Metazoan cell death mechanisms are diverse and include numerous non-apoptotic programs. One program called entosis involves the invasion of live cells into their neighbors and is known to occur in cancers. Here, we identify a developmental function for entosis: to clear the male-specific linker cell in C. elegans. The linker cell leads migration to shape the gonad and is removed to facilitate fusion of the gonad to the cloaca. We find that the linker cell is cleared in a manner involving cell-cell adhesions and cell-autonomous control of uptake through linker cell actin. Linker cell entosis generates a lobe structure that is deposited at the site of gonad-to-cloaca fusion and is removed during mating. Inhibition of lobe scission inhibits linker cell death, demonstrating that the linker cell invades its host while alive. Our findings demonstrate a developmental function for entosis: to eliminate a migrating cell and facilitate gonad-to-cloaca fusion, which is required for fertility.

Genetic instability of the mitochondrial genome (mtDNA) plays an important role in human aging and disease. Thus far, it has proven difficult to develop successful treatment strategies for diseases that are caused by mtDNA instability. To address this issue, we developed a model of mtDNA disease in the nematode C. elegans, an animal model that can rapidly be screened for genes and biological pathways that reduce mitochondrial pathology. These worms recapitulate all the major hallmarks of mtDNA disease in humans, including increased mtDNA instability, loss of respiration, reduced neuromuscular function, and a shortened lifespan. We found that these phenotypes could be rescued by intervening in numerous biological pathways, including IGF-1/insulin signaling, mitophagy, and the mitochondrial unfolded protein response, suggesting that it may be possible to ameliorate mtDNA disease through multiple molecular mechanisms.

Animals have evolved critical mechanisms to maintain cellular and organismal proteostasis during development, disease, and exposure to environmental stressors. The Unfolded Protein Response (UPR) is a conserved pathway that senses and responds to the accumulation of misfolded proteins in the endoplasmic reticulum (ER) lumen. We have previously demonstrated that the IRE-1-XBP-1 branch of the UPR is required to maintain Caenorhabditis elegans ER homeostasis during larval development in the presence of pathogenic Pseudomonas aeruginosa In this study, we identify loss-of-function mutations in four conserved transcriptional regulators that suppress the larval lethality of xbp-1 mutant animals caused by immune activation in response to infection by pathogenic bacteria: FKH-9, a forkhead family transcription factor; ARID-1, an ARID/Bright domain-containing transcription factor; HCF-1, a transcriptional regulator that associates with histone modifying enzymes; and SIN-3, a subunit of a histone deacetylase complex. Further characterization of FKH-9 suggests that loss of FKH-9 enhances resistance to the ER toxin tunicamycin and results in enhanced ER-associated degradation (ERAD). Increased ERAD activity of fkh-9 loss-of-function mutants is accompanied by a diminished capacity to degrade cytosolic proteasomal substrates and a corresponding increased sensitivity to the proteasomal inhibitor bortezomib. Our data underscore how the balance between ER and cytosolic proteostasis can be influenced by compensatory activation of ERAD during the physiological ER stress of infection and immune activation.

The accumulation of deleterious mitochondrial DNA (∆mtDNA) causes inherited mitochondrial diseases and ageing-associated decline in mitochondrial functions such as oxidative phosphorylation. Following mitochondrial perturbations, the bZIP protein ATFS-1 induces a transcriptional programme to restore mitochondrial function. Paradoxically, ATFS-1 is also required to maintain ∆mtDNAs in heteroplasmic worms. The mechanism by which ATFS-1 promotes ∆mtDNA accumulation relative to wild-type mtDNAs is unclear. Here we show that ATFS-1 accumulates in dysfunctional mitochondria. ATFS-1 is absent in healthy mitochondria owing to degradation by the mtDNA-bound protease LONP-1, which results in the nearly exclusive association between ATFS-1 and ∆mtDNAs in heteroplasmic worms. Moreover, we demonstrate that mitochondrial ATFS-1 promotes the binding of the mtDNA replicative polymerase (POLG) to ∆mtDNAs. Interestingly, inhibition of the mtDNA-bound protease LONP-1 increased ATFS-1 and POLG binding to wild-type mtDNAs. LONP-1 inhibition in Caenorhabditis elegans and human cybrid cells improved the heteroplasmy ratio and restored oxidative phosphorylation. Our findings suggest that ATFS-1 promotes mtDNA replication in dysfunctional mitochondria by promoting POLG-mtDNA binding, which is antagonized by LONP-1.

