C. elegans electrotaxis behavior is modulated by heat shock response and unfolded protein response signaling pathways

Taylor, Shane K. B.; Minhas, Muhammad H.; Tong, Justin; Selvaganapathy, P. Ravi; Mishra, Ram K.; Gupta, Bhagwati P.

doi:10.1038/s41598-021-82466-z

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Published: 04 February 2021

C. elegans electrotaxis behavior is modulated by heat shock response and unfolded protein response signaling pathways

Shane K. B. Taylor¹^na1,
Muhammad H. Minhas¹^na1,
Justin Tong¹,
P. Ravi Selvaganapathy²,
Ram K. Mishra³ &
…
Bhagwati P. Gupta¹

Scientific Reports volume 11, Article number: 3115 (2021) Cite this article

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Abstract

The nematode C. elegans is a leading model to investigate the mechanisms of stress-induced behavioral changes coupled with biochemical mechanisms. Our group has previously characterized C. elegans behavior using a microfluidic-based electrotaxis device, and showed that worms display directional motion in the presence of a mild electric field. In this study, we describe the effects of various forms of genetic and environmental stress on the electrotactic movement of animals. Using exposure to chemicals, such as paraquat and tunicamycin, as well as mitochondrial and endoplasmic reticulum (ER) unfolded protein response (UPR) mutants, we demonstrate that chronic stress causes abnormal movement. Additionally, we report that pqe-1 (human RNA exonuclease 1 homolog) is necessary for the maintenance of multiple stress response signaling and electrotaxis behavior of animals. Further, exposure of C. elegans to several environmental stress-inducing conditions revealed that while chronic heat and dietary restriction caused electrotaxis speed deficits due to prolonged stress, daily exercise had a beneficial effect on the animals, likely due to improved muscle health and transient activation of UPR. Overall, these data demonstrate that the electrotaxis behavior of worms is susceptible to cytosolic, mitochondrial, and ER stress, and that multiple stress response pathways contribute to its preservation in the face of stressful stimuli.

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Introduction

Organisms have evolved intricate molecular machineries in order to respond to adverse or stressful stimuli as their survival depends on their ability to continuously monitor the environment and mount appropriate responses. Since the initial work of Hans Selye in 1936, who studied stress response in rats and termed it ‘general adaptation syndrome’^1,2, the work in the field of stress biology has grown exponentially, leading to a better understanding of the connections between stress and diseases. Animals exposed to stressful conditions such as heat, toxins, starvation, and psychological stress activate signaling cascades leading to physiological changes. For instance, in the larvae of fruit fly Drosophila melanogaster, noxious stimuli induce a stereotyped escape response mediated by neuronal signaling that manifests as rolling and bending reactions³. Work in rats showed that when animals were separated from their mothers as pups they showed increased stress response to environmental conditions⁴. Studies in humans have shown that stress can induce intracellular signaling leading to changes in prefrontal cortex function, thereby impacting learning and memory⁵. In addition, there are numerous reports on the endocrine system responding to stress via signaling cascades mediated by hormones such as adrenaline and cortisol⁶.

Detrimental stress in eukaryotes can often lead to an accumulation of misfolded proteins. The stress caused by this accumulation is mitigated by the molecular machinery consisting of several proteins that constitute the UPR signaling network⁷. The three well-known UPR pathways function in the cytosol, mitochondria, and ER, respectively⁷. The cytosolic heat shock response (HSR), UPR^ER, and UPR^MT regulate both the expression and function of multiple chaperons to maintain homeostasis. These chaperons are members of the heat shock protein family and are transcriptionally regulated by the heat shock factor (HSF), which is a major player in the HSR. Together, UPR^MT, UPR^ER, and HSR allay cellular stress by attenuating translation to maintain protein homeostasis within the cell, lowering reactive oxygen species (ROS), and inducing cell death⁷.

UPR^MT is activated following mitochondrial dysfunction induced by misfolded protein accumulation within the mitochondria, and disrupts oxidative phosphorylation⁸. The response is similar across eukaryotes, although the regulation in mammals appears to be more complex⁸. Experiments in the nematode Caenorhabditis elegans have revealed that ATFS-1 (activating transcription factor associated with stress-1), an ATF5 homolog and bZip transcription factor family member, acts as a sensor protein⁹. ATFS-1 is normally degraded inside healthy mitochondria, but upon mitochondrial dysfunction is transported to the nucleus to regulate gene expression⁸. Failure to reduce mitochondrial stress can result in dysfunctional mitochondria which, if not cleared by the protective mechanism of mitophagy^8,10, can lead to several deleterious conditions, including premature aging, sarcopenia, and cardiovascular disease¹⁰.

Similar to mitochondrial stress, ER stress is triggered by the accumulation of misfolded proteins in the ER. The UPR^ER response is facilitated by three different transmembrane proteins: inositol-requiring enzyme 1 (IRE1), protein kinase RNA (PKR)-like ER kinase (PERK), and activating transcription factor 6 (ATF6), which then activate distinct signaling pathways. There are significant redundancies between the pathways, with overlapping transcriptional activity. All three pathways regulate the expression of chaperones, including GRP78/BiP¹¹. A key transcription factor that modulates UPR^ER signaling is the X-Box binding protein-1 (XBP1). While IRE1 is the main regulator of XBP1 activity, ATF6 and PERK also participate in IRE1-XBP1 signaling^11,12. Failure to regulate ER stress can lead to diseases such as neurodegeneration, metabolic disorders, and cancer^13,14.

Much of our knowledge of how stress affects behavior and the underlying signaling mechanisms have emerged from studies using a small set of model organisms, including C. elegans. This model nematode (worm) is a particularly attractive system due to its genetic tractability, ease of culture, and short life cycle, all of which serve to greatly expedite the rate of discovery¹⁵. Despite its relative simplicity, approximately half of C. elegans genes have homologs in humans and utilize many of the same processes¹⁶. Cellular stress can affect diverse processes in worms. Manipulations that increase mitochondrial and ER stress affect lifespan, fertility, susceptibility to diseases, and many behavioral processes^17,18,19. The small size of the worm greatly facilitates experimentation using microfluidic devices²⁰. Our lab has been investigating electrotaxis in C. elegans and how it is impacted by stress caused by genetic and environmental factors. In an earlier study, we demonstrated that a mild DC electric field in a microfluidic channel stimulates C. elegans to move towards the cathode in a directed manner, a response that is robust, highly repeatable, sensitive, and instantaneous²¹. Electrotactic movement serves as a powerful noninvasive response to evaluate the functional output of the nematode’s locomotory circuit following manipulation²². To understand whether the response is affected by treatments that increase stress, we exposed animals to different environmental conditions as well as investigated the impact of genetic mutations. Our findings show that chronic stress compromises electrotaxis speed whereas some forms of transient stress augments it. We found that worms exposed to chemicals that induce ER and mitochondrial stress exhibit reduced speed. A similar phenotype was also observed in mutant strains with disrupted mitochondrial and ER function. The essential role of HSR and UPR^MT is further supported by our data showing that animals having defects in both atfs-1 and hsf-1 function have a significantly slower movement response. Our analysis of the UPR^ER genes reveals that ire-1/xbp-1 and pek-1 pathways play essential roles in regulating the electrotaxis of C. elegans. We also report that a polyglutamine enhancer-1 protein, PQE-1, maybe involved in the maintenance of HSR and mitochondrial and ER stress, likely due to its role in regulating the global protein synthesis^23,24. To examine the sensitivity of electrotaxis speed to stress-inducing conditions, we exposed worms to chemical stressors, heat, different bacterial diets, and dietary restriction as well as daily exercise. The results showed that while chronic heat and reduced diet caused a decreased electrotactic movement, exercise had an increased response. Overall, our findings demonstrate that the electrotactic response of C. elegans is sensitive to cytosolic, mitochondrial, and ER stress. The results form the basis of future investigations of HSR, UPR^MT, and UPR^ER signaling utilizing microfluidic electrotaxis as a functional output of nematode locomotor circuits.

