The HEAT repeat protein HPO-27 is a lysosome fission factor.

Lysosomes are degradation and signalling centres crucial for homeostasis, development and ageing1. To meet diverse cellular demands, lysosomes remodel their morphology and function through constant fusion and fission2,3. Little is known about the molecular basis of fission. Here we identify HPO-27, a conserved HEAT repeat protein, as a lysosome scission factor in Caenorhabditis elegans. Loss of HPO-27 impairs lysosome fission and leads to an excessive tubular network that ultimately collapses. HPO-27 and its human homologue MROH1 are recruited to lysosomes by RAB-7 and enriched at scission sites. Super-resolution imaging, negative-staining electron microscopy and in vitro reconstitution assays reveal that HPO-27 and MROH1 self-assemble to mediate the constriction and scission of lysosomal tubules in worms and mammalian cells, respectively, and assemble to sever supported membrane tubes in vitro. Loss of HPO-27 affects lysosomal morphology, integrity and degradation activity, which impairs animal development and longevity. Thus, HPO-27 and MROH1 act as self-assembling scission factors to maintain lysosomal homeostasis and function.

Nematode eggshells: A new anatomical and terminological framework, with a critical review of relevant literature and suggested guidelines for the interpretation and reporting of eggshell imagery.

A literature review for a recent ultrastructural study of a trichinelloid eggshell revealed consistently occurring errors in the literature on nematode eggshell anatomy. Examples included nematodes of medical, veterinary, and agricultural importance in several orders. Previous researchers had warned of some of these errors decades ago, but a comprehensive solution was not offered until 2012 when a clarifying new anatomical and developmental interpretation of nematode eggshells was proposed by members of the Caenorhabditis elegans Research Community. However, their findings were explained using arcane acronyms and technical jargon intended for an audience of experimental molecular geneticists, and so their papers have rarely been cited outside the C. elegans community. Herein we (1) provide a critical review of nematode eggshell literature in which we correct errors and relabel imagery in important historical reports; (2) describe common reporting errors and their causes using language familiar to researchers having a basic understanding of microscopy and nematode eggs; (3) recommend a new hexalaminar anatomical and terminological framework for nematode eggshells based on the 2012 C. elegans framework; and (4) recommend new unambiguous terms appropriate for the embryonated/larvated eggs regularly encountered by practicing nematodologists to replace ambiguous or ontogenetically restricted terms in the 2012 C. elegans framework. We also (5) propose a resolution to conflicting claims made by the C. elegans team versus classical literature regarding Layer #3, (6) extend the C. elegans hexalaminar framework to include the polar plugs of trichinelloids, and (7) report new findings regarding trichinelloid eggshell structure.

Title: La coque des œufs des nématodes : un nouveau cadre anatomique et terminologique, avec une revue critique de la littérature pertinente et des lignes directrices suggérées pour l'interprétation et la communication de l'imagerie des coques des œufs. Abstract: Une revue de la littérature pour une étude ultrastructurale récente de la coque de l'œuf d'un trichinelloïde a révélé des erreurs récurrentes dans la littérature sur l'anatomie de la coque de l'œuf des nématodes. Les exemples comprenaient des nématodes d'importance médicale, vétérinaire et agricole dans plusieurs ordres. Des chercheurs avaient mis en garde contre certaines de ces erreurs il y a des décennies, mais une solution complète n'a été proposée qu'en 2012, lorsqu'une nouvelle interprétation anatomique et développementale clarifiant la structure des coques des œufs de nématodes a été proposée par des membres de la communauté de recherche de Caenorhabditis elegans. Cependant, leurs découvertes ont été expliquées à l'aide d'acronymes mystérieux et d'un jargon technique destiné à un public de généticiens moléculaires expérimentaux, et leurs articles ont donc rarement été cités en dehors de la communauté de C. elegans. Ici, nous (1) fournissons une revue critique de la littérature sur les coques des œufs de nématodes dans laquelle nous corrigeons les erreurs et réétiquetons les images dans des rapports historiques importants; (2) décrivons les erreurs de description courantes et leurs causes en utilisant un langage familier aux chercheurs ayant une compréhension de base de la microscopie et des œufs de nématodes; (3) recommandons un nouveau cadre anatomique et terminologique hexalaminaire pour les coques des œufs de nématodes basé sur le cadre de C. elegans de 2012; et (4) recommandons de nouveaux termes non ambigus appropriés pour les œufs embryonnés/larvés régulièrement rencontrés par les spécialistes de nématodes en exercice pour remplacer les termes ambigus ou à restriction ontogénétique dans le cadre de C. elegans de 2012. Nous proposons également (5) une résolution des affirmations contradictoires de l'équipe C. elegans par rapport à la littérature classique concernant la couche 3, (6) étendons le cadre hexalaminaire de C. elegans pour inclure les bouchons polaires des trichinelloïdes, et (7) signalons de nouvelles découvertes concernant la structure de la coque des œufs des trichinelloïdes.

