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RNA-binding proteins (RBPs) regulate translation and plasticity which are required for memory. RBP dysfunction has been linked to a range of neurological disorders where cognitive impairments are a key symptom. However, of the 2,000 RBPs in the human genome, many are uncharacterized with regards to neurological phenotypes. To address this, we used the model organism C. elegans to assess the role of 20 conserved RBPs in memory. We identified eight previously uncharacterized memory regulators, three of which are in the C. elegansY-Box (CEY) RBP family. Of these, we determined that cey-1 is the closest ortholog to the mammalian Y-Box (YBX) RBPs. We found that CEY-1 is both necessary in the nervous system for memory ability and sufficient to promote memory. Leveraging human datasets, we found both copy number variation losses and single nucleotide variants in YBX1 and YBX3 in individuals with neurological symptoms. We identified one predicted deleterious YBX3 variant of unknown significance, p.Asn127Tyr, in two individuals with neurological symptoms. Introducing this variant into endogenous cey-1 locus caused memory deficits in the worm. We further generated two humanized worm lines expressing human YBX3 or YBX1 at the cey-1 locus to test evolutionary conservation of YBXs in memory and the potential functional significance of the p.Asn127Tyr variant. Both YBX1/3 can functionally replace cey-1, and introduction of p.Asn127Tyr into the humanized YBX3 locus caused memory deficits. Our study highlights the worm as a model to reveal memory regulators and identifies YBX dysfunction as a potential new source of rare neurological disease. Author summary: Memory relies on RNA translation, often controlled by RNA-binding proteins (RBPs). RBP dysfunction has been linked to memory issues in many neurological diseases. Here, we take advantage of the model organism Caenorhabditis elegans to determine the role of 20 RBPs in memory. We identify eight new memory regulators, including members of the CEY RBP family, which are closely related to the human YBX RBPs. We found that CEY-1 is required for memory in C. elegans and enhances memory when overexpressed. Taking advantage of human genome sequencing data, we found three individuals with the same genetic variant in YBX3, two of which have neurological symptoms including intellectual disability. Introducing this same variant into the CEY-1 protein caused memory deficits, suggesting the variant may cause dysfunction in vivo. To confirm this in the context of the mammalian genes, we made C. elegans that express the YBX3 variant protein instead of CEY-1 to model the functional consequences of the human YBX3 variant. Again, we observed memory deficits. Our study shows the utility of C. elegans in discovering new memory-regulating RBPs and highlights a potentially conserved role for the CEY/YBX proteins in memory. Moreover, our findings suggest that dysfunction in the YBX RBPs may contribute to human neurological disease, beckoning future human and mammalian studies. [ABSTRACT FROM AUTHOR]

Although centrosomes help organize spindles in most cell types, oocytes of most species lack these structures. During acentrosomal spindle assembly in C. elegans oocytes, microtubule minus ends are sorted outwards away from the chromosomes where they form poles, but then these outward forces must be balanced to form a stable bipolar structure. Simultaneously, microtubule dynamics must be precisely controlled to maintain spindle length and organization. How forces and dynamics are tuned to create a stable bipolar structure is poorly understood. Here, we have gained insight into this question through studies of ZYG-8, a conserved doublecortin-family kinase; the mammalian homolog of this microtubule-associated protein is upregulated in many cancers and has been implicated in cell division, but the mechanisms by which it functions are poorly understood. We found that ZYG-8 depletion from oocytes resulted in overelongated spindles with pole and midspindle defects. Importantly, experiments with monopolar spindles revealed that ZYG-8 depletion led to excess outward forces within the spindle and suggested a potential role for this protein in regulating the force-generating motor BMK-1/kinesin-5. Further, we found that ZYG-8 is also required for proper microtubule dynamics within the oocyte spindle and that kinase activity is required for its function during both meiosis and mitosis. Altogether, our findings reveal new roles for ZYG-8 in oocytes and provide insights into how acentrosomal spindles are stabilized to promote faithful meiosis. Author summary: When a cell divides, a football-shaped spindle made up of dynamic microtubule polymers functions to partition chromosomes. Usually, structures called centrosomes organize microtubules at the two ends of the spindle. However, in female reproductive cells ("oocytes") of many organisms, the spindle is formed without the help of centrosomes. In this study, we used the model organism C. elegans to understand how this type of "acentrosomal" spindle forms and maintains its shape. Specifically, we investigated a protein called ZYG-8 and we found that when this protein was removed from oocytes, the spindles became overelongated and their typical football shape was disrupted. Follow-up studies revealed that ZYG-8 plays two important functions that promote acentrosomal spindle assembly and stability in oocytes: 1) ZYG-8 helps control the dynamic properties of the microtubule polymers so that they can be properly arranged into the spindle structure and 2) ZYG-8 tunes forces within the spindle, to help the structure maintain it shape. Taken together, our findings have increased our understanding of how spindles form in the absence of centrosomes, shedding light on how female reproductive cells divide. [ABSTRACT FROM AUTHOR]

Chemical mutagenesis-driven forward genetic screens are pivotal in unveiling gene functions, yet identifying causal mutations behind phenotypes remains laborious, hindering their high-throughput application. Here, we reveal a non-uniform mutation rate caused by Ethyl Methane Sulfonate (EMS) mutagenesis in the C. elegans genome, indicating that mutation frequency is influenced by proximate sequence context and chromatin status. Leveraging these factors, we developed a machine learning enhanced pipeline to create a comprehensive EMS mutagenesis probability map for the C. elegans genome. This map operates on the principle that causative mutations are enriched in genetic screens targeting specific phenotypes among random mutations. Applying this map to Whole Genome Sequencing (WGS) data of genetic suppressors that rescue a C. elegans ciliary kinesin mutant, we successfully pinpointed causal mutations without generating recombinant inbred lines. This method can be adapted in other species, offering a scalable approach for identifying causal genes and revitalizing the effectiveness of forward genetic screens. Author summary: Exploring gene functions through chemical mutagenesis-driven genetic screens is pivotal, yet the cumbersome task of identifying causative mutations remains a bottleneck, limiting their high-throughput potential. In this investigation, we uncovered a non-uniform mutation pattern induced by Ethyl Methane Sulfonate (EMS) mutagenesis in the C. elegans genome, highlighting the influence of proximate sequence context and chromatin status on mutation frequency. Leveraging these insights, we engineered a machine learning enhanced pipeline to construct a comprehensive EMS mutagenesis probability map for the C. elegans genome. This map operates on the principle that causative mutations are selectively enriched in genetic screens targeting specific phenotypes amid the backdrop of random mutations. Applying this mapping tool to Whole Genome Sequencing (WGS) data derived from genetic suppressors rescuing a C. elegans ciliary kinesin mutant, we achieved precise identification of causal mutations without resorting to the conventional generation of recombinant inbred lines. Our work not only advances understanding of mutation dynamics but also revitalizes the efficacy of forward genetic screens, contributing to the refinement of genetic exploration methodologies with implications for various organisms. [ABSTRACT FROM AUTHOR]

Protein tyrosine phosphatases non-receptor type (PTPNs) have been studied extensively in the context of the adaptive immune system; however, their roles beyond immunoregulation are less well explored. Here we identify novel functions for the conserved C. elegans phosphatase PTPN-22, establishing its role in nematode molting, cell adhesion, and cytoskeletal regulation. Through a non-biased genetic screen, we found that loss of PTPN-22 phosphatase activity suppressed molting defects caused by loss-of-function mutations in the conserved NIMA-related kinases NEKL-2 (human NEK8/NEK9) and NEKL-3 (human NEK6/NEK7), which act at the interface of membrane trafficking and actin regulation. To better understand the functions of PTPN-22, we carried out proximity labeling studies to identify candidate interactors of PTPN-22 during development. Through this approach we identified the CDC42 guanine-nucleotide exchange factor DNBP-1 (human DNMBP) as an in vivo partner of PTPN-22. Consistent with this interaction, loss of DNBP-1 also suppressed nekl-associated molting defects. Genetic analysis, co-localization studies, and proximity labeling revealed roles for PTPN-22 in several epidermal adhesion complexes, including C. elegans hemidesmosomes, suggesting that PTPN-22 plays a broad role in maintaining the structural integrity of tissues. Localization and proximity labeling also implicated PTPN-22 in functions connected to nucleocytoplasmic transport and mRNA regulation, particularly within the germline, as nearly one-third of proteins identified by PTPN-22 proximity labeling are known P granule components. Collectively, these studies highlight the utility of combined genetic and proteomic approaches for identifying novel gene functions. Author summary: The human protein, PTPN22, has been studied extensively within the context of the adaptive immune system and mutations that affect PTPN22 function are implicated in multiple autoimmune disorders. Curiously, PTPN22 is conserved throughout the animal kingdom, including in the invertebrate roundworm Caenorhabditis elegans, which lack an adaptive immune system. Using several non-biased approaches to uncover novel protein functions, we identified roles for the Caenorhabditis elegans PTPN22 ortholog, PTPN-22, in a several different cell biological processes including functions related to epithelial cell and tissue connectivity. These studies, along with our analysis of the cellular and subcellular locations in which PTPN-22 is present during development, indicate that the worm PTPN-22 carries out a diverse range of functions. Our studies indicate that, in addition to critical roles within the immune-system, PTPN22 family members may carry additional functions that are conserved throughout the animal kingdom, a finding supported by several studies carried out on human PTPN22 suggesting roles in cell-cell adhesion. [ABSTRACT FROM AUTHOR]