Regulation of immunity throughout an organism is critical for host defense. Previous studies in the nematode Caenorhabditis elegans have described an "ON/OFF" immune switch comprised of the antagonistic paralogs PALS-25 and PALS-22, which regulate resistance against intestinal and epidermal pathogens. Here, we identify and characterize a PALS-25 gain-of-function mutant protein with a premature stop (Q293*), which we find is freed from physical repression by its negative regulator, the PALS-22 protein. PALS-25(Q293*) activates two related gene expression programs, the Oomycete Recognition Response (ORR) against natural pathogens of the epidermis, and the Intracellular Pathogen Response (IPR) against natural intracellular pathogens of the intestine. A subset of ORR/IPR genes is upregulated in pals-25(Q293*) mutants, and they are resistant to oomycete infection in the epidermis, and microsporidia and virus infection in the intestine, but without compromising growth. Surprisingly, we find that activation of PALS-25 seems to primarily stimulate the downstream bZIP transcription factor ZIP-1 in the epidermis, with upregulation of gene expression in both the epidermis and in the intestine. Interestingly, we find that PALS-22/25-regulated epidermal-to-intestinal signaling promotes resistance to the N. parisii intestinal pathogen, demonstrating cross-tissue protective immune induction from one epithelial tissue to another in C. elegans.

Metazoans identify and eliminate bacterial pathogens in microbe-rich environments such as the intestinal lumen; however, the mechanisms are unclear. Host cells could potentially use intracellular surveillance or stress response programs to detect pathogens that target monitored cellular activities and then initiate innate immune responses. Mitochondrial function is evaluated by monitoring mitochondrial protein import efficiency of the transcription factor ATFS-1, which mediates the mitochondrial unfolded protein response (UPR(mt)). During mitochondrial stress, mitochondrial import is impaired, allowing ATFS-1 to traffic to the nucleus where it mediates a transcriptional response to re-establish mitochondrial homeostasis. Here we examined the role of ATFS-1 in Caenorhabditis elegans during pathogen exposure, because during mitochondrial stress ATFS-1 induced not only mitochondrial protective genes but also innate immune genes that included a secreted lysozyme and anti-microbial peptides. Exposure to the pathogen Pseudomonas aeruginosa caused mitochondrial dysfunction and activation of the UPR(mt). C. elegans lacking atfs-1 were susceptible to P. aeruginosa, whereas hyper-activation of ATFS-1 and the UPR(mt) improved clearance of P. aeruginosa from the intestine and prolonged C. elegans survival in a manner mainly independent of known innate immune pathways. We propose that ATFS-1 import efficiency and the UPR(mt) is a means to detect pathogens that target mitochondria and initiate a protective innate immune response.

Protein misfolding caused by exposure to arsenite is associated with transcriptional activation of the AIRAP gene. We report here that AIRAP is an arsenite-inducible subunit of the proteasome's 19S cap that binds near PSMD2 at the 19S base. Compared to the wild-type, knockout mouse cells or C. elegans lacking AIRAP accumulate more polyubiquitylated proteins and exhibit higher levels of stress when exposed to arsenite, and proteasomes isolated from arsenite-treated AIRAP knockout cells are relatively impaired in substrate degradation in vitro. AIRAP's association with the 19S cap reverses the stabilizing affect of ATP on the 26S proteasome during particle purification, and AIRAP-containing proteasomes, though constituted of 19S and 20S subunits, acquire features of hybrid proteasomes with both 19S and 11S regulatory caps. These features include enhanced cleavage of peptide substrates and suggest that AIRAP adapts the cell's core protein degradation machinery to counteract proteotoxicity induced by an environmental toxin.

Early host responses toward pathogens are essential for defense against infection. In Caenorhabditis elegans, the transcription factor, SKN-1, regulates cellular defenses during xenobiotic intoxication and bacterial infection. However, constitutive activation of SKN-1 results in pleiotropic outcomes, including a redistribution of somatic lipids to the germline, which impairs health and shortens lifespan. Here, we show that exposing C. elegans to Pseudomonas aeruginosa similarly drives the rapid depletion of somatic, but not germline, lipid stores. Modulating the epigenetic landscape refines SKN-1 activity away from innate immunity targets, which alleviates negative metabolic outcomes. Similarly, exposure to oxidative stress redirects SKN-1 activity away from pathogen response genes while restoring somatic lipid distribution. In addition, activating p38/MAPK signaling in the absence of pathogens, is sufficient to drive SKN-1-dependent loss of somatic fat. These data define a SKN-1- and p38-dependent axis for coordinating pathogen responses, lipid homeostasis, and survival and identify transcriptional redirection, rather than inactivation, as a mechanism for counteracting the pleiotropic consequences of aberrant transcriptional activity.

