Ciliary intrinsic mechanisms regulate dynamic ciliary extracellular vesicle release from sensory neurons.

Extracellular vesicles (EVs) are submicron membranous structures and key mediators of intercellular communication.1,2 Recent research has highlighted roles for cilia-derived EVs in signal transduction, underscoring their importance as bioactive extracellular organelles containing conserved ciliary signaling proteins.3,4 Members of the transient receptor potential (TRP) channel polycystin-2 (PKD-2) family are found in ciliary EVs of the green algae Chlamydomonas and the nematode Caenorhabditis elegans5,6 and in EVs in the mouse embryonic node and isolated from human urine.7,8 In C. elegans, PKD-2 is expressed in male-specific EV-releasing sensory neurons, which extend ciliary tips to ciliary pore and directly release EVs into the environment.6,9 Males release EVs in a mechanically stimulated manner, regulate EV cargo content in response to mating partners, and deposit PKD-2::GFP-labeled EVs on the vulval cuticle of hermaphrodites during mating.9,10 Combined, our findings suggest that ciliary EV release is a dynamic process. Herein, we identify mechanisms controlling dynamic EV shedding using time-lapse imaging. Cilia can sustain the release of PKD-2-labeled EVs for 2 h. This extended release doesn't require neuronal transmission. Instead, ciliary intrinsic mechanisms regulate PKD-2 ciliary membrane replenishment and dynamic EV release. The kinesin-3 motor kinesin-like protein 6 (KLP-6) is necessary for initial and extended EV release, while the transition zone protein NPHP-4 is required only for sustained EV release. The dynamic replenishment of PKD-2 at the ciliary tip is key to sustained EV release. Our study provides a comprehensive portrait of real-time ciliary EV release and mechanisms supporting cilia as proficient EV release platforms.

Polycystins recruit cargo to distinct ciliary extracellular vesicle subtypes.

Therapeutic use of tiny extracellular vesicles (EVs) requires understanding cargo loading mechanisms. Here, we used a modular proximity label approach to identify EV cargo associated with the transient potential channel (TRP) polycystin PKD-2 of C. elegans. Polycystins are conserved receptor-TRP channel proteins affecting cilium function; dysfunction causes polycystic kidney disease in humans and mating deficits in C. elegans. Polycystin-2 EV localization is conserved from algae to humans, hinting at an ancient and unknown function. We discovered that polycystins associate with and direct specific cargo to EVs: channel-like PACL-1, dorsal and ventral membrane C-type lectins PAMLs, and conserved tumor necrosis-associated factor (TRAF) signaling adaptors TRF-1 and TRF-2. Loading of these components relied on polycystin-1 LOV-1. Our modular EV-TurboID approach can be applied in both cell- and tissue-specific manners to define the composition of distinct EV subtypes, addressing a major challenge of the EV field.

Ciliary intrinsic mechanisms regulate dynamic ciliary extracellular vesicle release from sensory neurons.

Cilia-derived extracellular vesicles (EVs) contain signaling proteins and act in intercellular communication. Polycystin-2 (PKD-2), a transient receptor potential channel, is a conserved ciliary EVs cargo. Caenorhabditis elegans serves as a model for studying ciliary EV biogenesis and function. C. elegans males release EVs in a mechanically-induced manner and deposit PKD-2-labeled EVs onto the hermaphrodite vulva during mating, suggesting an active release process. Here, we study the dynamics of ciliary EV release using time-lapse imaging and find that cilia can sustain the release of PKD-2-labeled EVs for a two-hour duration. Intriguingly, this extended release doesn't require neuronal synaptic transmission. Instead, ciliary intrinsic mechanisms regulate PKD-2 ciliary membrane replenishment and dynamic EV release. The ciliary kinesin-3 motor KLP-6 is necessary for both initial and extended ciliary EV release, while the transition zone protein NPHP-4 is required only for sustained EV release. The dihydroceramide desaturase DEGS1/2 ortholog TTM-5 is highly expressed in the EV-releasing sensory neurons, localizes to cilia, and is required for sustained but not initial ciliary EV release, implicating ceramide in ciliary ectocytosis. The study offers a comprehensive portrait of real-time ciliary EV release, and mechanisms supporting cilia as proficient EV release platforms.

Tracking N- and C-termini of C. elegans polycystin-1 reveals their distinct targeting requirements and functions in cilia and extracellular vesicles.

