Introduction Sepsis is a highly morbid condition characterized by multi-organ dysfunction resulting from dysregulated inflammation in response to acute infection. Mitochondrial dysfunction may contribute to sepsis pathogenesis, but quantifying mitochondrial dysfunction remains challenging. Objective To assess the extent to which circulating markers of mitochondrial dysfunction are increased in septic shock, and their relationship to severity and mortality. Methods We performed both full-scan and targeted (known markers of genetic mitochondrial disease) metabolomics on plasma to determine markers of mitochondrial dysfunction which distinguish subjects with septic shock (n = 42) from cardiogenic shock without infection (n = 19), bacteremia without sepsis (n = 18), and ambulatory controls (n = 19) – the latter three being conditions in which mitochondrial function, proxied by peripheral oxygen consumption, is presumed intact. Results Nine metabolites were significantly increased in septic shock compared to all three comparator groups. This list includes N -formyl- l -methionine (f-Met), a marker of dysregulated mitochondrial protein translation, and N -lactoyl-phenylalanine (lac-Phe), representative of the N -lactoyl-amino acids (lac-AAs), which are elevated in plasma of patients with monogenic mitochondrial disease. Compared to lactate, the clinical biomarker used to define septic shock, there was greater separation between survivors and non-survivors of septic shock for both f-Met and the lac-AAs measured within 24 h of ICU admission. Additionally, tryptophan was the one metabolite significantly decreased in septic shock compared to all other groups, while its breakdown product kynurenate was one of the 9 significantly increased. Conclusion Future studies which validate the measurement of lac-AAs and f-Met in conjunction with lactate could define a sepsis subtype characterized by mitochondrial dysfunction.

Heteroplasmy occurs when wild-type and mutant mitochondrial DNA (mtDNA) molecules co-exist in single cells 1 . Heteroplasmy levels change dynamically in development, disease and ageing 2,3 , but it is unclear whether these shifts are caused by selection or drift, and whether they occur at the level of cells or intracellularly. Here we investigate heteroplasmy dynamics in dividing cells by combining precise mtDNA base editing (DdCBE) 4 with a new method, SCI-LITE (single-cell combinatorial indexing leveraged to interrogate targeted expression), which tracks single-cell heteroplasmy with ultra-high throughput. We engineered cells to have synonymous or nonsynonymous complex I mtDNA mutations and found that cell populations in standard culture conditions purge nonsynonymous mtDNA variants, whereas synonymous variants are maintained. This suggests that selection dominates over simple drift in shaping population heteroplasmy. We simultaneously tracked single-cell mtDNA heteroplasmy and ancestry, and found that, although the population heteroplasmy shifts, the heteroplasmy of individual cell lineages remains stable, arguing that selection acts at the level of cell fitness in dividing cells. Using these insights, we show that we can force cells to accumulate high levels of truncating complex I mtDNA heteroplasmy by placing them in environments where loss of biochemical complex I activity has been reported to benefit cell fitness. We conclude that in dividing cells, a given nonsynonymous mtDNA heteroplasmy can be harmful, neutral or even beneficial to cell fitness, but that the ‘sign’ of the effect is wholly dependent on the environment.

Iron-sulfur clusters (ISC) are essential cofactors that participate in electron transfer, environmental sensing, and catalysis. Amongst the most ancient ISC-containing proteins are the ferredoxin (FDX) family of electron carriers. Humans have two FDXs- FDX1 and FDX2, both of which are localized to mitochondria, and the latter of which is itself important for ISC synthesis. We have previously shown that hypoxia can eliminate the requirement for some components of the ISC biosynthetic pathway, but FDXs were not included in that study. Here, we report that FDX1, but not FDX2, is dispensable under 1% O2 in cultured human cells. We find that FDX1 is essential for production of the lipoic acid cofactor, which is synthesized by the ISC-containing enzyme lipoyl synthase. While hypoxia can rescue the growth phenotype of either FDX1 or lipoyl synthase KO cells, lipoylation in these same cells is not rescued, arguing against an alternative biosynthetic route or salvage pathway for lipoate in hypoxia. Our work reveals the divergent roles of FDX1 and FDX2 in mitochondria, identifies a role for FDX1 in lipoate synthesis, and suggests that loss of lipoic acid can be tolerated under low oxygen tensions in cell culture.

