Search

Anthelmintic drugs are used to treat parasitic roundworm and flatworm infections in humans and other animals.Caenorhabditis elegansis an established model to investigate anthelmintics used to treat roundworms. In this study, we useC. elegansto examine the mode of action and the mechanisms of resistance against the flatworm anthelmintic drug praziquantel (PZQ), used to treat trematode and cestode infections. We found that PZQ inhibited development and that this developmental delay varies by genetic background. Interestingly, both enantiomers of PZQ are equally effective againstC. elegans, but only the left-handed PZQ (S-PZQ) is effective against schistosome infections. We conducted a genome-wide association mapping with 74 wildC. elegansstrains to identify a region on chromosome IV that is correlated with differential PZQ susceptibility. Five candidate genes in this region:cct-8, znf-782, Y104H12D.4, Y104H12D.2, andcox-18, might underlie this variation. The genecct-8, a subunit of the protein folding complex TRiC, has variation that causes a putative protein coding change (G226V), which is correlated with reduced developmental delay. Gene expression analysis suggests that this variant correlates with slightly increased expression of bothcct-8andhsp-70. Acute exposure to PZQ caused increased expression ofhsp-70, indicating that altered TRiC function might be involved in PZQ responses. To test if this variant affects development upon exposure to PZQ, we used CRISPR-Cas9 genome editing to introduce the V226 allele into the N2 genetic background (G226) and the G226 allele into the JU775 genetic background (V226). These experiments revealed that this variant was not sufficient to explain the effects of PZQ on development. Nevertheless, this study shows thatC. eleganscan be used to study responses to PZQ to identify mode of action and resistance mechanisms. Additionally, we show that the TRiC complex requires further evaluation for PZQ responses inC. elegans.

During proteotoxic stress, a pathway known as the heat shock response is induced to maintain protein-folding homeostasis or proteostasis. Previously, we identified the Caenorhabditis elegans GATAD2 ortholog, dcp-66, as a novel regulator of the heat shock response. Here, we extend these findings to show that dcp-66 positively regulates the heat shock response at the cellular, molecular, and organismal levels. As GATAD2 is a subunit of the nucleosome remodeling and deacetylase chromatin remodeling complex, we examined other nucleosome remodeling and deacetylase subunits and found that the let-418 (CHD4) nucleosome repositioning core also regulates the heat shock response. However, let-418 acts as a negative regulator of the heat shock response, in contrast to positive regulation by dcp-66. The divergent effects of these two nucleosome remodeling and deacetylase subunits extend to the regulation of other stress responses including oxidative, genotoxic, and endoplasmic reticulum stress. Furthermore, a transcriptomic approach reveals additional divergently regulated pathways, including innate immunity and embryogenesis. Taken together, this work establishes new insights into the role of nucleosome remodeling and deacetylase subunits in organismal physiology. We incorporate these findings into a molecular model whereby different mechanisms of recruitment to promoters can result in the divergent effects of nucleosome remodeling and deacetylase subunits.

The bluegill sunfish Lepomis macrochirus and the closely related redear sunfish Lepomis microlophus have important ecological and recreational value and are widely used for research and aquaculture. While both species have been introduced outside of their native ranges, only the bluegill is considered invasive. Here, we report de novo transcriptome assemblies for these fish as a resource for sunfish biology. Comparative analyses of the transcriptomes revealed an unexpected, bluegill-specific expansion in the HSP70 and HSP90 molecular chaperone gene families. These expansions were not unique to the bluegill as expansions in HSP70s and HSP90s were identified in the genomes of other teleost fish using the NCBI RefSeq database. To determine whether gene family expansions are specific for thermal stress responses, GST and SOD gene families that are associated with oxidative stress responses were also analyzed. Species-specific expansions were also observed for these gene families in distinct fish species. Validating our approach, previously described expansions in the MHC gene family were also identified. Intriguingly, the number of HSP70 paralogs was positively correlated with thermotolerance range for each species, suggesting that these expansions can impact organismal physiology. Furthermore, fish that are considered invasive contained a higher average number of HSP70 paralogs than non-invasive fish. Invasive fish also had higher average numbers of HSP90, MHC and GST paralogs, but not SOD paralogs. Taken together, we propose that expansions in key cellular stress response gene families represent novel genetic signatures that correlate with invasive potential.