The cilium acts as an antenna receiving and sending signals, the latter via extracellular vesicles (EVs). In C. elegans and mammals, the Autosomal Dominant Polycystic Kidney Disease (ADPKD) gene products polycystin-1 (PC1) and polycystin-2 (PC2) localize to both cilia and EVs, act in the same genetic pathway, and function in a sensory capacity, suggesting ancient conservation. However, the functions of the polycystins on cilia and EVs remain enigmatic. We used our C. elegans model and endogenously fluorescent-tagged LOV-1/polycystin-1 to study LOV-1 processing, trafficking, transport, EV biogenesis, and function in living animals. Super resolution, real time imaging reveals that LOV-1 is processed into N-terminal (NTM) and C-terminal (CTM) forms via a conserved GPCR proteolytic site (GPS). The LOV-1 NTM is secreted into the extracellular matrix and not localized to ciliary tip EVs. In contrast, LOV-1 CTM and PKD-2 are co-trafficked, co-transported, and co-localized in cilia and on environmentally released ciliary EVs. LOV-1 CTM requires PKD-2 for ciliary EV localization, while PKD-2 localizes to ciliary EVs independent of LOV-1. We find that LOV-1 but not PKD-2 is required for chemosensation of an ascaroside mating pheromone. These findings indicate that the polycystins LOV-1 and PKD-2 function together and independently and provide insight to how cargo is selected and packaged in ciliary EVs.

Isolation, profiling, and tracking of extracellular vesicle cargo in Caenorhabditis elegans.

Extracellular vesicles (EVs) may mediate intercellular communication by carrying protein and RNA cargo. The composition, biology, and roles of EVs in physiology and pathology have been primarily studied in the context of biofluids and in cultured mammalian cells. The experimental tractability of C. elegans makes for a powerful in vivo animal system to identify and study EV cargo from its cellular source. We developed an innovative method to label, track, and profile EVs using genetically encoded, fluorescent-tagged EV cargo and conducted a large-scale isolation and proteomic profiling. Nucleic acid binding proteins (∼200) are overrepresented in our dataset. By integrating our EV proteomic dataset with single-cell transcriptomic data, we identified and validated ciliary EV cargo: CD9-like tetraspanin (TSP-6), ectonucleotide pyrophosphatase/phosphodiesterase (ENPP-1), minichromosome maintenance protein (MCM-3), and double-stranded RNA transporter SID-2. C. elegans EVs also harbor RNA, suggesting that EVs may play a role in extracellular RNA-based communication.

Sensory cilia act as a specialized venue for regulated extracellular vesicle biogenesis and signaling.

Ciliary extracellular vesicle (EV) shedding is evolutionarily conserved. In Chlamydomonas and C. elegans, ciliary EVs act as signaling devices.1-3 In cultured mammalian cells, ciliary EVs regulate ciliary disposal but also receptor abundance and signaling, ciliary length, and ciliary membrane dynamics.4-7 Mammalian cilia produce EVs from the tip and along the ciliary membrane.8,9 This study aimed to determine the functional significance of shedding at distinct locations and to explore ciliary EV biogenesis mechanisms. Using Airyscan super-resolution imaging in living C. elegans animals, we find that neuronal sensory cilia shed TRP polycystin-2 channel PKD-2::GFP-carrying EVs from two distinct sites: the ciliary tip and the ciliary base. Ciliary tip shedding requires distal ciliary enrichment of PKD-2 by the myristoylated coiled-coil protein CIL-7. Kinesin-3 KLP-6 and intraflagellar transport (IFT) kinesin-2 motors are also required for ciliary tip EV shedding. A big unanswered question in the EV field is how cells sort EV cargo. Here, we show that two EV cargoes- CIL-7 and PKD-2-localized and trafficked differently along cilia and were sorted to different environmentally released EVs. In response to mating partners, C. elegans males modulate EV cargo composition by increasing the ratio of PKD-2 to CIL-7 EVs. Overall, our study indicates that the cilium and its trafficking machinery act as a specialized venue for regulated EV biogenesis and signaling.

Age modulates liver responses to asparaginase-induced amino acid stress in mice.

