoresistance normally seen early in adulthood
Caenorhabditis elegans thermo-resistance declines following the onset
of reproduction. Young, wild-type (wt) adults (grown for 50 h at 25 °C)
were more resistant to HS than were day 2 adults (grown for 74 h at
25 °C; Fig. S1A and B). While 76.2 5.6% survived if challenged by HS
on day 1 of adulthood, only 12.6 2.3% survived if first challenged on
day 2 of adulthood. This shift in HS survival rate was rapid, starting
7–12 h after transition to adulthood (76.6 3.4% to 44.7 6%,
respectively, P < 0.005), when animals entered the reproductive phase
(Fig. 1A). To assess whether the onset of reproduction is linked to the
maintenance of proteostasis in aging, we monitored the effects of
different sterile mutations on thermo-resistance. Survival of gonadogenesis-defective mutants declined sharply on day 2 of adulthood
(Fig. 1B). Likewise, survival on day 2 of adulthood of both male and
female gamotogenesis-defective mutants was limited (Table S1). In
contrast, HS resistance and survival rates of GSC proliferation mutant
glp-1(e2141ts) (glp-1) animals remained high, namely 71.8 7.6% and
68.6 7.3% on days 2 and 3 of adulthood, respectively (Figs 1B, S1A,
and S1B). Prolonging stress resistance was dependent on GSC arrest, as
the survival rates of several different GSC proliferation mutants also
remained high (Fig. 1B and Table S1). Therefore, as with lifespan, GSC
proliferation (but not reproduction per se) is required for modulating
stress survival after the onset of reproduction. Specifically, GSC arrest
inhibits the rapid and acute decline in the organismal response to stress,
suggesting that signals from proliferating GSCs may modulate somatic
stress response activation upon transition to adulthood.
wt
gon-2 (C)
glp-1
glp-4
(B)
wt
glp-1
(E)
glp-1
wt
(D)
day 1
day 3
Fig. 1 Germline stem cell (GSC) proliferation mutants maintain the ability to mount a protective stress response during adulthood. (A) Age-synchronized wt animals were
subjected to heat-shock (HS) (6 h at 37 °C) at the indicated times, and survival was assayed. (B) Age-synchronized wt or mutant animals gon-2(q388ts) and GSC
proliferation-defective, glp-1(e2141) and glp-4(bn2), were exposed to HS (6 h at 37 °C) and survival was assayed. (C) Quantification of hsp-70 (left) and hsp-16.11 (right)
mRNA levels from age-synchronized wt or glp-1 mutant animals untreated or subjected to HS (90 min at 37 °C). The data presented are relative to those obtained
with untreated animals and normalized to day 1 of adulthood treated animals. (D) Confocal images of age-synchronized wt or glp-1 animals expressing phsp-16.2::GFP
and subjected to HS (90 min at 37 °C). Scale bar is 100 lm. (E) Age-synchronized wt or glp-1 animals expressing phsp-16.2::GFP were treated as in D, and the percent of
animals expressing green fluorescent protein (GFP) was scored.
ª 2013 The Anatomical Society and John Wiley & Sons Ltd
816 Germline stem cells regulate somatic proteostasis, N. Shemesh et al.
Inhibiting GSC proliferation restores efficient activation of
the HSR during adulthood
To determine whether stress survival is associated with differential
regulation of the HSR, we next compared the induction of representative
HS genes by monitoring changes in mRNA levels following HS on days 1
or 2 of adulthood. While the induction of hsp-70 and hsp-16.11 in glp-1
mutant animals remained high, the induction of these genes in wt or
gonadogenesis-defective mutant gon-2(q388) (gon-2) animals was
reduced by ~65% between the first and second days of adulthood
(Figs 1C and S1C). This finding suggests the existence of an early switch
in HSR regulation that is mediated by GSC proliferation.
To determine whether GSC proliferation comes at the cost of
maintaining a robust stress response in somatic tissues, we utilized a HS
transcriptional reporter in which the hsp-16.2 promoter regulates green
fluorescent protein (GFP) expression in a stress-dependent manner. We
then followed GFP expression patterns and expression levels in intact and
germline-less animals. When animals were challenged on day 1 of
adulthood, GFP fluorescence was detected in various somatic tissues,
predominantly in the intestinal cells of both wt and glp-1 mutant
individuals (Fig. 1D). While the ability to induce hsp-16.2-regulated GFP
expression in wt animals declined strongly by day 3 of adulthood, with
only 8.2 6.6% of the animals expressing GFP in the intestine
(P < 0.05), the ability to induce hsp-16.2-regulated GFP in somatic
tissues of glp-1 mutant animals remained high up to day 10 of
adulthood (Figs 1D,E, and S1D). Similar results were observed upon
expression of an hsp-70 (C12C8.1) promoter-regulated reporter and
depended on glp-1 loss-of-function (Fig. S1E). No GFP fluorescence was
detected in untreated wt or glp-1 animals, with hsp-16.2-regulated GFP
induction declining if the glp-1 animals were fertile (Fig. S1F and S1G),
suggesting that hsp-16.2-regulated GFP induction can be attributed to
glp-1 loss-of-function. These results suggest that cell-nonautonomous
signals from proliferating GSCs regulate the cellular HSR in somatic
tissues, whereas interfering with such signals prevents the decline in HSR
activation seen in adult animals, thereby increasing stress survival.