Results

Paraquat-induced oxidative stress reduces the electrotaxis speed of C. elegans

Oxidative stress is known to affect the behavioral responses of C. elegans^17,18,25. To investigate whether this form of stress can also alter electrotaxis in animals, we used paraquat (PQ), a herbicide that disrupts mitochondrial function by inducing the generation of superoxides^26,27. High concentrations of PQ have a detrimental effect on the lifespan of worms and cause damage to dopaminergic (DA) and other neurons by increasing ROS levels^{17,18,26,27,28}. We examined the effect of PQ-induced stress on electrotaxis by exposing animals to different doses of the chemical. While exposures to low or moderate concentrations ranging from 50 to 125 µM had no effect on the movement of day-1 adults, 250 µM of PQ caused a significant defect, resulting in a slower speed in the microfluidic channel (Fig. 1A). In addition to electrotaxis, PQ-exposed animals showed increased expression of mitochondrial chaperones. Specifically, the mitochondrial stress response reporters hsp-6::GFP (HSP70 family) and hsp-60::GFP (HSP60 family) were significantly upregulated (Fig. 1B,C).

To determine whether PQ affects other processes, we examined the expression of two other chaperones for ER and cytosolic stress, namely HSP-4 (BiP/GRP78 family member, ER stress) and HSP-16.2 (HSP16 family, cytosolic stress) following PQ treatment. Although the GFP reporters for both these chaperons showed a statistically significant increase in fluorescence, the fold change of the ER GFP reporter was much higher than that of mitochondrial reporters (Fig. 1D,E). Similar to changes in fluorescence reporters, the transcript analysis also showed higher levels of hsp-6 and hsp-16.2 following PQ exposure (Fig. 1F). We did not see a significant increase in hsp-4 transcript. These results are consistent with the previously reported finding that PQ induces both mitochondrial and ER stress in different animal models^29,30,31. Overall, PQ causes multiple stresses in C. elegans that contribute to a decrease in electrotaxis speed.

Mutations in genes regulating mitochondrial function affect electrotaxis

To further investigate whether the electrotactic response is perturbed by mitochondrial dysfunction, we conducted a survey of mutants affecting mitochondrial processes and UPR^MT. The set of genes consisted of an ortholog of ubiquinol-cytochrome C reductase (isp-1, Rieske iron sulfur protein subunit of mitochondrial complex III), a subunit of mitochondrial complex I (gas-1), a subunit of mitochondrial complex II (mev-1, cytochrome b), PTEN-induced kinase 1 (pink-1), mitochondrial uncoupling protein (UCP) ortholog (ucp-4), two mitochondrial superoxide dismutase orthologs (sod-2 and sod-3), coenzyme Q7 ortholog (clk-1), and atfs-1. Some of these genes are reported to affect motility of C. elegans on solid media and thrashing in liquid environment^32,33,34,35.

Analysis of the electrotactic response showed that most of the mutant animals, including mev-1(kn1), pink-1(ok3538), ucp-4(ok195), clk-1(e2519), sod-2(gk257), sod-3(tm760), and sod-2(gk257); sod-3(tm760) double, had no obvious defects; however, two particular mutants, isp-1(qm150) and gas-1(fc21), showed a significantly reduced electrotaxis speed (Fig. 2A). Although both genes encode components of the electron transport chain, isp-1(qm150) is long-lived with resistance to oxidative damage³⁶, while gas-1(fc21) is short-lived with increased ROS and sensitivity to oxidative damage³⁷, suggesting that neither lifespan differences nor ROS toxicity correlate with electrotaxis phenotypes. This notion is supported by the other mutant data, indicating that mev-1(kn1) has increased ROS and oxidative damage with short lifespan, clk-1(e2519) shows decreased oxidative damage and long lifespan, and sod mutants show increased oxidative damage with normal or extended lifespans, yet all exhibit similar electrotaxis speeds (Fig. 2A)^38,39.

We chose a subset of mitochondrial genes for further investigation. Specifically, we explored the possibility that mutant animals may not exhibit an electrotaxis phenotype on their own, but may do so when subjected to further stress. Consistent with this, mev-1(kn1) and pink-1(ok3538), which show sensitivity to PQ^18,38, displayed electrotaxis defects at exposure concentrations that do not alter the speed of control animals. Specifically, mev-1(kn1) animals exhibited speed deficits when cultured under chronic exposures to either 50 μM or 75 μM PQ (Fig. 2B). Similarly, pink-1(ok3538) animals were also affected, displaying significantly reduced electrotaxis speed at 125 μM PQ (Fig. 2C). Interestingly, ucp-4(ok195) adults displayed increased sensitivity to PQ under our chronic exposure paradigm, exhibiting slow electrotaxis at 75 μM (Fig. 2D). Thus, PQ-induced stress and mitochondrial defects perturb C. elegans electrotaxis speed.

To further investigate the effect of perturbations in UPR^MT on electrotaxis, we examined the phenotypes of two different atfs-1 alleles, a genetic null atfs-1(gk3094) and a gain-of-function atfs-1(et15). While the gk3094 mutation prevents the UPR^MT response, an opposite phenotype is caused by et15^40,41,42. Additionally, the HSR is activated in gk3094 animals⁴¹. Both atfs-1 mutants displayed significantly slower speeds than wild-type controls, although the null allele phenotype was most severe (Fig. 2E). We also knocked down atfs-1 in isp-1 and gas-1 mutant strains and found that while the electrotaxis speed of isp-1(qm150) was reduced, there was no change in gas-1(fc21) animals (Fig. 2F). Collectively, these data demonstrate that electrotaxis speed is sensitive to disruptions in mitochondrial function, UPR^MT, and HSR.

Impairment of UPR^ER causes electrotaxis speed deficits

Our finding that PQ induces the expression of ER chaperone hsp-4::GFP (Fig. 1C) led us to investigate the impact of ER stress on electrotaxis using tunicamycin, a chemical that promotes protein misfolding by inhibiting protein glycosylation^43,44. As shown in Fig. 3A, chronic exposure to tunicamycin at concentrations of 5 μg/mL or greater significantly reduced electrotaxis speed. These results are supported by the analysis of hsp-4::GFP reporter expression and hsp-4 transcript (Fig. 3B,C), suggesting the detrimental effect of tunicamycin exposure⁴⁰. As expected, hsp-6 level was unchanged (Fig. 3C). We did not test electrotactic responses of animals at concentrations higher than 10 μg/mL due to significant toxicity, as judged by the highly pronounced larval lethality and slow growth of escapers at this concentration (data not shown).

Our group had previously demonstrated the role of dopaminergic neurons in mediating electrotaxis²². Therefore, we examined whether tunicamycin-induced ER stress will affect these neurons in our assay. The analysis of dat-1::YFP reporter revealed visible defects in neuronal morphology in treated animals illustrated by dendritic/axonal abnormalities and loss of cell bodies (Fig. S1A). A similar result was obtained following PQ treatment (Fig. S1B). These data suggest that toxin-induced neuronal damage may contribute to electrotaxis deficits in animals.

We further characterized ER disruption by testing the phenotypes of UPR^ER pathway mutants, specifically ire-1, pek-1, atf-6, and xbp-1. The speed was significantly lower in ire-1(v33), xbp-1(zc12), and pek-1(ok275) but not atf-6(ok551) animals compared to the control, (Fig. 3D). Interestingly, the defect was most pronounced in xbp-1 mutants, which may be due to the gene being regulated by multiple UPR^ER arms. We also examined the sensitivity of UPR^ER mutants by exposing them to tunicamycin. Treatment with 2 μg/mL caused a significant decrease in the mobility of all three strains (Fig. 3E). A higher concentration (5 μg/mL) was found to be toxic to worms, resulting in lethality (data not shown). Taken together, the results show that electrotaxis is susceptible to ER stress-inducing conditions, and all three UPR^ER transducers function to facilitate this behavior.

Next, the role of pqe-1 was investigated based on its potential link to ER stress. Earlier, pqe-1 mutants were reported to cause an increase in transgene expression and enhance polyglutamine (poly-Q) neurotoxicity^23,24. We speculated that both these phenotypes may be caused by proteotoxic stress due to a failure to regulate global protein synthesis. In support of this, pqe-1(ok1983) animals showed an increase in the expression of hsp-4 chaperon and xbp-1 (Fig. 4A, Fig. S2A). Interestingly, hsp-6 and hsp16.2 levels were also upregulated (Fig. 4A). As expected, the electrotaxis speed of mutant animals was significantly reduced (Fig. 4B), and the phenotype was exacerbated by treatment with PQ and tunicamycin (Fig. 4C,D). The sensitivity towards tunicamycin was particularly pronounced; although wild-type animals showed no abnormal electrotaxis phenotype at 2 μg/mL tunicamycin, pqe-1 mutants exhibited a significantly reduced speed (compare Fig. 4D with 3A). Concentrations of tunicamycin above 2 μg/mL induced growth arrest and lethality in pqe-1(ok1983) worms (data not shown). The mutant animals also exhibited an increased degeneration of dopaminergic neurons and shorter lifespan (Fig. S2B,C).