Sex determination in the nematode C. elegans is controlled by the master regulator XOL-1 during embryogenesis. Expression of xol-1 is dependent on the ratio of X chromosomes and autosomes, which differs between XX hermaphrodites and XO males. In males, xol-1 is highly expressed and in hermaphrodites, xol-1 is expressed at very low levels. XOL-1 activity is known to be critical for the proper development of C. elegans males, but its low expression was considered to be of minimal importance in the development of hermaphrodite embryos. Our study reveals that XOL-1 plays an important role as a regulator of developmental timing during hermaphrodite embryogenesis. Using a combination of imaging and bioinformatics techniques, we found that hermaphrodite embryos have an accelerated rate of cell division, as well as a more developmentally advanced transcriptional program when xol-1 is lost. Further analyses reveal that XOL-1 is responsible for regulating the timing of initiation of dosage compensation on the X chromosomes, and the appropriate expression of sex-biased transcriptional programs in hermaphrodites. We found that xol-1 mutant embryos overexpress the H3K9 methyltransferase MET-2 and have an altered H3K9me landscape. Some of these effects of the loss of xol-1 gene were reversed by the loss of met-2. These findings demonstrate that XOL-1 plays an important role as a developmental regulator in embryos of both sexes, and that MET-2 acts as a downstream effector of XOL-1 activity in hermaphrodites. Author summary: Various organisms have differing ways of determining, at a molecular level, what the sex of a developing embryo is supposed to be. The two sexes in the nematode C. elegans, hermaphrodite and male, have different numbers of X chromosomes. Hermaphrodites have two X chromosomes and males only have one. This mismatch raises an additional problem as hermaphrodites will have twice the amount of genes expressed from the X compared to males. This is solved by a process called dosage compensation, which equalizes gene expression between the sexes. A molecular sensor called XOL-1 detects the number of X chromosomes in an embryo and kick-starts the process of proper sexual development and/or dosage compensation. XOL-1 was known to be very important for activating male development but was believed to not have any role in the development of the hermaphrodite sex. We show that XOL-1 has some crucial roles in hermaphrodite embryos in controlling the rate of development of the embryo, and regulating the timing of dosage compensation. We also show that MET-2, a protein that deposits repressive methyl marks on DNA-bound histone proteins, is involved in this process. [ABSTRACT FROM AUTHOR]

A recently reported Schizophrenia-associated genetic variant in the 3′UTR of the human furin gene, a homolog of C. elegans kpc-1, highlights an important role of the furin 3′UTR in neuronal development. We isolate three kpc-1 mutants that display abnormal dendrite arborization in PVD neurons and defective male mating behaviors. We show that the kpc-1 3′UTR participates in dendrite branching and self-avoidance. The kpc-1 3′UTR facilitates mRNA localization to branching points and contact points between sibling dendrites and promotes translation efficiency. A predicted secondary structural motif in the kpc-1 3′UTR is required for dendrite self-avoidance. Animals with over-expression of DMA-1, a PVD dendrite receptor, exhibit similar dendrite branching and self-avoidance defects that are suppressed with kpc-1 over-expression. Our results support a model in which KPC-1 proteins are synthesized at branching points and contact points to locally down-regulate DMA-1 receptors to promote dendrite branching and self-avoidance of a mechanosensory neuron important for male courtship. Author summary: Here, we report a non-coding sequence immediately downstream of the kpc-1 gene participating in the transport and, subsequently, the translation of the kpc-1 messenger RNA in the nerve process of a mechanosensory neuron, which is required to establish a functional neuronal circuit regulating the male mating behavior in C. elegans. Interestingly, a human Schizophrenia-associated mutation located in the non-coding sequence immediately downstream of the human furin gene, a kpc-1 homologous gene, was recently shown to contribute to the pathogenesis of Schizophrenia. By analogy, whether this human furin downstream mutation disrupts the transport and the translation of the furin mRNA in the nerve process of Schizophrenia-causing neurons during its differentiation remains to be seen. [ABSTRACT FROM AUTHOR]

Some types of collagens, including transmembrane MACIT collagens and C. elegans cuticle collagens, are N-terminally cleaved at a dibasic site that resembles the consensus for furin or other proprotein convertases of the subtilisin/kexin (PCSK) family. Such cleavage may release transmembrane collagens from the plasma membrane and affect extracellular matrix assembly or structure. However, the functional consequences of such cleavage are unclear and evidence for the role of specific PCSKs is lacking. Here, we used endogenous collagen fusions to fluorescent proteins to visualize the secretion and assembly of the first collagen-based cuticle in C. elegans and then tested the role of the PCSK BLI-4 in these processes. Unexpectedly, we found that cuticle collagens SQT-3 and DPY-17 are secreted into the extraembryonic space several hours before cuticle matrix assembly. Furthermore, this early secretion depends on BLI-4/PCSK; in bli-4 and cleavage-site mutants, SQT-3 and DPY-17 are not efficiently secreted and instead form large intracellular puncta. Their later assembly into cuticle matrix is reduced but not entirely blocked. These data reveal a role for collagen N-terminal processing in intracellular trafficking and the control of matrix assembly in vivo. Our observations also prompt a revision of the classic model for C. elegans cuticle matrix assembly and the pre-cuticle-to-cuticle transition, suggesting that cuticle layer assembly proceeds via a series of regulated steps and not simply by sequential secretion and deposition.

The pathway for axon regeneration in Caenorhabditis elegans is activated by SVH-1, a growth factor belonging to the HGF/plasminogen family. SVH-1 is a dual-function factor that acts as an HGF-like growth factor to promote axon regeneration and as a protease to regulate early development. It is important to understand how SVH-1 is converted from a protease to a growth factor for axon regeneration. In this study, we demonstrate that cytidine deaminase (CDD) SVH-17/CDD-2 plays a role in the functional conversion of SVH-1. We find that the codon exchange of His-755 to Tyr in the Asp–His–Ser catalytic triad of SVH-1 can suppress the cdd-2 defect in axon regeneration. Furthermore, the stem hairpin structure around the His-755 site in svh-1 mRNA is required for the activation of axon regeneration by SVH-1. These results suggest that CDD-2 promotes axon regeneration by transforming the function of SVH-1 from a protease to a growth factor through modification of svh-1 mRNA. Author summary: The axon regeneration pathway in C. elegans is activated by SVH-1, a growth factor that belongs to the HGF/plasminogen family. SVH-1 is a dual-functional factor that promotes axon regeneration as an HGF-like growth factor and regulates development as a plasminogen-like protease. It is crucial to understand the mechanism by which SVH-1 transforms from a protease to a growth factor during axon regeneration. In this study, we demonstrate that CDD-2, a cytidine deaminase, is critical for converting SVH-1 function from a protease to a growth factor by modifying svh-1 mRNA. [ABSTRACT FROM AUTHOR]

Bulky DNA adducts such as those induced by ultraviolet light are removed from the genomes of multicellular organisms by nucleotide excision repair, which occurs through two distinct mechanisms, global repair, requiring the DNA damage recognition-factor XPC (xeroderma pigmentosum complementation group C), and transcription-coupled repair (TCR), which does not. TCR is initiated when elongating RNA polymerase II encounters DNA damage, and thus analysis of genome-wide excision repair in XPC-mutants only repairing by TCR provides a unique opportunity to map transcription events missed by methods dependent on capturing RNA transcription products and thus limited by their stability and/or modifications (5'-capping or 3'-polyadenylation). Here, we have performed eXcision Repair-sequencing (XR-seq) in the model organism Caenorhabditis elegans to generate genome-wide repair maps in a wild-type strain with normal excision repair, a strain lacking TCR (csb-1), and a strain that only repairs by TCR (xpc-1). Analysis of the intersections between the xpc-1 XR-seq repair maps with RNA-mapping datasets (RNA-seq, long- and short-capped RNA-seq) reveal previously unrecognized sites of transcription and further enhance our understanding of the genome of thtableis important model organism. Author summary: Most RNA sequencing technologies are based on capturing either processed RNA or primary transcripts to define the RNA expression profile of cell lines or organisms. These methods suffer from RNA's inherent instability in revealing a complete view of RNA Polymerase II (RNAPII) transcription. Here, we used a C. elegans xpc-1 mutant which only performs transcription-coupled repair (TCR) to generate a complete RNAPII transcription profile by using the eXcision Repair-sequencing (XR-seq) technology to analyze repair of UV-induced DNA damage. XR-seq captures all the excision products from RNAPII-transcribed regions in the form of 21–28 nucleotide-long DNA oligomers and thus in a sense converts the unstable RNA signal to a more stable DNA signal enabling the generation of the most complete RNAPII transcript profile to date including heretofore unknown transcripts. [ABSTRACT FROM AUTHOR]

The piRNA pathway is a conserved germline-specific small RNA pathway that ensures genomic integrity and continued fertility. In C. elegans and other nematodes, Type-I piRNAs are expressed from >10,000 independently transcribed genes clustered within two discrete domains of 1.5 and 3.5 MB on Chromosome IV. Clustering of piRNA genes contributes to their germline-specific expression, but the underlying mechanisms are unclear. We analyze isolated germ nuclei to demonstrate that the piRNA genomic domains are located in a heterochromatin-like environment. USTC (Upstream Sequence Transcription Complex) promotes strong association of nucleosomes throughout piRNA clusters, yet organizes the local nucleosome environment to direct the exposure of individual piRNA genes. Localization of USTC to the piRNA domains depends upon the ATPase chromatin remodeler ISW-1, which maintains high nucleosome density across piRNA clusters and ongoing production of piRNA precursors. Overall, this work provides insight into how chromatin states coordinate transcriptional regulation over large genomic domains, with implications for global genome organization. Author summary: Here we address how gene expression is coordinated over long stretches of DNA that is embedded in a dense chromatin environment. We focus on a large DNA domain that contains thousands of tiny noncoding piRNA genes that are only expressed in germ cells. Our work demonstrates that a specific factor called USTC works with chromatin remodeling factors and with the core transcription complex to activate piRNA gene expression. The ISW-1 chromatin remodeler creates a dense environment that is favorable for USTC to be recruited to piRNA genes. USTC then clears the chromatin away from a space around each piRNA gene that allows for the factor TBP-1 to bind and presumably recruit the core transcriptional machinery to turn on the short piRNA. This work illuminates a mechanism by which both local and long-distance regulatory information can be integrated to coordinate gene expression. [ABSTRACT FROM AUTHOR]

