A mutation in F-actin polymerization factor suppresses the distal arthrogryposis type 5 PIEZO2 pathogenic variant in Caenorhabditis elegans.

The mechanosensitive PIEZO channel family has been linked to over 26 disorders and diseases. Although progress has been made in understanding these channels at the structural and functional levels, the underlying mechanisms of PIEZO-associated diseases remain elusive. In this study, we engineered four PIEZO-based disease models using CRISPR/Cas9 gene editing. We performed an unbiased chemical mutagen-based genetic suppressor screen to identify putative suppressors of a conserved gain-of-function variant pezo-1[R2405P] that in human PIEZO2 causes distal arthrogryposis type 5 (DA5; p. R2718P). Electrophysiological analyses indicate that pezo-1(R2405P) is a gain-of-function allele. Using genomic mapping and whole-genome sequencing approaches, we identified a candidate suppressor allele in the C. elegans gene gex-3. This gene is an ortholog of human NCKAP1 (NCK-associated protein 1), a subunit of the Wiskott-Aldrich syndrome protein (WASP)-verprolin homologous protein (WAVE/SCAR) complex, which regulates F-actin polymerization. Depletion of gex-3 by RNAi, or with the suppressor allele gex-3(av259[L353F]), significantly increased brood size and ovulation rate, as well as alleviating the crushed oocyte phenotype of the pezo-1(R2405P) mutant. Expression of GEX-3 in the soma is required to rescue the brood size defects in pezo-1(R2405P) animals. Actin organization and orientation were disrupted and distorted in the pezo-1 mutants. Mutation of gex-3(L353F) partially alleviated these defects. The identification of gex-3 as a suppressor of the pathogenic variant pezo-1(R2405P) suggests that the PIEZO coordinates with the cytoskeleton regulator to maintain the F-actin network and provides insight into the molecular mechanisms of DA5 and other PIEZO-associated diseases.

Allele-specific mitochondrial stress induced by Multiple Mitochondrial Dysfunctions Syndrome 1 pathogenic mutations modeled in Caenorhabditis elegans.

Multiple Mitochondrial Dysfunctions Syndrome 1 (MMDS1) is a rare, autosomal recessive disorder caused by mutations in the NFU1 gene. NFU1 is responsible for delivery of iron-sulfur clusters (ISCs) to recipient proteins which require these metallic cofactors for their function. Pathogenic variants of NFU1 lead to dysfunction of its target proteins within mitochondria. To date, 20 NFU1 variants have been reported and the unique contributions of each variant to MMDS1 pathogenesis is unknown. Given that over half of MMDS1 individuals are compound heterozygous for different NFU1 variants, it is valuable to investigate individual variants in an isogenic background. In order to understand the shared and unique phenotypes of NFU1 variants, we used CRISPR/Cas9 gene editing to recreate exact patient variants of NFU1 in the orthologous gene, nfu-1 (formerly lpd-8), in C. elegans. Five mutant C. elegans alleles focused on the presumptive iron-sulfur cluster interaction domain were generated and analyzed for mitochondrial phenotypes including respiratory dysfunction and oxidative stress. Phenotypes were variable between the mutant nfu-1 alleles and generally presented as an allelic series indicating that not all variants have lost complete function. Furthermore, reactive iron within mitochondria was evident in some, but not all, nfu-1 mutants indicating that iron dyshomeostasis may contribute to disease pathogenesis in some MMDS1 individuals.

A complement factor H homolog, heparan sulfation, and syndecan maintain inversin compartment boundaries in C. elegans cilia.

Age-related macular degeneration (AMD) is a leading cause of blindness among the elderly. Canonical disease models suggest that defective interactions between complement factor H (CFH) and cell surface heparan sulfate (HS) result in increased alternative complement pathway activity, cytolytic damage, and tissue inflammation in the retina. Although these factors are thought to contribute to increased disease risk, multiple studies indicate that noncanonical mechanisms that result from defective CFH and HS interaction may contribute to the progression of AMD as well. A total of 60 ciliated sensory neurons in the nematode Caenorhabditis elegans detect chemical, olfactory, mechanical, and thermal cues in the environment. Here, we find that a C. elegans CFH homolog localizes on CEP mechanosensory neuron cilia where it has noncanonical roles in maintaining inversin/NPHP-2 within its namesake proximal compartment and preventing inversin/NPHP-2 accumulation in distal cilia compartments in aging adults. CFH localization and maintenance of inversin/NPHP-2 compartment integrity depend on the HS 3-O sulfotransferase HST-3.1 and the transmembrane proteoglycan syndecan/SDN-1. Defective inversin/NPHP-2 localization in mouse and human photoreceptors with CFH mutations indicates that these functions and interactions may be conserved in vertebrate sensory neurons, suggesting that previously unappreciated defects in cilia structure may contribute to the progressive photoreceptor dysfunction associated with CFH loss-of-function mutations in some AMD patients.