Dissecting the signaling pathways required to reset the HSR
in adulthood
To understand the mechanism by which GSCs regulate the activation of
HS response in adult somatic tissues, we asked whether genes that are
required for GSC lifespan-extending signaling also play a role in resetting
proteostasis. As discussed above, several signaling pathways are
specifically required for GSC-dependent longevity. Inactivation of daf16 (and of its GSC-specific modifiers kri-1 and tcer-1), hsf-1, daf-12, daf9, daf-36, nhr-80, or pha-4 suppresses the enhanced longevity of glp-1
animals, with most of these pathways showing little effect on the
lifespan of wt animals (Antebi, 2012). We therefore examined whether
inactivation of these genes also affected somatic proteostasis. We
utilized the hsp-16.2-regulated GFP expression reporter and followed
GFP expression patterns in glp-1 animals treated with RNA interference
(RNAi) for genes involved in GSC-dependent lifespan regulation. When
challenged on day 3 of adulthood, 98.2 1.2% of the glp-1 animals
grown on control RNAi were able to induce hsp-16.2-regulated GFP in
the intestine. This ability to induce hsp-16.2-regulated GFP levels was not
affected by RNAi knockdown of daf-12, daf-9, daf-36, nhr-80, or pha-4
(Figs 2A and S2A). In agreement, HS survival rates of glp-1 animals
harboring a mutation in daf-12(rh61rh411), daf-9(daf-9(rh50)), daf-36
(daf-36(K114)) or nhr-80(tm1011) on day 2 of adulthood remained high
(Fig. 2B), while RNAi or mutations in any of the above genes alone did
(A)
(B)
Fig. 2 hsf-1 and daf-16 are required for germline-dependent regulation of the
heat-shock response (HSR) during adulthood. (A) Age-synchronized glp-1 animals
expressing phsp-16.2::GFP grown on indicated RNAi-expressing bacteria and
subjected to heat-shock (HS) (90 min at 37 °C) on day 3 of adulthood. The percent
of animals showing a fluorescent signal in the gut was scored. (B) Agesynchronized double mutant animals (as indicated) were subjected to HS (6 h at
37 °C) on day 2 of adulthood, and survival was assayed. glp-1 mutant animals are
presented as reference.
not affect HS activation or survival (Fig. S2B and S2C). To extend our
observations, we also examined the effect of RNAi-mediated knockdown
of daf-12, nhr-80, and pha-4 on the ability of glp-1 mutant animals to
respond to HS by examining the expression levels of the HS genes, hsp16
(hsp16.2 and hsp16.11) and hsp70 (C12C8.1 and F44E5.4), following HS
on the second day of adulthood. The induction of these genes was not
significantly affected (Fig. S2D–G). In contrast, RNAi-mediated
knockdown of the stress transcription factors daf-16 or hsf-1 reduced
hsp-16.2-regulated GFP induction in the intestine of glp-1 mutant
animals on day 3 of adulthood (33.3 7% and 4.7 2.7%, respectively, P < 0.005); while reducing hsf-1 levels affected all HS genes
examined, daf-16 knockdown affected only some (Figs 2A and S2).
However, the daf-16(mu86) deletion mutant repressed glp-1-mediated
thermo-resistance on day 2 of adulthood, reducing survival to
19.6 6.9% (P < 0.05; Fig. 2B). The effects of daf-16 and hsf-1
knockdown on the HSR of glp-1 animals were similar to those seen on
wt animals on day 1 of adulthood (37.3 4.1% and 16.1 3.8%,
respectively, Figs 2A and S2B). This finding suggests that signals from
proliferating GSCs converge on the cellular stress response of somatic
tissues and modulate DAF-16 and HSF-1 function in a cell-nonautonomous manner.
The function of daf-16 and hsf-1 in enhancing proteostasis is also
regulated cell-nonautonomously by the ILS signaling pathway (Libina
et al., 2003; Taylor & Dillin, 2011). We thus asked whether daf-16 and
hsf-1 regulation also occur independently of the ILS pathway. For hsf-1,
no GSC-specific effectors are known. In contrast, down-regulation of
kri-1 or tcer-1 was shown to affect daf-16 function in germline-less
animals but not in animals with decreased ILS signaling (Berman &
Kenyon, 2006; Ghazi et al., 2009). We therefore examined whether
knockdown of kri-1 or tcer-1 modulated HS activation and survival of
germline-less animals. Silencing of kri-1 or tcer-1 by RNAi reversed
hsp-16.2-dependent GFP induction in glp-1 animals by 68% and 56%,
respectively (P < 0.005), and reduced the induction of other HS genes,
yet did not affect the ability of wt animals to induce a HSR on day 1 of
adulthood (Figs 2A and S2). Likewise, a kri-1(ok1251) deletion mutant
completely suppressed glp-1-mediated thermo-resistance on day 2 of
ª 2013 The Anatomical Society and John Wiley & Sons Ltd
Germline stem cells regulate somatic proteostasis, N. Shemesh et al. 817
myo-3::GFP
glp-1
wt
(C)
day 1
(D)
day 3
day 5
day 7
(E)
glp-1
ª 2013 The Anatomical Society and John Wiley & Sons Ltd
(B)
wt
The ability of C. elegans to maintain meta-stable proteins that are
dependent on the cellular folding capacity for proper folding declines
early in adulthood. Because the inhibition of GSC proliferation affects
the expression of genes encoding for proteasome and autophagy
pathway components, as well as modulating the function of HSF-1 and
DAF-16 (Antebi, 2012), we asked whether signals from GSCs
compromise somatic proteostasis and lead to the age-dependent
misfolding of meta-stable proteins observed during C. elegans adulthood (Ben-Zvi et al., 2009). We first examined the function of perlecan
unc-52(e669,su250) (unc-52(ts)), a previously characterized folding
sensor. This sensor contains a temperature-sensitive missense mutation
that shows age-dependent paralysis due to disruption of body wall
muscle myofilament anchoring during adulthood (Ben-Zvi et al., 2009).
