The MAB-5/Hox family transcription factor is important for Caenorhabditis elegans innate immune response to Staphylococcus epidermidis infection.

Innate immunity functions as a rapid defense against broad classes of pathogenic agents. While the mechanisms of innate immunity in response to antigen exposure are well-studied, how pathogen exposure activates the innate immune responses and the role of genetic variation in immune activity is currently being investigated. Previously, we showed significant survival differences between the N2 and the CB4856 Caenorhabditis elegans isolates in response to Staphylococcus epidermidis infection. One of those differences was expression of the mab-5 Hox family transcription factor, which was induced in N2, but not CB4856, after infection. In this study, we use survival assays and RNA-sequencing to better understand the role of mab-5 in response to S. epidermidis. We found that mab-5 loss-of-function (LOF) mutants were more susceptible to S. epidermidis infection than N2 or mab-5 gain-of-function (GOF) mutants, but not as susceptible as CB4856 animals. We then conducted transcriptome analysis of infected worms and found considerable differences in gene expression profiles when comparing animals with mab-5 LOF to either N2 or mab-5 GOF. N2 and mab-5 GOF animals showed a significant enrichment in expression of immune genes and C-type lectins, whereas mab-5 LOF mutants did not. Overall, gene expression profiling in mab-5 mutants provided insight into MAB-5 regulation of the transcriptomic response of C. elegans to pathogenic bacteria and helps us to understand mechanisms of innate immune activation and the role that transcriptional regulation plays in organismal health.

Familial Alzheimer mutations stabilize synaptotoxic γ-secretase-substrate complexes.

Mutations that cause familial Alzheimer's disease (FAD) are found in amyloid precursor protein (APP) and presenilin, the catalytic component of γ-secretase, that together produce amyloid ß-peptide (Aß). Nevertheless, whether Aß is the primary disease driver remains controversial. We report here that FAD mutations disrupt initial proteolytic events in the multistep processing of APP substrate C99 by γ-secretase. Cryoelectron microscopy reveals that a substrate mimetic traps γ-secretase during the transition state, and this structure aligns with activated enzyme-substrate complex captured by molecular dynamics simulations. In silico simulations and in cellulo fluorescence microscopy support stabilization of enzyme-substrate complexes by FAD mutations. Neuronal expression of C99 and/or presenilin-1 in Caenorhabditis elegans leads to synaptic loss only with FAD-mutant transgenes. Designed mutations that stabilize the enzyme-substrate complex and block Aß production likewise led to synaptic loss. Collectively, these findings implicate the stalled process-not the products-of γ-secretase cleavage of substrates in FAD pathogenesis.

Optical trapping of sub-millimeter sized particles and microorganisms.

While optical tweezers (OT) are mostly used for confining smaller size particles, the counter-propagating (CP) dual-beam traps have been a versatile method for confining both small and larger size particles including biological specimen. However, CP traps are complex sensitive systems, requiring tedious alignment to achieve perfect symmetry with rather low trapping stiffness values compared to OT. Moreover, due to their relatively weak forces, CP traps are limited in the size of particles they can confine which is about 100 µm. In this paper, a new class of counter-propagating optical tweezers with a broken symmetry is discussed and experimentally demonstrated to trap and manipulate larger than 100 µm particles inside liquid media. Our technique exploits a single Gaussian beam folding back on itself in an asymmetrical fashion forming a CP trap capable of confining small and significantly larger particles (up to 250 µm in diameter) based on optical forces only. Such optical trapping of large-size specimen to the best of our knowledge has not been demonstrated before. The broken symmetry of the trap combined with the retro-reflection of the beam has not only significantly simplified the alignment of the system, but also made it robust to slight misalignments and enhances the trapping stiffness as shown later. Moreover, our proposed trapping method is quite versatile as it allows for trapping and translating of a wide variety of particle sizes and shapes, ranging from one micron up to a few hundred of microns including microorganisms, using very low laser powers and numerical aperture optics. This in turn, permits the integration of a wide range of spectroscopy techniques for imaging and studying the optically trapped specimen. As an example, we will demonstrate how this novel technique enables simultaneous 3D trapping and light-sheet microscopy of C. elegans worms with up to 450 µm length.