Structures of the TMC-1 complex illuminate mechanosensory transduction.

The initial step in the sensory transduction pathway underpinning hearing and balance in mammals involves the conversion of force into the gating of a mechanosensory transduction channel1. Despite the profound socioeconomic impacts of hearing disorders and the fundamental biological significance of understanding mechanosensory transduction, the composition, structure and mechanism of the mechanosensory transduction complex have remained poorly characterized. Here we report the single-particle cryo-electron microscopy structure of the native transmembrane channel-like protein 1 (TMC-1) mechanosensory transduction complex isolated from Caenorhabditis elegans. The two-fold symmetric complex is composed of two copies each of the pore-forming TMC-1 subunit, the calcium-binding protein CALM-1 and the transmembrane inner ear protein TMIE. CALM-1 makes extensive contacts with the cytoplasmic face of the TMC-1 subunits, whereas the single-pass TMIE subunits reside on the periphery of the complex, poised like the handles of an accordion. A subset of complexes additionally includes a single arrestin-like protein, arrestin domain protein (ARRD-6), bound to a CALM-1 subunit. Single-particle reconstructions and molecular dynamics simulations show how the mechanosensory transduction complex deforms the membrane bilayer and suggest crucial roles for lipid-protein interactions in the mechanism by which mechanical force is transduced to ion channel gating.

Connectomes across development reveal principles of brain maturation.

An animal's nervous system changes as its body grows from birth to adulthood and its behaviours mature1-8. The form and extent of circuit remodelling across the connectome is unknown3,9-15. Here we used serial-section electron microscopy to reconstruct the full brain of eight isogenic Caenorhabditis elegans individuals across postnatal stages to investigate how it changes with age. The overall geometry of the brain is preserved from birth to adulthood, but substantial changes in chemical synaptic connectivity emerge on this consistent scaffold. Comparing connectomes between individuals reveals substantial differences in connectivity that make each brain partly unique. Comparing connectomes across maturation reveals consistent wiring changes between different neurons. These changes alter the strength of existing connections and create new connections. Collective changes in the network alter information processing. During development, the central decision-making circuitry is maintained, whereas sensory and motor pathways substantially remodel. With age, the brain becomes progressively more feedforward and discernibly modular. Thus developmental connectomics reveals principles that underlie brain maturation.

Patched regulates lipid homeostasis by controlling cellular cholesterol levels.

Hedgehog (Hh) signaling is essential during development and in organ physiology. In the canonical pathway, Hh binding to Patched (PTCH) relieves the inhibition of Smoothened (SMO). Yet, PTCH may also perform SMO-independent functions. While the PTCH homolog PTC-3 is essential in C. elegans, worms lack SMO, providing an excellent model to probe non-canonical PTCH function. Here, we show that PTC-3 is a cholesterol transporter. ptc-3(RNAi) leads to accumulation of intracellular cholesterol and defects in ER structure and lipid droplet formation. These phenotypes were accompanied by a reduction in acyl chain (FA) length and desaturation. ptc-3(RNAi)-induced lethality, fat content and ER morphology defects were rescued by reducing dietary cholesterol. We provide evidence that cholesterol accumulation modulates the function of nuclear hormone receptors such as of the PPARα homolog NHR-49 and NHR-181, and affects FA composition. Our data uncover a role for PTCH in organelle structure maintenance and fat metabolism.