Asparaginase is an amino acid-depleting agent used to treat blood cancers. Metabolic complications due to asparaginase affect liver function in humans. To examine how the liver response to asparaginase changes during maturity to adulthood, here we treated juvenile (2-week), young adult (8-week), and mature adult (16-week) mice with drug or excipient for 1 week and conducted RNA-Seq and functional analyses. Asparaginase reduced body growth and liver mass in juveniles but not in the adult animals. Unbiased exploration of the effect of asparaginase on the liver transcriptome revealed that the integrated stress response (ISR) was the only molecular signature shared across the ages, corroborating similar eukaryotic initiation factor 2 phosphorylation responses to asparaginase at all ages. Juvenile livers exhibited steatosis and iron accumulation following asparaginase exposure along with a hepatic gene signature indicating that asparaginase uniquely affects lipid, cholesterol, and iron metabolism in juvenile mice. In contrast, asparaginase-treated adult mice displayed greater variability in liver function, which correlated with an acute-phase inflammatory response gene signature. Asparaginase-exposed adults also had a serine/glycine/one-carbon metabolism gene signature in liver that corresponded with reduced circulating glycine and serine levels. These results establish the ISR as a conserved response to asparaginase-mediated amino acid deprivation and provide new insights into the relationship between the liver transcriptome and hepatic function upon asparaginase exposure.

Time-resolved analysis of amino acid stress identifies eIF2 phosphorylation as necessary to inhibit mTORC1 activity in liver.

Amino acid availability is sensed by GCN2 (general control nonderepressible 2) and mechanistic target of rapamycin complex 1 (mTORC1), but how these two sensors coordinate their respective signal transduction events remains mysterious. In this study we utilized mouse genetic models to investigate the role of GCN2 in hepatic mTORC1 regulation upon amino acid stress induced by a single injection of asparaginase. We found that deletion of Gcn2 prevented hepatic phosphorylation of eukaryotic initiation factor 2α to asparaginase and instead unleashed mTORC1 activity. This change in intracellular signaling occurred within minutes and resulted in increased 5'-terminal oligopyrimidine mRNA translation instead of activating transcription factor 4 synthesis. Asparaginase also promoted hepatic mRNA levels of several genes which function as mTORC1 inhibitors, and these genes were blunted or blocked in the absence of Gcn2, but their timing could not explain the early discordant effects in mTORC1 signaling. Preconditioning mice with a chemical endoplasmic reticulum stress agent before amino acid stress rescued normal mTORC1 repression in the liver of Gcn2-/- mice but not in livers with both Gcn2 and the endoplasmic reticulum stress kinase, Perk, deleted. Furthermore, treating wildtype and Gcn2-/- mice with ISRIB, an inhibitor of PERK signaling, also failed to alter hepatic mTORC1 responses to asparaginase, although administration of ISRIB alone had an inhibitory GCN2-independent effect on mTORC1 activity. Taken together, the data show that activating transcription factor 4 is not required, but eukaryotic initiation factor 2α phosphorylation is necessary to prevent mTORC1 activation during amino acid stress.

Role of activating transcription factor 4 in the hepatic response to amino acid depletion by asparaginase.

The anti-leukemic agent asparaginase activates the integrated stress response (ISR) kinase GCN2 and inhibits signaling via mechanistic target of rapamycin complex 1 (mTORC1). The study objective was to investigate the protective role of activating transcription factor 4 (ATF4) in controlling the hepatic transcriptome and mediating GCN2-mTORC1 signaling during asparaginase. We compared global gene expression patterns in livers from wildtype, Gcn2 -/-, and Atf4 -/- mice treated with asparaginase or excipient and further explored selected responses in livers from Atf4 +/- mice. Here, we show that ATF4 controls a hepatic gene expression profile that overlaps with GCN2 but is not required for downregulation of mTORC1 during asparaginase. Ingenuity pathway analysis indicates GCN2 independently influences inflammation-mediated hepatic processes whereas ATF4 uniquely associates with cholesterol metabolism and endoplasmic reticulum (ER) stress. Livers from Atf4 -/- or Atf4 +/- mice displayed an amplification of the amino acid response and ER stress response transcriptional signatures. In contrast, reduction in hepatic mTORC1 signaling was retained in Atf4 -/- mice treated with asparaginase. CONCLUSIONS: GCN2 and ATF4 serve complementary roles in the hepatic response to asparaginase. GCN2 functions to limit inflammation and mTORC1 signaling whereas ATF4 serves to limit the amino acid response and prevent ER stress during amino acid depletion by asparaginase.