Age-dependent accumulation of tau aggregation in Caenorhabditis elegans.

Aging is the primary risk factor for Alzheimer's disease (AD) and related disorders (ADRDs). Tau aggregation is a hallmark of AD and other tauopathies. Even in normal aging, tau aggregation is found in brains, but in disease states, significantly more aggregated tau is present in brain regions demonstrating synaptic degeneration and neuronal loss. It is unclear how tau aggregation and aging interact to give rise to the phenotypes observed in disease states. Most AD/ADRD animal models have focused on late stages, after significant tau aggregation has occurred. There are fewer where we can observe the early aggregation events and progression during aging. In an attempt to address this gap, we created C. elegans models expressing a GFP-tagged version of the human tau protein. Here we examined how tau-gfp behaved during aging, comparing wild-type tau (hTau40), a disease-associated mutation (P301S), and an aggregation-prone variant (3PO). We measured age-dependent changes in GFP intensity and correlated those changes to normal aging in the nematode. We found differences in tau stability and accumulation depending on the tau variant expressed. hTau40GFP and P301SGFP were localized to axons and cell bodies, while 3POGFP was more concentrated within cell bodies. Expression of 3POGFP resulted in decreased lifespan and variations in locomotor rate, consistent with a pathological effect. Finally, we found that the human tau interacted genetically with the C. elegans ortholog of human tau, ptl-1, where the loss of ptl-1 significantly accelerated the time to death in animals expressing 3PO.

Distinct roles for two Caenorhabditis elegans acid-sensing ion channels in an ultradian clock.

Biological clocks are fundamental to an organism's health, controlling periodicity of behaviour and metabolism. Here, we identify two acid-sensing ion channels, with very different proton sensing properties, and describe their role in an ultradian clock, the defecation motor program (DMP) of the nematode Caenorhabditis elegans. An ACD-5-containing channel, on the apical membrane of the intestinal epithelium, is essential for maintenance of luminal acidity, and thus the rhythmic oscillations in lumen pH. In contrast, the second channel, composed of FLR-1, ACD-3 and/or DEL-5, located on the basolateral membrane, controls the intracellular Ca2+ wave and forms a core component of the master oscillator that controls the timing and rhythmicity of the DMP. flr-1 and acd-3/del-5 mutants show severe developmental and metabolic defects. We thus directly link the proton-sensing properties of these channels to their physiological roles in pH regulation and Ca2+ signalling, the generation of an ultradian oscillator, and its metabolic consequences.

Biological clocks regulate a myriad of processes that occur periodically, from sleeping and waking to how cells use nutrients and energy. One such clock is the one that controls intestinal movements and defecation in the nematode worm Caenorhabditis elegans, which consists of three muscle contractions occurring every 50 seconds. This rhythm is controlled by calcium and proton signalling in the cells of the intestine. The cells of the nematode intestine form a tube, through which gut contents pass. The inside of the tube is acidic, but acidity also plays a role on the outer face of the intestinal tube. In this area, nutrients are distributed and signals are conveyed to other tissues, such as muscles. In fact, acid ­ in the form of protons ­ secreted from the intestinal cells stimulates the muscles that contract in the biological clock that controls the worms' defecation. However, it is poorly understood how the worms control the release of these protons. Kaulich et al. identified two ion channels on the membranes of intestinal cells that become inhibited when the levels of acid surrounding them are high. These channels play distinct roles in controlling the contractions that move the contents of the roundworms' intestines along. The first channel contains a protein called ACD-5, and it is in the membrane of the intestinal cells that faces the inside of the intestinal tube. The second channel is formed by three proteins: FLR-1, ACD-3 and DEL-5. This channel is found on the other side of the intestinal cells, the region where nutrients are distributed and signals are conveyed to the rest of the body. To determine the role of each channel, Kaulich et al. genetically engineered the worms so they would not make the proteins that make up the channels, and imaged the live nematodes to see the effects of removing each channel. The inside of the intestines of worms lacking the ACD-5 containing channel was less acidic than that of normal worms, and the timing of the contractions that control defecation was also slightly altered. Removing the second channel (the one formed by three different proteins), however, had more dramatic effects: the worms were thin, developed more slowly, had less fat tissue and defecated very irregularly. Kaulich et al. imaged live worms to show that the second channel plays a major role in regulating oscillations in acidity both inside and outside cells, as well as controlling calcium levels. This demonstrates that this channel is responsible for the rhythmicity in the contractions that control defecation in the nematodes. Their findings provide important insights towards better understanding proton signalling and the role of acid-sensing ion channels in cellular contexts and biological clocks.