In Vivo Visualization and Quantification of Mitochondrial Morphology in C. elegans.

Caenorhabditis elegans is a highly versatile model system, intensively used for functional, genetic, cytometric, and integrative studies. Due to its simplicity and large muscle cell number, C. elegans has frequently been used to study mitochondrial deficiencies caused by disease or drug toxicity. Here we describe a robust and efficient method to visualize and quantify mitochondrial morphology in vivo. This method has many practical and technical advantages above traditional (manual) methods and provides a comprehensive analysis of mitochondrial morphology.

Prohibitin depletion extends lifespan of a TORC2/SGK-1 mutant through autophagy and the mitochondrial UPR.

Mitochondrial prohibitins (PHB) are highly conserved proteins with a peculiar effect on lifespan. While PHB depletion shortens lifespan of wild-type animals, it enhances longevity of a plethora of metabolically compromised mutants, including target of rapamycin complex 2 (TORC2) mutants sgk-1 and rict-1. Here, we show that sgk-1 mutants have impaired mitochondrial homeostasis, lipogenesis and yolk formation, plausibly due to alterations in membrane lipid and sterol homeostasis. Remarkably, all these features are suppressed by PHB depletion. Our analysis shows the requirement of SRBP1/SBP-1 for the lifespan extension of sgk-1 mutants and the further extension conferred by PHB depletion. Moreover, although the mitochondrial unfolded protein response (UPRmt ) and autophagy are induced in sgk-1 mutants and upon PHB depletion, they are dispensable for lifespan. However, the enhanced longevity caused by PHB depletion in sgk-1 mutants requires both, the UPRmt and autophagy, but not mitophagy. We hypothesize that UPRmt induction upon PHB depletion extends lifespan of sgk-1 mutants through autophagy and probably modulation of lipid metabolism.

Host CDK-1 and formin mediate microvillar effacement induced by enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) induces changes to the intestinal cell cytoskeleton and formation of attaching and effacing lesions, characterized by the effacement of microvilli and then formation of actin pedestals to which the bacteria are tightly attached. Here, we use a Caenorhabditis elegans model of EHEC infection to show that microvillar effacement is mediated by a signalling pathway including mitotic cyclin-dependent kinase 1 (CDK1) and diaphanous-related formin 1 (CYK1). Similar observations are also made using EHEC-infected human intestinal cells in vitro. Our results support the use of C. elegans as a host model for studying attaching and effacing lesions in vivo, and reveal that the CDK1-formin signal axis is necessary for EHEC-induced microvillar effacement.

Phosphoregulation of HORMA domain protein HIM-3 promotes asymmetric synaptonemal complex disassembly in meiotic prophase in Caenorhabditis elegans.

In the two cell divisions of meiosis, diploid genomes are reduced into complementary haploid sets through the discrete, two-step removal of chromosome cohesion, a task carried out in most eukaryotes by protecting cohesion at the centromere until the second division. In eukaryotes without defined centromeres, however, alternative strategies have been innovated. The best-understood of these is found in the nematode Caenorhabditis elegans: after the single off-center crossover divides the chromosome into two segments, or arms, several chromosome-associated proteins or post-translational modifications become specifically partitioned to either the shorter or longer arm, where they promote the correct timing of cohesion loss through as-yet unknown mechanisms. Here, we investigate the meiotic axis HORMA-domain protein HIM-3 and show that it becomes phosphorylated at its C-terminus, within the conserved "closure motif" region bound by the related HORMA-domain proteins HTP-1 and HTP-2. Binding of HTP-2 is abrogated by phosphorylation of the closure motif in in vitro assays, strongly suggesting that in vivo phosphorylation of HIM-3 likely modulates the hierarchical structure of the chromosome axis. Phosphorylation of HIM-3 only occurs on synapsed chromosomes, and similarly to other previously-described phosphorylated proteins of the synaptonemal complex, becomes restricted to the short arm after designation of crossover sites. Regulation of HIM-3 phosphorylation status is required for timely disassembly of synaptonemal complex central elements from the long arm, and is also required for proper timing of HTP-1 and HTP-2 dissociation from the short arm. Phosphorylation of HIM-3 thus plays a role in establishing the identity of short and long arms, thereby contributing to the robustness of the two-step chromosome segregation.