Obesity challenges the hepatoprotective function of the integrated stress response to asparaginase exposure in mice.

Obesity increases risk for liver toxicity by the anti-leukemic agent asparaginase, but the mechanism is unknown. Asparaginase activates the integrated stress response (ISR) via sensing amino acid depletion by the eukaryotic initiation factor 2 (eIF2) kinase GCN2. The goal of this work was to discern the impact of obesity, alone versus alongside genetic disruption of the ISR, on mechanisms of liver protection during chronic asparaginase exposure in mice. Following diet-induced obesity, biochemical analysis of livers revealed that asparaginase provoked hepatic steatosis that coincided with activation of another eIF2 kinase PKR-like endoplasmic reticulum kinase (PERK), a major ISR transducer to ER stress. Genetic loss of Gcn2 intensified hepatic PERK activation to asparaginase, yet surprisingly, mRNA levels of key ISR gene targets such as Atf5 and Trib3 failed to increase. Instead, mechanistic target of rapamycin complex 1 (mTORC1) signal transduction was unleashed, and this coincided with liver dysfunction reflected by a failure to maintain hydrogen sulfide production or apolipoprotein B100 (ApoB100) expression. In contrast, obese mice lacking hepatic activating transcription factor 4 (Atf4) showed an exaggerated ISR and greater loss of endogenous hydrogen sulfide but normal inhibition of mTORC1 and maintenance of ApoB100 during asparaginase exposure. In both genetic mouse models, expression and phosphorylation of Sestrin2, an ATF4 gene target, was increased by asparaginase, suggesting mTORC1 inhibition during asparaginase exposure is not driven via eIF2-ATF4-Sestrin2. In conclusion, obesity promotes a maladaptive ISR during asparaginase exposure. GCN2 functions to repress mTORC1 activity and maintain ApoB100 protein levels independently of Atf4 expression, whereas hydrogen sulfide production is promoted via GCN2-ATF4 pathway.

Role of GCN2-Independent Signaling Through a Noncanonical PERK/NRF2 Pathway in the Physiological Responses to Dietary Methionine Restriction.

Restricting availability of essential amino acids (EAAs) limits aminoacylation of tRNAs by their cognate EAAs and activates the nutrient-sensing kinase, general control nonderepressible 2 (GCN2). Activated GCN2 phosphorylates eukaryotic initiation factor 2 (eIF2), altering gene-specific translation and initiating a transcriptional program collectively described as the integrated stress response (ISR). Central GCN2 activation by EAA deprivation is also linked to an acute aversive feeding response. Dietary methionine restriction (MR) produces a well-documented series of physiological responses (increased energy intake and expenditure, decreased adiposity, and increased insulin sensitivity), but the role of GCN2 in mediating them is unknown. Using Gcn2(-/-) mice, we found that the absence of GCN2 had no effect on the ability of MR to reduce body weight or adiposity, increase energy intake and expenditure, increase hepatic transcription and release of fibroblast growth factor 21, or improve insulin sensitivity. Interestingly, hepatic eIF2 phosphorylation by MR was uncompromised in Gcn2(-/-) mice. Instead, protein kinase R-like endoplasmic reticulum (ER) kinase (PERK) was activated in both intact and Gcn2(-/-) mice. PERK activation corresponded with induction of the ISR and the nuclear respiratory factor 2 antioxidant program but not ER stress. These data uncover a novel glutathione-sensing mechanism that functions independently of GCN2 to link dietary MR to its metabolic phenotype.

Identification of a Mg2+-sensitive ORF in the 5'-leader of TRPM7 magnesium channel mRNA.

TRPM7 is an essential and ubiquitous channel-kinase regulating cellular influx of Mg2+. Although TRPM7 mRNA is highly abundant, very small amount of the protein is detected in cells, suggesting post-transcriptional regulation of trpm7 gene expression. We found that TRPM7 mRNA 5'-leader contains two evolutionarily conserved upstream open reading frames that act together to drastically inhibit translation of the TRPM7 reading frame at high magnesium levels and ensure its optimal translation at low magnesium levels, when the activity of the channel-kinase is most required. The study provides the first example of magnesium channel synthesis being controlled by Mg2+ in higher eukaryotes.