Animals integrate changes in external and internal environments to generate behavior. While neural circuits detecting external cues have been mapped, less is known about how internal states like hunger are integrated into behavioral outputs. Here, we use the nematode C. elegans to examine how changes in internal nutritional status affect chemosensory behaviors. We show that acute food deprivation leads to a reversible decline in repellent, but not attractant, sensitivity. This behavioral change requires two conserved transcription factors MML-1 (MondoA) and HLH-30 (TFEB), both of which translocate from the intestinal nuclei to the cytoplasm during food deprivation. Next, we identify the insulin-like peptide INS-31 as a candidate ligand relaying food-status signals from the intestine to other tissues. Further, we show that neurons likely use the DAF-2 insulin receptor and AGE-1/PI-3 Kinase, but not DAF-16/FOXO to integrate these intestine-released peptides. Altogether, our study shows how internal food status signals are integrated by transcription factors and intestine-neuron signaling to generate flexible behaviors via the gut-brain axis.

Nutrient availability regulates the C. elegans life cycle as well as mitochondrial physiology. Food deprivation significantly reduces mitochondrial genome (mtDNA) numbers and leads to aging-related phenotypes. Here we show that the bZIP (basic leucine zipper) protein ATFS-1, a mediator of the mitochondrial unfolded protein response (UPRmt), is required to promote growth and establish a functional germline after prolonged starvation. We find that recovery of mtDNA copy numbers and development after starvation requires mitochondrion-localized ATFS-1 but not its nuclear transcription activity. We also find that the insulin-like receptor DAF-2 functions upstream of ATFS-1 to modulate mtDNA content. We show that reducing DAF-2 activity represses ATFS-1 nuclear function while causing an increase in mtDNA content, partly mediated by mitochondrion-localized ATFS-1. Our data indicate the importance of the UPRmt in recovering mitochondrial mass and suggest that atfs-1-dependent mtDNA replication precedes mitochondrial network expansion after starvation.

As organisms develop, individual cells generate mitochondria to fulfill physiological requirements. However, it remains unknown how mitochondrial network expansion is scaled to cell growth. The mitochondrial unfolded protein response (UPRmt) is a signaling pathway mediated by the transcription factor ATFS-1 which harbors a mitochondrial targeting sequence (MTS). Here, using the model organism Caenorhabditis elegans we demonstrate that ATFS-1 mediates an adaptable mitochondrial network expansion program that is active throughout normal development. Mitochondrial network expansion requires the relatively inefficient MTS in ATFS-1, which allows the transcription factor to be responsive to parameters that impact protein import capacity of the mitochondrial network. Increasing the strength of the ATFS-1 MTS impairs UPRmt activity by increasing accumulation within mitochondria. Manipulations of TORC1 activity increase or decrease ATFS-1 activity in a manner that correlates with protein synthesis. Lastly, expression of mitochondrial-targeted GFP is sufficient to expand the muscle cell mitochondrial network in an ATFS-1-dependent manner. We propose that mitochondrial network expansion during development is an emergent property of the synthesis of highly expressed mitochondrial proteins that exclude ATFS-1 from mitochondrial import, causing UPRmt activation.

Prey respond to predators by altering their behavior to optimize their own fitness and survival. Specifically, prey are known to avoid predator-occupied territories to reduce their risk of harm or injury to themselves and their progeny. We probe the interactions between Caenorhabditis elegans and its naturally cohabiting predator Pristionchus uniformis to reveal the pathways driving changes in prey behavior. While C. elegans prefers to lay its eggs on a bacteria food lawn, the presence of a predator inside a lawn induces C. elegans to lay more eggs away from that lawn. We confirm that this change in egg laying is in response to bites from predators, rather than to predatory secretions. Moreover, predator-exposed prey continue to lay their eggs away from the dense lawn even after the predator is removed, indicating a form of learning. Next, we find that mutants in dopamine synthesis significantly reduce egg laying behavior off the lawn in both predator-free and predator-inhabited lawns, which we can rescue by transgenic complementation or supplementation with exogenous dopamine. Moreover, we find that dopamine is likely released from multiple dopaminergic neurons and requires combinations of both D1- (DOP-1) and D2-like (DOP-2 and DOP-3) dopamine receptors to alter predator-induced egg laying behavior, whereas other combinations modify baseline levels of egg laying behavior. Together, we show that dopamine signaling can alter both predator-free and predator-induced foraging strategies, suggesting a role for this pathway in defensive behaviors.

