7-Dehydrocholesterol is an endogenous suppressor of ferroptosis.

Ferroptosis is a form of cell death that has received considerable attention not only as a means to eradicate defined tumour entities but also because it provides unforeseen insights into the metabolic adaptation that tumours exploit to counteract phospholipid oxidation1,2. Here, we identify proferroptotic activity of 7-dehydrocholesterol reductase (DHCR7) and an unexpected prosurvival function of its substrate, 7-dehydrocholesterol (7-DHC). Although previous studies suggested that high concentrations of 7-DHC are cytotoxic to developing neurons by favouring lipid peroxidation3, we now show that 7-DHC accumulation confers a robust prosurvival function in cancer cells. Because of its far superior reactivity towards peroxyl radicals, 7-DHC effectively shields (phospho)lipids from autoxidation and subsequent fragmentation. We provide validation in neuroblastoma and Burkitt's lymphoma xenografts where we demonstrate that the accumulation of 7-DHC is capable of inducing a shift towards a ferroptosis-resistant state in these tumours ultimately resulting in a more aggressive phenotype. Conclusively, our findings provide compelling evidence of a yet-unrecognized antiferroptotic activity of 7-DHC as a cell-intrinsic mechanism that could be exploited by cancer cells to escape ferroptosis.

Dopey proteins are essential but overlooked regulators of membrane trafficking.

Dopey family proteins play crucial roles in diverse processes from morphogenesis to neural function and are conserved from yeast to mammals. Understanding the mechanisms behind these critical functions could have major clinical significance, as dysregulation of Dopey proteins has been linked to the cognitive defects in Down syndrome, as well as neurological diseases. Dopey proteins form a complex with the non-essential GEF-like protein Mon2 and an essential lipid flippase from the P4-ATPase family. Different combinations of Dopey, Mon2 and flippases have been linked to regulating membrane remodeling, from endosomal recycling to extracellular vesicle formation, through their interactions with lipids and other membrane trafficking regulators, such as ARL1, SNX3 and the kinesin-1 light chain KLC2. Despite these important functions and their likely clinical significance, Dopey proteins remain understudied and their roles elusive. Here, we review the major scientific discoveries relating to Dopey proteins and detail key open questions regarding their function to draw attention to these fascinating enigmas.

The power of imaging to understand extracellular vesicle biology in vivo.

Extracellular vesicles (EVs) are nano-sized lipid bilayer vesicles released by virtually every cell type. EVs have diverse biological activities, ranging from roles in development and homeostasis to cancer progression, which has spurred the development of EVs as disease biomarkers and drug nanovehicles. Owing to the small size of EVs, however, most studies have relied on isolation and biochemical analysis of bulk EVs separated from biofluids. Although informative, these approaches do not capture the dynamics of EV release, biodistribution, and other contributions to pathophysiology. Recent advances in live and high-resolution microscopy techniques, combined with innovative EV labeling strategies and reporter systems, provide new tools to study EVs in vivo in their physiological environment and at the single-vesicle level. Here we critically review the latest advances and challenges in EV imaging, and identify urgent, outstanding questions in our quest to unravel EV biology and therapeutic applications.

Regulation of neurogenesis by interkinetic nuclear migration through an apical-basal notch gradient.

The different cell types in the central nervous system develop from a common pool of progenitor cells. The nuclei of progenitors move between the apical and basal surfaces of the neuroepithelium in phase with their cell cycle, a process termed interkinetic nuclear migration (INM). In the retina of zebrafish mikre oko (mok) mutants, in which the motor protein Dynactin-1 is disrupted, interkinetic nuclei migrate more rapidly and deeply to the basal side and more slowly to the apical side. We found that Notch signaling is predominantly activated on the apical side in both mutants and wild-type. Mutant progenitors are, thus, less exposed to Notch and exit the cell cycle prematurely. This leads to an overproduction of early-born retinal ganglion cells (RGCs) at the expense of later-born interneurons and glia. Our data indicate that the function of INM is to balance the exposure of progenitor nuclei to neurogenic versus proliferative signals.

Trypanosomes can initiate nuclear export co-transcriptionally.