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.

Mutation of NEKL-4/NEK10 and TTLL genes suppress neuronal ciliary degeneration caused by loss of CCPP-1 deglutamylase function.

Ciliary microtubules are subject to post-translational modifications that act as a "Tubulin Code" to regulate motor traffic, binding proteins and stability. In humans, loss of CCP1, a cytosolic carboxypeptidase and tubulin deglutamylating enzyme, causes infantile-onset neurodegeneration. In C. elegans, mutations in ccpp-1, the homolog of CCP1, result in progressive degeneration of neuronal cilia and loss of neuronal function. To identify genes that regulate microtubule glutamylation and ciliary integrity, we performed a forward genetic screen for suppressors of ciliary degeneration in ccpp-1 mutants. We isolated the ttll-5(my38) suppressor, a mutation in a tubulin tyrosine ligase-like glutamylase gene. We show that mutation in the ttll-4, ttll-5, or ttll-11 gene suppressed the hyperglutamylation-induced loss of ciliary dye filling and kinesin-2 mislocalization in ccpp-1 cilia. We also identified the nekl-4(my31) suppressor, an allele affecting the NIMA (Never in Mitosis A)-related kinase NEKL-4/NEK10. In humans, NEK10 mutation causes bronchiectasis, an airway and mucociliary transport disorder caused by defective motile cilia. C. elegans NEKL-4 localizes to the ciliary base but does not localize to cilia, suggesting an indirect role in ciliary processes. This work defines a pathway in which glutamylation, a component of the Tubulin Code, is written by TTLL-4, TTLL-5, and TTLL-11; is erased by CCPP-1; is read by ciliary kinesins; and its downstream effects are modulated by NEKL-4 activity. Identification of regulators of microtubule glutamylation in diverse cellular contexts is important to the development of effective therapies for disorders characterized by changes in microtubule glutamylation. By identifying C. elegans genes important for neuronal and ciliary stability, our work may inform research into the roles of the tubulin code in human ciliopathies and neurodegenerative diseases.

Localized TWIST1 and TWIST2 basic domain substitutions cause four distinct human diseases that can be modeled in Caenorhabditis elegans.

Twist transcription factors, members of the basic helix-loop-helix family, play crucial roles in mesoderm development in all animals. Humans have two paralogous genes, TWIST1 and TWIST2, and mutations in each gene have been identified in specific craniofacial disorders. Here, we describe a new clinical entity, Sweeney-Cox syndrome, associated with distinct de novo amino acid substitutions (p.Glu117Val and p.Glu117Gly) at a highly conserved glutamic acid residue located in the basic DNA binding domain of TWIST1, in two subjects with frontonasal dysplasia and additional malformations. Although about one hundred different TWIST1 mutations have been reported in patients with the dominant haploinsufficiency Saethre-Chotzen syndrome (typically associated with craniosynostosis), substitutions uniquely affecting the Glu117 codon were not observed previously. Recently, subjects with Barber-Say and Ablepharon-Macrostomia syndromes were found to harbor heterozygous missense substitutions in the paralogous glutamic acid residue in TWIST2 (p.Glu75Ala, p.Glu75Gln and p.Glu75Lys). To study systematically the effects of these substitutions in individual cells of the developing mesoderm, we engineered all five disease-associated alleles into the equivalent Glu29 residue encoded by hlh-8, the single Twist homolog present in Caenorhabditis elegans. This allelic series revealed that different substitutions exhibit graded severity, in terms of both gene expression and cellular phenotype, which we incorporate into a model explaining the various human disease phenotypes. The genetic analysis favors a predominantly dominant-negative mechanism for the action of amino acid substitutions at this highly conserved glutamic acid residue and illustrates the value of systematic mutagenesis of C. elegans for focused investigation of human disease processes.