Innate immunity in Caenorhabditis elegans requires a conserved PMK-1 p38 mitogen-activated protein kinase (MAPK) pathway that regulates the basal and pathogen-induced expression of immune effectors. The mechanisms by which PMK-1 p38 MAPK regulates the transcriptional activation of the C. elegans immune response have not been identified. Furthermore, in mammalian systems the genetic analysis of physiological targets of p38 MAPK in immunity has been limited. Here, we show that C. elegans ATF-7, a member of the conserved cyclic AMP-responsive element binding (CREB)/activating transcription factor (ATF) family of basic-region leucine zipper (bZIP) transcription factors and an ortholog of mammalian ATF2/ATF7, has a pivotal role in the regulation of PMK-1-mediated innate immunity. Genetic analysis of loss-of-function alleles and a gain-of-function allele of atf-7, combined with expression analysis of PMK-1-regulated genes and biochemical characterization of the interaction between ATF-7 and PMK-1, suggest that ATF-7 functions as a repressor of PMK-1-regulated genes that undergoes a switch to an activator upon phosphorylation by PMK-1. Whereas loss-of-function mutations in atf-7 can restore basal expression of PMK-1-regulated genes observed in the pmk-1 null mutant, the induction of PMK-1-regulated genes by pathogenic Pseudomonas aeruginosa PA14 is abrogated. The switching modes of ATF-7 activity, from repressor to activator in response to activated PMK-1 p38 MAPK, are reminiscent of the mechanism of regulation mediated by the corresponding ancestral Sko1p and Hog1p proteins in the yeast response to osmotic stress. Our data point to the regulation of the ATF2/ATF7/CREB5 family of transcriptional regulators by p38 MAPK as an ancient conserved mechanism for the control of innate immunity in metazoans, and suggest that ATF2/ATF7 may function in a similar manner in the regulation of mammalian innate immunity.

Pathogens attack host cells by deploying toxins that perturb core host processes. Recent findings from the nematode C. elegans and other metazoans indicate that surveillance or "effector-triggered" pathways monitor functioning of these core processes and mount protective responses when they are perturbed. Despite a growing number of examples of surveillance immunity, the signaling components remain poorly defined. Here, we show that CEBP-2, the C. elegans ortholog of mammalian CCAAT-enhancer-binding protein gamma, is a key player in surveillance immunity. We show that CEBP-2 acts together with the bZIP transcription factor ZIP-2 in the protective response to translational block by P. aeruginosa Exotoxin A as well as perturbations of other processes. CEBP-2 serves to limit pathogen burden, promote survival upon P. aeruginosa infection, and also promote survival upon Exotoxin A exposure. These findings may have broad implications for the mechanisms by which animals sense pathogenic attack and mount protective responses.

In Caenorhabditis elegans, the programmed repression of the heat shock response (HSR) accompanies the transition to reproductive maturity, leaving cells vulnerable to environmental stress and protein aggregation with age. To identify the factors driving this event, we performed an unbiased genetic screen for suppressors of stress resistance and identified the mitochondrial electron transport chain (ETC) as a central regulator of the age-related decline of the HSR and cytosolic proteostasis. Mild downregulation of ETC activity, either by genetic modulation or exposure to mitochondria-targeted xenobiotics, maintained the HSR in adulthood by increasing HSF-1 binding and RNA polymerase II recruitment at HSF-1 target genes. This resulted in a robust restoration of cytoplasmic proteostasis and increased vitality later in life, without detrimental effects on fecundity. We propose that low levels of mitochondrial stress regulate cytoplasmic proteostasis and healthspan during aging by coordinating the long-term activity of HSF-1 with conditions preclusive to optimal fitness.

Cryptic transcription occurs widely across the eukaryotic genome; however, its regulation during vertebrate development is not understood. Here, we show that two class I histone deacetylases, Hdac1 and Hdac2, silence cryptic transcription to promote mitochondrial function in developing murine hearts. Mice lacking Hdac1 and Hdac2 in heart exhibit defective developmental switch from anaerobic to mitochondrial oxidative phosphorylation (OXPHOS), severe defects in mitochondrial mass, mitochondrial function, and complete embryonic lethality. Hdac1/Hdac2 promotes the transition to OXPHOS by enforcing transcriptional fidelity of metabolic gene programs. Mechanistically, Hdac1/Hdac2 deacetylates histone residues including H3K23, H3K14, and H4K16 to suppress cryptic transcriptional initiation within the coding regions of actively transcribed metabolic genes. Thus, Hdac1/2-mediated epigenetic silencing of cryptic transcription is essential for mitochondrial function during early vertebrate development.