The Transforming Growth Factor beta (TGF-β) family consists of numerous secreted peptide growth factors that play significant roles in cell function, tissue patterning, and organismal homeostasis, including wound repair and immunity. Typically studied as homodimers, these ligands have the potential to diversify their functions through ligand interactions that may enhance, repress, or generate novel functions. In the nematode Caenorhabditis elegans, there are only five TGF-β ligands, providing an opportunity to dissect ligand interactions in fewer combinations than in vertebrates. As in vertebrates, these ligands can be divided into bone morphogenetic protein (BMP) and TGF-β/Activin subfamilies that predominantly signal through discrete signaling pathways. The BMP subfamily ligand DBL-1 has been well studied for its role in the innate immune response in C. elegans. Here we show that all five TGF-β ligands play a role in survival on bacterial pathogens. We also demonstrate that multiple TGF-β ligand pairs act nonredundantly as part of this response. We show that the two BMP-like ligands–DBL-1 and TIG-2 –function independently of each other in the immune response, while TIG2/BMP and the TGF-β/Activin-like ligand TIG-3 function together. Structural modeling supports the potential for TIG-2 and TIG-3 to form heterodimers. Additionally, we identify TIG-2 and TIG-3 as members of a rare subset of TGF-β ligands lacking the conserved cysteine responsible for disulfide linking mature dimers. Finally, we show that canonical DBL1/BMP receptor and Smad signal transducers function in the response to bacterial pathogens, while components of the DAF7 TGF-β/Activin signaling pathway do not play a major role in survival. These results demonstrate a novel potential for BMP and TGF-β/Activin subfamily ligands to interact and may provide a mechanism for distinguishing the developmental and homeostatic functions of these ligands from an acute response such as the innate immune response to bacterial pathogens. Author summary: The first line of defense upon exposure to a pathogen consists of innate immunity, which includes barrier functions and cell-cell communication. These cell-cell communication signaling pathways are highly conserved across species, enabling the use of simpler genetically tractable model organisms for their study. One such signaling pathway is the Transforming Growth Factor beta (TGF-β) pathway, which is conserved from invertebrates to vertebrates. TGF-β signaling ligands can be broadly divided into two major groups: the TGF-β/Activin group and the bone morphogenetic protein (BMP) group. There is compelling evidence that heterodimers have biological activities that differ from homodimers, but to this time, heterodimers have only been observed within subfamilies and not across subfamilies. In humans, there are 33 TGF-β ligands. In comparison, there are only five ligands in the nematode C. elegans, providing an opportunity to dissect ligand interactions in fewer combinations than in vertebrates. In this work, we show that all five ligands contribute to survival on bacterial pathogens to different extents and with specificity concerning the pathogen. Strikingly, genetic evidence and structural modeling support the existence of cross-subfamily interaction between BMP and TGF-β/Activin ligands. [ABSTRACT FROM AUTHOR]

The Integrator is a multi-subunit protein complex that catalyzes the maturation of snRNA transcripts via 3' cleavage, a step required for snRNA incorporation with snRNP for spliceosome biogenesis. Here we developed a GFP based in vivo snRNA misprocessing reporter as a readout of Integrator function and performed a genome-wide RNAi screen for Integrator regulators. We found that loss of the Argonaute encoding csr-1 gene resulted in widespread 3' misprocessing of snRNA transcripts that is accompanied by a significant increase in alternative splicing. Loss of the csr-1 gene down-regulates the germline expression of Integrator subunits 4 and 6 and is accompanied by a reduced protein translation efficiency of multiple Integrator catalytic and non-catalytic subunits. Through isoform and motif mutant analysis, we determined that CSR-1's effect on snRNA processing is dependent on its catalytic slicer activity but does not involve the CSR-1a isoform. Moreover, mRNA-sequencing revealed high similarity in the transcriptome profile between csr-1 and Integrator subunit knockdown via RNAi. Together, our findings reveal CSR-1 as a new regulator of the Integrator complex and implicate a novel role of this Argonaute protein in snRNA 3' processing. Author summary: Small nuclear RNA molecules are an important structural component of the spliceosome, which is the cellular machinery that performs RNA splicing. Proper RNA splicing is important for ensuring proteins are accurately produced within the cell, and this process is broadly important for contributing to basic physiological processes such as development. Inside the cell, small nuclear RNA is processed by a protein complex called the Integrator, and mutations to the Integrator affect RNA splicing and can lead to neurodevelopmental defects. Here, we used the roundworm Caenorhabditis elegans to describe how an Argonaute encoding gene called csr-1 is required for maintaining Integrator protein expression and that loss of csr-1 gene expression contributes to the dysregulation of small nuclear RNA processing. These results provide new insights into our understanding of fundamental factors that regulate small nuclear RNA processing in cells, which are directly important in RNA splicing control. [ABSTRACT FROM AUTHOR]

Synaptic vesicle proteins (SVps) are transported by the motor UNC-104/KIF1A. We show that SVps travel in heterogeneous carriers in C. elegans neuronal processes, with some SVp carriers co-transporting lysosomal proteins (SV-lysosomes). LRK-1/LRRK2 and the clathrin adaptor protein complex AP-3 play a critical role in the sorting of SVps and lysosomal proteins away from each other at the SV-lysosomal intermediate trafficking compartment. Both SVp carriers lacking lysosomal proteins and SV-lysosomes are dependent on the motor UNC-104/KIF1A for their transport. In lrk-1 mutants, both SVp carriers and SV-lysosomes can travel in axons in the absence of UNC-104, suggesting that LRK-1 plays an important role to enable UNC-104 dependent transport of synaptic vesicle proteins. Additionally, LRK-1 acts upstream of the AP-3 complex and regulates its membrane localization. In the absence of the AP-3 complex, the SV-lysosomes become more dependent on the UNC-104-SYD-2/Liprin-α complex for their transport. Therefore, SYD-2 acts to link upstream trafficking events with the transport of SVps likely through its interaction with the motor UNC-104. We further show that the mistrafficking of SVps into the dendrite in lrk-1 and apb-3 mutants depends on SYD-2, likely by regulating the recruitment of the AP-1/UNC-101. SYD-2 acts in concert with AP complexes to ensure polarized trafficking & transport of SVps. Author summary: Synaptic vesicles (SVs) at neuronal synapses have a defined membrane composition and size. We show that SV proteins (SVps) are transported in carriers with heterogenous composition, with a small subset containing lysosomal proteins (SV-lysosomes). We propose that the SV-lysosomes are perhaps post-Golgi trafficking intermediates from which the SV proteins are sorted away from lysosomal proteins through the action of LRK-1, the ortholog of LRRK2, which is associated with both familial and sporadic Parkinson's disease, and the AP-3 complex, which is associated with Hermansky-Pudlak syndrome. These sorting events could be necessary events for the formation of SV precursors. SVp carriers and SV-lysosomes both depend on the UNC-104/KIF1A kinesin motor and the active zone protein SYD-2/Liprin-α for their transport. However, in the absence of the AP-3 complex, altered trafficking of SV and lysosomal proteins likely causes the SV-lysosomes to become more dependent on the UNC-104-SYD-2 complex for their transport, while the SVp carriers lacking lysosomal proteins become partially independent of UNC-104. We therefore propose that SYD-2 can act as a link between the upstream trafficking events and the subsequent transport of SVps likely through its interaction with the motor UNC-104. We also show that SYD-2 genetically interacts with and regulates the localization of the AP-1 complex to facilitate polarised distribution of SVp carriers to axons. Thus, our study highlights novel roles for SYD-2 in several early steps of SVp trafficking. [ABSTRACT FROM AUTHOR]

Oocyte meiotic spindles mediate the expulsion of ¾ of the genome into polar bodies to generate diploid zygotes in nearly all animal species. Failures in this process result in aneuploid or polyploid offspring that are typically inviable. Accurate meiotic chromosome segregation and polar body extrusion require the spindle to elongate while maintaining its structural integrity. Previous studies have implicated three hypothetical activities during this process, including microtubule crosslinking, microtubule sliding and microtubule polymerization. However, how these activities regulate spindle rigidity and elongation as well as the exact proteins involved in the activities remain unclear. We discovered that C. elegans meiotic anaphase spindle integrity is maintained through redundant microtubule crosslinking activities of the Kinesin-5 family motor BMK-1, the microtubule bundling protein SPD-1/PRC1, and the Kinesin-4 family motor, KLP-19. Using time-lapse imaging, we found that single depletion of KLP-19KIF4A, SPD-1PRC1 or BMK-1Eg5 had minimal effects on anaphase B spindle elongation velocity. In contrast, double depletion of SPD-1PRC1 and BMK-1Eg5 or double depletion of KLP-19KIF4A and BMK-1Eg5 resulted in spindles that elongated faster, bent in a myosin-dependent manner, and had a high rate of polar body extrusion errors. Bending spindles frequently extruded both sets of segregating chromosomes into two separate polar bodies. Normal anaphase B velocity was observed after double depletion of KLP-19KIF4A and SPD-1PRC1. These results suggest that KLP-19KIF4A and SPD-1PRC1 act in different pathways, each redundant with a separate BMK-1Eg5 pathway in regulating meiotic spindle elongation. Depletion of ZYG-8, a doublecortin-related microtubule binding protein, led to slower anaphase B spindle elongation. We found that ZYG-8DCLK1 acts by excluding SPD-1PRC1 from the spindle. Thus, three mechanistically distinct microtubule regulation modules, two based on crosslinking, and one based on exclusion of crosslinkers, power the mechanism that drives spindle elongation and structural integrity during anaphase B of C.elegans female meiosis.