We investigated whether reducing protein translation would lower proteotoxicity leading to a rescue of the electrotaxis defect of pqe-1 mutant animals. This was tested by knocking down the eIF4G homolog, ifg-1, that encodes a component of the eIF4 complex⁴⁵. The results showed that ifg-1 RNAi was unable to suppress the phenotype of pqe-1 mutant (Figs. 4E, S3). Overall, these observations provide the first evidence that pqe-1 plays an essential role in maintaining multiple stress response pathways and electrotactic response of C. elegans.

Chronic heat impairs the electrotactic response

Studies have shown that heat treatments can have both positive and negative effects on animals due to activated HSR^7,46 and UPR^ER^7,47. We investigated the electrotactic movement of young adults that were subjected to acute and chronic heat conditions. Worms exposed to two types of heat pulses, namely 5 h at 25 °C and 1 h at 33 °C, showed no change in their speed (Fig. 5A). Three additional treatments with a longer exposure time (i.e., 8 h each at 15 °C, 25 °C, and 28 °C), did not affect the speed either (Fig. 5B); however, all three heat shock chaperons (hsp-4, hsp-6, and hsp-16.2) were significantly upregulated (Figs. 5C, S4), suggesting the activation of UPR pathways. By contrast, chronic heat treatment at 28 °C for 3 days significantly reduced the speed (Fig. 5D). This demonstrates that unlike shorter duration (up to 8 h) of heat pulses, prolonged heat stress affects the electrotaxis speed of C. elegans.

One of the ways by which heat can induce stress is by interfering with the function of the heat shock transcription factor (HSF)⁷. In C. elegans, the HSF ortholog hsf-1 is transcriptionally activated by a variety of stress conditions, including heat exposure⁴⁸. In support of hsf-1′s involvement, we found that hsf-1(sy441) mutants have a significant electrotaxis speed deficit (Fig. 5E), which was further enhanced by a 1 h heat pulse at 33 °C but not by 5 h at 25 °C (Fig. 5F). There was no change in hsp-6 and hsp-16.2 levels in hsf-1 mutant animals, although hsp-4 was significantly down-regulated (Fig. 5G). Given that hsf-1 regulates the expression of a large number of genes⁴⁹, we speculate that other heat shock chaperon(s) might mediate hsf-1 function in electrotaxis. We also found that the hsf-1(sy441) phenotype was exacerbated by PQ-induced stress and L1 starvation (Figs. S5, S6), which indicates that hsf-1 is a key regulator of diverse stress-induced responses.

Electrotactic movement decreases with reduced diet but increases after exercise

Another form of stress that we tested is starvation. Initially, the effects of two different starvation exposure protocols on electrotaxis were investigated. In one case, L1 larvae were subjected to starvation for 168 h and subsequently allowed to resume development in the presence of food. In the second case, day-1 adults were exposed to acute starvation conditions. Both nutrient deprivation treatments showed that the effect on electrotaxis was comparable to that of the controls (Fig. 6A,B).

We also investigated a chronic form of nutrient deprivation that involves reduced daily caloric intake. This treatment, termed ‘dietary restriction (DR),’ has been shown to be beneficial in animals such as lifespan extension, increased metabolic fitness, and reduced severity of age-related physiological decline^50,51,52. To this end eat-2 mutants were examined. eat-2(ad1116) animals have a longer lifespan that is attributed to chronic DR due to their slower pharyngeal pumping³³. We found that the mutants had a significantly slower speed, suggesting that DR affects electrotaxis behavior (Fig. 6C). Since eat-2 mutants show higher fluorescence based on hsp-4::GFP reporter analysis⁵³ but no change in hsp-6::GFP⁵⁴, we conclude that the sensitivity of electrotactic response to dietary restriction is likely due to the detrimental effect of persistent ER stress.

In addition to starvation and DR, we explored the effect of dietary changes on electrotaxis. Two of the bacterial strains that were initially tested were Streptomyces venezuelae and Bacillus thuringiensis. The metabolites secreted by these bacteria cause intracellular stress in C. elegans^55,56. We found that neither bacteria had an impact on electrotaxis (Fig. S7). Next, worms were fed with two plant pathogens, Agrobacterium tumefaciens and Pectobacterium carotovorum^57,58,59 and a nonpathogenic fungus, Cryptococcus aquaticus. Plant pathogens have been reported to have a detrimental effect on the lifespan of worms⁶⁰. None of these cultures had an obvious effect on electrotactic movement (Fig. S8). We also tested three commonly used E. coli strains, HB101, HT115, and DH5α, but, once again, observed no change in movement (Fig. S8). These results led us to conclude that electrotaxis behavior is unaffected by the above selected set of microorganisms, although the possibility of reduced nutrient intake in some or all cases cannot be ruled out. In the future, a larger set of microbial diets should be tested to investigate their effect on animals more thoroughly.

Finally, we investigated the effect of physical activity on the electrotactic movement of animals. The beneficial effects of exercise in humans and model organisms are well documented. In addition to improving muscle fitness, exercise also promotes neuronal health^61,62,63. Since swimming activity in C. elegans induces known features of exercise reported in mammalian systems^62,64,65, worms were allowed to swim in M9 for 30 min daily, starting at the L3 larval stage, for a period of 7 days (see Methods for details). The results showed that animals had a significantly faster electrotaxis speed compared to controls of the same age, although they were slower compared to day-1 controls (Fig. 7A). Consistent with this, all three heat shock chaperons, hsp-4, hsp-6, and hsp-16.2, were upregulated (Fig. 7B). Additionally, we found that exercise caused a significant increase in the proportion of animals with tubular muscle mitochondria^62,66 (Fig. 7C,D). There was no change in dopaminergic neurons (Fig. S9).

Discussion

The experiments described herein are the first to demonstrate that the electrotactic response of C. elegans is affected by mutations and environmental conditions that increase cytosolic, mitochondrial, and ER stress. We found that the electrotaxis speed was altered by treatment with PQ and tunicamycin, two chemicals that are known to negatively impact the locomotory behavior of worms on solid media^17,67. PQ exposure causes generation of ROS, which activates the expression of multiple chaperones such as hsp-4 (ER stress), hsp-6 and hsp-60 (both mitochondrial stress), and hsp-16.2 (cytosolic stress). The effects of PQ on mitochondrial and ER stress are well documented; hence, the effects of PQ on electrotaxis are likely due to increased stress associated with these two organelles^13,30,68.

Independent of ROS, our data show that mutations that cause mitochondrial stress also lead to decreased electrotaxis speed. The slow speed of atfs-1(gf) mutants indicates that the UPR^MT contributes to maintaining the wild-type behavior. Analysis of ucp-4, mev-1, and pink-1 mutants revealed that exposure to a relatively low concentration of PQ induced speed defects. In mammalian cells, mutations in UCP genes are associated with increased ROS and oxidative stress^69,70,71. Interestingly, Iser and colleagues (2005) reported earlier that ucp-4(ok195) worms exhibit wild-type lifespan and survival in the presence of PQ, although the animals display an elevated ATP level^36,72. Our finding that ucp-4 mutants have defects in electrotaxis suggests that the movement response of animals may be sensitive to ROS levels. Future studies directly measuring oxidative damage following PQ exposure in different genetic backgrounds will be helpful in illuminating the relationship between ROS and electrotaxis.

isp-1 and gas-1 mutants exhibit reduced electrotaxis speed. While mutations in both these genes are also associated with electron transport hindrance and increased ROS generation, the isp-1 mutant actually exhibits oxidative stress resistance due to low oxygen consumption and increased expression of mitochondrial superoxide dismutase SOD-3³⁶. On the other hand, gas-1 mutants are hypersensitive to oxidative stresses such as PQ^36,37. The lifespan phenotypes of isp-1 and gas-1 mutants are also opposite, wherein isp-1 animals have a long lifespan and gas-1 are short-lived, possibly due to differences in ROS production and ROS-associated enzymatic activity³⁶. These data suggest that electrotaxis speed is independent of pathways involved in lifespan maintenance. The isp-1(qm150) and gas-1(fc21) also both activate the UPR^MT^73,74, which led us to investigate the role of atfs-1. We found that atfs-1 null mutant and gain-of-function mutant had a slower speed in our assay. Moreover, RNAi knock-down of atfs-1 reduced the speed of isp-1 mutant but not gas-1 mutant animals. Together with known roles of atfs-1 in regulating the expression of mitochondrial and cytosolic heat shock chaperons, these results support the conclusion that multiple stress response signaling affect the electrotaxis behavior of C. elegans.