While unc-52(ts) mutant animals grown under permissive conditions
(during adulthood) were partially paralyzed on day 4 of adulthood
(48 7.7%), the motility of unc-52(ts) in a glp-1 or mes-1 mutant
background was significantly improved (7.4 3% and 7.9 3.2%,
respectively, P < 0.005). In contrast, the motility of animals expressing
unc-52(ts) in the gon-2 mutant background was not significantly
different (55.3 8.1%) from that of unc-52(ts) mutant animals
(Fig. 3A).
We then examined the folding of the naturally occurring metastable proteins, myosin and dynamin, which show age-dependent
misfolding and aggregation later in adulthood (Ben-Zvi et al., 2009;
David et al., 2010). We first monitored the age-dependent myofilament disruption (sarcopenia) and motility decline associated with
myosin misfolding (Ben-Zvi et al., 2009). As expected, by the seventh
day of adulthood, the myofilaments of wt animals were mostly
disrupted (83.6 5.3%). In contrast, the integrity of the myofilaments
of glp-1 animals was well maintained (14.3 1.8%, P < 0.005;
Fig. 3B and C). Accordingly, the motility of wt animals quickly
declined, whereas the glp-1 mutant animals were mostly unaffected
(Fig. 3D). Germline arrest also altered the reported age-dependent
mislocalization of the metastable protein dynamin-1 (DYN-1), a protein
that participates in neuronal synaptic vesicle recycling (Ben-Zvi et al.,
2009). While DYN-1 was mostly mislocalized by the fifth day of
adulthood in wt animals (90 5.8%, P < 0.05), such mislocalization
was rarely observed in glp-1 animals (14 4.7%; Fig. 3E). Thus,
inhibition of GSC proliferation before transition to adulthood abrogated age-dependent decline in protein quality control in muscle,
neuronal and intestinal cells (Figs 1E and 3), resulting in the maintenance of robust proteostasis and stress resistance in these somatic
tissues during adulthood.
unc-52 ( ts )
unc-52 ( ts );gon-2
unc-52 ( ts );glp-1
unc-52 ( ts );mes-1
glp-1
Inhibiting GSC proliferation postpones the onset of ageassociated misfolding of meta-stable proteins
(A)
wt
adulthood, reducing survival to 19.7 11.3% (P < 0.05; Fig. 2B). Our
data suggest that GSC signaling modulates DAF-16 (and possibly HSF-1)
function upon transition to adulthood. This claim is supported by the
observation that germline ablation can extend the lifespan of daf-2
mutants (Hsin & Kenyon, 1999). Indeed, microarray analysis of DAF-16
targets identified many nonoverlapping targets between genes showing
GSC-regulated expression and genes previously identified in a daf-2(-)
expression analysis (McCormick et al., 2012). Likewise, whereas hsp16.2
induction was shown to be independent of daf-16 in a daf-2(-)
background (McColl et al., 2010), hsp16.2 was affected by GSCregulated DAF-16 function (Figs 2A, S2A and S2D). Nonetheless, we
cannot rule out a possible contribution of ILS-dependent regulation of
DAF-16 and HSF-1 upon transition to adulthood.
Fig. 3 Inhibition of germline stem cell (GSC) proliferation regulates protein quality
control in the soma. (A) Age-synchronized animals (as indicated) were scored for stiffbody paralysis on day 4 of adulthood. (B) Percent of affected cells quantified from
confocal images presented in C. (C) Confocal images of age-synchronized wt or
glp-1 animals expressing pmyo-3::MYO-3::GFP. Scale bar is 10 lm. (D) Percent of
paralyzed, age-synchronized wt or glp-1 animals. (E) The percent of age-synchronized
wt or glp-1 animals showing DYN-1 mislocalization was scored.
Inhibiting GSC proliferation postpones the onset of protein
misfolding and toxicity in a polyglutamine disease model
A suggested consequence of proteostatic decline in adulthood is the
onset of aging-associated protein misfolding diseases. Accordingly, we
monitored protein aggregation propensity and cellular toxicity of
polyglutamine-expansion (polyQ) proteins in glp-1 mutant animals. By
the second day of adulthood, glp-1 animals expressing polyQ-yellow
fluorescent protein (YFP) with 35 repeats (Q35) in body wall muscle
developed 45% less visible foci than did Q35-expressing animals
(P < 0.005; Fig. 4A and B). In agreement, when Q35 aggregation was
monitored using semi-denaturing detergent–agarose gel electrophoresis
(SDD–AGE), 67% less insoluble high molecular weight (HMW) species
that could only be dissolved by boiling were detected in Q35;glp-1 than
in Q35 animals already at day 2 of adulthood (Fig. 4C and D), even
818 Germline stem cells regulate somatic proteostasis, N. Shemesh et al.
though no effect in terms of Q35 protein levels were noted (Fig. S3).
When motility was examined as a measure of toxicity, the onset of Q35mediated paralysis was significantly delayed in glp-1 animals. By the sixth
day of adulthood, only 18.3 4.2% of Q35;glp-1 animals were
paralyzed, as compared to 88.1 5.1% of Q35 animals (P < 0.05;
Fig. 4E). Thus, GSC arrest modulates the onset and progression of
protein aggregation in a polyQ disease model.