The nuclear envelope serves as important messenger RNA (mRNA) surveillance system. In yeast and human, several control systems act in parallel to prevent nuclear export of unprocessed mRNAs. Trypanosomes lack homologues to most of the involved proteins and their nuclear mRNA metabolism is non-conventional exemplified by polycistronic transcription and mRNA processing by trans-splicing. We here visualized nuclear export in trypanosomes by intra- and intermolecular multi-colour single molecule FISH. We found that, in striking contrast to other eukaryotes, the initiation of nuclear export requires neither the completion of transcription nor splicing. Nevertheless, we show that unspliced mRNAs are mostly prevented from reaching the nucleus-distant cytoplasm and instead accumulate at the nuclear periphery in cytoplasmic nuclear periphery granules (NPGs). Further characterization of NPGs by electron microscopy and proteomics revealed that the granules are located at the cytoplasmic site of the nuclear pores and contain most cytoplasmic RNA-binding proteins but none of the major translation initiation factors, consistent with a function in preventing faulty mRNAs from reaching translation. Our data indicate that trypanosomes regulate the completion of nuclear export, rather than the initiation. Nuclear export control remains poorly understood, in any organism, and the described way of control may not be restricted to trypanosomes.

Extracellular vesicle budding is inhibited by redundant regulators of TAT-5 flippase localization and phospholipid asymmetry.

Cells release extracellular vesicles (EVs) that mediate intercellular communication and repair damaged membranes. Despite the pleiotropic functions of EVs in vitro, their in vivo function is debated, largely because it is unclear how to induce or inhibit their formation. In particular, the mechanisms of EV release by plasma membrane budding or ectocytosis are poorly understood. We previously showed that TAT-5 phospholipid flippase activity maintains the asymmetric localization of the lipid phosphatidylethanolamine (PE) in the plasma membrane and inhibits EV budding by ectocytosis in Caenorhabditis elegans However, no proteins that inhibit ectocytosis upstream of TAT-5 were known. Here, we identify TAT-5 regulators associated with retrograde endosomal recycling: PI3Kinase VPS-34, Beclin1 homolog BEC-1, DnaJ protein RME-8, and the uncharacterized Dopey homolog PAD-1. PI3Kinase, RME-8, and semiredundant sorting nexins are required for the plasma membrane localization of TAT-5, which is important to maintain PE asymmetry and inhibit EV release. PAD-1 does not directly regulate TAT-5 localization, but is required for the lipid flipping activity of TAT-5. PAD-1 also has roles in endosomal trafficking with the GEF-like protein MON-2, which regulates PE asymmetry and EV release redundantly with sorting nexins independent of the core retromer. Thus, in addition to uncovering redundant intracellular trafficking pathways, our study identifies additional proteins that regulate EV release. This work pinpoints TAT-5 and PE as key regulators of plasma membrane budding, further supporting the model that PE externalization drives ectocytosis.

Extracellular vesicles.

Extracellular vesicles (EVs) encompass a diverse array of membrane-bound organelles released outside cells in response to developmental and physiological cell needs. EVs play important roles in remodeling the shape and content of differentiating cells and can rescue damaged cells from toxic or dysfunctional content. EVs can send signals and transfer metabolites between tissues and organisms to regulate development, respond to stress or tissue damage, or alter mating behaviors. While many EV functions have been uncovered by characterizing ex vivo EVs isolated from body fluids and cultured cells, research using the nematode Caenorhabditis elegans has provided insights into the in vivo functions, biogenesis, and uptake pathways. The C. elegans EV field has also developed methods to analyze endogenous EVs within the organismal context of development and adult physiology in free-living, behaving animals. In this review, we summarize major themes that have emerged for C. elegans EVs and their relevance to human health and disease. We also highlight the diversity of biogenesis mechanisms, locations, and functions of worm EVs and discuss open questions and unexplored topics tenable in C. elegans, given the nematode model is ideal for light and electron microscopy, genetic screens, genome engineering, and high-throughput omics.

Phagolysosomes break down the membrane of a non-apoptotic corpse independent of macroautophagy.

Cell corpses must be cleared in an efficient manner to maintain tissue homeostasis and regulate immune responses. Ubiquitin-like Atg8/LC3 family proteins promote the degradation of membranes and internal cargo during both macroautophagy and corpse clearance, raising the question how macroautophagy contributes to corpse clearance. Studying the clearance of non-apoptotic dying polar bodies in Caenorhabditis elegans embryos, we show that the LC3 ortholog LGG-2 is enriched in the polar body phagolysosome independent of membrane association or autophagosome formation. We demonstrate that ATG-16.1 and ATG-16.2, which promote membrane association of lipidated Atg8/LC3 proteins, redundantly promote polar body membrane breakdown in phagolysosomes independent of their role in macroautophagy. We also show that the lipid scramblase ATG-9 is needed for autophagosome formation in early embryos but is dispensable for timely polar body membrane breakdown or protein cargo degradation. These findings demonstrate that macroautophagy is not required to promote polar body degradation, in contrast to recent findings with apoptotic corpse clearance in C. elegans embryos. Determining how membrane association of Atg8/LC3 promotes the breakdown of different types of cell corpses in distinct cell types or metabolic states is likely to give insights into the mechanisms of immunoregulation during normal development, physiology, and disease.