The translation initiation factor eIF3 is a multi-subunit protein complex that coordinates the assembly of the 43S pre-initiation complex in eukaryotes. Prior studies have demonstrated that not all subunits of eIF3 are essential for the initiation of translation, suggesting that some subunits may serve regulatory roles. Here, we show that loss-of-function mutations in the genes encoding the conserved eIF3k and eIF3l subunits of the translation initiation complex eIF3 result in a 40% extension in lifespan and enhanced resistance to endoplasmic reticulum (ER) stress in Caenorhabditis elegans. In contrast to previously described mutations in genes encoding translation initiation components that confer lifespan extension in C. elegans, loss-of-function mutations in eif-3.K or eif-3.L are viable, and mutants show normal rates of growth and development, and have wild-type levels of bulk protein synthesis. Lifespan extension resulting from EIF-3.K or EIF-3.L deficiency is suppressed by a mutation in the Forkhead family transcription factor DAF-16. Mutations in eif-3.K or eif-3.L also confer enhanced resistance to ER stress, independent of IRE-1-XBP-1, ATF-6, and PEK-1, and independent of DAF-16. Our data suggest a pivotal functional role for conserved eIF3k and eIF3l accessory subunits of eIF3 in the regulation of cellular and organismal responses to ER stress and aging.

Maintenance of protein homeostasis, or proteostasis, is crucial for organismal health. Disruption of proteostasis can lead to the accumulation of protein aggregates, which are associated with aging and many human diseases such as Alzheimer's disease [1-3]. Through analysis of the C. elegans host response to intracellular infection, we describe here a novel response pathway that enhances proteostasis capacity and appears to act in parallel to well-studied proteostasis pathways. These findings are based on analysis of the transcriptional response to infection by the intracellular pathogen Nematocida parisii [4]. The response to N. parisii is strikingly similar to the response to infection by the Orsay virus, another natural intracellular pathogen of C. elegans, and is distinct from responses to extracellular pathogen infection [4-6]. We have therefore named this common transcriptional response the intracellular pathogen response (IPR), and it includes upregulation of several predicted ubiquitin ligase complex components such as the cullin cul-6. Through a forward genetic screen we found pals-22, a gene of previously unknown function, to be a repressor of the cul-6/cullin gene and other IPR gene expression. Interestingly, pals-22 mutants have increased thermotolerance and reduced levels of stress-induced polyglutamine aggregates, likely due to upregulated IPR gene expression. We found the enhanced stress resistance of pals-22 mutants to be dependent on cul-6, suggesting that pals-22 mutants have increased activity of a CUL-6/cullin-containing ubiquitin ligase complex. pals-22 mutant phenotypes appear independent of the well-studied heat shock and insulin signaling pathways, indicating that the IPR is a distinct pathway that protects animals from proteotoxic stress.

Animal foraging is an essential and evolutionarily conserved behavior that occurs in social and solitary contexts, but the underlying molecular pathways are not well defined. We discover that conserved autism-associated genes (NRXN1(nrx-1), NLGN3(nlg-1), GRIA1,2,3(glr-1), GRIA2(glr-2), and GLRA2,GABRA3(avr-15)) regulate aggregate feeding in C. elegans, a simple social behavior. NRX-1 functions in chemosensory neurons (ADL and ASH) independently of its postsynaptic partner NLG-1 to regulate social feeding. Glutamate from these neurons is also crucial for aggregate feeding, acting independently of NRX-1 and NLG-1. Compared to solitary counterparts, social animals show faster presynaptic release and more presynaptic release sites in ASH neurons, with only the latter requiring nrx-1. Disruption of these distinct signaling components additively converts behavior from social to solitary. Aggregation induced by circuit activation is also dependent on nrx-1. Collectively, we find that aggregate feeding is tuned by conserved autism-associated genes through complementary synaptic mechanisms, revealing molecular principles driving social feeding.

Ultrasound has been shown to affect the function of both neurons and non-neuronal cells, but, the underlying molecular machinery has been poorly understood. Here, we show that at least two mechanosensitive proteins act together to generate C. elegans behavioral responses to ultrasound stimuli. We first show that these animals generate reversals in response to a single 10 msec pulse from a 2.25 MHz ultrasound transducer. Next, we show that the pore-forming subunit of the mechanosensitive channel TRP-4, and a DEG/ENaC/ASIC ion channel MEC-4, are both required for this ultrasound-evoked reversal response. Further, the trp-4;mec-4 double mutant shows a stronger behavioral deficit compared to either single mutant. Finally, overexpressing TRP-4 in specific chemosensory neurons can rescue the ultrasound-triggered behavioral deficit in the mec-4 null mutant, suggesting that both TRP-4 and MEC-4 act together in affecting behavior. Together, we demonstrate that multiple mechanosensitive proteins likely cooperate to transform ultrasound stimuli into behavioral changes.