Misfolding of polyQ proteins was shown to impact many different
aspects of the protein quality control machinery, including chaperones,
proteasome-mediated degradation, and autophagy (Tyedmers et al.,
2010). To examine whether GSC signaling pathways similarly regulate
age-dependent toxicity of polyQ proteins, we monitored the roles of
the signaling pathways specifically required for GSC-dependent
longevity in modulating the Q35-dependent paralysis of germline-less
animals. Similar to the regulation of HSR, the delay in the onset of
Q35-mediated paralysis in glp-1 animals was largely unaffected
by RNAi of daf-12, daf-9, and daf-36 on day 6 of adulthood, as
compared to animals fed control RNAi (11.7 2.5%, 14.4 1.3%,
20.7 2.2% and 13.1 2.2% respectively). By contrast, daf-16, kri1, and tcer-1 RNAi significantly inhibited glp-1-dependent rescue of
Q35-mediated toxicity (35.7 3.3%, 54.2 9% and 46 2.4%,
respectively, P < 0.005; Fig. 4F). Likewise, knockdown of hsf-1 resulted
in a severe phenotype with 65.7 4% (P < 0.005) paralysis on day 5
of adulthood and 100% lethality on day 6 of adulthood (data not
shown). In contrast to the regulation of HSR, treatment with RNAi for
nhr-80 or pha-4 also inhibited glp-1-dependent rescue of Q35mediated toxicity (33 2.2% and 27.6 2.3%, respectively,
P < 0.005), suggesting that the specific requirements of a given
protein determines its susceptibility to GSC-dependent signaling
pathways. This is further supported by our observation that knockdown
of hsf-1, daf-12, and daf-9 (and to a lesser extent, daf-36, nhr-80 and
pha-4) inhibited glp-1-dependent rescue of unc-52(ts) paralysis, while
(A)
Q35
Q35;glp-1
Discussion
Cell-nonautonomous regulation of cellular quality can restore
proteostasis in adulthood
The question of how dynamic is the functional capacity of cellular
protein quality control networks during the lifespan of an organism
remains open. Several lines of evidence suggest that the machineries
responsible for general maintenance of proteostasis undergo significant
changes when animals enter fertile adulthood. The ability to activate
cellular stress responses, such as the heat-shock response and the
unfolded protein response, is disrupted following transition to adulthood (Ben-Zvi et al., 2009). Likewise, the activation of c-Jun N-terminal
kinase (JNK) pathway was shown to enhance DAF-16 transcriptional
activity during development but to inhibit such activity after the
transition to adulthood (Twumasi-Boateng et al., 2012). Finally,
epidermal growth factor signaling (EGF) was shown to change the
expression of proteostasis components, such as elements of the
ubiquitin-proteasome system and chaperone genes, early in adulthood
(Liu et al., 2011). This switch is associated with altered requirements of
stress transcription factors at the transition to adulthood (Cohen et al.,
2010; Volovik et al., 2012). Thus, there is a differential regulation of
protein quality control networks at the transition to adulthood. A
corresponding change is observed in the functional capacity of the
(C)
Q35;glp-1
Q35
(B)
knockdown of daf-16, kri-1, and tcer-1 did not affect the motility of
unc-52(ts) animals (Fig. S4). We therefore propose that the effect of
GSCs on proteostasis is due to a regulatory switch mechanism that
links GSC status with the maintenance of somatic proteostasis by
regulating a set of quality control machineries and cellular stress
responses, each affecting different cellular functions, yet all required
for longevity.
(D)
Q35
Q35;glp-1
(E)
(F)
Fig. 4 Inhibiting germline stem cell (GSC) proliferation delays the onset of polyQ-dependent aggregation and toxicity. (A) Images of representative age-synchronized wt or
glp-1 animals expressing Q35 on day 2 of adulthood. Scale bar represents 100 lm. (B) The number of visible foci scored in age-synchronized Q35 or Q35;glp-1 animals.
(C) Extracts of age-synchronized Q35 or Q35;glp-1 animals incubated in 2% SDS at room temperature ( ) or heated at 98 °C (+) were separated on a SDD-AGE gel, and
the formation of HMW species was detected with anti-green fluorescent protein (GFP) antibodies. (D) Quantification of Q35-derived HMW species seen in C. Results are
the average of two independent experiments. (E) The percent of paralyzed animals scored in age-synchronized Q35 or Q35;glp-1 animals. (F) Age-synchronized Q35;glp-1
animals were grown on the indicated RNAi-expressing bacteria and scored for the percent of paralyzed animals on day 6 of adulthood.
ª 2013 The Anatomical Society and John Wiley & Sons Ltd
Germline stem cells regulate somatic proteostasis, N. Shemesh et al. 819
cellular quality control machinery that progressively loses the ability to
maintain metastable proteins during adulthood (Ben-Zvi et al., 2009;
David et al., 2010; Fig. 5A). Our findings suggest that the switch
between the two functional states of proteostatic networks that
occurs once the animals enter reproductive adulthood is regulated by
signals from proliferating GSCs. These signals, which are mediated via
different signaling pathways and which affect the composition and
function of the proteostasis network (Lapierre et al., 2011; Vilchez
et al., 2012), limit the ability of somatic cells to maintain proteostasis
(Fig. 5B and C).
What are the consequences of reduced proteostasis capacity in
adulthood? For an aggregation-prone protein that is highly dependent
on the proteostasis network (Tyedmers et al., 2010), like Q35, such
reduced capacity resulted in the fast accumulation of aggregates as early
as day 2 of adulthood (Fig. 4). For metastable proteins that are
responsive to the load of misfolded proteins in the cell (Gidalevitz et al.,
2010), misfolding was observed during the first few days of adulthood,
earlier than when the corresponding wt proteins were affected (Ben-Zvi
et al., 2009). In agreement, analysis of aggregate accumulation during
adulthood identified several proteins that aggregated early in adulthood
and showed that wt proteins increasingly aggregated during adulthood
(David et al., 2010). When proteostasis was modified by GSC arrest early
in adulthood, the beneficial effects of maintaining robust proteostasis
were observed throughout adulthood, with the onset of protein
misfolding and aggregation being delayed for aggregation-prone
metastable and wt proteins. We therefore propose that a switch in
proteostasis composition and function early in adulthood can result in
progressive damage accumulation during adulthood, with severity of the
phenotype depending on the genetic polymorphism of the organism
(Gidalevitz et al., 2006, 2010).
