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HSF-1–mediated cytoskeletal integrity determines thermotolerance and life span

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Science  17 Oct 2014:
Vol. 346, Issue 6207, pp. 360-363
DOI: 10.1126/science.1253168

Abstract

The conserved heat shock transcription factor–1 (HSF-1) is essential to cellular stress resistance and life-span determination. The canonical function of HSF-1 is to regulate a network of genes encoding molecular chaperones that protect proteins from damage caused by extrinsic environmental stress or intrinsic age-related deterioration. In Caenorhabditis elegans, we engineered a modified HSF-1 strain that increased stress resistance and longevity without enhanced chaperone induction. This health assurance acted through the regulation of the calcium-binding protein PAT-10. Loss of pat-10 caused a collapse of the actin cytoskeleton, stress resistance, and life span. Furthermore, overexpression of pat-10 increased actin filament stability, thermotolerance, and longevity, indicating that in addition to chaperone regulation, HSF-1 has a prominent role in cytoskeletal integrity, ensuring cellular function during stress and aging.

Cytoskeleton protects from stress and aging

The transcription factor HSF-1 has an unexpected second function that allows it to extend longevity in worms. Baird et al. expressed a modified form of HSF-1 in nematodes. The modified protein could not activate genes encoding protein chaperones. Such chaperones are thought to protect many cellular proteins from heat shock and other damage during aging However, the modified protein still extended the worm life span by regulating the transcription of other genes. One gene it regulated was pat-10, which encodes a troponin-like calcium binding protein. Overexpression of PAT-10 also extended worm life span, apparently by changing the stability of the actin cytoskeleton.

Science, this issue p. 360

The survival of an organism is intricately linked to its ability to maintain cellular quality control, including organelle integrity, lipid homeostasis, proper protein folding, and cellular communication. The organismal response to unpredictable environmental changes is critical to mitigate damages caused by stress. Genes encoding the heat shock protein (HSP) family of molecular chaperones show the largest transcriptional increase in response to thermal stress, suggesting that these proteins are part of a fundamental defense against proteotoxic stress. Consistent with this hypothesis, ectopic expression of the master transcriptional regulator of HSPs, HSF-1, is sufficient to confer resistance to thermal stress and increase life span in the nematode Caenorhabditis elegans (1). Furthermore, hsf-1 overexpression can alleviate toxicity associated with diseases caused by misfolded or aggregated proteins (2).

However, chaperones may be dispensable for thermotolerance and longevity. Neither a hypomorphic mutation of hsf-1, nor preventing the up-regulation of HSPs affects thermotolerance of C. elegans (3, 4). However, other studies using the same hsf-1 mutant show decreased heat resistance (5). The conflicting data may result from differences in experimental design, but it is clear that HSF-1 function is not fully explained by chaperones mediating stress resistance and life-span determination.

To test for protective mechanisms independent of enhancing chaperone induction, we generated transgenic nematodes overexpressing full-length hsf-1 [hsf-1(FL)] or a hsf-1 C-terminal truncation [hsf-1(CT)]. The hsf-1(CT) variant was designed to mimic the C-terminal missense mutation found in the hsf-1(sy441) mutant, a widely used allele that decreases stress-induced HSP transcription via the removal of a transactivation domain (6). hsf-1(FL) was overexpressed in the N2 wild-type (WT) background, and hsf-1(CT) was overexpressed in the hsf-1(sy441) mutant. Therefore, the hsf-1(CT) strain mirrored the overexpression of hsf-1(FL) but contained no endogenous copies of full-length hsf-1 (fig. S1). Both transgenes were expressed threefold higher than endogenous hsf-1 (Fig. 1A).

Fig. 1 hsf-1(CT) increases life span and thermotolerance without enhancing chaperone induction.

(A) Equal overexpression of hsf-1(FL) and hsf-1(CT) determined by means of quantitative polymerase chain reaction (PCR). (B) Western blot of HSP-16 before and after heat shock (HS). (C to F) Quantitative PCR of (C) hsp-16.2, (D) hsp-17, (E) hsp-70a (C12C8.1), and (F) hsp-70b (F44E5.4) show enhanced induction in hsf-1(FL). (G) Thermotolerance assay of WT, hsf-1(FL), and hsf-1(CT) worms shifted from 20° to 34° for 13 hours. (H) Life-span survival curves of WT, hsf-1(FL), and hsf-1(CT) strains. (I) Life-span survival curves of WT and hsf-1(CT) strains with the DNA-binding domain deleted (CT-DBDΔ). *P < 0.005; error bars indicate SEM.

Analysis of protein and transcript abundance confirmed that overexpression of hsf-1(FL) enhanced heat-inducible expression of all HSPs tested, whereas hsf-1(CT) caused no difference from wild type (Fig. 1, B to F, and fig. S2). Yet, both hsf-1(FL) and hsf-1(CT) transgenic worms had increased thermotolerance and life span (Fig. 1, G and H). The life-span extension of hsf-1(CT) was unexpected, so we tested whether this phenotype was dependent on a functional DNA-binding domain. Increased longevity was abolished upon removal of the DNA binding domain (hsf-1(CT-DBDΔ) (Fig. 1I). Thus, increased life span and thermotolerance did not correlate with enhanced HSP transcription.