The effect of ROS induced by PQ and mitochondrial mutants could also impact UPR^ER. Thus, we investigated the responses of animals treated with tunicamycin and observed defects in electrotaxis speed that were accompanied by an increase in the expression of the ER chaperone GRP78/BiP homolog hsp-4. Similar electrotaxis phenotypes were also observed in UPR^ER mutants, indicating that the ER genes play a crucial role in mediating normal movement behavior. The analysis of mutants affecting the three major arms of UPR^ER revealed that while ire-1, pek-1, and xbp-1 are needed to maintain a normal response, atf-6 is not essential. Differences between UPR^ER pathway components in mediating some of the biological processes have been reported earlier. Shen et al. (2001) found that while knockdown of pek-1 and ire-1 affected larval development, there was no such phenotype in the case of atf-6⁷⁵. Additionally, ire-1 and xbp-1 mutants have a short lifespan and slower development, but pek-1 and atf-6 mutants appear normal^56,76. Finally, animals lacking ire-1, xbp-1, and atf-6 function show high sensitivity to pore forming bacterial toxin Cry5B but pek-1 does not appear to be involved⁴⁶. In spite of these differences, we found that increasing the ER stress load by tunicamycin treatment exacerbated the electrotaxis defects of all three UPR^ER pathway mutants.

In addition to the known UPR^ER genes, we also tested the less well-characterized gene pqe-1. Mutations in pqe-1 are thought to cause proteotoxic ER stress, possibly due to the upregulation of global protein synthesis⁷⁷. Consistent with this idea, our results showed that pqe-1 mutants have an abnormal electrotactic response. Studies in yeast have shown that the REXO1 (RNA exonuclease homolog 1) domain of PQE-1 functions in the processing of the 3′ ends of rRNA and tRNA^78,79, which may suggest a role of pqe-1 in the regulation of protein translation. Since pqe-1 localizes to the cell nucleus^23,24, it is possible that it affects genes whose products modulate the function of translation machinery. To investigate this further, we knocked-down the eIF4G homolog, ifg-1, a component of the initiation factor 4F (eIF4F) complex⁴⁵, but saw no suppression of the pqe-1 mutant phenotype. This result combined with high sensitivity of mutant animals to tunicamycin and changes in the expression of multiple chaperons and xbp-1 lead us to suggest that pqe-1 plays an essential role in maintaining both HSR and organelle-specific stress response (UPR) pathways in C. elegans.

One of the ways by which stress can affect electrotaxis is by causing damage to neurons. Consistent with this, animals exposed to PQ and tunicamycin exhibited degenerated dopaminergic neurons (Fig. S1). A similar phenotype was also observed in pqe-1 mutants (Fig. S2). These findings, along with our previous work demonstrating the role of dopaminergic neuron signaling in mediating electrotaxis behavior²², led us to conclude that neuronal defects may in part contribute to the electrotaxis phenotype in animals as a result of stress-inducing conditions.

In addition to examining the effects of genetic mutations and compounds, we investigated whether environmental perturbations alter the electrotactic response. Among other effects , heat is reported to induce mitochondrial stress⁷. High temperatures have also been shown to modulate ER and cytosolic stress. This may involve multiple mechanisms, such as activation or repression of PERK depending on the severity of the heat regimen⁴⁷ and changes in global protein synthesis and apoptosis⁴⁷. We exposed animals to acute heat and found that their electrotaxis was unaffected. Chronic heat, however, severely reduced the speed, suggesting that behavior is affected by prolonged heat exposure, which is consistent with cellular stress playing a role in modulating electrotaxis. The resistance to heat-induced stress appears to depend on the transcription factor hsf-1, since hsf-1 mutants exhibit electrotaxis defects and are sensitive to heat treatments.

As diet can also play a role in the UPR maintenance^53,80, we subjected worms to different forms of starvation. When C. elegans are starved during the L1 larval stage, they enter L1 arrest, which halts the development and reproductive growth, enhances stress resistance, modifies feeding behavior, and alters metabolic flux^81,82. Starvation during the L4 larval and adult stages has beneficial effects such as extended life span, germline cell reduction, delayed reproduction, and thermotolerance⁸³. At the cellular level, starvation induces both mitochondrial and ER stress^53,80,84. Mitochondria become fragmented in starved animals and are cleared through mitophagy^10,80,85. Zhang et al.⁸⁶ reported that starvation caused ER stress and led to activation of PERK/eIF2α in an astrocyte cell line. Other groups have shown changes in the regulation of different ER proteins, such as chaperones⁸⁴. Our work demonstrates that short-term DR does not affect the electrotactic response. However, eat-2 mutants that are chronically starved, and therefore mimic a long-term DR condition, show a significantly slower electrotaxis speed. It is conceivable that a reduction in speed may aid in the long life span of eat-2 mutants as the animals are slow feeders⁵³. This line of reasoning agrees with previous findings that young adults lacking eat-2 function move slower on solid media³³. Furthermore, eat-2 mutants show increased expression of hsp-4⁵³ but not hsp-6⁵⁴, suggesting higher ER stress levels.

The last stress condition we investigated was a daily exercise regimen. The benefits of exercise on muscular and neuronal health are well demonstrated. Healthier muscles through exercise allow animals to increase their athletic capacity⁶³. At the molecular level, exercise activates the UPR^ER response as a protective mechanism⁸⁷. Beneficial effects of exercise have also been demonstrated in C. elegans, such as a longer lifespan and improved neuronal and muscular health^62,64,65,88. Laranjeiro et al. (2017) reported that the ER chaperone hsp-4 is downregulated in animals subjected to exercise, which suggests a conserved role of UPR^ER in exercise-induced benefits⁶⁴. Our lab showed earlier that defects in muscles reduce the electrotaxis speed of C. elegans²². Consistent with the above studies, we found that daily exercise treatment significantly improved the electrotaxis speed of C. elegans and resulted in increased expression of heat shock chaperons as well as better preservation of mitochondrial morphology.

In summary, the work described in this paper shows that electrotactic movement of worms is reduced by mutations in genes that affect HSR, UPR^MT, and UPR^ER as well as following chronic exposure to chemicals, heat, and reduced diet. In contrast, short bursts of daily exercise are beneficial since they resulted in a higher speed of animals and better preservation of mitochondrial morphology. Collectively, these findings lead us to conclude that conditions that elevate cellular stress for prolonged periods cause detrimental effects, whereas transient stress imparts a beneficial effect on health. The results established that a microfluidic-based assay is a reliable output of locomotory circuits and demonstrate that electrotaxis can be used to study stress response pathways in C. elegans.

Materials and methods

Strains and culturing

This study used the following C. elegans strains: N2: wild-type Bristol isolate; MQ887: isp-1(qm150); CW152: gas-1(fc21); TK22: mev-1(kn1); CY121: ucp-4(ok195); DY356: pink-1(ok3538); GA184: sod-2(gk257); GA186: sod-3(tm760); GA480: sod-2(gk257); sod-3(tm760); QC115: atfs-1(et15); VC3201: atfs-1(gk3094); RB545: pek-1(ok275); RB772: atf-6(ok551); RE666: ire-1(v33); SJ17 xbp-1(zc12) III; zcIs4 V; SJ4100: zcIs13(hsp-6::GFP); SJ4058: zcIs9(hsp-60::GFP); PS3551: hsf-1(sy441); DY693: pqe-1(ok1983);zcIs4 V; RB1611: pqe-1(ok1983); SJ4005: zcIs4(hsp-4::GFP); DY542: pqe-1(ok1983); bhEx138[pGLC72(Cel-dat-1p::YFP)]; CL2070: dvIs70[hsp-16.2::GFP + rol-6(su1006)]; DY353: bhEx138[pGLC72(Cel-dat-1p::YFP)]; SJ4103: zcIs14[myo-3::GFP(mit)] and DY356 (5x outcrossed RB2547): pink-1(ok3538). RB2547 and all other strains were originally obtained from the Caenorhabditis Genetics Center (University of Minnesota, St. Paul, MN)⁸⁹.