Different signaling pathways mediate cross talk between
reproduction and somatic maintenance
Active signaling cascades initiated by proliferating GSCs were first
identified as modulators of lipid metabolism and lifespan (Hsin &
Kenyon, 1999; Arantes-Oliveira et al., 2002). While inactivation of any
of the GSC signaling pathways has only mild effects on the lifespan of wt
animals, all pathways are required for GSC-dependent extension of
lifespan (Antebi, 2012). Whereas changes in lipid metabolism in GSCarrested animals are mostly regulated by the daf-12, nhr-80, and pha-4
signaling pathways (Goudeau et al., 2011; Lapierre et al., 2011;
McCormick et al., 2012), GSC-dependent regulation of autophagy
requires pha-4, yet is independent of daf-16. At the same time, GSCdependent regulation of proteasome function requires daf-16 and daf12 but not hsf-1 (Lapierre et al., 2011; Vilchez et al., 2012). Here, we
show that different GSC signaling pathways are required to modulate
somatic proteostasis. While both daf-16 and hsf-1 are required for the
activation of HSR in adulthood (Fig. 2), the reduced toxicity of Q35;glp-1
animals is mediated by hsf-1, daf-16, nhr-80, and pha-4 (Fig. 4),
whereas unc-52(ts)-dependent paralysis is modulated by hsf-1, daf-12,
and daf-9 (Fig. S4). Autophagy and proteasomal function in germlineless animals was shown to be regulated by pha-4 and daf-16,
respectively (Lapierre et al., 2011; Vilchez et al., 2012), suggesting that
different pathways affect the expression of a specific set of proteostasis
modulators and therefore those proteins that depend on these machineries for their function. This premise implies that GSC signaling pathways
do not overlap and cannot functionally complement each other. Indeed,
daf-16 and daf-12 were shown to regulate a unique set of genes with
very little overlap in their expression patterns, when regulated by GSC
signaling (McCormick et al., 2012). We thus propose that GSC signaling
(A)
(B)
daf-12 (daf-9, daf-36)
nhr-80
pha-4
daf-16 (kri-1, tcer-1)
hsf-1
...
(C)
daf-12 (daf-9, daf-36)
nhr-80
pha-4
daf-16 (kri-1, tcer-1)
hsf-1
...
(D)
daf-12 (daf-9, daf-36)
nhr-80
pha-4
daf-16 (kri-1, tcer-1)
hsf-1
...
Fig. 5 Signaling from germline stem cells (GSCs) activates a switch between two states of somatic proteostasis. (A) Proteostasis of young adult animals is robust. (B)
Following the onset of reproduction, signals from GSCs activate a regulatory switch that changes proteostatic capacity in the soma. Somatic proteostasis thus becomes
limiting, resulting in the age-dependent accumulation of damaged proteins. (C) Inhibiting GSC proliferation induces sterility and allows the organism to maintain robust
proteostasis, as seen in young animals by activating different signaling pathways downstream. (D) Inactivating these pathways affects different aspects of proteostasis.
ª 2013 The Anatomical Society and John Wiley & Sons Ltd
820 Germline stem cells regulate somatic proteostasis, N. Shemesh et al.
activates several different regulatory programs that differentially modulate somatic functions, such as metabolism, cellular quality control, and
responses to various stress conditions, resulting in a progressive loss of
the organism’s ability to maintain somatic proteostasis and other repair
and defense functions. The combined actions of all of these pathways
are required for lifespan enhancement, although activation of only some
of the pathways may suffice to improve the health of the organism
during adulthood (Fig. 5C and D).
One potential interpretation of our results is that GSC signaling
regulates proteostasis capacity of somatic cells by modulating the cellautonomous function of various transcription factors. However, several
signaling pathways that are modulated by GSC arrest were shown to
function only in specific tissues. For example, nhr-80 was shown to
function in intestine and neurons (Goudeau et al., 2011), while kri-1 and
tcer-1 were shown to modulate DAF-16 function specifically in intestine
(Berman & Kenyon, 2006). Thus, it remains to be examined whether the
different signaling pathways function downstream of germline signaling
to modulate proteostasis in somatic cells in a cell-autonomous manner or
by modulating cell-nonautonomous signaling between different somatic
tissues.
Uncoupling somatic proteostasis maintenance from GSC
signaling
The link between GSC proliferation and somatic function suggests that
the commitment to reproduction is reported to the soma, resulting in
altered metabolism and cellular repair that can affect the rate of aging.
This interpretation is supported by the fact that different mutations in
DNA damage checkpoint genes affect somatic stress resistance and
lifespan (Olsen et al., 2006). Given that reproduction is also affected by
environmental conditions, it is likely that, in return, signals from the
soma can impact GSC proliferation. This idea is further supported by the
finding that cell-nonautonomous signaling via kri-1 regulates germline
cell death in response to DNA damage (Ito et al., 2010). Thus, kri-1 can
integrate signals from both the germline and the soma to determine
GSC reproduction potential and somatic cellular repair. It is of note that
the reproduction potential of GSCs can modulate both protein homeostasis (this work), repair and defense responses (Arantes-Oliveira et al.,
2002; Alper et al., 2010), and lipid metabolism (Wang et al., 2008;
Lapierre et al., 2011), thus serving to link reproduction, metabolism, and
somatic repair.
The ability demonstrated in this study to restore somatic proteostatic
function in adulthood strongly indicates that C. elegans possesses a
highly efficient cellular repair system that is actively down-regulated
once reproduction is initiated. Identifying these signals and then
uncoupling somatic proteostasis from the reproductive system may
restore the intrinsic ability of the cell to maintain its proteome. Defining
the cell-nonautonomous signals that regulate proteostasis could therefore offer novel approaches for the treatment of age-dependent diseases
with different etiologies but similar underlying biology.