Striated muscle laminopathies caused by missense mutations in the nuclear lamin gene LMNA are characterized by cardiac dysfunction and often skeletal muscle defects. Attempts to predict which LMNA variants are pathogenic and to understand their physiological effects lag behind variant discovery. We created Caenorhabditis elegans models for striated muscle laminopathies by introducing pathogenic human LMNA variants and variants of unknown significance at conserved residues within the lmn-1 gene. Severe missense variants reduced fertility and/or motility in C. elegans. Nuclear morphology defects were evident in the hypodermal nuclei of many lamin variant strains, indicating a loss of nuclear envelope integrity. Phenotypic severity varied within the two classes of missense mutations involved in striated muscle disease, but overall, variants associated with both skeletal and cardiac muscle defects in humans lead to more severe phenotypes in our model than variants predicted to disrupt cardiac function alone. We also identified a separation of function allele, lmn-1(R204W), that exhibited normal viability and swimming behavior but had a severe nuclear migration defect. Thus, we established C. elegans avatars for striated muscle laminopathies and identified LMNA variants that offer insight into lamin mechanisms during normal development.

The tumor suppressor BRCA1-BARD1 complex regulates many cellular processes; of critical importance to its tumor suppressor function is its role in genome integrity. Although RING E3 ubiquitin ligase activity is the only known enzymatic activity of the complex, the in vivo requirement for BRCA1-BARD1 E3 ubiquitin ligase activity has been controversial. Here we probe the role of BRCA1-BARD1 E3 ubiquitin ligase activity in vivo using C. elegans. Genetic, cell biological, and biochemical analyses of mutants defective for E3 ligase activity suggest there is both E3 ligase-dependent and independent functions of the complex in the context of DNA damage repair and meiosis. We show that E3 ligase activity is important for nuclear accumulation of the complex and specifically to concentrate at meiotic recombination sites but not at DNA damage sites in proliferating germ cells. While BRCA1 alone is capable of monoubiquitylation, BARD1 is required with BRCA1 to promote polyubiquitylation. We find that the requirement for E3 ligase activity and BARD1 in DNA damage signaling and repair can be partially alleviated by driving the nuclear accumulation and self-association of BRCA1. Our data suggest that in addition to E3 ligase activity, BRCA1 may serve a structural role for DNA damage signaling and repair while BARD1 plays an accessory role to enhance BRCA1 function.

Axon regeneration requires actomyosin interaction, which generates contractile force and pulls the regenerating axon forward. In Caenorhabditis elegans, TLN-1/talin promotes axon regeneration through multiple down-stream events. One is the activation of the PAT-3/integrin–RHO-1/RhoA GTPase–LET-502/ROCK (Rho-associated coiled-coil kinase)–regulatory non-muscle myosin light-chain (MLC) phosphorylation signaling pathway, which is dependent on the MLC scaffolding protein ALP-1/ALP-Enigma. The other is mediated by the F-actin-binding protein DEB-1/vinculin and is independent of the MLC phosphorylation pathway. In this study, we identified the svh-7/rtkn-1 gene, encoding a homolog of the RhoA-binding protein Rhotekin, as a regulator of axon regeneration in motor neurons. However, we found that RTKN-1 does not function in the RhoA–ROCK–MLC phosphorylation pathway in the regulation of axon regeneration. We show that RTKN-1 interacts with ALP-1 and the vinculin-binding protein SORB-1/vinexin, and that SORB-1 acts with DEB-1 to promote axon regeneration. Thus, RTKN-1 links the DEB-1–SORB-1 complex to ALP-1 and physically connects phosphorylated MLC on ALP-1 to the actin cytoskeleton. These results suggest that TLN-1 signaling pathways coordinate MLC phosphorylation and recruitment of phosphorylated MLC to the actin cytoskeleton during axon regeneration. Author summary: Axon regeneration requires actomyosin interactions, whereby actomyosin generates contractile forces to advance the regenerating axon. In C. elegans, TLN-1/talin promotes axon regeneration through the PAT-3/integrin–RHO-1/RhoA–LET-502/ROCK–MLC phosphorylation signaling pathway, which is dependent on the MLC phosphorylation scaffold protein ALP-1/ALP-Enigma. TLN-1 also activates the F-actin-binding protein DEB-1/vinculin in axon regeneration, but this pathway activates SORB-1/vinexin but does not induce MLC phosphorylation. Here, we identified the svh-7/rtkn-1 gene, which encodes a homolog of the RhoA-binding protein Rhotekin, as a regulator of axon regeneration. However, we show that RTKN-1 does not function in the RhoA–ROCK–MLC phosphorylation pathway in the regulation of axon regeneration. Instead, RTKN-1 can connect phosphorylated MLC on ALP-1 to the actin cytoskeleton by physically linking the DEB-1–SORB-1 complex to ALP-1. This study suggests that the TLN-1–DEB-1–SORB-1–RTKN-1 axis promotes axon regeneration by recruiting myosin to the actin cytoskeleton via ALP-1. [ABSTRACT FROM AUTHOR]

The Muscleblind-like (MBNL) family is a highly conserved set of RNA-binding proteins (RBPs) that regulate RNA metabolism during the differentiation of various animal tissues. Functional insufficiency of MBNL affects muscle and central nervous system development, and contributes to the myotonic dystrophies (DM), a set of incurable multisystemic disorders. Studies on the regulation of MBNL genes are essential to provide insight into the gene regulatory networks controlled by MBNL proteins and to understand how dysregulation within these networks causes disease. In this study, we demonstrate the evolutionary conservation of an autoregulatory mechanism that governs the function of MBNL proteins by generating two distinct protein isoform types through alternative splicing. Our aim was to further our understanding of the regulatory principles that underlie this conserved feedback loop in a whole-organismal context, and to address the biological significance of the respective isoforms. Using an alternative splicing reporter, our studies show that, during development of the Caenorhabditis elegans central nervous system, the orthologous mbl-1 gene shifts production from long protein isoforms that localize to the nucleus to short isoforms that also localize to the cytoplasm. Using isoform-specific CRISPR/Cas9-generated strains, we showed that expression of short MBL-1 protein isoforms is required for healthy neuromuscular function and neurodevelopment, while expression of long MBL-1 protein isoforms is dispensable, emphasizing a key role for cytoplasmic functionalities of the MBL-1 protein. Furthermore, RNA-seq and lifespan analyses indicated that short MBL-1 isoforms are crucial regulators of miRNA expression and, in consequence, required for normal lifespan. In conclusion, this study provides support for the disruption of cytoplasmic RNA metabolism as a contributor in myotonic dystrophy and paves the way for further exploration of miRNA regulation through MBNL proteins during development and in disease models. Author summary: Alternative splicing is a molecular process by which one gene can produce multiple protein isoforms. These isoforms often have different properties, and normal cell function and development requires expression of the appropriate isoform. The regulation of alternative splicing is carried out by a set of specialized proteins, one of which is the Muscleblind-like protein (MBNL). This protein is essential during the development and differentiation of many animal tissues. We found that this protein regulates the alternative splicing of its own gene in animals as divergent as nematode and human. We showed that this leads to the timely expression of MBNL protein isoforms that localize to the cytoplasm of the cell. In the cytoplasm, these isoforms acquire new functions unrelated to splicing and regulate molecular processes that control the expression of many other genes. These functions are required for normal development of the nervous system and lifespan in nematodes. This finding is particularly important because it helps us understand the regulation and function of MBNL proteins in animal development as well as identify potential disease mechanisms in myotonic dystrophies, neurodegenerative disorders associated with MBNL dysfunction. [ABSTRACT FROM AUTHOR]

Organismal responses to temperature fluctuations include an evolutionarily conserved cytosolic chaperone machinery as well as adaptive alterations in lipid constituents of cellular membranes. Using C. elegans as a model system, we asked whether adaptable lipid homeostasis is required for survival during physiologically relevant heat stress. By systematic analyses of lipid composition in worms during and before heat stress, we found that unsaturated fatty acids are reduced in heat-stressed animals. This is accompanied by the transcriptional downregulation of fatty acid desaturase enzymes encoded by fat-1, fat-3, fat-4, fat-5, fat-6, and fat-7 genes. Conversely, overexpression of the Δ9 desaturase FAT-7, responsible for the synthesis of PUFA precursor oleic acid, and supplementation of oleic acid causes accelerated death of worms during heat stress. Interestingly, heat stress causes permeability defects in the worm's cuticle. We show that fat-7 expression is reduced in the permeability defective collagen (PDC) mutant, dpy-10, known to have enhanced heat stress resistance (HSR). Further, we show that the HSR of dpy-10 animals is dependent on the upregulation of PTR-23, a patched-like receptor in the epidermis, and that PTR-23 downregulates the expression of fat-7. Consequently, abrogation of ptr-23 in wild type animals affects its survival during heat stress. This study provides evidence for the negative regulation of fatty acid desaturase expression in the soma of C. elegans via the non-canonical role of a patched receptor signaling component. Taken together, this constitutes a skin-gut axis for the regulation of lipid desaturation to promote the survival of worms during heat stress. Author summary: Temperature fluctuation is a major environmental stressor for all living organisms. Here, we describe a mechanism that allows multicellular organisms to coordinate heat stress response between different tissues to ensure better survival. We find that a receptor in the skin of the worm controls stress response in adults. The receptor regulates lipid metabolism to enhance cellular health and organismal survival during chronic heat stress. [ABSTRACT FROM AUTHOR]