Wild-type Caenorhabditis elegans isolates exhibit distinct gene expression profiles in response to microbial infection.

The soil-dwelling nematode Caenorhabditis elegans serves as a model system to study innate immunity against microbial pathogens. C. elegans have been collected from around the world, where they, presumably, adapted to regional microbial ecologies. Here we use survival assays and RNA-sequencing to better understand how two isolates from disparate climates respond to pathogenic bacteria. We found that, relative to N2 (originally isolated in Bristol, UK), CB4856 (isolated in Hawaii), was more susceptible to the Gram-positive microbe, Staphylococcus epidermidis, but equally susceptible to Staphylococcus aureus as well as two Gram-negative microbes, Providencia rettgeri and Pseudomonas aeruginosa. We performed transcriptome analysis of infected worms and found gene-expression profiles were considerably different in an isolate-specific and microbe-specific manner. We performed GO term analysis to categorize differential gene expression in response to S. epidermidis. In N2, genes that encoded detoxification enzymes and extracellular matrix proteins were significantly enriched, while in CB4856, genes that encoded detoxification enzymes, C-type lectins, and lipid metabolism proteins were enriched, suggesting they have different responses to S. epidermidis, despite being the same species. Overall, discerning gene expression signatures in an isolate by pathogen manner can help us to understand the different possibilities for the evolution of immune responses within organisms.

Draft Genome Sequence of Novel Staphylococcus epidermidis Strain EVL2000, Exhibiting Pathogenicity against Caenorhabditis elegans.

Staphylococcus epidermidis is a frequent cause of nosocomial infections occurring after the insertion of indwelling medical devices. Here, we report the 2.5-Mb draft genome of S. epidermidis strain EVL2000, which was identified during an examination of nematode susceptibility to microbial pathogens.

The Immunoglobulin Superfamily Members syg-2 and syg-1 Regulate Neurite Development in C. elegans.

Neurons form elaborate networks by guiding axons and dendrites to appropriate destinations. Neurites require information about the relative body axes during the initial projection from the cell body, and failure to receive or interpret those cues correctly can result in outgrowth errors. We identified a mutation in the Ig superfamily member syg-2 in a screen for animals with anterior/posterior (A/P) axon guidance defects. We found that syg-2 and its cognate Ig family member syg-1 appear to function in a linear genetic pathway to control the outgrowth of GABAergic axons. We determined that this pathway works in parallel to Wnt signaling. Specifically, mutations in syg-2 or syg-1 selectively affected the embryonically derived Dorsal D-type (DD) GABAergic neurons. We found no evidence that these mutations affected the Ventral D-type neurons (VD) that form later, during the first larval stage. In addition, mutations in syg-1 or syg-2 could result in the DD neurons forming multiple processes, becoming bipolar, rather than the expected pseudounipolar morphology. Given SYG-2's essential function in synaptogenesis of the hermaphrodite-specific neurons (HSNs), we also examined DD neuron synapses in syg-2 mutants. We found syg-2 mutants had a decreased number of synapses formed, but synaptic morphology was largely normal. These results provide further evidence that the GABAergic motorneurons use multiple guidance pathways during development.

Terminal web and vesicle trafficking proteins mediate nematode single-cell tubulogenesis.

Single-celled tubules represent a complicated structure that forms during development, requiring extension of a narrow cytoplasm surrounding a lumen exerting osmotic pressure that can burst the luminal membrane. Genetic studies on the excretory canal cell of Caenorhabditis elegans have revealed many proteins that regulate the cytoskeleton, vesicular transport, and physiology of the narrow canals. Here, we show that ßH-spectrin regulates the placement of intermediate filament proteins forming a terminal web around the lumen, and that the terminal web in turn retains a highly conserved protein (EXC-9/CRIP1) that regulates apical endosomal trafficking. EXC-1/IRG, the binding partner of EXC-9, is also localized to the apical membrane and affects apical actin placement and RAB-8-mediated vesicular transport. The results suggest that an intermediate filament protein acts in a novel pathway to direct the traffic of vesicles to locations of lengthening apical surface during single-celled tubule development.