Activation of a cytoprotective cellular pathway known as the heat shock response (HSR) is a promising strategy for the treatment of Alzheimer's disease and other neurodegenerative diseases. Geranylgeranylacetone (GGA) is a commonly used anti-ulcer drug in Japan that has been shown to activate the HSR. Here, we establish

Organocatalysis is an emerging field, in which small metal-free organic structures catalyze a diversity of reactions with a remarkable stereoselectivity. The ability to selectively switch on such pathways upon demand has proven to be a valuable tool in biological systems. Light as a trigger provides the ultimate spatial and temporal control of activation. However, there have been limited examples of phototriggered catalytic systems. Herein, we describe the synthesis and application of a caged proline system that can initiate organocatalysis upon irradiation. The caged proline was generated using the highly efficient 4-carboxy-5,7-dinitroindolinyl (CDNI) photocleavable protecting group in a four-step synthesis. Advantages of this system include water solubility, biocompatibility, high quantum yield for catalyst release, and responsiveness to two-photon excitation. We showed the light-triggered catalysis of a crossed aldol reaction, a Mannich reaction, and a self-aldol condensation reaction. We also demonstrated light-initiated catalysis, leading to the formation of a biocide in situ, which resulted in the growth inhibition of E. coli, with as little as 3 min of irradiation. This technique can be broadly applied to other systems, by which the formation of active forms of drugs can be catalytically assembled remotely via two-photon irradiation.

SF3B1 is a core component of the U2 spliceosome that is frequently mutated in cancer. We have previously shown that titrating the activity of SF3B1, using the inhibitor pladienolide B (PB), affects distinct steps of the heat shock response (HSR). Here, we identify other genes that are sensitive to different levels of SF3B1 (5 vs. 100 nM PB) using RNA sequencing. Significant changes to mRNA splicing were identified at both low PB and high PB concentrations. Changes in expression were also identified in the absence of alternative splicing, suggesting that SF3B1 influences other gene expression pathways. Surprisingly, gene expression changes identified in low PB are not predictive of changes in high PB. Specific pathways were identified with differential sensitivity to PB concentration, including nonsense-mediated decay and protein-folding homeostasis, both of which were validated using independent reporter constructs. Strikingly, cells exposed to low PB displayed enhanced protein-folding capacity relative to untreated cells. These data reveal that the transcriptome is exquisitely sensitive to SF3B1 and suggests that the activity of SF3B1 is finely regulated to coordinate mRNA splicing, gene expression and cellular physiology.

The advantages of active learning approaches have prompted national recommendations for the development of inquiry-based laboratories to replace traditional laboratory classes. However, there is little consensus for the most-effective implementation strategies. Frequently, a single inquiry-based exercise is incorporated at the end of a traditional course and students have little opportunity to repeat the experience before moving on to new courses. To test whether multiple-rounds of inquiry would be beneficial, we incorporated three rounds of inquiry-based experiments during a redesign of a traditional upper-level undergraduate developmental biology laboratory class. After the second and third round of inquiry, students gave slideshow presentations of their projects and received peer and instructor feedback. We then designed and validated a scoring rubric to assess student use of scientific skills. Substantial improvements were observed in five of seven categories of scientific skills when comparing student performance from the third round of projects to the second round. Surprisingly, prior knowledge of the rubric did not diminish these gains. Anecdotal evidence and responses to student questionnaires revealed substantial levels of student interest and engagement in the course. Overall, these results indicate that incorporating iterative rounds of inquiry-based laboratories is a promising strategy for teaching scientific skills, enhancing student engagement, and promoting learning.