Except where indicated, animals were grown and maintained at 20 °C on nematode growth medium (NGM) agar plates containing E. coli OP50 culture using previously described methods¹⁵. All experiments used age-synchronous populations obtained by bleach treatment¹⁵. Experiments involving wild-type and untreated controls were done using young adults unless stated otherwise. Growth times for all animals bearing mutations or undergoing treatments that affect developmental rate were appropriately adjusted.

Assays and treatments

The following methods were used to analyze worms in this study. Additional details are provided in supplementary methods.

Chemical treatments

Paraquat dichloride was obtained from Sigma-Aldrich (St. Louis, MO, USA) and tunicamycin was obtained from Bioshop Canada Inc (Burlington, ON, CA). Paraquat treatments were first prepared as 20 × solutions in M9 buffer; subsequently, 1 × treatment plates were produced by spreading 500 μL of the 20 × solutions across the surface of plates containing 10 mL of NGM agar. Tunicamycin treatments were first prepared as 5 mg/mL stock solutions in 100% DMSO, then diluted with water to make 20 × solutions; subsequently, 1 × treatment plates were produced by spreading 500 μL of the 20 × solutions across the surface of plates containing 10 mL of NGM agar. For chemical treatments, synchronized worms were grown on chemical-containing plates from L1 until adulthood. Young day-1 adults, based on direct observation, were used for experiments.

Heat stress

Animals were first grown at 20 °C until young adulthood. Plates were then sealed with parafilm and exposed to specific temperatures in a water bath for desired durations. Control plates were also sealed but kept at 20 °C. After the treatment, parafilm was removed and animals were allowed to recover at 20 °C for 30 min to an hour before electrotaxis assays. For 28 °C treatment, synchronized larvae were allowed to grow at 20 °C till day-1 adulthood and then shifted to 28 °C for 3 days.

Feeding bacterial and fungal cultures

C. elegans plates were seeded with overnight grown cultures of microorganisms. For E. coli, plant pathogens and fungus, L1 animals were transferred on plates and allowed to grow till adulthood. Day-1 adults were used for electrotaxis assays. In the case of Streptomyces venezuelae and Bacillius thuringiensis, L1 larvae were first fed with E. coli OP50 till adulthood. One day-old adults were transferred on maltose yeast extract medium (MYM) plates containing culture of S. venezuelae for 24 h. Bacillus thuringiensis culture was grown on NGM agar and fed to one day-old adults for 24 h. Electrotaxis was performed the following day. Control worms were grown in the presence of OP50 culture on NGM-Agar and MYM-Agar plates.

Starvation

Animals were first grown on OP50 until day-1 adulthood. They were then transferred to an unseeded NGM agar plate (no food) for 8 h to mimic acute starvation. Following the starvation period, electrotaxis assay was performed.

Exercise

Worms were subjected to exercise starting the L3 larval stage until day-6 adulthood (for a total of 7 days). Two different treatments were performed, each of which consisted of 30 min of daily swimming, followed by recovery on LB-agar plates. The first exercise regimen involved a belly dancer shaker in which worms, suspended in M9, were subjected to 100 rpm rotation. The second regimen consisted of keeping worms in M9 on the countertop without shaking, thereby allowing them to perform natural swimming.

Microchannel fabrication and electrotaxis assay

Microfluidic channels were fabricated as previously described^21,90. The channel design was printed on a transparency sheet using high-resolution photoplotting to create a photomask, which was then used in conjunction with SU-8 100 negative photoresist (MicroChem Corp., MA, USA) to lithographically pattern the design onto a silicon wafer. Microchannels were then casted by pouring polydimethylsiloxane (PDMS) pre-polymer (Sylgard 184 Kit, Dow Corning Corp., MI, USA; 10:1 ratio of base and cross-linker) onto the resultant master mold and allowing 24 h for curing. The channel was then excised from the PDMS replica and fluid access ports were punched into each end. Next, the channel, a blank PDMS strip and a glass slide were oxidized via exposure to oxygen plasma for 40 s at 40 W power and stuck together to seal the microchannel. Lastly, plastic tubing and insulated copper wire were affixed to the punched reservoirs and secured with PDMS pre-polymer.

The electrotaxis assay protocol has also been described^21,90. A syringe was attached to one of the inlet/outlet tubes of the PDMS microchannel to facilitate worm loading at the other tube. A power supply was connected via insulated copper wiring to the electrodes of the microchannel device to provide worms with electrical stimulus. A microscope, camera and monitor allowed visualization and recording of the electrotaxis experiment.

In preparation for the assay, worms were washed off of their culture plates, cleaned, and suspended in M9 buffer. Animals were then aspirated into the channel using the syringe pump. Both tubes were then laid flat at the same elevation to eliminate pressure-induced flow. Next, a 3 V/cm DC electric field was applied, and the worm’s resultant behaviour recorded by camera.

Electrotaxis was carried out for up to 5 min. Animals were allowed to travel a minimum distance of 5 mm in one direction, towards the cathode, after which the field polarity was reversed to induce a turning response. Locomotory data was extracted from recorded videos using custom MATLAB-based worm tracking software. Electrotaxis speed data was plotted in box plots.

Fluorescence microscopy and quantification

Animals were mounted on 2% agar pad containing glass slides. Before placing the cover slip, they were anesthetized using a 15 μL drop of 30 mM NaN₃ mixed with a drop of M9. GFP fluorescence was visualized using a Zeiss Observer Z1 microscope equipped with an Apotome 2 and X-Cite R 120LED fluorescence illuminator. Fluorescence intensity was quantified using NIH ImageJ (http://rsbweb.nih.gov/ij/). Neurodegeneration was manually scored by counting the number of cell bodies of DAergic neurons and by assessing neurite morphologies and trajectories. Worms consist of two pairs of cephalic neurons (CEPs), a pair of anterior deirid neurons (ADE) and one pair of posterior dieirid neurons (PDE).

RNAi

The atfs-1 RNAi plasmid (pGLC171) was constructed by inserting a 759 bp genomic fragment into the L4440 vector using the restriction enzymes KpnI and XbaI. The fragment was obtained by PCR using the forward primer GL1649 (5'-AAGGGTACCCACTACTTGGAGAGCGACGAC-3') and reverse primer GL1650 (5'-AAGTCTAGACTACTTCTTGGAACTCCCTGC-3'). We used the RNAi gene silencing protocol described by Wu et al.⁴¹ Since authors reported that knock-down in parental isp-1 mutant worms caused an arrest of F1 larvae, L4-stage animals were treated with RNAi and day-2 old adults were analyzed. Synchronized worms were grown on OP50 until L4 stage and then transferred to RNAi plates containing either L4440 empty plasmid or atfs-1 RNAi plasmid. atfs-1 knock-down was confirmed by qPCR, which showed a range of ~ 2 × to 10 × reduction in different batches. For the ifg-1 RNAi, animals were fed with bacteria from Ahringer library. In this RNAi paradigm synchronized animals were grown on OP50 until day-1 adult stage and then transferred to ifg-1 RNAi plates for 48 h and analyzed right after.

Quantitative reverse transcription PCR (RT-qPCR)

For RT-qPCR experiments, synchronized cultures were prepared by bleaching adult hermaphrodites twice in two successive generations to obtain highly synchronous cultures. After the second round of bleaching, eggs were grown until the day-1 adulthood. Total RNA was extracted from these animals using trizol (Catalog Number T9424, Sigma-Aldrich, Canada), according to the manufacturer’s instructions. The SensiFAST cDNA Synthesis Kit (Catalog Number BIO-65053, MeridianBioscience, Canada) was used to obtain cDNA according to manufacturer’s instructions. RT-qPCR was performed (in triplicate) using the Bio-Rad cycler CFX 96 machine. The primers are listed in the Supplementary table (Table S1). The reaction was set up using SensiFAST SYBR Green Kit (Catalog Number BIO-98005, BIOLINE, USA) according to the manufacturer’s instructions. The expression levels of the analyzed genes were normalized to pmp-3.