Fluorescent dATP for DNA Synthesis In Vivo.

Fluorescent nucleoside triphosphates are powerful probes of DNA synthesis, but their potential use in living animals has been previously underexplored. Here, we report the synthesis and characterization of 7-deaza-(1,2,3-triazole)-2'-deoxyadenosine-5'-triphosphate (dATP) derivatives of tetramethyl rhodamine ("TAMRA-dATP"), cyanine ("Cy3-dATP"), and boron-dipyrromethene ("BODIPY-dATP"). Upon microinjection into live zebrafish embryos, all three compounds were incorporated into the DNA of dividing cells; however, their impact on embryonic toxicity was highly variable, depending on the exact structure of the dye. TAMRA-EdATP exhibited superior characteristics in terms of its high brightness, low toxicity, and rapid incorporation and depletion kinetics in both a vertebrate (zebrafish) and a nematode (Caenorhabditis elegans). TAMRA-EdATP allows for unprecedented, real-time visualization of DNA replication and chromosome segregation in vivo.

Gap junctions: historical discoveries and new findings in the Caenorhabditiselegans nervous system.

Gap junctions are evolutionarily conserved structures at close membrane contacts between two cells. In the nervous system, they mediate rapid, often bi-directional, transmission of signals through channels called innexins in invertebrates and connexins in vertebrates. Connectomic studies from Caenorhabditis elegans have uncovered a vast number of gap junctions present in the nervous system and non-neuronal tissues. The genome also has 25 innexin genes that are expressed in spatial and temporal dynamic pattern. Recent findings have begun to reveal novel roles of innexins in the regulation of multiple processes during formation and function of neural circuits both in normal conditions and under stress. Here, we highlight the diverse roles of gap junctions and innexins in the C. elegans nervous system. These findings contribute to fundamental understanding of gap junctions in all animals.

Loss of the seipin gene perturbs eggshell formation in Caenorhabditiselegans.

Seipin, an evolutionary conserved protein, plays pivotal roles during lipid droplet (LD) biogenesis and is associated with various human diseases with unclear mechanisms. Here, we analyzed Caenorhabditis elegans mutants deleted of the sole SEIPIN gene, seip-1 Homozygous seip-1 mutants displayed penetrant embryonic lethality, which is caused by the disruption of the lipid-rich permeability barrier, the innermost layer of the C. elegans embryonic eggshell. In C. elegans oocytes and embryos, SEIP-1 is associated with LDs and is crucial for controlling LD size and lipid homeostasis. The seip-1 deletion mutants reduced the ratio of polyunsaturated fatty acids (PUFAs) in their embryonic fatty acid pool. Interestingly, dietary supplementation of selected n-6 PUFAs rescued the embryonic lethality and defective permeability barrier. Accordingly, we propose that SEIP-1 may maternally regulate LD biogenesis and lipid homeostasis to orchestrate the formation of the permeability barrier for eggshell synthesis during embryogenesis. A lipodystrophy allele of seip-1 resulted in embryonic lethality as well and could be rescued by PUFA supplementation. These experiments support a great potential for using C. elegans to model SEIPIN-associated human diseases.

Cryo-electron microscopy structure of CLHM1 ion channel from Caenorhabditis elegans.

Calcium homeostasis modulators (CALHMs/CLHMs) comprise a family of pore-forming protein complexes assembling into voltage-gated, Ca2+ -sensitive, nonselective channels. These complexes contain an ion-conduction pore sufficiently wide to permit the passing of ATP molecules serving as neurotransmitters. While their function and structure information is accumulating, the precise mechanisms of these channel complexes remain to be full understood. Here, we present the structure of the Caenorhabditis elegans CLHM1 channel in its open state solved through single-particle cryo-electron microscopy at 3.7-Å resolution. The transmembrane region of the channel structure of the dominant class shows an assembly of 10-fold rotational symmetry in one layer, and its cytoplasmic region is involved in additional twofold symmetrical packing in a tail-to-tail manner. Furthermore, we identified a series of amino acid residues critical for the regulation of CeCLHM1 channel using functional assays, electrophysiological analyses as well as structural-based analysis. Our structure and function analyses provide new insights into the mechanisms of CALHM channels.