Dopey-dependent regulation of extracellular vesicles maintains neuronal morphology.

Mature neurons maintain their distinctive morphology for extended periods in adult life. Compared to developmental neurite outgrowth, axon guidance, and target selection, relatively little is known of mechanisms that maintain mature neuron morphology. Loss of function in C. elegans DIP-2, a member of the conserved lipid metabolic regulator Dip2 family, results in progressive overgrowth of neurites in adults. We find that dip-2 mutants display specific genetic interactions with sax-2, the C. elegans ortholog of Drosophila Furry and mammalian FRY. Combined loss of DIP-2 and SAX-2 results in severe disruption of neuronal morphology maintenance accompanied by increased release of neuronal extracellular vesicles (EVs). By screening for suppressors of dip-2 sax-2 double mutant defects we identified gain-of-function (gf) mutations in the conserved Dopey family protein PAD-1 and its associated phospholipid flippase TAT-5/ATP9A. In dip-2 sax-2 double mutants carrying either pad-1(gf) or tat-5(gf) mutation, EV release is reduced and neuronal morphology across multiple neuron types is restored to largely normal. PAD-1(gf) acts cell autonomously in neurons. The domain containing pad-1(gf) is essential for PAD-1 function, and PAD-1(gf) protein displays increased association with the plasma membrane and inhibits EV release. Our findings uncover a novel functional network of DIP-2, SAX-2, PAD-1, and TAT-5 that maintains morphology of neurons and other types of cells, shedding light on the mechanistic basis of neurological disorders involving human orthologs of these genes.

PAR-3 mediates the initial clustering and apical localization of junction and polarity proteins during C. elegans intestinal epithelial cell polarization.

The apicobasal polarity of epithelial cells is critical for organ morphogenesis and function, and loss of polarity can promote tumorigenesis. Most epithelial cells form when precursor cells receive a polarization cue, develop distinct apical and basolateral domains and assemble junctions near their apical surface. The scaffolding protein PAR-3 regulates epithelial cell polarity, but its cellular role in the transition from precursor cell to polarized epithelial cell has not been determined in vivo. Here, we use a targeted protein-degradation strategy to remove PAR-3 from C. elegans embryos and examine its cellular role as intestinal precursor cells become polarized epithelial cells. At initial stages of polarization, PAR-3 accumulates in cortical foci that contain E-cadherin, other adherens junction proteins, and the polarity proteins PAR-6 and PKC-3. Using live imaging, we show that PAR-3 foci move apically and cluster, and that PAR-3 is required to assemble E-cadherin into foci and for foci to accumulate at the apical surface. We propose that PAR-3 facilitates polarization by promoting the initial clustering of junction and polarity proteins that then travel and accumulate apically. Unexpectedly, superficial epidermal cells form apical junctions in the absence of PAR-3, and we show that PAR-6 has a PAR-3-independent role in these cells to promote apical junction maturation. These findings indicate that PAR-3 and PAR-6 function sequentially to position and mature apical junctions, and that the requirement for PAR-3 can vary in different types of epithelial cells.

Visualizing Phagocytic Cargo In Vivo from Engulfment to Resolution in Caenorhabditis elegans.

The nematode Caenorhabditis elegans offers many experimental advantages to study conserved mechanisms of phagocytosis and phagocytic clearance. These include the stereotyped timing of phagocytic events in vivo for time-lapse imaging, the availability of transgenic reporters labeling molecules involved in different steps of phagocytosis, and the transparency of the animal for fluorescence imaging. Further, the ease of forward and reverse genetics in C. elegans has enabled many of the initial discoveries of proteins involved in phagocytic clearance. In this chapter, we focus on phagocytosis by the large undifferentiated blastomeres of C. elegans embryos, which engulf and eliminate diverse phagocytic cargo from the corpse of the second polar body to cytokinetic midbody remnants. We describe the use of fluorescent time-lapse imaging to observe the distinct steps of phagocytic clearance and methods to normalize this process to distinguish defects in mutant strains. These approaches have enabled us to reveal new insights from the initial signaling to induce phagocytosis up until the final resolution of phagocytic cargo in phagolysosomes.

The ATPase activity of the phosphatidylethanolamine flippase TAT-5 inhibits extracellular vesicle budding from the plasma membrane.