The integrated stress response (ISR) comprises the eIF2α kinases PERK, GCN2, HRI, and PKR, which induce translational and transcriptional signaling in response to diverse insults. Deficiencies in PERK signaling lead to mitochondrial dysfunction and contribute to the pathogenesis of numerous diseases. We define the potential for pharmacologic activation of compensatory eIF2α kinases to rescue ISR signaling and promote mitochondrial adaptation in PERK-deficient cells. We show that the HRI activator BtdCPU and GCN2 activator halofuginone promote ISR signaling and rescue ER stress sensitivity in PERK-deficient cells. However, BtdCPU induces mitochondrial depolarization, leading to mitochondrial fragmentation and activation of the OMA1-DELE1-HRI signaling axis. In contrast, halofuginone promotes mitochondrial elongation and adaptive mitochondrial respiration, mimicking regulation induced by PERK. This shows halofuginone can compensate for deficiencies in PERK signaling and promote adaptive mitochondrial remodeling, highlighting the potential for pharmacologic ISR activation to mitigate mitochondrial dysfunction and motivating the pursuit of highly selective ISR activators.

Mitochondria are dynamic organelles that continually respond to cellular stress. Recent studies have demonstrated that mitochondrial stress is relayed from mitochondria to the cytosol by the release of a proteolytic fragment of DELE1 that binds to the eIF2α kinase HRI to initiate integrated stress response (ISR) signaling. We report the cryo-electron microscopy structure of the C-terminal cleavage product of human DELE1, which assembles into a high-order oligomer. The oligomer consists of eight DELE1 monomers that assemble with D 4 symmetry via two sets of hydrophobic inter-subunit interactions. We identified the key residues involved in DELE1 oligomerization, and confirmed their role in stabilizing the octamer in vitro and in cells using mutagenesis. We further show that assembly-impaired DELE1 mutants are compromised in their ability to induce HRI-dependent ISR activation in cell culture models. Together, our findings provide molecular insights into the activity of DELE1 and how it signals to promote ISR activity following mitochondrial insult.

SARS-CoV-2 infection of human cells is initiated by the binding of the viral Spike protein to its cell-surface receptor ACE2. We conducted a targeted CRISPRi screen to uncover druggable pathways controlling Spike protein binding to human cells. Here we show that the protein BRD2 is required for ACE2 transcription in human lung epithelial cells and cardiomyocytes, and BRD2 inhibitors currently evaluated in clinical trials potently block endogenous ACE2 expression and SARS-CoV-2 infection of human cells, including those of human nasal epithelia. Moreover, pharmacological BRD2 inhibition with the drug ABBV-744 inhibited SARS-CoV-2 replication in Syrian hamsters. We also found that BRD2 controls transcription of several other genes induced upon SARS-CoV-2 infection, including the interferon response, which in turn regulates the antiviral response. Together, our results pinpoint BRD2 as a potent and essential regulator of the host response to SARS-CoV-2 infection and highlight the potential of BRD2 as a therapeutic target for COVID-19. Samelson et al. report that BRD2 inhibition suppresses SARS-CoV-2 infection in epithelial cells and Syrian hamsters via reducing the transcription of host cell surface receptor ACE2.

In the eukaryotic cytosol, the Hsp70 and the Hsp90 chaperone machines work in tandem with the maturation of a diverse array of client proteins. The transfer of nonnative clients between these systems is essential to the chaperoning process, but how it is regulated is still not clear. We discovered that NudC is an essential transfer factor with an unprecedented mode of action: NudC interacts with Hsp40 in Hsp40-Hsp70-client complexes and displaces Hsp70. Then, the interaction of NudC with Hsp90 allows the direct transfer of Hsp40-bound clients to Hsp90 for further processing. Consistent with this mechanism, NudC increases client activation in vitro as well as in cells and is essential for cellular viability. Together, our results show the complexity of the cooperation between the major chaperone machineries in the eukaryotic cytosol.