The heat shock response (HSR) is a cellular stress response induced by cytosolic protein misfolding that functions to restore protein folding homeostasis, or proteostasis. Caenorhabditis elegans occupies a unique and powerful niche for HSR research because the HSR can be assessed at the molecular, cellular, and organismal levels. Therefore, changes at the molecular level can be visualized at the cellular level and their impacts on physiology can be quantitated at the organismal level. While assays for measuring the HSR are straightforward, variations in the timing, temperature, and methodology described in the literature make it challenging to compare results across studies. Furthermore, these issues act as a barrier for anyone seeking to incorporate HSR analysis into their research. Here, a series of protocols is presented for measuring induction of the HSR in a robust and reproducible manner with RT-qPCR, fluorescent reporters, and an organismal thermorecovery assay. Additionally, we show that a widely used thermotolerance assay is not dependent on the well-established master regulator of the HSR, HSF-1, and therefore should not be used for HSR research. Finally, variations in these assays found in the literature are discussed and best practices are proposed to help standardize results across the field, ultimately facilitating neurodegenerative disease, aging, and HSR research.

The heat shock response (HSR) is a cellular stress response induced by cytosolic protein misfolding that functions to restore protein folding homeostasis, or proteostasis. Caenorhabditis elegans occupies a unique and powerful niche for HSR research because the HSR can be assessed at the molecular, cellular, and organismal levels. Therefore, changes at the molecular level can be visualized at the cellular level and their impacts on physiology can be quantitated at the organismal level. While assays for measuring the HSR are straightforward, variations in the timing, temperature, and methodology described in the literature make it challenging to compare results across studies. Furthermore, these issues act as a barrier for anyone seeking to incorporate HSR analysis into their research. Here, a series of protocols is presented for measuring induction of the HSR in a robust and reproducible manner with RT-qPCR, fluorescent reporters, and an organismal thermorecovery assay. Additionally, we show that a widely used thermotolerance assay is not dependent on the well-established master regulator of the HSR, HSF-1, and therefore should not be used for HSR research. Finally, variations in these assays found in the literature are discussed and best practices are proposed to help standardize results across the field, ultimately facilitating neurodegenerative disease, aging, and HSR research.

The heat shock response (HSR) is a cellular stress response that senses protein misfolding and restores protein folding homeostasis, or proteostasis. We previously identified an HSR regulatory network in Caenorhabditis elegans consisting of highly conserved genes that have important cellular roles in maintaining proteostasis. Unexpectedly, the effects of these genes on the HSR are distinctly tissue-specific. Here, we explore this apparent discrepancy and find that muscle-specific regulation of the HSR by the TRiC/CCT chaperonin is not driven by an enrichment of TRiC/CCT in muscle, but rather by the levels of one of its most abundant substrates, actin. Knockdown of actin subunits reduces induction of the HSR in muscle upon TRiC/CCT knockdown; conversely, overexpression of an actin subunit sensitizes the intestine so that it induces the HSR upon TRiC/CCT knockdown. Similarly, intestine-specific HSR regulation by the signal recognition particle (SRP), a component of the secretory pathway, is driven by the vitellogenins, some of the most abundant secretory proteins. Together, these data indicate that the specific protein folding requirements from the unique cellular proteomes sensitizes each tissue to disruption of distinct subsets of the proteostasis network. These findings are relevant for tissue-specific, HSR-associated human diseases such as cancer and neurodegenerative diseases. Additionally, we characterize organismal phenotypes of actin overexpression including a shortened lifespan, supporting a recent hypothesis that maintenance of the actin cytoskeleton is an important factor for longevity.

Inshore-offshore migration occurs frequently in seahorse species, either because of prey opportunities or because they are driven by reproduction, and variations in water temperature may dramatically change migratory seahorse behavior and physiology. The present study investigated the behavioral and physiological responses of the lined seahorse Hippocampus erectus under thermal stress and evaluated the potential effects of different temperatures on its reproduction. The results showed that the thermal tolerance of the seahorses was time dependent. Acute thermal stress (30°C, 2–10 h) increased the basal metabolic rate (breathing rate) and the expression of stress response genes (Hsp genes) significantly and further stimulated seahorse appetite. Chronic thermal treatment (30°C, 4 weeks) led to a persistently higher basal metabolic rate, higher stress response gene expression and higher mortality rates, indicating that the seahorses could not acclimate to chronic thermal stress and might experience massive mortality rates due to excessively high basal metabolic rates and stress damage. Additionally, no significant negative effects on gonad development or reproductive endocrine regulation genes were observed in response to chronic thermal stress, suggesting that seahorse reproductive behavior could adapt to higher-temperature conditions during migration and within seahorse breeding grounds. In conclusion, this simulation experiment indicates that temperature variations during inshore-offshore migration have no effect on reproduction, but promote significantly high basal metabolic rates and stress responses. Therefore, we suggest that the observed high tolerance of seahorse reproduction is in line with the inshore-offshore reproductive migration pattern of lined seahorses. This article has an associated First Person interview with the first author of the paper.