Cells release extracellular vesicles (EVs) from their surface, but the mechanisms that govern EV release by plasma membrane budding are poorly understood. The lipid flippase TAT-5 inhibits EV release from the plasma membrane in C. elegans , but how the level of flippase activity regulates EV release was unknown. We generated point mutations in the DGET motif of TAT-5 predicted to lead to a partial or complete loss of ATPase activity. We discovered that tat-5(E246Q) mutants were sterile, while tat-5(D244T) mutants produced embryos that arrested during development. Using degron-based reporters, we found that EV release was increased in tat-5(D244T) mutant embryos and that phagocytosis was also disrupted. These data suggest that a low level of flippase activity can promote fertility, while a higher level of flippase activity is required to inhibit EV release, allow phagocytosis, and carry out embryonic development.

A BORC-dependent molecular pathway for vesiculation of cell corpse phagolysosomes.

Phagocytic clearance is important to provide cells with metabolites and regulate immune responses, but little is known about how phagolysosomes finally resolve their phagocytic cargo of cell corpses, cell debris, and pathogens. While studying the phagocytic clearance of non-apoptotic polar bodies in C. elegans, we previously discovered that phagolysosomes tubulate into small vesicles to facilitate corpse clearance within 1.5 h. Here, we show that phagolysosome vesiculation depends on amino acid export by the solute transporter SLC-36.1 and the activation of TORC1. We demonstrate that downstream of TORC1, BLOC-1-related complex (BORC) is de-repressed by Ragulator through the BORC subunit BLOS-7. In addition, the BORC subunit SAM-4 is needed continuously to recruit the small GTPase ARL-8 to the phagolysosome for tubulation. We find that disrupting the regulated GTP-GDP cycle of ARL-8 reduces tubulation by kinesin-1, delays corpse clearance, and mislocalizes ARL-8 away from lysosomes. We also demonstrate that mammalian phagocytes use BORC to promote phagolysosomal degradation, confirming the conserved importance of TOR and BORC. Finally, we show that HOPS is required after tubulation for the rapid degradation of cargo in small phagolysosomal vesicles, suggesting that additional rounds of lysosome fusion occur. Thus, by observing single phagolysosomes over time, we identified the molecular pathway regulating phagolysosome vesiculation that promotes efficient resolution of phagocytosed cargos.

The g protein-coupled receptor agtrl1b regulates early development of myocardial progenitors.

While many factors that modulate the morphogenesis and patterning of the embryonic heart have been identified, relatively little is known about the molecular events that regulate the differentiation of progenitor cells fated to form the myocardium. Here, we show that zebrafish grinch (grn) mutants form a reduced number of myocardial progenitor cells, which results in a profound deficit in cardiomyocyte numbers in the most severe cases. We show that grn encodes the G protein-coupled receptor (GPCR) Agtrl1b, a known regulator of adult cardiovascular physiology. Ectopic expression of Apelin, an Agtrl1b ligand, results in the complete absence of cardiomyocytes. Data from transplantation and transgenic approaches indicate that Agtrl1 signaling plays a cell-autonomous role in myocardial specification, with activity being required coincident with the onset of gastrulation movements. These results support a model in which agtrl1b regulates the migration of cells fated to form myocardial progenitors.

Conserved N- and C-terminal motifs of PAD-1 are required to inhibit extracellular vesicle release.

Cells release extracellular vesicles (EVs) carrying cargos that can influence development and disease, but the mechanisms that govern EV release by plasma membrane budding are poorly understood. We previously showed that the Dopey protein PAD-1 inhibits EV release from the plasma membrane in C. elegans . However, PAD-1 is large, and the domains required to regulate EV release were unknown. Here, we reveal that the conserved N-terminal EWAD motif and C-terminal leucine zippers are required to inhibit EV release from the plasma membrane. Revealing a role for these domains is an important first step to identifying how EV release is regulated.

The developmental and physiological roles of phagocytosis in Caenorhabditis elegans.

Phagocytosis is an essential process by which cellular debris and pathogens are cleared from the environment. Cells extend their plasma membrane to engulf objects and contain them within a limiting membrane for isolation from the cytosol or for intracellular degradation in phagolysosomes. The basic mechanisms of phagocytosis and intracellular clearance are well conserved between animals. Indeed, much of our understanding is derived from studies on the nematode worm, Caenorhabditis elegans. Here, we review the latest progress in understanding the mechanisms and functions of phagocytic clearance from C. elegans studies. In particular, we highlight new insights into phagocytic signaling pathways, phagosome formation and phagolysosome resolution, as well as the challenges in studying these cyclic processes.