Experimental procedures
experiment. The first day of adulthood (day 1) was set at 50 h after
temperature shift, before the onset of egg laying. Animals were
moved every 1–2 days during the reproductive period to avoid
progeny contamination. HS-treated animals were discarded after
scoring.
Statistical analysis
Experiments were repeated at least three times, and >30 animals per
experimental condition were scored. Data are presented as
means SEM. P-values were calculated using the Wilcoxon–Mann–
Whitney rank sum test to compare two independent populations. (*)
denotes P < 0.05 and (**) denotes P < 0.005. For Figs 1E, 3D, and 4E,
data were compared with age-matched wt animals. For Figs 2A, 4F, S2,
and S4, data were compared with empty vector control. For all other
figures, data were compared with wt animals on day 1 of adulthood.
Heat-shock treatment
Unless otherwise stated, a total of 30–50 age-synchronized animals
grown at 25 °C were used for each assay. Animals were moved to
fresh plates, and the plates were then sealed and placed in a 37 °C
bath for 90 min. Animals were frozen or fixed immediately following
stress.
Thermo-resistance assay
Animals were picked at the indicated ages and transferred to a 24-well
plate containing HS buffer (100 mM Tris–HCl, pH 7.4, 17 mM NaCl and
1% cholesterol supplemented with bacteria). These animals were then
subjected to a 37 °C HS for 6 h. HS buffer was supplemented with
SYTOX orange (Invitrogen, Carlsbad, CA, USA), and animal survival was
scored by monitoring dye uptake, using a Leica M165 FC fluorescent
stereoscope with a TXR filter. Fluorescent animals were scored as dead.
For comparison, kinetic thermo-resistance assays were performed in
which >70 animals/strain were subjected to a 37 °C HS, and survival
was tested by manual scoring (Fig. S1A and S1B). In agreement with
previously reported data (Libina et al., 2003; Wang et al., 2008), when
the HS treatment was extended (9 h at 37 °C), glp-1 animals were
significantly more thermo-resistant than were wt animals (42.2 2.9%
and 21 2.3%, respectively, P < 0.005). However, given that the HSR
in wt animals declines in this timeframe (Fig. 1A), we monitored survival
after 6 h.
Fluorescence stress reporters
Animals expressing GFP under control of the hsp-16.2 promoter
(phsp-16.2::GFP) or mCherry fluorescent protein under control of
the hsp-70 (C12C8.1) promoter (phsp-70::mCherry) were crossed with
glp-1(e2141ts) animals. Animals expressing GFP or mCherry were
subjected to HS, and fluorescence was monitored 18–24 h later.
Animals expressing GFP or mCherry in the gut were scored as
HS-induced.
Nematodes and growth conditions
A list of strains used in this work and name abbreviations is given in
Tables S2 and S3 (Supporting information). Nematodes were grown on
NGM plates seeded with the Escherichia coli OP50 or OP50-1 strains at
15 °C. Unless otherwise stated, 15–30 embryos, laid at 15 °C, were
transferred to fresh plates and grown at 25 °C for the duration of the
RNA interference
A total of 30–50 eggs were placed on E. coli strain HT115(DE3)
transformed with the indicated RNAi vectors (obtained from the
Ahringer or Vidal RNAi libraries), as previously described (Ben-Zvi et al.,
2009). RNAi knockdown of mRNA levels was controlled by comparing
ª 2013 The Anatomical Society and John Wiley & Sons Ltd
Germline stem cells regulate somatic proteostasis, N. Shemesh et al. 821
the mRNA levels of the target gene with mRNA levels of animals fed on
strain HT115(DE3) bacteria containing the empty vector (pL4440).
60 9 1.0 numerical aperture objective with a 488-nm line for excitation.
More than 210 cells per experimental condition were scored.
RNA levels
Immunostaining
Total RNA was extracted from wild-type or glp-1 animals untreated or
subjected to HS (see HS treatment, above). RNA was extracted using the
TRIzol reagent (Invitrogen). For cDNA synthesis, mRNA was reversetranscribed using the iScriptTM cDNA Synthesis Kit (Bio-Rad, Hercules, CA,
USA). Quantitative PCR was performed on a C1000 Thermal Cycler
(Bio-Rad) with SsoFas EvaGreen Supermix (Bio-Rad).
Immunofluorescence studies were performed as previously described
(Gidalevitz et al., 2006). Animals were stained with anti-dynamin-1
antibodies (Hybridoma Bank). Secondary DyLight 488 goat anti-mouse
IgG antibodies (Jackson Immuno-Research, West Grove, PA, USA) were
used. Animals were imaged using an Olympus Fluoview FV1000 confocal
microscope through a 60 9 1.0 numerical aperture objective with a
488-nm line for excitation.
Protein expression levels
Synchronized nematodes were collected and lyzed in SDS sample buffer.
Samples were analyzed by immunoblot, using anti-paramyosin antibody
5-23 (Hybridoma Bank, Iowa city, IA, USA) and anti-GFP antibodies
(Enco Scientific, Petach Tikvah, Israel). Peroxidase-conjugated AffiniPure
goat anti-rabbit and goat anti-mouse antibodies (Jackson ImmunoResearch, West Grove, PA, USA) were used as secondary antibodies.
Aggregation quantification
Animals expressing punc-54::Q35::YFP were crossed with glp-1(e2141ts)
animals. For quantification, the number of bright foci of age-synchronized animals expressing Q35-YFP was counted.
Acknowledgments
Some nematode strains used in this work were provided by the
Caenorhabditis Genetics Center, which is funded by the NIH National
Center for Research Resources (NCRR). The monoclonal antibodies
developed by M. Nonet and H.F. Epstein were obtained from the
Developmental Studies Hybridoma Bank developed under the auspices
of the NICHD and maintained by the Department of Biology, University
of Iowa. N. Shemesh was supported by Fay and Bert Harbour award.