To find other cellular networks that contribute to HSF-1–mediated stress resistance and longevity assurance, we performed quantitative transcriptomic and proteomic analyses comparing hsf-1(FL) and hsf-1(CT) strains with WT and hsf-1(sy441) strains. We filtered for transcripts or proteins that showed increased abundance, under basal or heat-stressed conditions, exclusively in the heat-protected strains (fig. S3). The 98 genes that met our filtering criteria were enriched for functions in development, cytoskeleton organization, complex assembly, and immune defense response (fig. S4).

Reduced expression of genes essential to thermotolerance should lower survival under heat stress. Therefore, we performed a RNA interference (RNAi)–based thermotolerance screen on the genes that passed our filtering criteria (fig. S5 and table S1). From the screen, we identified a troponin-like calcium-binding protein, PAT-10, as essential for thermotolerance (Fig. 2A). Transcription of pat-10 was heat-inducible in all strains (Fig. 2B). Furthermore, hsf-1 overexpression strains showed an increase in pat-10 transcripts under basal and heat-stress conditions (Fig. 2B). After examining the upstream promoter region of pat-10, a putative binding site for HSF-1 (7, 8) was identified within 500 base pairs of the transcription start site (fig. S6). Additionally, hsf-1 RNAi blocked the up-regulation of pat-10 upon heat shock (Fig. 2C). Therefore, pat-10 appears to be a direct target of HSF-1 transcriptional regulation.

Fig. 2 pat-10 is necessary and sufficient for thermotolerance and longevity.

(A) Thermotolerance of WT and hsf-1(CT) strains treated with pat-10 or lev-11 RNAi. (B) Quantitative PCR of pat-10 with and without heat shock (HS). (C) Effect of hsf-1 RNAi on pat-10 transcription upon heat shock. (D) Quantified expression of pat-10 in the pat-10 OE strain. (E) Effect of pat-10 overexpression or pat-10 RNAi on thermotolerance. (F) Life-span survival curves for WT or pat-10–overexpressing strains treated with control or pat-10 RNAi. (G) Life-span survival curves for WT or hsf-1–overexpressing strains treated with control or pat-10 RNAi. *P < 0.05; error bars indicate SEM.

Because loss of pat-10 expression reduced thermotolerance, we tested whether ectopic overexpression of pat-10 could render animals more thermotolerant. Twofold overexpression of pat-10 (Fig. 2D) significantly increased heat protection (Fig. 2E) and extended life span (Fig. 2F). Furthermore, RNAi of pat-10 eliminated the increased thermotolerance (Fig. 2E) and life span (Fig. 2F) of the pat-10 overexpression strain. pat-10 RNAi also abolished the extended life spans of the hsf-1 overexpression strains (Fig. 2G). Thus, pat-10 appears to be necessary and sufficient for increased thermotolerance and longevity. Additionally, the beneficial effects of pat-10 overexpression were not due to increases in basal HSP transcription (fig. S7). One function of pat-10 is its role in the troponin complex (911), which is necessary for the contraction of body wall muscles. However, RNAi toward the worm homolog of tropomyosin—lev-11, a partner with pat-10 in the troponin complex—did not affect heat resistance (Fig. 2A). This suggests that the role of pat-10 in muscle contraction does not influence thermotolerance.

Loss of pat-10 also disrupts actin cytoskeleton dynamics and endocytosis (1013). To address these potential mechanisms of protection, we used green fluorescent protein (GFP)–labeled muscle filaments to assess actin organization (14). Upon heat shock, muscle filaments became unorganized and damaged, leading to impaired motility (Fig. 3, A and B). However, pat-10 overexpression was sufficient to prevent heat-induced muscle and motility deterioration (Fig. 3, A and B). Furthermore, heat shock decreased the ratio of the filamentous (F) actin to globular (G) actin in WT worms, whereas the protected pat-10 overexpression animals maintained F actin upon exposure to heat stress (Fig. 3C and fig. S8). With regard to aging, the ratio of F to G actin also decreased with age, and pat-10 overexpression lessened this decline (Fig. 3D and fig. S8). Hence, under conditions of acute stress or gradual age-related deterioration, the integrity of the actin cytoskeleton is correlated with organismal survival, and overexpression of pat-10 can abrogate the collapse of actin filaments.

Fig. 3 pat-10 overexpression improves actin cytoskeletal integrity and cellular trafficking.

(A) GFP-tagged myosin heavy chain in muscle detected by means of fluorescent microscopy, before and after heat shock (HS). (B) Worm thrashes per minute in liquid to monitor motility after heat shock. (C and D) Abundance of filamentous (F) actin or globular (G) actin after (C) heat shock or (D) aging. Ponceau S staining shown as a loading control. (E) Microscopy showing ssGFP derived from muscle cells (m), endocytosed by coelomocytes (c) to be degraded. (F) Normalized ssGFP fluorescence quantification with and without pat-10 overexpression. (G) Effect of blocking coelomocytic endocytosis on GFP fluorescence in the ssGFP reporter strain. (H) Thermotolerance after RNAi of cup-4. *P < 0.05; error bars indicate SEM. Scale bars, 10 μm.