Novel functions for the RNA-binding protein ETR-1 in Caenorhabditis elegans reproduction and engulfment of germline apoptotic cell corpses.

RNA-binding proteins (RBPs) are essential regulators of gene expression that act through a variety of mechanisms to ensure the proper post-transcriptional regulation of their target RNAs. RBPs in multiple species have been identified as playing crucial roles during development and as having important functions in various adult organ systems, including the heart, nervous, muscle, and reproductive systems. ETR-1, a highly conserved ELAV-Type RNA-binding protein belonging to the CELF/Bruno protein family, has been previously reported to be involved in C. elegans muscle development. Animals depleted of ETR-1 have been previously characterized as arresting at the two-fold stage of embryogenesis. In this study, we show that ETR-1 is expressed in the hermaphrodite somatic gonad and germ line, and that reduction of ETR-1 via RNA interference (RNAi) results in reduced hermaphrodite fecundity. Detailed characterization of this fertility defect indicates that ETR-1 is required in both the somatic tissue and the germ line to ensure wild-type reproductive levels. Additionally, the ability of ETR-1 depletion to suppress the published WEE-1.3-depletion infertility phenotype is dependent on ETR-1 being reduced in the soma. Within the germline of etr-1(RNAi) hermaphrodite animals, we observe a decrease in average oocyte size and an increase in the number of germline apoptotic cell corpses as evident by an increased number of CED-1::GFP and acridine orange positive apoptotic germ cells. Transmission Electron Microscopy (TEM) studies confirm the significant increase in apoptotic cells in ETR-1-depleted animals, and reveal a failure of the somatic gonadal sheath cells to properly engulf dying germ cells in etr-1(RNAi) animals. Through investigation of an established engulfment pathway in C. elegans, we demonstrate that co-depletion of CED-1 and ETR-1 suppresses both the reduced fecundity and the increase in the number of apoptotic cell corpses observed in etr-1(RNAi) animals. Combined, this data identifies a novel role for ETR-1 in hermaphrodite gametogenesis and in the process of engulfment of germline apoptotic cell corpses.

From phenologs to silent suppressors: Identifying potential therapeutic targets for human disease.

Orthologous phenotypes, or phenologs, are seemingly unrelated phenotypes generated by mutations in a conserved set of genes. Phenologs have been widely observed and accepted by those who study model organisms, and allow one to study a set of genes in a model organism to learn more about the function of those genes in other organisms, including humans. At the cellular and molecular level, these conserved genes likely function in a very similar mode, but are doing so in different tissues or cell types and can result in different phenotypic effects. For example, the RAS-RAF-MEK-MAPK pathway in animals is a highly conserved signaling pathway that animals adopted for numerous biological processes, such as vulval induction in Caenorhabditis elegans and cell proliferation in mammalian cells; but this same gene set has been co-opted to function in a variety of cellular contexts. In this review, I give a few examples of how suppressor screens in model organisms (with a emphasis on C. elegans) can identify new genes that function in a conserved pathway in many other organisms. I also demonstrate how the identification of such genes can lead to important insights into mammalian biology. From such screens, an occasional silent suppressor that does not cause a phenotype on its own is found; such suppressors thus make for good candidates as therapeutic targets.

Identification of genetic suppressors for a BSCL2 lipodystrophy pathogenic variant in Caenorhabditis elegans.

Seipin (BSCL2), a conserved endoplasmic reticulum protein, plays a critical role in lipid droplet (LD) biogenesis and in regulating LD morphology, pathogenic variants of which are associated with Berardinelli-Seip congenital generalized lipodystrophy type 2 (BSCL2). To model BSCL2 disease, we generated an orthologous BSCL2 variant, seip-1(A185P), in Caenorhabditis elegans. In this study, we conducted an unbiased chemical mutagenesis screen to identify genetic suppressors that restore embryonic viability in the seip-1(A185P) mutant background. A total of five suppressor lines were isolated and recovered from the screen. The defective phenotypes of seip-1(A185P), including embryonic lethality and impaired eggshell formation, were significantly suppressed in each suppressor line. Two of the five suppressor lines also alleviated the enlarged LDs in the oocytes. We then mapped a suppressor candidate gene, lmbr-1, which is an ortholog of human limb development membrane protein 1 (LMBR1). The CRISPR/Cas9 edited lmbr-1 suppressor alleles, lmbr-1(S647F) and lmbr-1(P314L), both significantly suppressed embryonic lethality and defective eggshell formation in the seip-1(A185P) background. The newly identified suppressor lines offer valuable insights into potential genetic interactors and pathways that may regulate seipin in the lipodystrophy model.