In the presence of different physiological and environmental stresses, cells rapidly initiate stress responses to re-establish cellular homeostasis. Stress responses usually orchestrate both transcriptional and translational programs via distinct mechanisms. With the advance of transcriptomics and proteomics technologies, transcriptional and translational outputs to a particular stress condition have become easier to measure; however, these technologies lack the ability to reveal the upstream regulatory pathways. Unbiased genetic screens based on a transcriptional or translational reporter are powerful approaches to identify regulatory factors of a specific stress response. CRISPR/Cas-based technologies, together with next-generation sequencing, enable genome-scale pooled screens to systematically elucidate gene function in mammalian cells, with a significant reduction in the rate of off-target effects compared to the previously used RNAi technology. Here, we describe our fluorescence-activated cell sorting (FACS)-based CRISPR interference (CRISPRi) screening platform using a translational reporter to identify novel genetic factors of the mitochondrial stress response in mammalian cells. This protocol provides a general framework for scientists who wish to establish a reporter-based CRISPRi screening platform to address questions in their area of research.

In mammalian cells, mitochondrial dysfunction triggers the integrated stress response, in which the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) results in the induction of the transcription factor ATF41–3. However, how mitochondrial stress is relayed to ATF4 is unknown. Here we show that HRI is the eIF2α kinase that is necessary and sufficient for this relay. In a genome-wide CRISPR interference screen, we identified factors upstream of HRI: OMA1, a mitochondrial stress-activated protease; and DELE1, a little-characterized protein that we found was associated with the inner mitochondrial membrane. Mitochondrial stress stimulates OMA1-dependent cleavage of DELE1 and leads to the accumulation of DELE1 in the cytosol, where it interacts with HRI and activates the eIF2α kinase activity of HRI. In addition, DELE1 is required for ATF4 translation downstream of eIF2α phosphorylation. Blockade of the OMA1–DELE1–HRI pathway triggers an alternative response in which specific molecular chaperones are induced. The OMA1–DELE1–HRI pathway therefore represents a potential therapeutic target that could enable fine-tuning of the integrated stress response for beneficial outcomes in diseases that involve mitochondrial dysfunction. A genome-wide CRISPR interference screen shows that a signalling pathway involving OMA1, DELE1 and the eIF2α kinase HRI relays mitochondrial stress to the cytosol to trigger the integrated stress response.

Dysregulated metabolism accelerates reduced decision-making and locomotor ability during aging. To identify mechanisms for delaying behavioral decline, we investigated how C. elegans males sustain their copulatory behavior during early to mid-adulthood. We found that in mid-agedmales, gluco-/glyceroneogenesis, promoted by phosphoenolpyruvate carboxykinase (PEPCK), sustains competitive reproductive behavior. C. elegans' PEPCK paralogs, pck-1 and pck-2, increase in expression during the first 2 days of adulthood. Insufficient PEPCK expression correlates with reduced egl-2-encoded ether-a-go-go K+ channel expression and premature hyper-excitability of copulatory circuits. For copulation, pck-1 is required in neurons, whereas pck-2 is required in the epidermis. However, PCK-2 is more essential, because we found that epidermal PCK-2 likely supplements the copulation circuitry with fuel. We identified the subunit A of succinate dehydrogenase SDHA-1 as a potent modulator of PEPCK expression. We postulate that during mid-adulthood, reduction in mitochondrial physiology signals the upregulation of cytosolic PEPCK to sustain the male's energy demands.