Kin-recognition is observed across diverse species forming an important behavioral adaptation influencing organismal interactions. In many species, the molecular mechanisms involved are difficult to characterize, but in the nematode Pristionchus pacificus molecular components regulating its kin-recognition system have been identified. These determine its predatory behaviors towards other con-specifics which prevents the killing and cannibalization of kin. Importantly, their impact on other interactions including collective behaviors is unknown. Here, we explored a high altitude adapted clade of this species which aggregates abundantly under laboratory conditions, to investigate the influence of the kin-recognition system on their group behaviours. By utilizing pairwise aggregation assays between distinct strains of P. pacificus with differing degrees of genetic relatedness, we observe aggregation between kin but not distantly related strains. In assays between distantly related strains, the aggregation ratio is frequently reduced. Furthermore, abolishing predation behaviors through CRISPR/Cas9 induced mutations in Ppa-nhr-40 result in rival strains successfully aggregating together. Finally, as Caenorhabditis elegans are found naturally occurring with P. pacificus, we also explored aggregation events between these species. Here, aggregates were dominated by P. pacificus with the presence of only a small number of predators proving sufficient to disrupt C. elegans aggregation dynamics. Thus, aggregating strains of P. pacificus preferentially group with kin, revealing competition and nepotism as previously unknown components influencing collective behaviors in nematodes. Author summary: Many species are able to recognize their relatives from other organisms and this influences how they behave with one another. In some species, this kin-recognition ability results in cooperation while in others it can induce more competitive and aggressive behaviors. Importantly, in many species we do not know the mechanisms which regulate these interactions. In the predatory nematode (round worm) Pristionchus pacificus, we have previously observed that they attack and kill other nematode larvae but their kin-recognition abilities prevent them from harming their close relatives. Importantly, in P. pacificus we have an abundance of genetic and molecular tools and can begin to understand how they are able to carry out this recognition behavior. Here, we have shown that their kin-recognition and predatory abilities also influence other interactions including how these nematodes group together. Many nematode species form aggregates to help them find food or to avoid harmful environmental conditions. In P. pacificus, we have observed that they preferentially aggregate with their close relatives and avoid grouping with more distantly related strains. This may ensure that they give their relatives an advantage within their ecological niche. Therefore, our work will help us understand more about the significance of kin-recognition behaviors and their evolution. [ABSTRACT FROM AUTHOR]

This document is a correction notice for an article published in PLoS Genetics. Concerns were raised about several figures in the article, including duplication of panels and incorrect labeling. The corresponding author provided updated versions of the figures and underlying data from replicate experiments. An expert reviewer confirmed that the conclusions of the article are still supported. The primary data underlying the article are no longer available, except for some data underlying one figure. [Extracted from the article]

MicroRNAs (miRNAs) regulate gene expression by base-pairing to target sequences in messenger RNAs (mRNAs) and recruiting factors that induce translational repression and mRNA decay. In animals, nucleotides 2–8 at the 5' end of the miRNA, called the seed region, are often necessary and sometimes sufficient for functional target interactions. MiRNAs that contain identical seed sequences are grouped into families where individual members have the potential to share targets and act redundantly. A rare exception seemed to be the miR-238/239ab family in Caenorhabditis elegans, as previous work indicated that loss of miR-238 reduced lifespan while deletion of the miR-239ab locus resulted in enhanced longevity and thermal stress resistance. Here, we re-examined these potentially opposing roles using new strains that individually disrupt each miRNA sister. We confirmed that loss of miR-238 is associated with a shortened lifespan but could detect no longevity or stress phenotypes in animals lacking miR-239a or miR-239b, individually or in combination. Additionally, dozens of genes were mis-regulated in miR-238 mutants but almost no gene expression changes were detected in either miR-239a or miR-239b mutants compared to wild type animals. We present evidence that the lack of redundancy between miR-238 and miR-239ab is independent of their sequence differences; miR-239a or miR-239b could substitute for the longevity role of miR-238 when expressed from the miR-238 locus. Altogether, these studies disqualify miR-239ab as negative regulators of aging and demonstrate that expression, not sequence, dictates the specific role of miR-238 in promoting longevity. Author summary: MicroRNAs (miRNAs) are tiny non-coding RNAs that function in diverse biological pathways. To exert their regulatory influence, miRNAs bind to specific target RNAs through partial base-pairing. A critical aspect of this miRNA-target engagement is the seed sequence, nucleotides 2–8 of the miRNA. MiRNAs that share seed sequences are grouped into families and presumed to have similar functions. Yet, other factors, such as non-seed sequences in the miRNA and its expression level, can also contribute to target regulation and result in distinct roles for miRNAs within a family. To better understand how miRNA family members can have specific functions, we focused on miR-238 and its sisters, miR-239a and miR-239b, because these miRNAs had previously been reported to play opposing longevity roles in the nematode C. elegans. Using new genetic tools, we found that loss of miR-238 alone leads to the misregulation of many genes and a reduced lifespan. However, the lack of miR-239a, miR-239b, or both sisters had almost no effect on gene expression or longevity compared to wild type animals. Strikingly, though, miR-239a or miR-239b could substitute for the aging role of miR-238 when expressed from the miR-238 locus. Thus, expression, not sequence, is the predominant distinguishing feature of mir-238 that bestows upon it a role in aging not shared with the other family members. [ABSTRACT FROM AUTHOR]

The cuticles of ecdysozoan animals are barriers to material loss and xenobiotic insult. Key to this barrier is lipid content, the establishment of which is poorly understood. Here, we show that the p-glycoprotein PGP-14 functions coincidentally with the sphingomyelin synthase SMS-5 to establish a polar lipid barrier within the pharyngeal cuticle of the nematode C. elegans. We show that PGP-14 and SMS-5 are coincidentally expressed in the epithelium that surrounds the anterior pharyngeal cuticle where PGP-14 localizes to the apical membrane. pgp-14 and sms-5 also peak in expression at the time of new cuticle synthesis. Loss of PGP-14 and SMS-5 dramatically reduces pharyngeal cuticle staining by Nile Red, a key marker of polar lipids, and coincidentally alters the nematode's response to a wide-range of xenobiotics. We infer that PGP-14 exports polar lipids into the developing pharyngeal cuticle in an SMS-5-dependent manner to safeguard the nematode from environmental insult. Author summary: An infinite number of small molecules have the potential to threaten life. Not surprisingly then, animals have evolved multiple mechanisms to defend against such threats. One defense mechanism employed by many animals is the creation of an outer protective layer called the cuticle. Lipids within the cuticle act as a barrier to retard small molecule passage into the underlying tissues. Water-loving small molecules cannot traverse a lipid barrier and fat-loving molecules can get trapped within the barrier. How this lipid barrier is established is incompletely understood. Here, we describe our discovery of a conserved protein called PGP-14 that is expressed in the tissue that makes the cuticle that protects the mouth and pharynx of the nematode worm C. elegans. We show that PGP-14 peaks in expression at the end of new cuticle synthesis and is necessary for lipid deposition within it. Without PGP-14, many small molecules adversely accumulate within the animal and consequently kill it. Hence, PGP-14 is a key component employed by the animal to help protect it from small molecule threats. [ABSTRACT FROM AUTHOR]

Heterotrimeric G (αβγ) proteins are canonical transducers of G-protein-coupled receptor (GPCR) signaling and play critical roles in communication between cells and their environment. Many GPCRs and heterotrimeric G proteins localize to primary cilia and modulate cilia morphology via mechanisms that are not well understood. Here, we show that RIC-8, a cytosolic guanine nucleotide exchange factor (GEF) and chaperone for Gα protein subunits, shapes cilia membrane morphology in a subset of Caenorhabditis elegans sensory neurons. Consistent with its role in ciliogenesis, C. elegans RIC-8 localizes to cilia in different sensory neuron types. Using domain mutagenesis, we demonstrate that while the GEF function alone is not sufficient, both the GEF and Gα-interacting chaperone motifs of RIC-8 are required for its role in cilia morphogenesis. We identify ODR-3 as the RIC-8 Gα client and demonstrate that RIC-8 functions in the same genetic pathway with another component of the non-canonical G protein signaling AGS-3 to shape cilia morphology. Notably, despite defects in AWC cilia morphology, ags-3 null mutants exhibit normal chemotaxis toward benzaldehyde unlike odr-3 mutant animals. Collectively, our findings describe a novel function for the evolutionarily conserved protein RIC-8 and non-canonical RIC-8-AGS-3-ODR-3 signaling in cilia morphogenesis and uncouple Gα ODR-3 functions in ciliogenesis and olfaction. Author summary: Primary cilia are specialized cellular compartments that mediate communication between cells and their environment. In humans, deficits in cilia assembly and function manifest in genetic diseases called ciliopathies. While the molecular machinery of all major signaling pathways engaged in cell-cell and cell-environment communication is present inside cilia of different cell types, the mechanisms by which these signaling factors modulate cilia structure or cilia-dependent cellular functions are not well understood. In this study, we identify a new ciliary signaling module comprised of RIC-8 and Gα ODR-3 –key transducer of olfactory signaling–and find that RIC-8-dependent signaling is critically important for formation of specialized ciliary structures in C. elegans neurons. Our findings provide new insight into mechanisms of cilia assembly and highlight distinct functions of Gα ODR-3 in sensory transduction and cilia formation. [ABSTRACT FROM AUTHOR]

Regulation of immunity throughout an organism is critical for host defense. Previous studies in the nematode Caenorhabditis elegans have described an "ON/OFF" immune switch comprised of the antagonistic paralogs PALS-25 and PALS-22, which regulate resistance against intestinal and epidermal pathogens. Here, we identify and characterize a PALS-25 gain-of-function mutant protein with a premature stop (Q293*), which we find is freed from physical repression by its negative regulator, the PALS-22 protein. PALS-25(Q293*) activates two related gene expression programs, the Oomycete Recognition Response (ORR) against natural pathogens of the epidermis, and the Intracellular Pathogen Response (IPR) against natural intracellular pathogens of the intestine. A subset of ORR/IPR genes is upregulated in pals-25(Q293*) mutants, and they are resistant to oomycete infection in the epidermis, and microsporidia and virus infection in the intestine, but without compromising growth. Surprisingly, we find that activation of PALS-25 seems to primarily stimulate the downstream bZIP transcription factor ZIP-1 in the epidermis, with upregulation of gene expression in both the epidermis and in the intestine. Interestingly, we find that PALS-22/25-regulated epidermal-to-intestinal signaling promotes resistance to the N. parisii intestinal pathogen, demonstrating cross-tissue protective immune induction from one epithelial tissue to another in C. elegans.