Data analysis

GraphPad Prism 7 was used to plot the graphs. For all assays, data from repeat experiments were pooled and analyzed together. Statistical analyses were done using GraphPad Prism 7 except for RT-qPCR experiments that were analyzed using CFX Maestro 3.1 software (Bio-Rad, Canada; https://www.bio-rad.com/en-ca/product/cfx-maestro-software-for-cfx-real-time-pcr-instruments). P values less than 0.05 were considered statistically significant.

Data availability

All data generated or analysed during this study are included in this published article (and its Supplementary Information files). Additional details are available from the corresponding author on reasonable request.

References

Fink, G. In retrospect: eighty years of stress. Nature 539, 175–176 (2016).
Article ADS PubMed CAS Google Scholar
Selye, H. A syndrome produced by diverse nocuous agents. Nature 138, 32 (1936).
Article ADS Google Scholar
Yoshino, J., Morikawa, R. K., Hasegawa, E. & Emoto, K. Neural circuitry that evokes escape behavior upon activation of nociceptive sensory neurons in drosophila larvae. Curr. Biol. 27, 2499-2504.e3 (2017).
Article CAS PubMed Google Scholar
Ladd, C. O. et al. Long-term behavioral and neuroendocrine adaptations to adverse early experience. Prog. Brain Res. 122, 81–103 (2000).
Article CAS PubMed Google Scholar
Arnsten, A. F. T. Stress signalling pathways that impair prefrontal cortex structure and function. Nat. Rev. Neurosci. 10, 410–422 (2009).
Article CAS PubMed PubMed Central Google Scholar
McEwen, B. S. Mood disorders and allostatic load. Biol. Psychiat. 54, 200–207 (2003).
Article PubMed Google Scholar
Higuchi-Sanabria, R., Frankino, P. A., Paul, J. W., Tronnes, S. U. & Dillin, A. A futile battle? Protein quality control and the stress of aging. Dev. Cell 44, 139–163 (2018).
Article CAS PubMed PubMed Central Google Scholar
Shpilka, T. & Haynes, C. M. The mitochondrial UPR: mechanisms, physiological functions and implications in ageing. Nat. Rev. Mol. Cell Biol. 19, 109–120 (2018).
Article CAS PubMed Google Scholar
Fiorese, C. J. et al. The transcription factor ATF5 mediates a mammalian mitochondrial UPR. Curr. Biol. 26, 2037–2043 (2016).
Article CAS PubMed PubMed Central Google Scholar
Melber, A. & Haynes, C. M. UPR mt regulation and output: a stress response mediated by mitochondrial-nuclear communication. Cell Res. 28, 281–295 (2018).
Article CAS PubMed PubMed Central Google Scholar
Remondelli, P. & Renna, M. The endoplasmic reticulum unfolded protein response in neurodegenerative disorders and its potential therapeutic significance. Front. Mol. Neurosci. 10, 187 (2017).
Article PubMed PubMed Central CAS Google Scholar
Tsuru, A., Imai, Y., Saito, M. & Kohno, K. Novel mechanism of enhancing IRE1α-XBP1 signalling via the PERK-ATF4 pathway. Sci. Rep. 6, 1–8 (2016).
Article CAS Google Scholar
Salminen, A. & Kaarniranta, K. ER stress and hormetic regulation of the aging process. Ageing Res. Rev. 9, 211–217 (2010).
Article CAS PubMed Google Scholar
Wang, S. & Kaufman, R. J. The impact of the unfolded protein response on human disease. J. Cell Biol. 197, 857–867 (2012).
Article CAS PubMed PubMed Central Google Scholar
Brenner, S. The genetics of caenorhabditis elegans. Genetics 77, 71–94 (1974).
Article CAS PubMed PubMed Central Google Scholar
Corsi, A. K., Wightman, B. & Chalfie, M. A transparent window into biology: a primer on Caenorhabditis elegans. Genetics 200, 387–407 (2015).
Article CAS PubMed PubMed Central Google Scholar
Gourgou, E. & Chronis, N. Chemically induced oxidative stress affects ASH neuronal function and behavior in C. elegans. Sci. Rep. 6, 1–9 (2016).
Article CAS Google Scholar
Wu, S. et al. Mutation of hop-1 and pink-1 attenuates vulnerability of neurotoxicity in C. elegans: the role of mitochondria-associated membrane proteins in Parkinsonism. Exp. Neurol. 309, 67–78 (2018).
Article CAS PubMed PubMed Central Google Scholar
Cristina, D., Cary, M., Lunceford, A., Clarke, C. & Kenyon, C. A regulated response to impaired respiration slows behavioral rates and increases lifespan in caenorhabditis elegans. PLoS Genet. 5, e1000450 (2009).
Article PubMed PubMed Central CAS Google Scholar
Gupta, B. P. & Rezai, P. Microfluidic approaches for manipulating, imaging, and screening C. elegans. Micromachines 7 (2016).
Rezai, P., Siddiqui, A., Selvaganapathy, P. R. & Gupta, B. P. Electrotaxis of Caenorhabditis elegans in a microfluidic environment. Lab Chip 10, 220–226 (2010).
Article CAS PubMed Google Scholar
Salam, S. et al. A microfluidic phenotype analysis system reveals function of sensory and dopaminergic neuron signaling in C. elegans electrotactic swimming behavior. Worm 2, e24558 (2013).
Article PubMed PubMed Central Google Scholar
Faber, P. W., Voisine, C., King, D. C., Bates, E. A. & Hart, A. C. Glutamine/proline-rich PQE-1 proteins protect Caenorhabditis elegans neurons from huntingtin polyglutamine neurotoxicity. Proc. Natl. Acad. Sci. USA 99, 17131–17136 (2002).
Article ADS CAS PubMed PubMed Central Google Scholar
Yamada, K., Tsuchiya, J. I. & Iino, Y. Mutations in the pqe-1 gene enhance transgene expression in Caenorhabditis elegans. G3 Genes Genomes Genet. 2, 741–751 (2012).
CAS Google Scholar
Kumsta, C., Thamsen, M. & Jakob, U. Effects of oxidative stress on behavior, physiology, and the redox thiol proteome of Caenorhabditis elegans. Antioxid. Redox Signal. 14, 1023–1037 (2011).
Article CAS PubMed PubMed Central Google Scholar
Lee, S. J., Hwang, A. B. & Kenyon, C. Inhibition of respiration extends C. elegans life span via reactive oxygen species that increase HIF-1 activity. Curr. Biol. 20, 2131–2136 (2010).
Article CAS PubMed PubMed Central Google Scholar
Yang, W. & Hekimi, S. A mitochondrial superoxide signal triggers increased longevity in Caenorhabditis elegans. PLoS Biol. 8, e1000556 (2010).
Article PubMed PubMed Central CAS Google Scholar
Ortiz-Ortiz, M. A. et al. Curcumin enhances paraquat-induced apoptosis of N27 mesencephalic cells via the generation of reactive oxygen species. Neurotoxicology 30, 1008–1018 (2009).
Article CAS PubMed PubMed Central Google Scholar
Zhang, X. F., Thompson, M. & Xu, Y. H. Multifactorial theory applied to the neurotoxicity of paraquat and paraquat-induced mechanisms of developing Parkinson’s disease. Lab. Investig. 96, 496–507 (2016).
Article CAS PubMed Google Scholar
Chinta, S. J. et al. Coupling endoplasmic reticulum stress to the cell death program in dopaminergic cells: effect of paraquat. NeuroMol. Med. 10, 333–342 (2008).
Article CAS Google Scholar
Cao, S. S. & Kaufman, R. J. Endoplasmic reticulum stress and oxidative stress in cell fate decision and human disease. Antioxid. Redox Signal. 21, 396–413 (2014).
Article CAS PubMed PubMed Central Google Scholar
Cooper, J. F. et al. Activation of the mitochondrial unfolded protein response promotes longevity and dopamine neuron survival in Parkinson’s disease models. Sci. Rep. 7, 1–16 (2017).
Article CAS Google Scholar
Bansal, A., Zhu, L. J., Yen, K. & Tissenbaum, H. A. Uncoupling lifespan and healthspan in caenorhabditis elegans longevity mutants. Proc. Natl. Acad. Sci. USA 112, E277–E286 (2015).
Article ADS CAS PubMed PubMed Central Google Scholar
Jafari, G. et al. Tether mutations that restore function and suppress pleiotropic phenotypes of the C. elegans ISP-1(qm150) Rieske iron-sulfur protein. Proc. Natl. Acad. Sci. USA 112, E6148–E6157 (2015).
Article CAS PubMed PubMed Central Google Scholar
Rollins, J. A., Howard, A. C., Dobbins, S. K., Washburn, E. H. & Rogers, A. N. Assessing health span in Caenorhabditis elegans: lessons from short-lived mutants. J. Gerontol. Ser. A. Biol. Sci. Med. Sci. 72, 473–480 (2017).
Article CAS Google Scholar
Feng, J., Bussière, F. & Hekimi, S. Mitochondrial electron transport is a key determinant of life span in Caenorhabditis elegans. Dev. Cell 1, 633–644 (2001).
Article CAS PubMed Google Scholar
Hartman, P. S., Ishii, N., Kayser, E. B., Morgan, P. G. & Sedensky, M. M. Mitochondrial mutations differentially affect aging, mutability and anesthetic sensitivity in Caenorhabditis elegans. Mech. Ageing Dev. 122, 1187–1201 (2001).
Article CAS PubMed Google Scholar
Sedensky, M. M. & Morgan, P. G. Mitochondrial respiration and reactive oxygen species in C. elegans. Exp. Gerontol. 41, 957–967 (2006).
Article CAS PubMed Google Scholar
Van Raamsdonk, J. M. & Hekimi, S. Deletion of the mitochondrial superoxide dismutase sod-2 extends lifespan in Caenorhabditis elegans. PLoS Genet. 5 (2009).
Bennett, C. F. et al. Activation of the mitochondrial unfolded protein response does not predict longevity in Caenorhabditis elegans. Nat. Commun. 5, 1–10 (2014).
Article CAS Google Scholar
Wu, Z. et al. Mitochondrial unfolded protein response transcription factor ATFS-1 promotes longevity in a long-lived mitochondrial mutant through activation of stress response pathways. BMC Biol. 16, 147 (2018).
Article CAS PubMed PubMed Central Google Scholar
Rauthan, M., Ranji, P., Pradenas, N. A., Pitot, C. & Pilon, M. The mitochondrial unfolded protein response activator ATFS-1 protects cells from inhibition of the mevalonate pathway. Proc. Natl. Acad. Sci. USA 110, 5981–5986 (2013).
Article ADS CAS PubMed PubMed Central Google Scholar
Banerjee, A. et al. Unfolded protein response is required in nu/nu mice microvasculature for treating breast tumor with tunicamycin. J. Biol. Chem. 286, 29127–29138 (2011).
Article CAS PubMed PubMed Central Google Scholar
Kornfeld, R. & Kornfeld, S. Assembly of asparagine-linked oligosaccharides. Annu. Rev. Biochem. 54, 631–664 (1985).
Article CAS PubMed Google Scholar
Pan, K. Z. et al. Inhibition of mRNA translation extends lifespan in Caenorhabditis elegans. Aging Cell 6, 111–119 (2007).
Article CAS PubMed Google Scholar