The Hox Gene egl-5 Acts as a Terminal Selector for VD13 Development via Wnt Signaling.

Nervous systems are comprised of diverse cell types that differ functionally and morphologically. During development, extrinsic signals, e.g., growth factors, can activate intrinsic programs, usually orchestrated by networks of transcription factors. Within that network, transcription factors that drive the specification of features specific to a limited number of cells are often referred to as terminal selectors. While we still have an incomplete view of how individual neurons within organisms become specified, reporters limited to a subset of neurons in a nervous system can facilitate the discovery of cell specification programs. We have identified a fluorescent reporter that labels VD13, the most posterior of the 19 inhibitory GABA (γ-amino butyric acid)-ergic motorneurons, and two additional neurons, LUAL and LUAR. Loss of function in multiple Wnt signaling genes resulted in an incompletely penetrant loss of the marker, selectively in VD13, but not the LUAs, even though other aspects of GABAergic specification in VD13 were normal. The posterior Hox gene, egl-5, was necessary for expression of our marker in VD13, and ectopic expression of egl-5 in more anterior GABAergic neurons induced expression of the marker. These results suggest egl-5 is a terminal selector of VD13, subsequent to GABAergic specification.

Vitamin B12 Regulates Glial Migration and Synapse Formation through Isoform-Specific Control of PTP-3/LAR PRTP Expression.

Vitamin B12 is known to play critical roles during the development and aging of the brain, and vitamin B12 deficiency has been linked to neurodevelopmental and degenerative disorders. However, the underlying molecular mechanisms of how vitamin B12 affects the development and maintenance of the nervous system are still unclear. Here, we report that vitamin B12 can regulate glial migration and synapse formation through control of isoform-specific expression of PTP-3/LAR PRTP (leukocyte-common antigen-related receptor-type tyrosine-protein phosphatase). We found the uptake of diet-supplied vitamin B12 in the intestine to be critical for the expression of a long isoform of PTP-3 (PTP-3A) in neuronal and glial cells. The expression of PTP-3A cell autonomously regulates glial migration and synapse formation through interaction with an extracellular matrix protein NID-1/nidogen 1. Together, our findings demonstrate that isoform-specific regulation of PTP-3/ LAR PRTP expression is a key molecular mechanism that mediates vitamin-B12-dependent neuronal and glial development.

The C. elegans CHP1 homolog, pbo-1, functions in innate immunity by regulating the pH of the intestinal lumen.

Caenorhabditis elegans are soil-dwelling nematodes and models for understanding innate immunity and infection. Previously, we developed a novel fluorescent dye (KR35) that accumulates in the intestine of C. elegans and reports a dynamic wave in intestinal pH associated with the defecation motor program. Here, we use KR35 to show that mutations in the Ca2+-binding protein, PBO-1, abrogate the pH wave, causing the anterior intestine to be constantly acidic. Surprisingly, pbo-1 mutants were also more susceptible to infection by several bacterial pathogens. We could suppress pathogen susceptibility in pbo-1 mutants by treating the animals with pH-buffering bicarbonate, suggesting the pathogen susceptibility is a function of the acidity of the intestinal pH. Furthermore, we use KR35 to show that upon infection by pathogens, the intestinal pH becomes neutral in a wild type, but less so in pbo-1 mutants. C. elegans is known to increase production of reactive oxygen species (ROS), such as H2O2, in response to pathogens, which is an important component of pathogen defense. We show that pbo-1 mutants exhibited decreased H2O2 in response to pathogens, which could also be partially restored in pbo-1 animals treated with bicarbonate. Ultimately, our results support a model whereby PBO-1 functions during infection to facilitate pH changes in the intestine that are protective to the host.

Behavior: Should I Stay or Should I Go?

Laboratory-conditioned 'wild-type' Caenorhabditis elegans are different from wild-isolated strains. Now it seems some differences lead lab-conditioned animals to act irrationally after experiencing starvation early in development. This increases the repertoire of behaviors we can study with these fascinating animals.