Accurate chromosome segregation requires a cohesin-mediated physical attachment between chromosomes that are to be segregated apart, and a bipolar spindle with microtubule plus ends emanating from exactly two poles toward the paired chromosomes. We asked whether the striking bipolar structure of C. elegans meiotic chromosomes is required for bipolarity of acentriolar female meiotic spindles by time-lapse imaging of mutants that lack cohesion between chromosomes. Both a spo-11 rec-8 coh-4 coh-3 quadruple mutant and a spo-11 rec-8 double mutant entered M phase with separated sister chromatids lacking any cohesion. However, the quadruple mutant formed an apolar spindle whereas the double mutant formed a bipolar spindle that segregated chromatids into two roughly equal masses. Residual non-cohesive COH-3/4-dependent cohesin on separated sister chromatids of the double mutant was sufficient to recruit haspin-dependent Aurora B kinase, which mediated bipolar spindle assembly in the apparent absence of chromosomal bipolarity. We hypothesized that cohesin-dependent Aurora B might activate or inhibit spindle assembly factors in a manner that would affect their localization on chromosomes and found that the chromosomal localization patterns of KLP-7 and CLS-2 correlated with Aurora B loading on chromosomes. These results demonstrate that cohesin is essential for spindle assembly and chromosome segregation independent of its role in sister chromatid cohesion.

A family of giant KASH proteins, including C. elegans ANC-1 and mammalian Nesprin-1 and -2, are involved in organelle anchoring and are associated with multiple neurodevelopmental disorders including autism, bipolar disorder, and schizophrenia. However, little is known about how these proteins function in neurons. Moreover, the role of organelle anchoring in axon development is poorly understood. Here, we report that ANC-1 functions with the SLT-1 extracellular guidance cue to polarize ALM axon growth. This role for ANC-1 is specific to its longer ANC-1A and ANC-1C isoforms, suggesting that it is mechanistically distinct from previously described roles for ANC-1. We find that ANC-1 is required for the localization of a cluster of mitochondria to the base of the proximal axon. Furthermore, genetic and pharmacological studies indicate that ANC-1 functions with mitochondria to promote polarization of ALM axon growth. These observations reveal a mechanism whereby ANC-1 functions through mitochondria to polarize axon growth in response to SLT-1.

Meiotic homolog synapsis is essential to ensure accurate segregation of chromosomes during meiosis. In C. elegans, proper regulation of synapsis and a checkpoint that monitors synapsis relies on the spindle checkpoint components, Mad1 and Mad2, and Pairing Centers (PCs), cis-acting loci that interact with the nuclear envelope to mobilize chromosomes within the nucleus. Here, we test what specific functions of Mad1 and Mad2 are required to regulate and monitor synapsis. We find that a mutation that prevents Mad1s localization to the nuclear periphery abolishes the synapsis checkpoint but has no effect on Mad2s localization to the nuclear periphery or synapsis. By contrast, a mutation that prevents Mad1s interaction with Mad2 abolishes the synapsis checkpoint, delays synapsis and fails to localize Mad2 to the nuclear periphery. These data indicate that Mad1s primary role in regulating synapsis is through control of Mad2 and that Mad2 can bind other factors at the nuclear periphery. We also tested whether Mad2s ability to adopt a specific conformation associated with its activity during spindle checkpoint function is required for its role in meiosis. A mutation that prevents Mad2 from adopting its active conformer fails to localize to the nuclear periphery, abolishes the synapsis checkpoint and exhibits substantial defects in meiotic synapsis. Thus, Mad2, and its regulation by Mad1, is an important regulator of meiotic synapsis in C. elegans.

During C. elegans oocyte meiosis I cytokinesis and polar body extrusion, cortical actomyosin is locally remodeled to assemble a contractile ring that forms within and remains part of a much larger and actively contractile cortical actomyosin network. This network both mediates contractile ring dynamics and generates shallow ingressions throughout the oocyte cortex during polar body extrusion. Based on our analysis of requirements for CLS-2, a member of the CLASP family of proteins that stabilize microtubules, we recently proposed that a balance of actomyosin-mediated tension and microtubule-mediated stiffness limits membrane ingression throughout the oocyte during meiosis I polar body extrusion. Here, using live cell imaging and fluorescent protein fusions, we show that CLS-2 is part of a group of kinetochore proteins, including the scaffold KNL-1 and the kinase BUB-1, that also co-localize during meiosis I to structures called linear elements, which are present within the assembling oocyte spindle and also are distributed throughout the oocyte in proximity to, but appearing to underlie, the actomyosin cortex. We further show that KNL-1 and BUB-1, like CLS-2, promote the proper organization of sub-cortical microtubules and also limit membrane ingression throughout the oocyte. Moreover, nocodazole or taxol treatment to destabilize or stabilize oocyte microtubules leads to, respectively, excess or decreased membrane ingression throughout the oocyte. Furthermore, taxol treatment, and genetic backgrounds that elevate the levels of cortically associated microtubules, both suppress excess membrane ingression in cls-2 mutant oocytes. We propose that linear elements influence the organization of sub-cortical microtubules to generate a stiffness that limits cortical actomyosin-driven membrane ingression throughout the oocyte during meiosis I polar body extrusion. We discuss the possibility that this regulation of sub-cortical microtubule dynamics facilitates actomyosin contractile ring dynamics during C. elegans oocyte meiosis I cell division. Author summary: Animal oocytes undergo a sequence of two specialized cell divisions called meiosis I and II in which three quarters of the genome content is expelled into two small, extracellular, membrane-bound compartments called polar bodies. These two meiotic divisions reduce the genome content of the oocyte from a diploid state, with two copies of each chromosome, to a haploid state, with a single copy of each chromosome. As a result, fertilization by a sperm restores the normal diploid genome content. We provide evidence that tubular cytoskeletal polymers called microtubules are enriched near the inner surface the of oocyte cell membrane, where they provide a stiffness that resists membrane ingressions driven by the force-generating cortical actomyosin cytoskeleton. These membrane ingressions occur throughout the oocyte during meiotic polar body extrusion. Our results suggest that a balance of microtubule-mediated stiffness and actomyosin-mediated membrane ingression throughout the oocyte may be important for the proper assembly and function of the contractile ring that pinches off polar bodies at one end of the oocyte during oocyte meiotic cell division. [ABSTRACT FROM AUTHOR]

Adenylosuccinate lyase deficiency is an ultrarare congenital metabolic disorder associated with muscle weakness and neurobehavioral dysfunction. Adenylosuccinate lyase is required for de novo purine biosynthesis, acting twice in the pathway at non-sequential steps. Genetic models can contribute to our understanding of the etiology of disease phenotypes and pave the way for development of therapeutic treatments. Here, we establish the first model to specifically study neurobehavioral aspects of adenylosuccinate lyase deficiency. We show that reduction of adsl-1 function in C. elegans is associated with a novel learning phenotype in a gustatory plasticity assay. The animals maintain capacity for gustatory plasticity, evidenced by a change in their behavior in response to cue pairing. However, their behavioral output is distinct from that of control animals. We link substrate accumulation that occurs upon adsl-1 deficiency to an unexpected perturbation in tyrosine metabolism and show that a lack of tyramine mediates the behavioral changes through action on the metabotropic TYRA-2 tyramine receptor. Our studies reveal a potential for wider metabolic perturbations, beyond biosynthesis of purines, to impact behavior under conditions of adenylosuccinate lyase deficiency. Author summary: Adenylosuccinate lyase deficiency is an ultra-rare metabolic disorder associated with muscle and neural dysfunction, including muscle weakness, psychomotor delay, and repetitive vocalizations and behaviors. The disorder has been modeled successfully in the nematode C. elegans in terms of recapitulating the metabolic signatures and the muscular deficits. However, a model for studying neurobehavioral aspects of the disorder has not been established. We have used C. elegans to explore the cause of neurobehavioral phenotypes associated with adenylosuccinate lyase deficiency. We found that reduction of adsl-1 function in C. elegans results in an exaggerated response in a gustatory plasticity assay. Naïve control animals are attracted to salt but become indifferent to salt after being starved in the presence of salt. When adsl-1 function is compromised, animals change behavior after starvation-cue pairing but show a repulsive response in contrast to the indifferent response of control animals. The adsl-1 animals are capable of learning from experience but their response to that learning is distinct. We also discovered that this abnormal behavior is caused by a deficiency in production of the metabolite tyramine and is shared by other mutants with tyramine signaling deficits. Finally, we demonstrate that it is likely the accumulation of a toxic substrate that causes the phenotype as opposed to perturbation of purine metabolism. Development of this model and these findings have exciting implications for therapeutic development for adenylosuccinate lyase deficiency. [ABSTRACT FROM AUTHOR]