The unfolded protein response (UPR) detects and restores deficits in the endoplasmic reticulum (ER) protein folding capacity. Ceapins specifically inhibit the UPR sensor ATF6α, an ER-tethered transcription factor, by retaining it at the ER through an unknown mechanism. Our genome-wide CRISPR interference (CRISPRi) screen reveals that Ceapins function is completely dependent on the ABCD3 peroxisomal transporter. Proteomics studies establish that ABCD3 physically associates with ER-resident ATF6α in cells and in vitro in a Ceapin-dependent manner. Ceapins induce the neomorphic association of ER and peroxisomes by directly tethering the cytosolic domain of ATF6α to ABCD3’s transmembrane regions without inhibiting or depending on ABCD3 transporter activity. Thus, our studies reveal that Ceapins function by chemical-induced misdirection which explains their remarkable specificity and opens up new mechanistic routes for drug development and synthetic biology.

In mammalian cells, mitochondrial dysfunction triggers the integrated stress response (ISR), in which eIF2α phosphorylation upregulates the transcription factor ATF4. However, how mitochondrial stress is relayed to the ISR is unknown. We found that HRI is the eIF2α kinase necessary and sufficient for this relay. Using an unbiased CRISPRi screen, we identified factors upstream of HRI: OMA1, a mitochondrial stress-activated protease, and DELE1, a little-characterized protein we found to be associated with the inner mitochondrial membrane. Mitochondrial stress stimulates the OMA1-dependent cleavage of DELE1, leading to its accumulation in the cytosol, where it interacts with HRI and activates its eIF2α kinase activity. Blockade of the OMA1-DELE1-HRI pathway is beneficial during some, but not all types of mitochondrial stress, and leads to an alternative response that induces specific molecular chaperones. Therefore, this pathway is a potential therapeutic target enabling fine-tuning of the ISR for beneficial outcomes in diseases involving mitochondrial dysfunction.

Although diet affects growth and behaviour, the adaptive mechanisms that coordinate these processes in non-optimal food sources are unclear. Here we show that the C. elegans tmc-1 channel, which is homologous to the mammalian tmc deafness genes, attenuates development and inhibits sexual behaviour in non-optimal food, the synthetic CeMM medium. In CeMM medium, signalling from the pharyngeal MC neurons and body wall muscles slows larval development. However, in the non-standard diet, mutation in tmc-1 accelerates development, by impairing the excitability of these cells. The tmc-1 larva can immediately generate ATP when fed CeMM, and their fast development requires insulin signalling. Our findings suggest that the tmc-1 channel indirectly affects metabolism in wild-type animals. In addition to regulating the development, we show that mutating tmc-1 can relax diet-induced inhibition of male sexual behaviour, thus indicating that a single regulator can be genetically modified to promote growth rate and reproductive success in new environments.

The decline of aging C. elegans male’s mating behavior is correlated with the increased excitability of the cholinergic circuitry that executes copulation. In this study, we show that the mating circuits’ functional durability depends on the metabolic regulator SIR-2.1, a NAD+-dependent histone deacetylase. Aging sir-2.1(0) males display accelerated mating behavior decline due to premature hyperexcitability of cholinergic circuits used for intromission and ejaculation. In sir-2.1(0) males, the hypercontraction of the spicule-associated muscles pinch the vas deferens opening, thus blocking sperm release. The hyperexcitability is aggravated by reactive oxygen species (ROS). Our genetic, pharmacological, and behavioral analyses suggest that in sir-2.1(0) and older wild-type males, enhanced catabolic enzymes expression, coupled with the reduced expression of ROS-scavengers contribute to the behavioral decline. However, as a compensatory response to reduce altered catabolism/ROS production, anabolic enzymes expression levels are also increased, resulting in higher gluconeogenesis and lipid synthesis.

Deteriorative changes in behavioral functions are natural processes that accompany aging. In advanced aged C. elegans nematodes, gross decline in general behaviors, such as locomotion and feeding, is correlated with degeneration of muscle structure and contractile function. In this study, we characterized the age-related changes in C. elegans male mating behavior to determine possible causes that ultimately lead to age-related muscle frailty. Unlike the kinetics of general behavioral decline, we found that mating behavior deteriorates early in adulthood, with no obvious muscle fiber disorganization or sperm dysfunction. Through direct mating behavior observations, Ca2+ imaging, and pharmacological tests, we found that the muscular components used for mating become more excitable as the males age. Interestingly, manipulating either the expression of acetylcholine receptor (AChR) genes or dietary-mediated ether-a-go-go family K+ channel function can reduce the muscle excitability of older males and concurrently improve mating behavior, suggesting a correlation between these biological processes.