RNAi-induced loss of pat-10 disrupts endocytosis through impairment of the actin cytoskeleton (12, 13, 15). To assay the role of pat-10 in endocytosis, we used a secretion and endocytosis reporter designed to actively secrete GFP (ssGFP) from muscle cells into the pseudocoelomic fluid, where it is endocytosed by the coelomocyte cells and degraded (fig. S9A) (16). Therefore, the ssGFP reports upon effective muscular secretion and endocytosis by coelomocytes. Fitting the hypothesis that pat-10 overexpression improves transport and cellular processing through improved subcellular scaffolding, the pat-10 OE strain had a decrease in overall ssGFP fluorescence (Fig. 3, E and F). The decrease in ssGFP resulted from improved secretion and uptake, as shown by the absence of fluorescence in the muscle and pseudocoelomic fluid (Fig. 3E). This decrease was not due to an overall decrease in expression of GFP (fig. S9B). Conversely, RNAi of pat-10 increased overall fluorescence through decreased muscle secretion and coelomocytic endocytosis (Fig. 3, E and G). To fully block coelomocytic uptake and degradation of ssGFP, RNAi of cup-4, a ligand-gated ion channel required in endocytosis (17), showed an even higher increase in fluorescence (Fig. 3G) and also reduced thermotolerance in the wild type (Fig. 3H). Collectively, these data indicate pat-10 has an active role in cytoskeletal maintenance, which is critical to cellular transport.

To test for conservation, we disrupted the actin cytoskeleton in human embryonic kidney (HEK) 293T cells using cytochalasin D, which blocks the addition of actin monomers to filaments (18), or latrunculin A, which binds actin monomers and prevents polymerization (Fig. 4A) (19). Inhibiting filamentous actin formation with either cytochalasin D or latrunculin A significantly reduced thermotolerance in human cells without causing death at permissive temperatures (Fig. 4B and fig. S10). Similar to our C. elegans data, these findings reiterate the importance of the actin cytoskeleton during times of cellular stress.

Fig. 4 Impairing actin dynamics decreases thermotolerance in mammalian cell culture.

(A) Microscopy of HEK293T cells treated with cytochalasin D or latrunculin A [phalloidin stain of actin in red, 4ʹ,6-diamidino-2-phenylindole (DAPI) stain of DNA in blue]. (B) Thermotolerance of HEK293T cells after a heat shock at 45°C for 2 hours treated with cytochalasin D or latrunculin A. (C) Proposed model of the dual pathways of HSF-1–mediated health assurance. *P < 0.05; error bars indicate SEM. Scale bars, 5 μm.

Elevated levels of hsf-1 have been shown to benefit multiple organisms, yet its oncogenic properties are a major therapeutic drawback (20, 21). Because the inducible chaperone network promotes survival and proliferation of metastasizing cells (22), the ability to harness protective, nonchaperone components within the HSF-1 signal transduction cascade appears essential for future drug development. Identification of pat-10 as a modifier of thermotolerance and longevity may apply to mammalian systems without the typical oncogenic dangers associated with increased chaperone levels.

The hsf-1(CT) strain was still able to mount a transcriptional response to heat shock, albeit reduced in complexity of hsf-1(FL). The molecular mechanism remains unclear by which hsf-1(CT) regulates transcription without the C-terminal activation domain, but possible explanations include HSF-1 containing multiple activation domains. Alternatively, the hsf-1(CT) modification may alter affinities to DNA-binding sites or different cofactors, which would modify the transcriptional profile.

Our findings underscore the importance of maintaining filamentous actin, as opposed to total levels of actin. We propose a model in which HSF-1 regulates chaperones and actin cytoskeletal genes in parallel to promote thermotolerance and longevity (Fig. 4C). In the absence of chaperone induction, stabilization of the actin cytoskeleton is sufficient to promote survival under conditions of cellular stress and aging.

Supplementary Materials

www.sciencemag.org/content/346/6207/360/suppl/DC1

Materials and Methods

Figs. S1 to S10

Tables S1

References (2340)

References and Notes

  1. Acknowledgments: Bioinformatic analysis is included in the supplementary materials. The following funding sources supported this research: Howard Hughes Medical Institute, National Center for Research Resources (5P41RR011823-17), National Institute of General Medical Sciences (8 P41 GM103533-17), and National Institute on Aging (R01AG027463-04). N.A.B. was funded by a postdoctoral fellowship to the Salk Center for Nutritional Genomics from the Leona M. & Harry B. Helmsley Charitable Trust; P.M.D. was funded by George E. Hewitt Foundation for Medical Research and National Institute of Aging (1K99AG042495-01A1). We thank the laboratory of G. Lithgow for generously sharing the HSP-16 antibody and J. Durieux for helping design figure illustrations. Some of the nematode strains used in this work were provided by the Caenorhabditis Genetics Center (University of Minnesota), which is supported by the NIH–Office of Research Infrastructure Programs (P40 OD010440).
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