A.B.-Z. was supported by the Israeli Council for Higher Education Alon
Fellowship, by a Marie Curie International Reintegration grant, and by a
grant from the Binational Science Foundation.
Author contributions
SDD–AGE
For semi-denaturing agarose gel electrophoresis, 120 animals were
collected and extracts were prepared by mechanical disruption under
moderately denaturing conditions. Samples were diluted in SDD–AGE
buffer (50 mM Tris–HCl, pH 6.8, 2% (w/v) SDS, 0.025% bromophenol
blue, 5% glycerol), incubated at room temperature or heated at 98 °C
for 10 min, and loaded onto a 1.5% agarose gel as previously described
(Eremenko et al., 2013). Samples were analyzed by immunoblot using
anti-GFP antibodies (Enco Scientific).
Paralysis assay
A total of 15–30 age-synchronized animals were used for each assay.
Animals were grown at 25 °C for the duration of the experiment. Animals
were moved every day, and paralysis was scored by monitoring animals’
movement 10 min after transfer to a new plate. Animals that did not move
were scored as paralyzed.
Stiff-body paralysis assay
Animals expressing mutant unc-52(ts) were grown at 25 °C until day 1
of adulthood, then shifted to 15 °C, and paralysis-scored.
Myosin-filament organization
Animals expressing GFP-tagged myosin heavy chain A (MYO-3::GFP)
were crossed with glp-1(e2141ts) animals. wt or glp-1 embryos
expressing MYO-3::GFP were grown at 25 °C until the indicated age.
To assess MYO-3::GFP mislocalization, synchronized adults expressing
the transgene were fixed (Gidalevitz et al., 2006), and GFP fluorescence
was monitored. Animals were imaged using an Olympus Fluoview
FV1000 confocal microscope (Olympus, Tokyo, Japan) through a
ª 2013 The Anatomical Society and John Wiley & Sons Ltd
AB, N. Shemesh, and N. Shai designed the study, performed the
experimental work, and wrote the manuscript.
References
Akerfelt M, Morimoto RI, Sistonen L (2010) Heat shock factors: integrators of cell
stress, development and lifespan. Nat. Rev. Mol. Cell Biol. 11, 545–555.
Alper S, McElwee MK, Apfeld J, Lackford B, Freedman JH, Schwartz DA (2010) The
Caenorhabditis elegans germ line regulates distinct signaling pathways to
control lifespan and innate immunity. J. Biol. Chem. 285, 1822–1828.
Antebi A (2012) Regulation of longevity by the reproductive system. Exp. Gerontol.
doi: 10.1016/j.exger.2012.09.009 (in press).
Arantes-Oliveira N, Apfeld J, Dillin A, Kenyon C (2002) Regulation of life-span by
germ-line stem cells in Caenorhabditis elegans. Science 295, 502–505.
Arias E, Cuervo AM (2011) Chaperone-mediated autophagy in protein quality
control. Curr. Opin. Cell Biol. 23, 184–189.
Ben-Zvi A, Miller EA, Morimoto RI (2009) Collapse of proteostasis represents an
early molecular event in Caenorhabditis elegans aging. Proc. Natl Acad. Sci. USA
106, 14914–14919.
Berman JR, Kenyon C (2006) Germ-cell loss extends C. elegans life span through
regulation of DAF-16 by kri-1 and lipophilic-hormone signaling. Cell 124, 1055–
1068.
Cohen E, Du D, Joyce D, Kapernick EA, Volovik Y, Kelly JW, Dillin A (2010)
Temporal requirements of insulin/IGF-1 signaling for proteotoxicity protection.
Aging Cell 9, 126–134.
David DC, Ollikainen N, Trinidad JC, Cary MP, Burlingame AL, Kenyon C (2010)
Widespread protein aggregation as an inherent part of aging in C. elegans. PLoS
Biol. 8, e1000450.
Eremenko E, Ben-Zvi A, Morozova-Roche LA, Raveh D (2013) Aggregation of
human S100A8 and S100A9 amyloidogenic proteins perturbs proteostasis in a
yeast model. PLoS One 8, e58218.
Finley D (2009) Recognition and processing of ubiquitin-protein conjugates by the
proteasome. Annu. Rev. Biochem. 78, 477–513.
Gerisch B, Rottiers V, Li D, Motola DL, Cummins CL, Lehrach H, Mangelsdorf DJ,
Antebi A (2007) A bile acid-like steroid modulates Caenorhabditis elegans
lifespan through nuclear receptor signaling. Proc. Natl Acad. Sci. USA 104,
5014–5019.
822 Germline stem cells regulate somatic proteostasis, N. Shemesh et al.
Ghazi A, Henis-Korenblit S, Kenyon C (2009) A transcription elongation factor that
links signals from the reproductive system to lifespan extension in Caenorhabditis elegans. PLoS Genet. 5, e1000639.
Gidalevitz T, Ben-Zvi A, Ho KH, Brignull HR, Morimoto RI (2006) Progressive
disruption of cellular protein folding in models of polyglutamine diseases.
Science 311, 1471–1474.
Gidalevitz T, Kikis EA, Morimoto RI (2010) A cellular perspective on conformational
disease: the role of genetic background and proteostasis networks. Curr. Opin.
Struct. Biol. 20, 23–32.
Goudeau J, Bellemin S, Toselli-Mollereau E, Shamalnasab M, Chen Y, Aguilaniu H
(2011) Fatty acid desaturation links germ cell loss to longevity through NHR-80/
HNF4 in C. elegans. PLoS Biol. 9, e1000599.
Hansen M, Hsu AL, Dillin A, Kenyon C (2005) New genes tied to endocrine,
metabolic, and dietary regulation of lifespan from a Caenorhabditis elegans
genomic RNAi screen. PLoS Genet. 1, 119–128.