mTORC1 (mechanistic target of rapamycin complex 1) is a metabolic sensor that promotes growth when nutrients are abundant. Ubiquitous inhibition of mTORC1 extends lifespan in multiple organisms but also disrupts several anabolic processes resulting in stunted growth, slowed development, reduced fertility, and disrupted metabolism. However, it is unclear if these pleotropic effects of mTORC1 inhibition can be uncoupled from longevity. Here, we utilize the auxin-inducible degradation (AID) system to restrict mTORC1 inhibition to C. elegans neurons. We find that neuron-specific degradation of RAGA-1, an upstream activator of mTORC1, or LET-363, the ortholog of mammalian mTOR, is sufficient to extend lifespan in C. elegans. Unlike raga-1 loss of function genetic mutations or somatic AID of RAGA-1, neuronal AID of RAGA-1 robustly extends lifespan without impairing body size, developmental rate, brood size, or neuronal function. Moreover, while degradation of RAGA-1 in all somatic tissues alters the expression of thousands of genes, demonstrating the widespread effects of mTORC1 inhibition, degradation of RAGA-1 in neurons only results in around 200 differentially expressed genes with a specific enrichment in metabolism and stress response. Notably, our work demonstrates that targeting mTORC1 specifically in the nervous system in C. elegans uncouples longevity from growth and reproductive impairments, and that many canonical effects of low mTORC1 activity are not required to promote healthy aging. These data challenge previously held ideas about the mechanisms of mTORC1 lifespan extension and underscore the potential of promoting longevity by neuron-specific mTORC1 modulation. Author summary: Inhibition of the metabolic pathway mTORC1 has been shown to extend lifespan in many organisms and has gained traction as a possible anti-aging therapeutic. However, in addition to lifespan extension, chronic and body-wide mTORC1 inhibition results in a downregulation of anabolic processes that leads to unwanted side effects such as impaired growth, development, and reproduction in C. elegans, Drosophila melanogaster, and mice. An open question in the field is if mTORC1 inhibition can be restricted to specific tissues to promote longevity while minimizing the anabolic trade-offs. In this study, we test this hypothesis using the model organism C. elegans and the auxin-inducible degradation (AID) system which provides many advantages over previous techniques used to study the tissue-specificity of mTORC1 in aging. We find that degradation of mTORC1 pathway components specifically in the neurons robustly extends lifespan while preserving normal growth, development, and reproduction. Furthermore, we find that animals with neuronal mTORC1 inhibition maintain functional sensory neurons and have changes in the expression of genes related to stress response and metabolism. This work challenges previously held theories that anabolic trade-offs are required for mTORC1 longevity and highlights a critical role for neuronal mTORC1 in the regulation of organismal aging. [ABSTRACT FROM AUTHOR]

The nematode Caenorhabditis elegans memorizes various external chemicals, such as ions and odorants, during feeding. Here we find that C. elegans is attracted to the monosaccharides glucose and fructose after exposure to these monosaccharides in the presence of food; however, it avoids them without conditioning. The attraction to glucose requires a gustatory neuron called ASEL. ASEL activity increases when glucose concentration decreases. Optogenetic ASEL stimulation promotes forward movements; however, after glucose conditioning, it promotes turning, suggesting that after glucose conditioning, the behavioral output of ASEL activation switches toward glucose. We previously reported that chemotaxis toward sodium ion (Na+), which is sensed by ASEL, increases after Na+ conditioning in the presence of food. Interestingly, glucose conditioning decreases Na+ chemotaxis, and conversely, Na+ conditioning decreases glucose chemotaxis, suggesting the reciprocal inhibition of learned chemotaxis to distinct chemicals. The activation of PKC-1, an nPKC ε/η ortholog, in ASEL promotes glucose chemotaxis and decreases Na+ chemotaxis after glucose conditioning. Furthermore, genetic screening identified ENSA-1, an ortholog of the protein phosphatase inhibitor ARPP-16/19, which functions in parallel with PKC-1 in glucose-induced chemotactic learning toward distinct chemicals. These findings suggest that kinase–phosphatase signaling regulates the balance between learned behaviors based on glucose conditioning in ASEL, which might contribute to migration toward chemical compositions where the animals were previously fed. Author summary: Caenorhabditis elegans responds to compounds that taste salty, bitter, sour, etc. However, its response to sweet compounds is unclear. Here, we show that C. elegans responds to glucose through a chemosensory neuron called ASEL. C. elegans avoids high concentrations of glucose and learns to approach glucose after feeding in the presence of high glucose, dependent on the action of ASEL. The ASEL neuron has been reported to respond to salt and promotes salt attraction after feeding in the presence of high salt. We find that the feeding-associated attractive responses to glucose and salt are antagonistic. When encountered with a mixture of salt and glucose during feeding, C. elegans changes its chemotactic response toward those chemicals according to the balance of each chemical in the mixture. C. elegans may memorize the concentrations of the chemical mixture during feeding and migrates to the chemical composition previously fed, which may promote opportunities for obtaining food. Furthermore, we find that kinase–phosphatase signaling, which modulates neurotransmission, in ASEL is required for chemotaxis based on information processing of salt and glucose. [ABSTRACT FROM AUTHOR]

Regulation of the microtubule cytoskeleton is crucial for the development and maintenance of neuronal architecture, and recent studies have highlighted the significance of regulated RNA processing in the establishment and maintenance of neural circuits. In a genetic screen conducted using mechanosensory neurons of C. elegans, we identified a mutation in muscleblind-1/mbl-1 as a suppressor of loss of kinesin-13 family microtubule destabilizing factor klp-7. Muscleblind-1(MBL-1) is an RNA-binding protein that regulates the splicing, localization, and stability of RNA. Our findings demonstrate that mbl-1 is required cell-autonomously for axon growth and proper synapse positioning in the posterior lateral microtubule (PLM) neuron. Loss of mbl-1 leads to increased microtubule dynamics and mixed orientation of microtubules in the anterior neurite of PLM. These defects are also accompanied by abnormal axonal transport of the synaptic protein RAB-3 and reduction of gentle touch sensation in mbl-1 mutant. Our data also revealed that mbl-1 is genetically epistatic to mec-7 (β tubulin) and mec-12 (α tubulin) in regulating axon growth. Furthermore, mbl-1 is epistatic to sad-1, an ortholog of BRSK/Brain specific-serine/threonine kinase and a known regulator of synaptic machinery, for synapse formation at the correct location of the PLM neurite. Notably, the immunoprecipitation of MBL-1 resulted in the co-purification of mec-7, mec-12, and sad-1 mRNAs, suggesting a direct interaction between MBL-1 and these transcripts. Additionally, mbl-1 mutants exhibited reduced levels and stability of mec-7 and mec-12 transcripts. Our study establishes a previously unknown link between RNA-binding proteins and cytoskeletal machinery, highlighting their crucial roles in the development and maintenance of the nervous system. Author summary: Both RNA-binding proteins and microtubule cytoskeleton play important roles in the development and maintenance of a functional nervous system. A misregulation of mRNA processing and microtubule dynamics often contributes to neurodevelopmental and neurodegenerative disorders. Through a combination of genetic approaches and live cell imaging in the nematode C. elegans, we have uncovered a molecular connection between the RNA binding protein Muscleblind-1 (MBNL) and the microtubule cytoskeleton, specifically in neurons responsible for gentle touch sensation. Our investigations have revealed that Muscleblind-1/MBL-1 is involved in the optimal expression of the mRNAs that encode for the building blocks of microtubules, namely, the tubulin subunits MEC-7 (β-tubulin) and MEC-12 (α-tubulin) in touch neurons. Additionally, MBL-1 binds to another mRNA, sad-1, which encodes a kinase protein essential for synapse formation. In the absence of mbl-1, the microtubule cytoskeleton becomes unstable, leading to significant disruptions in the transport system within the neurite. Consequently, this impairment results in a reduction of sensory function performed by this set of neurons. The findings from our study provide a mechanistic insight into how RNA-binding proteins and the microtubule cytoskeleton are linked in neurons. [ABSTRACT FROM AUTHOR]

Caenorhabditis nematodes form an excellent model for studying how the mode of reproduction affects genetic diversity, as some species reproduce via outcrossing whereas others can self-fertilize. Currently, chromosome-level patterns of diversity and recombination are only available for self-reproducing Caenorhabditis, making the generality of genomic patterns across the genus unclear given the profound potential influence of reproductive mode. Here we present a whole-genome diversity landscape, coupled with a new genetic map, for the outcrossing nematode C. remanei. We demonstrate that the genomic distribution of recombination in C. remanei, like the model nematode C. elegans, shows high recombination rates on chromosome arms and low rates toward the central regions. Patterns of genetic variation across the genome are also similar between these species, but differ dramatically in scale, being tenfold greater for C. remanei. Historical reconstructions of variation in effective population size over the past million generations echo this difference in polymorphism. Evolutionary simulations demonstrate how selection, recombination, mutation, and selfing shape variation along the genome, and that multiple drivers can produce patterns similar to those observed in natural populations. The results illustrate how genome organization and selection play a crucial role in shaping the genomic pattern of diversity whereas demographic processes scale the level of diversity across the genome as a whole. Author summary: The mode of reproductive exchange among individuals has a profound effect on genetic diversity. In self-reproducing organisms, absence of genetic interchange between individuals reduces the effective population size and increases linkage among segregating sites at different genes, leading to lower diversity than outcrossing species. Caenorhabditis nematodes offer an exceptional system for studying the genomic effects of different systems of mating. While selfing species such as C. elegans have been studied, we present the first recombination map and genome-wide landscape of polymorphism for an outcrossing member of the genus, C. remanei. We find that, similar to C. elegans, C. remanei has high recombination rates on chromosome arms and low rates in central regions. The genomic diversity landscapes of these species are qualitatively similar, with higher diversity in the regions of higher recombination. However, C. remanei exhibits tenfold greater diversity than C. elegans due to their much larger effective population size and the decreased impact of linked selection as an outcrossing species. We use evolutionary simulations to show the influence of genomic and demographic processes work on these patterns. This work illustrates how understanding complex interactions among genetics, genomics, and reproduction is fundamental to describing patterns of genetic variation within natural populations. [ABSTRACT FROM AUTHOR]