Labbadia, J. et al. Mitochondrial stress restores the heat shock response and prevents proteostasis collapse during aging. Cell Rep. 21, 1481–1494 (2017).
Article CAS PubMed PubMed Central Google Scholar
Park, S. et al. Modulation of protein synthesis by eIF2α phosphorylation protects cell from heat stress-mediated apoptosis. Cells 7, 254 (2018).
Article CAS PubMed Central Google Scholar
Morley, J. F. & Morimoto, R. I. Regulation of longevity in Caenorhabditis elegans by heat shock factor and molecular chaperones. Mol. Biol. Cell 15, 657–664 (2004).
Article CAS PubMed PubMed Central Google Scholar
Brunquell, J., Morris, S., Lu, Y., Cheng, F. & Westerheide, S. D. The genome-wide role of HSF-1 in the regulation of gene expression in Caenorhabditis elegans. BMC Genomics 17, 559 (2016).
Article PubMed PubMed Central CAS Google Scholar
Kapahi, P., Kaeberlein, M. & Hansen, M. Dietary restriction and lifespan: lessons from invertebrate models. Ageing Res. Rev. 39, 3–14 (2017).
Article PubMed Google Scholar
Honjoh, S., Yamamoto, T., Uno, M. & Nishida, E. Signalling through RHEB-1 mediates intermittent fasting-induced longevity in C. elegans. Nature 457, 726–730 (2009).
Article ADS CAS PubMed Google Scholar
Robertson, L. T. & Mitchell, J. R. Benefits of short-term dietary restriction in mammals. Exp. Gerontol. 48, 1043–1048 (2013).
Article CAS PubMed PubMed Central Google Scholar
Matai, L. et al. Dietary restriction improves proteostasis and increases life span through endoplasmic reticulum hormesis. Proc. Natl. Acad. Sci. USA 116, 17383–17392 (2019).
Article CAS PubMed PubMed Central Google Scholar
Durieux, J., Wolff, S. & Dillin, A. The cell-non-autonomous nature of electron transport chain-mediated longevity. Cell 144, 79–91 (2011).
Article CAS PubMed PubMed Central Google Scholar
Ray, A., Martinez, B. A., Berkowitz, L. A., Caldwell, G. A. & Caldwell, K. A. Mitochondrial dysfunction, oxidative stress, and neurodegeneration elicited by a bacterial metabolite in a C. elegans Parkinson’s model. Cell Death Dis. 5, e984 (2014).
Article CAS PubMed PubMed Central Google Scholar
Bischof, L. J. et al. Activation of the unfolded protein response is required for defenses against bacterial pore-forming toxin in vivo. PLoS Pathog. 4 (2008).
Kwan, G., Charkowski, A. O. & Barak, J. D. Salmonella enterica suppresses Pectobacterium carotovorum subsp. carotovorum population and soft rot progression by acidifying the microaerophilic environment. MBio 4 (2013).
Gohlke, J. & Deeken, R. Plant responses to Agrobacterium tumefaciens and crown gall development. Front. Plant Sci. 5, 155 (2014).
Article PubMed PubMed Central Google Scholar
Ahn, S.-J., Donahue, K., Koh, Y., Martin, R. R. & Choi, M.-Y. Microbial-based double-stranded RNA production to develop cost-effective RNA interference application for insect pest management. Int. J. Insect Sci. 11, 117954331984032 (2019).
Article Google Scholar
Couillault, C. & Ewbank, J. J. Diverse bacteria are pathogens of Caenorhabditis elegans. Infect. Immun. 70, 4705–4707 (2002).
Article CAS PubMed PubMed Central Google Scholar
Piazza, N., Gosangi, B., Devilla, S., Arking, R. & Wessells, R. Exercise-training in young drosophila melanogaster reduces age-related decline in mobility and cardiac performance. PLoS ONE 4, e5886 (2009).
Article ADS PubMed PubMed Central CAS Google Scholar
Laranjeiro, R. et al. Swim exercise in Caenorhabditis elegans extends neuromuscular and gut healthspan, enhances learning ability, and protects against neurodegeneration. Proc. Natl. Acad. Sci. USA 116, 23829–23839 (2019).
Article CAS PubMed PubMed Central Google Scholar
Liu, Y. et al. The beneficial effects of physical exercise in the brain and related pathophysiological mechanisms in neurodegenerative diseases. Lab. Invest. 99, 943–957 (2019).
Article PubMed Google Scholar
Laranjeiro, R., Harinath, G., Burke, D., Braeckman, B. P. & Driscoll, M. Single swim sessions in C. elegans induce key features of mammalian exercise. BMC Biol. 15, 30 (2017).
Article PubMed PubMed Central CAS Google Scholar
Hartman, J. H. et al. Swimming exercise and transient food deprivation in Caenorhabditis elegans promote mitochondrial maintenance and protect against chemical-induced mitotoxicity. Sci. Rep. 8 (2018).
Regmi, S. G., Rolland, S. G. & Conradt, B. Age-dependent changes in mitochondrial morphology and volume are not predictors of lifespan. Aging (Albany. NY) 6, 118–130 (2014).
Article Google Scholar
Kim, H. M., Do, C. H. & Lee, D. H. Taurine reduces ER stress in C. elegans. J. Biomed. Sci. 17, 1–6 (2010).
Article CAS Google Scholar
Huang, C. L., Lee, Y. C., Yang, Y. C., Kuo, T. Y. & Huang, N. K. Minocycline prevents paraquat-induced cell death through attenuating endoplasmic reticulum stress and mitochondrial dysfunction. Toxicol. Lett. 209, 203–210 (2012).
Article CAS PubMed Google Scholar
Kim-Han, J. S., Reichert, S. A., Quick, K. L. & Dugan, L. L. BMCP1: a mitochondrial uncoupling protein in neurons which regulates mitochondrial function and oxidant production. J. Neurochem. 79, 658–668 (2008).
Article Google Scholar
Krauss, S. et al. Superoxide-mediated activation of uncoupling protein 2 causes pancreatic β cell dysfunction. J. Clin. Invest. 112, 1831–1842 (2003).
Article CAS PubMed PubMed Central Google Scholar
Vidal-Puig, A. J. et al. Energy metabolism in uncoupling protein 3 gene knockout mice. J. Biol. Chem. 275, 16258–16266 (2000).
Article CAS PubMed Google Scholar
Iser, W. B., Kim, D., Bachman, E. & Wolkow, C. Examination of the requirement for ucp-4, a putative homolog of mammalian uncoupling proteins, for stress tolerance and longevity in C. elegans. Mech. Ageing Dev. 126, 1090–1096 (2005).
Article CAS PubMed PubMed Central Google Scholar
Pujol, C., Bratic-Hench, I., Sumakovic, M., Hench, J. & Mourier, A. Succinate dehydrogenase upregulation destabilize complex i and limits the lifespan of gas-1 mutant. PLoS ONE 8, 59493 (2013).
Article ADS CAS Google Scholar
Nargund, A. M., Pellegrino, M. W., Fiorese, C. J., Baker, B. M. & Haynes, C. M. Mitochondrial import efficiency of ATFS-1 regulates mitochondrial UPR activation. Science 337, 587–590 (2012).
Article ADS CAS PubMed PubMed Central Google Scholar
Shen, X. et al. Complementary signaling pathways regulate the unfolded protein response and are required for C. elegans development. Cell 107, 893–903 (2001).
Article CAS PubMed Google Scholar
Henis-Korenblit, S. et al. Insulin/IGF-1 signaling mutants reprogram ER stress response regulators to promote longevity. Proc. Natl. Acad. Sci. USA 107, 9730–9735 (2010).
Article ADS CAS PubMed PubMed Central Google Scholar
Yamada, K., Tsuchiya, J. I. & Iino, Y. Mutations in the pqe1 gene enhance transgene expression in Caenorhabditis elegans. G3 Genes Genomes Genet. 2, 741–751 (2012).
CAS Google Scholar
Ozanick, S. G. et al. Rex1p deficiency leads to accumulation of precursor initiator tRNA Met and polyadenylation of substrate RNAs in Saccharomyces cerevisiae. Nucl. Acids Res. 37, 298–308 (2009).
Article CAS PubMed Google Scholar
Copela, L. A., Fernandez, C. F., Sherrer, R. L. & Wolin, S. L. Competition between the Rex1 exonuclease and the La protein affects both Trf4p-mediated RNA quality control and pre-tRNA maturation. RNA 14, 1214–1227 (2008).
Article CAS PubMed PubMed Central Google Scholar
Raut, G. K., Chakrabarti, M., Pamarthy, D. & Bhadra, M. P. Glucose starvation-induced oxidative stress causes mitochondrial dysfunction and apoptosis via Prohibitin 1 upregulation in human breast cancer cells. Free Radic. Biol. Med. 145, 428–441 (2019).
Article CAS PubMed Google Scholar
Baugh, L. R. To grow or not to grow: Nutritional control of development during Caenorhabditis elegans L1 Arrest. Genetics 194, 539–555 (2013).
Article CAS PubMed PubMed Central Google Scholar
Kang, C., You, N. J. & Avery, L. Dual roles of autophagy in the survival of Caenorhabditis elegans during starvation. Genes Dev. 21, 2161–2171 (2007).
Article CAS PubMed PubMed Central Google Scholar
Angelo, G. & Van Gilst, M. R. Starvation protects germline stem cells and extends reproductive longevity in C. elegans. Science. 326, 954–958 (2009).
Article ADS CAS PubMed Google Scholar
Zhang, Y. et al. Inhibition of starvation-triggered endoplasmic reticulum stress, autophagy, and apoptosis in ARPE-19 cells by taurine through modulating the expression of calpain-1 and calpain-2. Int. J. Mol. Sci. 18, 2146 (2017).
Article CAS PubMed Central Google Scholar
Hibshman, J. D. et al. Nonselective autophagy reduces mitochondrial content during starvation in Caenorhabditis elegans. Am. J. Physiol. Cell Physiol. 315, C781–C792 (2018).
Article CAS PubMed PubMed Central Google Scholar
Zhang, A. et al. EIF2α and caspase-12 activation are involved in oxygen-glucose-serum deprivation/restoration-induced apoptosis of spinal cord astrocytes. Neurosci. Lett. 478, 32–36 (2010).
Article CAS PubMed Google Scholar
Estébanez, B., De Paz, J. A., Cuevas, M. J. & González-Gallego, J. Endoplasmic reticulum unfolded protein response, aging and exercise: An update. Front. Physiol. 9 (2018).
Chuang, H. S., Kuo, W. J., Lee, C. L., Chu, I. H. & Chen, C. S. Exercise in an electrotactic flow chamber ameliorates age-related degeneration in Caenorhabditis elegans. Sci. Rep. 6, 1–11 (2016).
Article CAS Google Scholar
The C. elegans deletion mutant consortium. Large-scale screening for targeted knockouts in the caenorhabditis elegans genome. G3 Genes, Genomes, Genet. 2, 1415–1425 (2012).
Tong, J., Rezai, P., Salam, S., Selvaganapathy, P. R. & Gupta, B. P. Microfluidic-based electrotaxis for on-demand quantitative analysis of Caenorhabditis elegans’ locomotion. J. Vis. Exp. https://doi.org/10.3791/50226 (2013).
Article PubMed PubMed Central Google Scholar