The heat shock response (HSR) is a well-conserved, cytoprotective stress response that activates the HSF1 transcription factor. During severe stress, cells inhibit mRNA splicing which also serves a cytoprotective function via inhibition of gene expression. Despite their functional interconnectedness, there have not been any previous reports of crosstalk between these two pathways. In a genetic screen, we identified SF3B1, a core component of the U2 snRNP subunit of the spliceosome, as a regulator of the heat shock response in Caenorhabditis elegans. Here, we show that this regulatory connection is conserved in cultured human cells and that there are at least two distinct pathways by which SF3B1 can regulate the HSR. First, inhibition of SF3B1 with moderate levels of Pladienolide B, a previously established small molecule inhibitor of SF3B1, affects the transcriptional activation of HSF1, the transcription factor that mediates the HSR. However, both higher levels of Pladienolide B and SF3B1 siRNA knockdown also change the concentration of HSF1, a form of HSR regulation that has not been previously documented during normal physiology but is observed in some forms of cancer. Intriguingly, mutations in SF3B1 have also been associated with several distinct types of cancer. Finally, we show that regulation of alternative splicing by SF3B1 is sensitive to temperature, providing a new mechanism by which temperature stress can remodel the transcriptome.

SUMMARY The heat shock response (HSR) is a homeostatic response that maintains the proper protein-folding environment in the cell. This response is universal, and many of its components are well conserved from bacteria to humans. In this review, we focus on the regulation of one of the most well-characterized HSRs, that of Escherichia coli . We show that even for this simple model organism, we still do not fully understand the central component of heat shock regulation, a chaperone-mediated negative feedback loop. In addition, we review other components that contribute to the regulation of the HSR in E. coli and discuss how these additional components contribute to regulation. Finally, we discuss recent genomic experiments that reveal additional functional aspects of the HSR.

Protein quality control is accomplished by inducing chaperones and proteases in response to an altered cellular folding state. In Escherichia coli , expression of chaperones and proteases is positively regulated by σ 32 . Chaperone-mediated negative feedback control of σ 32 activity allows this transcription factor to sense the cellular folding state. We identified point mutations in σ 32 altered in feedback control. Surprisingly, such mutants are resistant to inhibition by both the DnaK/J and GroEL/S chaperones in vivo and also show dramatically increased stability. Further characterization of the most defective mutant revealed that it has almost normal binding to chaperones and RNA polymerase and is competent for chaperone-mediated inactivation in vitro . We suggest that the mutants identify a regulatory step downstream of chaperone binding that is required for both inactivation and degradation of σ 32 .

Hfq, a chaperone for small noncoding RNAs, regulates many processes in Escherichia coli , including the σ S -mediated general stress response. Here we used microarray analysis to identify the changes in gene expression resulting from lack of Hfq. We identify several potential new targets for Hfq regulation, including genes encoding outer membrane proteins, enzymes, factors, and transporters. Many of these genes are involved in amino acid uptake and biosynthesis, sugar uptake and metabolism, and cell energetics. In addition, we find altered regulation of the σ E - and σ 32 -mediated stress responses, which we analyze further. We show that cells lacking Hfq induce the σ E -mediated envelope stress response and are defective in σ E -mediated repression of outer membrane proteins. We also show that the σ 32 -mediated cytoplasmic stress response is repressed in cells lacking Hfq due to increased expression of DnaK. Furthermore, we show that cells lacking Hfq are defective in the “long-term adaptation” of σ 32 to chronic chaperone overexpression. Together, our results indicate that Hfq may play a general role in stress response regulation in E. coli .