Tubular Excretory Canal Structure Depends on Intermediate Filaments EXC-2 and IFA-4 in Caenorhabditis elegans.

The excretory canals of Caenorhabditis elegans are a model for understanding the maintenance of apical morphology in narrow single-celled tubes. Light and electron microscopy shows that mutants in exc-2 start to form canals normally, but these swell to develop large fluid-filled cysts that lack a complete terminal web at the apical surface, and accumulate filamentous material in the canal lumen. Here, whole-genome sequencing and gene rescue show that exc-2 encodes intermediate filament protein IFC-2 EXC-2/IFC-2 protein, fluorescently tagged via clustered regularly interspaced short palindromic repeats/Cas9, is located at the apical surface of the canals independently of other intermediate filament proteins. EXC-2 is also located in several other tissues, though the tagged isoforms are not seen in the larger intestinal tube. Tagged EXC-2 binds via pulldown to intermediate filament protein IFA-4, which is also shown to line the canal apical surface. Overexpression of either protein results in narrow but shortened canals. These results are consistent with a model whereby three intermediate filaments in the canals-EXC-2, IFA-4, and IFB-1-restrain swelling of narrow tubules in concert with actin filaments that guide the extension and direction of tubule outgrowth, while allowing the tube to bend as the animal moves.

Four specific immunoglobulin domains in UNC-52/Perlecan function with NID-1/Nidogen during dendrite morphogenesis in Caenorhabditis elegans.

The extracellular matrix is essential for various aspects of nervous system patterning. For example, sensory dendrites in flies, worms and fish have been shown to rely on coordinated interactions of tissues with extracellular matrix proteins. Here we show that the conserved basement membrane protein UNC-52/Perlecan is required for establishing the correct number of the highly ordered dendritic trees in the somatosensory neuron PVD in Caenorhabditis elegans This function is dependent on four specific immunoglobulin domains, but independent of the known functions of UNC-52 in mediating muscle-skin attachment. Intriguingly, the four conserved immunoglobulin domains in UNC-52 are necessary to correctly localize the basement membrane protein NID-1/Nidogen. Genetic experiments further show that unc-52, nid-1 and genes of the netrin axon guidance signaling cassette share a common pathway to establish the correct number of somatosensory dendrites. Our studies suggest that, in addition to its role in mediating muscle-skin attachment, UNC-52 functions through immunoglobulin domains to establish an ordered lattice of basement membrane proteins, which may control the function of morphogens during dendrite patterning.

Facilitation of Endosomal Recycling by an IRG Protein Homolog Maintains Apical Tubule Structure in Caenorhabditis elegans.

Determination of luminal diameter is critical to the function of small single-celled tubes. A series of EXC proteins, including EXC-1, prevent swelling of the tubular excretory canals in Caenorhabditis elegans In this study, cloning of exc-1 reveals it to encode a homolog of mammalian IRG proteins, which play roles in immune response and autophagy and are associated with Crohn's disease. Mutants in exc-1 accumulate early endosomes, lack recycling endosomes, and exhibit abnormal apical cytoskeletal structure in regions of enlarged tubules. EXC-1 interacts genetically with two other EXC proteins that also affect endosomal trafficking. In yeast two-hybrid assays, wild-type and putative constitutively active EXC-1 binds to the LIM-domain protein EXC-9, whose homolog, cysteine-rich intestinal protein, is enriched in mammalian intestine. These results suggest a model for IRG function in forming and maintaining apical tubule structure via regulation of endosomal recycling.

The Caenorhabditis elegans Ephrin EFN-4 Functions Non-cell Autonomously with Heparan Sulfate Proteoglycans to Promote Axon Outgrowth and Branching.

The Eph receptors and their cognate ephrin ligands play key roles in many aspects of nervous system development. These interactions typically occur within an individual tissue type, serving either to guide axons to their terminal targets or to define boundaries between the rhombomeres of the hindbrain. We have identified a novel role for the Caenorhabditis elegans ephrin EFN-4 in promoting primary neurite outgrowth in AIY interneurons and D-class motor neurons. Rescue experiments reveal that EFN-4 functions non-cell autonomously in the epidermis to promote primary neurite outgrowth. We also find that EFN-4 plays a role in promoting ectopic axon branching in a C. elegans model of X-linked Kallmann syndrome. In this context, EFN-4 functions non-cell autonomously in the body-wall muscle and in parallel with HS modification genes and HSPG core proteins. This is the first report of an epidermal ephrin providing a developmental cue to the nervous system.