International Cohort of Neonatal Timothy Syndrome.

INTRODUCTION: Timothy syndrome (TS) is an extremely rare, multisystem disorder classically associated with long QT, syndactyly, ventricular arrhythmias, and hypoglycaemia. A neonatal diagnosis allows maximal medical and device therapy to be implemented to avoid malignant arrhythmias and sudden cardiac death. METHODS: This was a retrospective case series study of type I TS (TS1) patients using data from the Timothy Syndrome Foundation's international registry, encompassing patients with a genetic diagnosis (CACNA1C variant G406R in exon 8A) recruited over a 28-year period. RESULTS: Forty-four cases of TS1 were included (26 male; 60%). Mean gestational age (GA) was 35.6 weeks (range 28 weeks - term), with 43% of patients born less than 37 weeks GA. In TS1 patients presenting with foetal bradycardia, mean GA was significantly lower (34.2 weeks, p < 0.05). Foetal bradycardia secondary to atrioventricular block was present in 20 patients (45%), resulting in premature delivery in 14 patients (32%). Fifteen patients (34%) were diagnosed with TS1 as neonates. Long QT at birth helped secure a diagnosis in 25 patients (57%). Syndactyly was seen in most patients (n = 40, 91%). Twenty patients died, with an average age of death of 2.3 years (range 1 month-6 years). Of the 7 patients who died before the first year of life (16%), the average age of death was 2.5 months. CONCLUSION: TS is associated with high early mortality. TS should be considered in paediatric patients presenting with long QT and syndactyly. Recognition of TS in the neonatal period allows for early intervention to prevent life-threatening arrhythmias.

A Natural History Study of Timothy Syndrome.

Timothy syndrome (OMIM #601005) is a rare disease caused by variants in the gene CACNA1C . Timothy syndrome patients were first identified as having a cardiac presentation of Long QT and syndactyly of the fingers and/or toes, and an identical variant in CACNA1C , Gly406Arg. However, since this original identification, more individuals harboring diverse variants in CACNA1C have been identified and have presented with various cardiac and extra-cardiac symptoms. Furthermore, it has remained underexplored whether individuals harboring canonical Gly406Arg variants in mutually exclusive exon 8A (Timothy syndrome 1) or exon 8 (Timothy syndrome 2) have additional symptoms. Here, we describe the first Natural History Study for Timothy syndrome, providing a thorough resource describing the current understanding of disease manifestation in Timothy syndrome patients. Parents of Timothy syndrome children were queried regarding a wide-ranging set of symptoms and features via a survey. Importantly, we find that in addition to cardiac concerns, Timothy syndrome patients commonly share extra-cardiac features including neurodevelopmental impairments, hypoglycemia, and respiratory problems. Our work expands the current understanding of the disorder to better inform the care of Timothy syndrome patients.

Protein phosphatase 5 is a negative regulator of separase function during cortical granule exocytosis in C. elegans.

Mutations in the Caenorhabditis elegans separase gene, sep-1, are embryonic lethal. Newly fertilized mutant embryos have defects in polar body extrusion, fail to undergo cortical granule exocytosis, and subsequently fail to complete cytokinesis. Chromosome nondisjunction during the meiotic divisions is readily apparent after depletion of sep-1 by RNAi treatment, but much less so in hypomorphic mutant embryos. To identify factors that influence the activity of separase in cortical granule exocytosis and cytokinesis, we carried out a genetic suppressor screen. A mutation in the protein phosphatase 5 (pph-5) gene was identified as an extragenic suppressor of sep-1. This mutation suppressed the phenotypes of hypomorphic separase mutants but not RNAi depleted animals. Depletion of pph-5 caused no phenotypes on its own, but was effective in restoring localization of mutant separase to vesicles and suppressing cortical granule exocytosis and cytokinesis phenotypes. The identification of PPH-5 as a suppressor of separase suggests that a new phospho-regulatory pathway plays an important role in regulating anaphase functions of separase.