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Acknowledgements

We thank Baekjun Sung for help with initial data collection, Devon Jones for adapting and fixing microfluidic channels, Sangeena Salam for some of the early experimental ideas and designs and Meghan Pepler for supplying the Streptomyces venezuelae culture. Some of the C. elegans strains were obtained from the Caenorhabditis Genetics Center (CGC), which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). We also acknowledge the C. elegans Deletion Mutant Consortium for generating the deletion strains used in this study and the Bacillus Genetics Stock Center for providing the Bacillus thuringiensis culture. This work was supported by the NSERC Discovery Grant to BG and a joint CIHR CHRP award to PRS, BG and RKM.

Author information

These authors contributed equally: Shane K. B. Taylor and Muhammad H. Minhas.

Authors and Affiliations

Department of Biology, McMaster University, Hamilton, ON, Canada
Shane K. B. Taylor, Muhammad H. Minhas, Justin Tong & Bhagwati P. Gupta
Department of Mechanical Engineering, McMaster University, Hamilton, ON, Canada
P. Ravi Selvaganapathy
Department of Psychiatry and Behavioural Neurosciences, McMaster University, Hamilton, ON, Canada
Ram K. Mishra

Authors

Shane K. B. Taylor
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Muhammad H. Minhas
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Justin Tong
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P. Ravi Selvaganapathy
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Ram K. Mishra
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Bhagwati P. Gupta
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Contributions

B.G. conceived and supervised the project. S.K.B.T., M.H.M., and J.T. performed the experiments and analyzed data. B.G., S.K.B.T., and J.T. prepared the outline. B.G., S.K.B.T., J.T. and M.H.M. wrote the manuscript. S.K.B.T. and M.H.M. created the figures. P.R.S. and R.M. provided input throughout the project and reviewed the manuscript.

Corresponding author

Correspondence to Bhagwati P. Gupta.

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Taylor, S.K.B., Minhas, M.H., Tong, J. et al. C. elegans electrotaxis behavior is modulated by heat shock response and unfolded protein response signaling pathways. Sci Rep 11, 3115 (2021). https://doi.org/10.1038/s41598-021-82466-z

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Received: 11 August 2020
Accepted: 18 January 2021
Published: 04 February 2021
DOI: https://doi.org/10.1038/s41598-021-82466-z

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