Teething during sleep: Ultrastructural analysis of pharyngeal muscle and cuticular grinder during the molt in Caenorhabditis elegans.

Complex extracellular structures exist throughout phylogeny, but the dynamics of their formation and dissolution are often opaque. One example is the pharyngeal grinder of the nematode Caenorhabditis elegans, an extracellular structure that ruptures bacteria during feeding. During each larval transition stage, called lethargus, the grinder is replaced with one of a larger size. Here, we characterize at the ultrastructural level the deconstruction of the larval grinder and the construction of the adult grinder during the fourth larval stage (L4)-to-adult transition. Early in L4 lethargus, pharyngeal muscle cells trans-differentiate from contractile to secretory cells, as evidenced by the appearance of clear and dense core vesicles and disruptions in sarcomere organization. This is followed, within minutes, by the dissolution of the L4 grinder and the formation and maturation of the adult grinder. Components of the nascent adult grinder are deposited basally, and are separated from the dissolving larval grinder by a visible apical layer. The complete grinder is a lamellated extracellular matrix comprised of five layers. Following grinder formation, pharyngeal muscle cells regain ultrastructural contractile properties, and muscle contractions resume. Our findings add to our understanding of how complex extracellular structures assemble and dissemble.

Expansion microscopy of C. elegans.

We recently developed expansion microscopy (ExM), which achieves nanoscale-precise imaging of specimens at ~70 nm resolution (with ~4.5x linear expansion) by isotropic swelling of chemically processed, hydrogel-embedded tissue. ExM of C. elegans is challenged by its cuticle, which is stiff and impermeable to antibodies. Here we present a strategy, expansion of C. elegans (ExCel), to expand fixed, intact C. elegans. ExCel enables simultaneous readout of fluorescent proteins, RNA, DNA location, and anatomical structures at resolutions of ~65-75 nm (3.3-3.8x linear expansion). We also developed epitope-preserving ExCel, which enables imaging of endogenous proteins stained by antibodies, and iterative ExCel, which enables imaging of fluorescent proteins after 20x linear expansion. We demonstrate the utility of the ExCel toolbox for mapping synaptic proteins, for identifying previously unreported proteins at cell junctions, and for gene expression analysis in multiple individual neurons of the same animal.

Fabrication of sharp silicon arrays to wound Caenorhabditis elegans.

Understanding how animals respond to injury and how wounds heal remains a challenge. These questions can be addressed using genetically tractable animals, including the nematode Caenorhabditis elegans. Given its small size, the current methods for inflicting wounds in a controlled manner are demanding. To facilitate and accelerate the procedure, we fabricated regular arrays of pyramidal features ("pins") sharp enough to pierce the tough nematode cuticle. The pyramids were made from monocrystalline silicon wafers that were micro-structured using optical lithography and alkaline wet etching. The fabrication protocol and the geometry of the pins, determined by electron microscopy, are described in detail. We also used electron microscopy to characterize the different types of injury caused by these pins. Upon wounding, C. elegans expresses genes encoding antimicrobial peptides. A comparison of the induction of antimicrobial peptide gene expression using traditional needles and the pin arrays demonstrates the utility of this new method.

Coordination of a Single Calcium Ion in the EF-hand Maintains the Off State of the Stromal Interaction Molecule Luminal Domain.