Evaluation of the Fluids Mixing Enclosure System for Life Science Experiments During a Commercial Caenorhabditis elegans Spaceflight Experiment.

The Student Spaceflight Experiments Program (SSEP) is a United States national science, technology, engineering, and mathematics initiative that aims to increase student interest in science by offering opportunities to perform spaceflight experiments. The experiment detailed here was selected and flown aboard the third SSEP mission and the first SSEP mission to the International Space Station (ISS). Caenorhabditis elegans is a small, transparent, self-fertilizing hermaphroditic roundworm that is commonly used in biological experiments both on Earth and in Low Earth Orbit. Past experiments have found decreased expression of mRNA for several genes whose expression can be controlled by the FOXO transcription factor DAF-16. We flew a daf-16 mutant and control worms to determine if the effects of spaceflight on C. elegans are mediated by DAF-16. The experiment used a Type Two Fluids Mixing Enclosure (FME), developed by Nanoracks LLC, and was delivered to the ISS aboard the SpaceX Dragon and returned aboard the Russian Soyuz. The short time interval between experiment selection and the flight rendered preflight experiment verification tests impossible. In addition, published research regarding the viability of the FME in life science experiments was not available. The experiment was therefore structured in such a way as to gather the needed data. Here we report that C. elegans can survive relatively short storage and activation in the FME but cannot produce viable populations for post-flight analysis on extended missions. The FME appears to support short-duration life science experiments, potentially on supply or crew exchange missions, but not on longer ISS expeditions. Additionally, the flown FME was not properly activated, reportedly due to a flaw in training procedures. We suggest that a modified transparent FME could prevent similar failures in future flight experiments.

Transmembrane protein 120A (TMEM-120A/TACAN) coordinates with PIEZO channel during Caenorhabditis elegans reproductive regulation.

Membrane protein TMEM120A (also known as TACAN) was presumed to be both a mechanically activated molecule and a lipid-modifying enzyme. TMEM120A has been identified as a negative regulator of the essential excitatory mechanosensitive protein PIEZO2. However, the extent to which TMEM120A mediates PIEZO2's activity during physiological processes remains largely unknown. In this study, we used the Caenorhabditis elegans reproductive tract to explore the functional contribution of tmem-120, the sole TMEM120A/B ortholog, and its genetic interaction with pezo-1 in vivo. tmem-120 was expressed throughout the C. elegans development, particularly in the germline, embryos, and spermatheca. A tmem-120 mutant with a full-length deletion (tmem-120Δ) displayed deformed germline, maternal sterility, and a reduced brood size. In vivo live imaging revealed that pinched zygotes were frequently observed in the uterus of tmem-120Δ mutant animals, suggesting damage during spermathecal contraction. We then employed the auxin-inducible degradation system to degrade TMEM-120 protein in all somatic tissues or the germline, both of which resulted in reduced brood sizes. These findings suggested that multiple inputs of tmem-120 from different tissues regulate reproduction. Lastly, the loss of tmem-120 alleviated the brood size reduction and defective sperm navigation behavior in the pezo-1Δ mutant. Overall, our findings reveal a role for tmem-120 in regulating reproductive physiology in C. elegans, and suggest an epistatic interaction between pezo-1 and tmem-120 when governing proper reproduction.

copa-1 mutants experience heightened endoplasmic reticulum stress sensitivity in a C. elegans COPA Syndrome model.

COPA Syndrome is a rare, autosomal dominant autoimmune/autoinflammatory disease caused by mutations in COPA , which codes for the alpha subunit of the Coat Protein Complex I (COPI). COPI coated vesicles move proteins in retrograde from the Golgi Apparatus to the Endoplasmic Reticulum. At the cellular level, COPA mutations cause ER stress, though the downstream genetic mechanisms of COPA Syndrome remain undefined. Here, we model COPA Syndrome in Caenorhabditis elegans , using CRISPR/Cas9 to generate patient alleles in copa-1 , the C. elegans COPA ortholog. The two alleles made thus far are superficially wild type under normal growth conditions. However, these animals demonstrate an increased ER stress sensitivity.