Prolonged nutrient limitation has been extensively studied due to its positive effects on life span. However, less is understood of how brief periods of starvation can have lasting consequences. In this study, we used genetics, biochemistry, pharmacology and behavioral analysis to show that after a limited period of starvation, the synthesis of egl-2-encoded ether-a-go-go (EAG) K+ channels and its C-terminal modifications by unc-43-encoded CaMKII have a perduring effect on C. elegans male sexual behavior. EGL-2 and UNC-43 interactions, induced after food deprivation, maintain reduced excitability in muscles involved in sex. In young adult males, spastic contractions occur in cholinergic-activated sex muscles that lack functional unc-103-encoded ERG-like K+ channels. Promoting EGL-2 and UNC-43 interactions in unc-103 mutant adult males by starving them for a few hours reduce spastic muscle contractions over multiple days. Although transient starvation during early adulthood has a hormetic effect of suppressing mutation-induced muscle contractions, the treatment reduces the ability of young wild-type (WT) males to compete with well-fed cohorts in siring progeny.

Penetration of a male copulatory organ into a suitable mate is a conserved and necessary behavioral step for most terrestrial matings; however, the detailed molecular and cellular mechanisms for this distinct social interaction have not been elucidated in any animal. During mating, the Caenorhabditis elegans male cloaca is maintained over the hermaphrodite's vulva as he attempts to insert his copulatory spicules. Rhythmic spicule thrusts cease when insertion is sensed. Circuit components consisting of sensory/motor neurons and sex muscles for these steps have been previously identified, but it was unclear how their outputs are integrated to generate a coordinated behavior pattern. Here, we show that cholinergic signaling between the cloacal sensory/motor neurons and the posterior sex muscles sustains genital contact between the sexes. Simultaneously, via gap junctions, signaling from these muscles is transmitted to the spicule muscles, thus coupling repeated spicule thrusts with vulval contact. To transit from rhythmic to sustained muscle contraction during penetration, the SPC sensory-motor neurons integrate the signal of spicule's position in the vulva with inputs from the hook and cloacal sensilla. The UNC-103 K+ channel maintains a high excitability threshold in the circuit, so that sustained spicule muscle contraction is not stimulated by fewer inputs. We demonstrate that coordination of sensory inputs and motor outputs used to initiate, maintain, self-monitor, and complete an innate behavior is accomplished via the coupling of a few circuit components.

DNA synthesis during S-phase and upon DNA repair is accompanied by chromatin assembly. The chromatin assembly factor CAF-1 has been biochemically well-characterized to deposit histones onto newly synthesized DNA. To gain insights into the in vivo functions of CAF-1 in Drosophila, we generated null mutants of the largest subunit of dCAF-1, dCAF-1-p180. We show that, unlike CAF-1 mutant yeast, dCAF-1-p180 mutant flies are hemizygous lethal. Removal of maternal dCAF-1-p180 activity by germline clones blocks oogenesis. Tissue-specific deletion of dCAF-1-p180 in the eye primordia disrupts eye development. In addition, reduction of dCAF-1-p180 activity suppresses gene silencing at heterochromatin and antagonizes Polycomb-mediated cell fate determination. Furthermore, heterozygous dCAF-1-p180 mutant flies display an increased sensitivity to gamma-irradiation and a reduced efficiency in recombinational double strand break (DSB) repair. Our experiments also show that human hCAF-1-p150 can rescue the dCAF-1-p180 mutant flies, demonstrating a functional conservation of eukaryotic CAF-1 activities in vivo. Together, our results establish that dCAF-1-p180 is an essential gene for Drosophila development and further underscore the importance of dCAF-1 in regulating gene expression and DNA repair in vivo.