Store operated calcium (Ca2+) entry (SOCE) is the process whereby endoplasmic reticulum (ER) Ca2+ store depletion causes Orai1-composed Ca2+ channels on the plasma membrane (PM) to open, mediating a rise in cytosolic Ca2+ levels. Stromal interaction molecules (STIMs) are the proteins that directly sense ER Ca2+ content and gate Orai1 channels due to store depletion. The trigger for STIM activation is Ca2+ unbinding from the ER lumen-oriented domains, which consist of a nonconserved amino (N) terminal region and EF-hand and sterile α motif (SAM) domains (EF-SAM), highly conserved from humans to Caenorhabditis elegans. Solution NMR structures of the human EF-SAM domains have been determined at high Ca2+ concentrations; however, no direct structural view of the Ca2+ binding mode has been elucidated. Further, no atomic resolution data currently exists on EF-SAM at low Ca2+ levels. Here, we determined the X-ray crystal structure of the C. elegans STIM luminal domain, revealing that EF-SAM binds a single Ca2+ ion with pentagonal bipyramidal geometry and an ancillary α-helix formed by the N-terminal region acts as a brace to stabilize EF-SAM. Using solution NMR, we observed EF-hand domain unfolding and a conformational exchange between folded and unfolded states involving the ancillary α-helix and the canonical EF-hand in low Ca2+. Remarkably, we also detected an α-helix (+Ca2+) to ß-strand (-Ca2+) transition at the terminal SAM domain α-helix. Collectively, our analyses indicate that one canonically bound Ca2+ ion is sufficient to stabilize the quiescent luminal domain structure, precluding unfolding, conformational exchange, and secondary structure transformation.

C. elegans pronuclei fuse after fertilization through a novel membrane structure.

After fertilization, parental genomes are enclosed in two separate pronuclei. In Caenorhabditis elegans, and possibly other organisms, when the two pronuclei first meet, the parental genomes are separated by four pronuclear membranes. To understand how these membranes are breached to allow merging of parental genomes we used focused ion beam scanning electron microscopy (FIB-SEM) to study the architecture of the pronuclear membranes at nanometer-scale resolution. We find that at metaphase, the interface between the two pronuclei is composed of two membranes perforated by fenestrations ranging from tens of nanometers to several microns in diameter. The parental chromosomes come in contact through one of the large fenestrations. Surrounding this fenestrated, two-membrane region is a novel membrane structure, a three-way sheet junction, where the four membranes of the two pronuclei fuse and become two. In the plk-1 mutant, where parental genomes fail to merge, these junctions are absent, suggesting that three-way sheet junctions are needed for formation of a diploid genome.

Three-dimensional virtual refocusing of fluorescence microscopy images using deep learning.

We demonstrate that a deep neural network can be trained to virtually refocus a two-dimensional fluorescence image onto user-defined three-dimensional (3D) surfaces within the sample. Using this method, termed Deep-Z, we imaged the neuronal activity of a Caenorhabditis elegans worm in 3D using a time sequence of fluorescence images acquired at a single focal plane, digitally increasing the depth-of-field by 20-fold without any axial scanning, additional hardware or a trade-off of imaging resolution and speed. Furthermore, we demonstrate that this approach can correct for sample drift, tilt and other aberrations, all digitally performed after the acquisition of a single fluorescence image. This framework also cross-connects different imaging modalities to each other, enabling 3D refocusing of a single wide-field fluorescence image to match confocal microscopy images acquired at different sample planes. Deep-Z has the potential to improve volumetric imaging speed while reducing challenges relating to sample drift, aberration and defocusing that are associated with standard 3D fluorescence microscopy.

Caenorhabditis elegans revisited by atomic force microscopy - Ultra-structural changes of the cuticle, but not in the intestine after treatment with Combretum mucronatum extract.

Assessing the internal morphology of Caenorhabditis elegans by a topographical technique like atomic force microscopy (AFM) is a challenging process. As a prerequisite for a successful image acquisition, direct contact between the structure of interest and the AFM probe needs to be established. To gain this insight into the morphology of cuticle and intestine in C. elegans before and after treatment with a tannin-enriched hydro-ethanolic extract from Combretum mucronatum, we developed an approach based on polyethylene glycol embedding, ultra-sectioning, de-embedding and hexamethyldisilazane-dehydration prior to measuring in ambient conditions by intermittent contact mode AFM. The used experimental protocol allowed a facile and fast insight into the ultrastructure of treated versus untreated C. elegans individuals, directly leading to the identification of treatment-associated morphological alterations in the cuticle but not the intestine of C. elegans. Additionally, the presented ultra-microtomy based protocol could allow future insight into virtually any tissue or organism by AFM.