Identification of Genetic Suppressors for a Berardinelli-Seip Congenital Generalized Lipodystrophy Type 2 (BSCL2) Pathogenic Variant in C. elegans.

Maintaining the metabolic homeostasis of fatty acids is crucial for human health. Excess fatty acids are stored in lipid droplets (LDs), the primary energy reservoir that helps regulate fat and lipid homeostasis in nearly all cell types. Seipin (BSCL2), a conserved endoplasmic reticulum protein, plays a critical role in LD biogenesis and regulating LD morphology. Pathogenic variants of seipin are associated with multiple human genetic diseases, including Berardinelli-Seip Congenital Generalized Lipodystrophy Type 2 (BSCL2). However, the cellular and molecular mechanisms by which dysfunctional seipin leads to these diseases remain unclear. To model BSCL2 disease, we generated an orthologous BSCL2 pathogenic variant seip-1(A185P) using CRISPR/Cas9 genome editing in Caenorhabditis elegans . This variant led to severe developmental and cellular defects, including embryonic lethality, impaired eggshell formation, and abnormally enlarged LDs. We set out to identify genetic determinants that could suppress these defective phenotypes in the seip-1(A185P) mutant background. To this end, we conducted an unbiased chemical mutagenesis screen to identify genetic suppressors that restore embryonic viability in the seip-1(A185P) mutant background. A total of five suppressor lines were isolated and recovered from the screen. The defective phenotypes of seip-1(A185P) , including embryonic lethality and impaired eggshell formation, were significantly suppressed in each suppressor line. Two of the five suppressor lines also alleviated the enlarged LDs in the oocytes. We then mapped a suppressor candidate gene, R05D3.2 (renamed as lmbr-1 ), which is an ortholog of human LMBR1 (limb development membrane protein 1). The CRISPR/Cas9 edited lmbr-1 suppressor alleles, lmbr-1(Ser647Phe) and lmbr-1(Pro314Leu) , both significantly suppressed embryonic lethality and defective eggshell formation in the seip-1(A185P) background. The newly identified suppressor lines offer valuable insights into potential genetic interactors and pathways that may regulate seipin in the lipodystrophy model.

A missense mutation in the C. elegans src-2 tyrosine-protein kinase reduces brood size and enhances embryonic morphogenesis defects in src-1(RNAi) conditions.

Goldenhar Syndrome is a rare congenital disorder characterized by hemifacial microsomia. Although select mutations have been mapped for this disorder, the genetic etiologies in the majority of cases remain unknown. A recent clinical report of a Goldenhar Syndrome patient identified a homozygous missense mutation in FRK , a gene associated with various types of cancer. In this work, we precisely modeled the disease-associated missense mutation in the C. elegans FRK ortholog src-2 , using CRISPR/Cas9 gene editing, and investigated the physiological role of this mutation and the src-2 gene. In addition, we generated a conserved variant in src-1 ( FYN ortholog) to assess the functional redundancy of the conserved variant. The putative pathogenic variants src-1 (Val190Ile) or src-2 (Val170Ile) caused only subtle phenotypes, suggesting that these mutations alone are not sufficient to explain the facial deformities observed in the Goldenhar Syndrome patient. However, the src-2 (Val170Ile) mutant exhibited reduced brood size and moderately enhanced embryonic developmental phenotypes, including epidermal and neuronal patterning defects, in the src-1 (RNAi) condition, indicating that the src-2 (Val170Ile) locus could play a supportive role during developmental processes. Overall, however, these studies showed that src-1 /FYN is essential for regulating embryogenesis and morphogenesis, while src-2 /FRK is largely dispensable for normal embryonic development, suggesting FYN , not FRK , is the dominant non-receptor protein kinase during embryonic development in C. elegans .

Mutation in F-actin Polymerization Factor Suppresses Distal Arthrogryposis Type 5 (DA5) PIEZO2 Pathogenic Variant in Caenorhabditis elegans.