Haynes CM, Ron D (2010) The mitochondrial UPR – protecting organelle protein
homeostasis. J. Cell Sci. 123, 3849–3855.
Hsin H, Kenyon C (1999) Signals from the reproductive system regulate the
lifespan of C. elegans. Nature 399, 362–366.
Ito S, Greiss S, Gartner A, Derry WB (2010) Cell-nonautonomous regulation of C.
elegans germ cell death by kri-1. Curr. Biol. 20, 333–338.
Lapierre LR, Gelino S, Melendez A, Hansen M (2011) Autophagy and lipid
metabolism coordinately modulate life span in germline-less C. elegans. Curr.
Biol. 21, 1507–1514.
Libina N, Berman JR, Kenyon C (2003) Tissue-specific activities of C. elegans
DAF-16 in the regulation of lifespan. Cell 115, 489–502.
Liu G, Rogers J, Murphy CT, Rongo C (2011) EGF signalling activates the
ubiquitin proteasome system to modulate C. elegans lifespan. EMBO J. 30,
2990–3003.
McColl G, Rogers AN, Alavez S, Hubbard AE, Melov S, Link CD, Bush AI, Kapahi P,
Lithgow GJ (2010) Insulin-like signaling determines survival during stress via
posttranscriptional mechanisms in C. elegans. Cell Metab. 12, 260–272.
McCormick M, Chen K, Ramaswamy P, Kenyon C (2012) New genes that extend
Caenorhabditis elegans’ lifespan in response to reproductive signals. Aging Cell
11, 192–202.
Olsen A, Vantipalli MC, Lithgow GJ (2006) Checkpoint proteins control survival of
the postmitotic cells in Caenorhabditis elegans. Science 312, 1381–1385.
Taylor RC, Dillin A (2011) Aging as an event of proteostasis collapse. Cold Spring
Harb. Perspect. Biol. 3, a004440.
Twumasi-Boateng K, Wang TW, Tsai L, Lee KH, Salehpour A, Bhat S, Tan MW,
Shapira M (2012) An age-dependent reversal in the protective capacities of JNK
signaling shortens Caenorhabditis elegans lifespan. Aging Cell 11, 659–667.
Tyedmers J, Mogk A, Bukau B (2010) Cellular strategies for controlling protein
aggregation. Nat. Rev. Mol. Cell Biol. 11, 777–788.
Vilchez D, Morantte I, Liu Z, Douglas PM, Merkwirth C, Rodrigues AP, Manning G,
Dillin A (2012) RPN-6 determines C. elegans longevity under proteotoxic stress
conditions. Nature 489, 263–268.
Volovik Y, Maman M, Dubnikov T, Bejerano-Sagie M, Joyce D, Kapernick EA,
Cohen E, Dillin A (2012) Temporal requirements of heat shock factor-1 for
longevity assurance. Aging Cell 11, 491–499.
Walter P, Ron D (2011) The unfolded protein response: from stress pathway to
homeostatic regulation. Science 334, 1081–1086.
Wang MC, O’Rourke EJ, Ruvkun G (2008) Fat metabolism links germline stem cells
and longevity in C. elegans. Science 322, 957–960.
Supporting Information
Additional Supporting Information may be found in the online version of this
article at the publisher’s web-site.
Fig. S1 (A–B) Age-synchronized wt or glp-1 animals were subjected to HS
(37 °C) on (A) day 1 or (B) day 2 of adulthood and survival was assayed by
manual scoring at the indicated times. (C) Quantification of hsp-70 (left) and
hsp-16.11 (right) mRNA levels from age-synchronized gon-2 mutant animals
untreated or subjected to HS (90 min at 37C) as in Fig. 1C. (D) Extracts of
animals grown as described in the legend to Fig. 1E were probed with antiGFP (bottom) and anti-paramyosin (top) antibodies. (E) Confocal images of
age-synchronized wt or glp-1 animals expressing pC12C8.1::mCherry,
following HS (09 min at 37C) on either day 1 or day 3 of adulthood. (F)
Confocal images of untreated age-synchronized wt or glp-1 animals
expressing phsp-16.2:GFP on either day 1 or day 3 of adulthood. (G)
Confocal images of age-synchronized wt or glp-1 animals expressing phsp16.2::GFP. Animals were grown at 15C and subjected to HS (90 min at
34C) on either day 2 or day 5 of adulthood.
Fig. S2 (A) Images of animals described in the legend to Fig. 2A. (B) Agesynchronized wt animals expressing phsp-16.2::GFP were grown on the
indicated RNAi-expressing bacteria and subjected to HS (90 min at 37C) on
day 1 of adulthood. (C) Age-synchronized mutant animals (as indicated) were
exposed to a HS (6h at 37C) at day 2 of adulthood and survival was assayed.
(D-G) Quantification of (D) hsp-16.2 (E) hsp-16.11 (F) C12C8.1 and (G)
F44E5.4 mRNA levels from age-synchronized glp-1 mutant animals grown on
the indicated RNAi-expressing bacteria and subjected to HS (90 min at 37C)
on day 2 of adulthood.
Fig. S3 Extracts of age-synchronized (day 2 of adulthood) Q35 or Q35;glp-1
animals were probed with anti-GFP (bottom) and anti-paramyosin (top)
antibodies.
Fig. S4 Age-synchronized unc-52(ts);glp-1 animals were grown on the
indicated RNAi-expressing bacteria and scored for the percent of stiff-body
paralyzed animals on day 4 of adulthood.
Table S1 Effect of reproduction mutations on HS survival.
Table S2 List of strains and abbreviations used in this work.
Table S3 List of crosses used in this work.
ª 2013 The Anatomical Society and John Wiley & Sons Ltd