Loss of the Major Phosphatidylserine or Phosphatidylethanolamine Flippases Differentially Affect Phagocytosis.

The lipids phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEth) are normally asymmetrically localized to the cytosolic face of membrane bilayers, but can both be externalized during diverse biological processes, including cell division, cell fusion, and cell death. Externalized lipids in the plasma membrane are recognized by lipid-binding proteins to regulate the clearance of cell corpses and other cell debris. However, it is unclear whether PtdSer and PtdEth contribute in similar or distinct ways to these processes. We discovered that disruption of the lipid flippases that maintain PtdSer or PtdEth asymmetry in the plasma membrane have opposite effects on phagocytosis in Caenorhabditis elegans embryos. Constitutive PtdSer externalization caused by disruption of the major PtdSer flippase TAT-1 led to increased phagocytosis of cell debris, sometimes leading to two cells engulfing the same debris. In contrast, PtdEth externalization caused by depletion of the major PtdEth flippase TAT-5 or its activator PAD-1 disrupted phagocytosis. These data suggest that PtdSer and PtdEth externalization have opposite effects on phagocytosis. Furthermore, externalizing PtdEth is associated with increased extracellular vesicle release, and we present evidence that the extent of extracellular vesicle accumulation correlates with the extent of phagocytic defects. Thus, a general loss of lipid asymmetry can have opposing impacts through different lipid subtypes simultaneously exerting disparate effects.

Transcriptional adaptation in Caenorhabditis elegans.

Transcriptional adaptation is a recently described phenomenon by which a mutation in one gene leads to the transcriptional modulation of related genes, termed adapting genes. At the molecular level, it has been proposed that the mutant mRNA, rather than the loss of protein function, activates this response. While several examples of transcriptional adaptation have been reported in zebrafish embryos and in mouse cell lines, it is not known whether this phenomenon is observed across metazoans. Here we report transcriptional adaptation in C. elegans, and find that this process requires factors involved in mutant mRNA decay, as in zebrafish and mouse. We further uncover a requirement for Argonaute proteins and Dicer, factors involved in small RNA maturation and transport into the nucleus. Altogether, these results provide evidence for transcriptional adaptation in C. elegans, a powerful model to further investigate underlying molecular mechanisms.

Degron-tagged reporters probe membrane topology and enable the specific labelling of membrane-wrapped structures.

Visualization of specific organelles in tissues over background fluorescence can be challenging, especially when reporters localize to multiple structures. Instead of trying to identify proteins enriched in specific membrane-wrapped structures, we use a selective degradation approach to remove reporters from the cytoplasm or nucleus of C. elegans embryos and mammalian cells. We demonstrate specific labelling of organelles using degron-tagged reporters, including extracellular vesicles, as well as individual neighbouring membranes. These degron-tagged reporters facilitate long-term tracking of released cell debris and cell corpses, even during uptake and phagolysosomal degradation. We further show that degron protection assays can probe the topology of the nuclear envelope and plasma membrane during cell division, giving insight into protein and organelle dynamics. As endogenous and heterologous degrons are used in bacteria, yeast, plants, and animals, degron approaches can enable the specific labelling and tracking of proteins, vesicles, organelles, cell fragments, and cells in many model systems.

The P4-ATPase ATP9A is a novel determinant of exosome release.

Extracellular vesicles (EVs) released by cells have a role in intercellular communication to regulate a wide range of biological processes. Two types of EVs can be recognized. Exosomes, which are released from multi-vesicular bodies upon fusion with the plasma membrane, and ectosomes, which directly bud from the plasma membrane. How cells regulate the quantity of EV release is largely unknown. One of the initiating events in vesicle biogenesis is the regulated transport of phospholipids from the exoplasmic to the cytosolic leaflet of biological membranes. This process is catalyzed by P4-ATPases. The role of these phospholipid transporters in intracellular vesicle transport has been established in lower eukaryotes and is slowly emerging in mammalian cells. In Caenorhabditis elegans (C. elegans), deficiency of the P4-ATPase member TAT-5 resulted in enhanced EV shedding, indicating a role in the regulation of EV release. In this study, we investigated whether the mammalian ortholog of TAT-5, ATP9A, has a similar function in mammalian cells. We show that knockdown of ATP9A expression in human hepatoma cells resulted in a significant increase in EV release that was independent of caspase-3 activation. Pharmacological blocking of exosome release in ATP9A knockdown cells did significantly reduce the total number of EVs. Our data support a role for ATP9A in the regulation of exosome release from human cells.