The heat shock response (HSR) is essential to survive acute proteotoxic stress and has been studied extensively in unicellular organisms and tissue culture cells, but to a lesser extent in intact metazoan animals. To identify the regulatory pathways that control the HSR in Caenorhabditis elegans, we performed a genome-wide RNAi screen and identified 59 genes corresponding to 7 positive activators required for the HSR and 52 negative regulators whose knockdown leads to constitutive activation of the HSR. These modifiers function in specific steps of gene expression, protein synthesis, protein folding, trafficking, and protein clearance, and comprise the metazoan heat shock regulatory network (HSN). Whereas the positive regulators function in all tissues of C. elegans, nearly all of the negative regulators exhibited tissue-selective effects. Knockdown of the subunits of the proteasome strongly induces HS reporter expression only in the intestine and spermatheca but not in muscle cells, while knockdown of subunits of the TRiC/CCT chaperonin induces HS reporter expression only in muscle cells. Yet, both the proteasome and TRiC/CCT chaperonin are ubiquitously expressed and are required for clearance and folding in all tissues. We propose that the HSN identifies a key subset of the proteostasis machinery that regulates the HSR according to the unique functional requirements of each tissue., Author Summary The heat shock response (HSR) is an essential stress response that functions to maintain protein folding homeostasis, or proteostasis, and whose critical role in human diseases is recently becoming apparent. Previously, most of our understanding of the HSR has come from cultured cells and unicellular organisms. Here we present the identification of the heat shock regulatory network (HSN) in Caenorhabditis elegans, an intact, multicellular organism, using genome-wide RNAi screening. We identify 59 positive and negative regulators of the HSR, all of which have a previously established role in proteostasis, linking the function of the HSR to its regulation. Some HSN genes were previously established in other systems, many were indirectly linked to HSR, and others are novel. Unexpectedly, almost all negative regulators of the HSR act in distinct, tissue-selective patterns, despite their broad expression and universal cellular requirements. Therefore, our data indicate that the HSN consists of a specific subset of the proteostasis machinery that functions to link the proteostasis network to HSR regulation in a tissue-selective manner.

The heat shock response (HSR) is an essential cellular and organismal protective mechanism against acute forms of physiological and environmental stress. Induction of the HSR by exposure to acute stress conditions such as elevated temperature, oxidants, and heavy metals involves activation of heat shock transcription factors (HSFs) and elevated expression of genes encoding heat shock proteins and molecular chaperones that restore proteostasis and prevent the further accumulation of misfolded and aggregated proteins. Here, we examine the stress-sensing mechanisms that regulate HSFs and the role of post-translational modifications that regulate HSF activity. In addition to its role in acute stress, the expression of molecular chaperones and heat shock genes is important for development and is protective when challenged by chronic proteostasis imbalance during aging and diseases of protein conformation.

The heat shock response controls levels of chaperones and proteases to ensure a proper cellular environment for protein folding. In Escherichia coli, this response is mediated by the bacterial-specific transcription factor, σ32. The DnaK chaperone machine regulates both the amount and activity of σ32, thereby coupling σ32 function to the cellular protein folding state. In this manuscript, we analyze the ability of other major chaperones in E. coli to regulate σ32, and we demonstrate that the GroEL/S chaperonin is an additional regulator of σ32. We show that increasing the level of GroEL/S leads to a decrease in σ32 activity in vivo and this effect can be eliminated by co-overexpression of a GroEL/S-specific substrate. We also show that depletion of GroEL/S in vivo leads to up-regulation of σ32 by increasing the level of σ32. In addition, we show that changing the levels of GroEL/S during stress conditions leads to measurable changes in the heat shock response. Using purified proteins, we show that that GroEL binds to σ32 and decreases σ32-dependent transcription in vitro, suggesting that this regulation is direct. We discuss why using a chaperone network to regulate σ32 results in a more sensitive and accurate detection of the protein folding environment.