A Synthetic Lethal Screen Identifies a Role for Lin-44/Wnt in C. elegans Embryogenesis.

BACKGROUND: The C. elegans proteins PTP-3/LAR-RPTP and SDN-1/Syndecan are conserved cell adhesion molecules. Loss-of-function (LOF) mutations in either ptp-3 or sdn-1 result in low penetrance embryonic developmental defects. Work from other systems has shown that syndecans can function as ligands for LAR receptors in vivo. We used double mutant analysis to test whether ptp-3 and sdn-1 function in a linear genetic pathway during C. elegans embryogenesis. RESULTS: We found animals with LOF in both sdn-1 and ptp-3 exhibited a highly penetrant synthetic lethality (SynLet), with only a small percentage of animals surviving to adulthood. Analysis of the survivors demonstrated that these animals had a synergistic increase in the penetrance of embryonic developmental defects. Together, these data strongly suggested PTP-3 and SDN-1 function in parallel during embryogenesis. We subsequently used RNAi to knockdown ~3,600 genes predicted to encode secreted and/or transmembrane molecules to identify genes that interacted with ptp-3 or sdn-1. We found that the Wnt ligand, lin-44, was SynLet with sdn-1, but not ptp-3. We used 4-dimensional time-lapse analysis to characterize the interaction between lin-44 and sdn-1. We found evidence that loss of lin-44 caused defects in the polarization and migration of endodermal precursors during gastrulation, a previously undescribed role for lin-44 that is strongly enhanced by the loss of sdn-1. CONCLUSIONS: PTP-3 and SDN-1 function in compensatory pathways during C. elegans embryonic and larval development, as simultaneous loss of both genes has dire consequences for organismal survival. The Wnt ligand lin-44 contributes to the early stages of gastrulation in parallel to sdn-1, but in a genetic pathway with ptp-3. Overall, the SynLet phenotype provides a robust platform to identify ptp-3 and sdn-1 interacting genes, as well as other genes that function in development, yet might be missed in traditional forward genetic screens.

The fat-like cadherin CDH-4 acts cell-non-autonomously in anterior-posterior neuroblast migration.

Directed migration of neurons is critical in the normal and pathological development of the brain and central nervous system. In Caenorhabditis elegans, the bilateral Q neuroblasts, QR on the right and QL on the left, migrate anteriorly and posteriorly, respectively. Initial protrusion and migration of the Q neuroblasts is autonomously controlled by the transmembrane proteins UNC-40/DCC, PTP-3/LAR, and MIG-21. As QL migrates posteriorly, it encounters and EGL-20/Wnt signal that induces MAB-5/Hox expression that drives QL descendant posterior migration. QR migrates anteriorly away from EGL-20/Wnt and does not activate MAB-5/Hox, resulting in anterior QR descendant migration. A forward genetic screen for new mutations affecting initial Q migrations identified alleles of cdh-4, which caused defects in both QL and QR directional migration similar to unc-40, ptp-3, and mig-21. Previous studies showed that in QL, PTP-3/LAR and MIG-21 act in a pathway in parallel to UNC-40/DCC to drive posterior QL migration. Here we show genetic evidence that CDH-4 acts in the PTP-3/MIG-21 pathway in parallel to UNC-40/DCC to direct posterior QL migration. In QR, the PTP-3/MIG-21 and UNC-40/DCC pathways mutually inhibit each other, allowing anterior QR migration. We report here that CDH-4 acts in both the PTP-3/MIG-21 and UNC-40/DCC pathways in mutual inhibition in QR, and that CDH-4 acts cell-non-autonomously. Interaction of CDH-4 with UNC-40/DCC in QR but not QL represents an inherent left-right asymmetry in the Q cells, the nature of which is not understood. We conclude that CDH-4 might act as a permissive signal for each Q neuroblast to respond differently to anterior-posterior guidance information based upon inherent left-right asymmetries in the Q neuroblasts.