The mechanosensitive PIEZO channel family has been linked to over 26 disorders and diseases. Although progress has been made in understanding these channels at the structural and functional levels, the underlying mechanisms of PIEZO-associated diseases remain elusive. In this study, we engineered four PIEZO-based disease models using CRISPR/Cas9 gene editing. We performed an unbiased chemical mutagen-based genetic suppressor screen to identify putative suppressors of a conserved gain-of-function variant pezo-1[R2405P] that in human PIEZO2 causes distal arthrogryposis type 5 (DA5; p. R2718P). Electrophysiological analyses indicate that pezo-1(R2405P) is a gain-of-function allele. Using genomic mapping and whole genome sequencing approaches, we identified a candidate suppressor allele in the C. elegans gene gex-3. This gene is an ortholog of human NCKAP1 (NCK-associated protein 1), a subunit of the Wiskott-Aldrich syndrome protein (WASP)-verprolin homologous protein (WAVE/SCAR) complex, which regulates F-actin polymerization. Depletion of gex-3 by RNAi, or with the suppressor allele gex-3(av259[L353F]) , significantly restored the small brood size and low ovulation rate, as well as alleviated the crushed oocyte phenotype of the pezo-1(R2405P) mutant. Auxin-inducible degradation of GEX-3 revealed that only somatic-specific degradation of GEX-3 restored the reduced brood size in the pezo-1(R2405P) mutants. Additionally, actin organization and orientation were disrupted and distorted in the pezo-1 mutants. Mutation of gex-3(L353F) partially alleviated these defects. The identification of gex-3 as a suppressor of the pathogenic variant pezo-1(R2405P) suggests that the cytoskeleton plays an important role in regulating PIEZO channel activity and provides insight into the molecular mechanisms of DA5 and other PIEZO-associated diseases.

Using CRISPR knock-in of fluorescent tags to examine isoform-specific expression of EGL-19 in C. elegans.

L-type voltage-gated calcium channels (VGCCs) regulate calcium influx and excitation-contraction coupling in many types of muscle cells. Thus, VGCC mutations can cause skeletal and cardiac muscle diseases in humans, such as Duchenne muscular dystrophy and Timothy syndrome. To better understand the genetics and native expression of VGCCs, we have chosen to use the microscopic roundworm, C. elegans . The egl-19 locus is the sole L-type VGCC gene and it encodes three distinct isoforms (a, b, and c). Isoform c is curious because the protein is truncated, lacking the transmembrane domains that form the physical calcium channel. In this study, we have characterized egl-19 expression using CRISPR/Cas9 genome editing to 'knock-in' fluorescent tags of differing colors, allowing us to distinguish the expression pattern of each isoform. Not surprisingly, we found that EGL-19 is expressed in all types of muscle. In addition, we provide evidence that the truncated c isoform is expressed. Finally, although we find evidence that specific isoforms can have unique subcellular distributions, we also observed some expression patterns that appear to be artifacts. Overall, our results show interesting patterns of egl-19 expression, but also highlight the need for caution when interpreting expression of reporter genes even when they represent endogenous tags.

Patient-specific variants of NFU1/NFU-1 disrupt cholinergic signaling in a model of multiple mitochondrial dysfunctions syndrome 1.

Neuromuscular dysfunction is a common feature of mitochondrial diseases and frequently presents as ataxia, spasticity and/or dystonia, all of which can severely impact individuals with mitochondrial diseases. Dystonia is one of the most common symptoms of multiple mitochondrial dysfunctions syndrome 1 (MMDS1), a disease associated with mutations in the causative gene (NFU1) that impair iron-sulfur cluster biogenesis. We have generated Caenorhabditis elegans strains that recreated patient-specific point variants in the C. elegans ortholog (nfu-1) that result in allele-specific dysfunction. Each of these mutants, Gly147Arg and Gly166Cys, have altered acetylcholine signaling at neuromuscular junctions, but opposite effects on activity and motility. We found that the Gly147Arg variant was hypersensitive to acetylcholine and that knockdown of acetylcholine release rescued nearly all neuromuscular phenotypes of this variant. In contrast, we found that the Gly166Cys variant caused predominantly postsynaptic acetylcholine hypersensitivity due to an unclear mechanism. These results are important for understanding the neuromuscular conditions of MMDS1 patients and potential avenues for therapeutic intervention.