Metabolic pathways are known to sense the environmental stimuli and result in physiological adjustments. The responding processes need to be tightly controlled. Here, we show that upon encountering P. aeruginosa, C. elegans upregulate the transcription factor ets-4, but this upregulation is attenuated by the ribonuclease, rege-1. As such, mutants with defective REGE-1 ribonuclease activity undergo ets-4-dependent early death upon challenge with P. aeruginosa. Furthermore, mRNA-seq analysis revealed associated global changes in two key metabolic pathways, the IIS (insulin/IGF signaling) and TOR (target of rapamycin) kinase signaling pathways. In particular, failure to degrade ets-4 mRNA in activity-defective rege-1 mutants resulted in upregulation of class II longevity genes, which are suppressed during longevity, and activation of TORC1 kinase signaling pathway. Genetic inhibition of either pathway way was sufficient to abolish the poor survival phenotype in rege-1 worms. Further analysis of ETS-4 ChIP data from ENCODE and characterization of one upregulated Class II gene, ins-7, support that the Class II genes are activated by ETS-4. Interestingly, deleting an upregulated Class II gene, acox-1.5, a peroxisome β-oxidation enzyme, largely rescues the fat lost phenotype and survival difference between rege-1 mutants and wild-types. Thus, rege-1 appears to be crucial for animal survival due to its tight regulation of physiological responses to environmental stimuli. This function is reminiscent of its mammalian ortholog, Regnase-1, which modulates the intestinal mTORC1 signaling pathway. Author summary: Eukaryotes rely on tightly regulated insulin-IGF signaling and TOR pathways to maintain proper cellular processes such as metabolism, aging, and pathogen defense, which allow them to sense and respond to changes in nutrient availability and environmental stress. While Regnase-1 is known for its role in regulating immune response in mammals, the importance of its ortholog, rege-1, in C. elegans was previously unclear. Our study revealed that rege-1 participates in regulating the TOR signaling pathway, which subsequently affects C. elegans lifespan and pathogen defense. By post-transcriptionally regulating the ets-4 transcription factor, rege-1 is able to regulate a set of longevity-suppressed genes, including those involved in the peroxisome fatty acid β-oxidation pathway, likely affecting the level of acetyl-COA and modulating the TOR signaling pathway. Our findings indicate that defective rege-1 leads to an excess of ets-4, which activates the lipid β-oxidation pathway and further activates the TOR pathway, ultimately reducing lifespan and survival through pathogen and fat loss. [ABSTRACT FROM AUTHOR]

Dysregulation of collagen production and secretion contributes to aging and tissue fibrosis of major organs. How procollagen proteins in the endoplasmic reticulum (ER) route as specialized cargos for secretion remains to be fully elucidated. Here, we report that TMEM39, an ER-localized transmembrane protein, regulates production and secretory cargo trafficking of procollagen. We identify the C. elegans ortholog TMEM-39 from an unbiased RNAi screen and show that deficiency of tmem-39 leads to striking defects in cuticle collagen production and constitutively high ER stress response. RNAi knockdown of the tmem-39 ortholog in Drosophila causes similar defects in collagen secretion from fat body cells. The cytosolic domain of human TMEM39A binds to Sec23A, a vesicle coat protein that drives collagen secretion and vesicular trafficking. TMEM-39 regulation of collagen secretion is independent of ER stress response and autophagy. We propose that the roles of TMEM-39 in collagen secretion and ER homeostasis are likely evolutionarily conserved.

Some organisms in nature have developed the ability to enter a state of suspended metabolism called cryptobiosis when environmental conditions are unfavorable. This state-transition requires execution of a combination of genetic and biochemical pathways that enable the organism to survive for prolonged periods. Recently, nematode individuals have been reanimated from Siberian permafrost after remaining in cryptobiosis. Preliminary analysis indicates that these nematodes belong to the genera Panagrolaimus and Plectus. Here, we present precise radiocarbon dating indicating that the Panagrolaimus individuals have remained in cryptobiosis since the late Pleistocene (~46,000 years). Phylogenetic inference based on our genome assembly and a detailed morphological analysis demonstrate that they belong to an undescribed species, which we named Panagrolaimus kolymaensis. Comparative genome analysis revealed that the molecular toolkit for cryptobiosis in P. kolymaensis and in C. elegans is partly orthologous. We show that biochemical mechanisms employed by these two species to survive desiccation and freezing under laboratory conditions are similar. Our experimental evidence also reveals that C. elegans dauer larvae can remain viable for longer periods in suspended animation than previously reported. Altogether, our findings demonstrate that nematodes evolved mechanisms potentially allowing them to suspend life over geological time scales. Author summary: Survival in extreme environments for prolonged periods is a challenge that only a few organisms, are capable of. It is not well understood, which molecular and biochemical pathways are utilized by such cryptobiotic organisms, and how long they might suspend life. Here, we show that a soil nematode Panagrolaimus kolymaensis, suspended life for 46,000 years in the Siberian permafrost. Through comparative analysis, we find that P. kolymaensis and model organism C. elegans utilize similar adaptive mechanisms to survive harsh environmental conditions for prolonged periods. Our findings here are important for the understanding of evolutionary processes because generation times could be stretched from days to millennia, and long-term survival of individuals of species can lead to the refoundation of otherwise extinct lineages. [ABSTRACT FROM AUTHOR]

While it has been appreciated for decades that prophase-arrested oocytes are transcriptionally silenced on a global level, the molecular pathways that promote silencing have remained elusive. Previous work in C. elegans has shown that both topoisomerase II (TOP-2) and condensin II collaborate with the H3K9me heterochromatin pathway to silence gene expression in the germline during L1 starvation, and that the PIE-1 protein silences the genome in the P-lineage of early embryos. Here, we show that all three of these silencing systems, TOP-2/condensin II, H3K9me, and PIE-1, are required for transcriptional repression in oocytes. We find that H3K9me3 marks increase dramatically on chromatin during silencing, and that silencing is under cell cycle control. We also find that PIE-1 localizes to the nucleolus just prior to silencing, and that nucleolar dissolution during silencing is dependent on TOP-2/condensin II. Our data identify both the molecular components and the trigger for genome silencing in oocytes and establish a link between PIE-1 nucleolar residency and its ability to repress transcription. Author summary: In most animals, oogenesis involves a genome-wide repression of transcription as oocytes approach maturation and fertilization. While transcriptional repression in fully grown oocytes was first documented in the early 1960s, the molecular players that promote genome silencing had yet to be identified. Here, we study the round worm C. elegans and we find that three previously defined genome silencing systems are all operational in pre-maturation oocytes and are all required for silencing. The first system, topoisomerase II acting together with the condensin II complex, promotes a hyper compaction of oocyte chromatin, which likely explains its role in genome silencing. The second, components of a system that modifies chromatin to produce heterochromatin, also drives compaction of the oocyte chromatin. Interestingly, the third system—the PIE-1 protein—is required for silencing but not for chromatin compaction. Our data show that while chromatin compaction is required for genome silencing in oocytes, it is not sufficient. Our studies open the door to more mechanistic analysis of how these pathways can silence gene expression on a global level. [ABSTRACT FROM AUTHOR]

The successful delivery of genetic material to gametes requires tightly regulated interactions between the parental chromosomes. Central to this regulation is a conserved chromosomal interface called the synaptonemal complex (SC), which brings the parental chromosomes in close proximity along their length. While many of its components are known, the interfaces that mediate the assembly of the SC remain a mystery. Here, we survey findings from different model systems while focusing on insight gained in the nematode C. elegans. We synthesize our current understanding of the structure, dynamics, and biophysical properties of the SC and propose mechanisms for SC assembly. [ABSTRACT FROM AUTHOR]

Genetic robustness can be achieved via several mechanisms including transcriptional adaptation (TA), a sequence similarity-driven process whereby mutant mRNA degradation products modulate, directly or indirectly, the expression of so-called adapting genes. To identify the sequences required for this process, we utilized a transgenic approach in Caenorhabditis elegans, combining an overexpression construct for a mutant gene (act-5) and a fluorescent reporter for the corresponding adapting gene (act-3). Analyzing a series of modifications for each construct, we identified, in the 5' regulatory region of the act-3 locus, a 25-base pair (bp) element which exhibits 60% identity with a sequence in the act-5 mRNA and which, in the context of a minimal promoter, is sufficient to induce ectopic expression of the fluorescent reporter. The 25 nucleotide (nt) element in the act-5 mRNA lies between the premature termination codon (PTC) and the next exon/exon junction, suggesting the importance of this region of the mutant mRNA for TA. Additionally, we found that single-stranded RNA injections of this 25 nt element from act-5 into the intestine of wild-type larvae led to higher levels of adapting gene (act-3) mRNA. Different models have been proposed to underlie the modulation of gene expression during TA including chromatin remodeling, the inhibition of antisense RNAs, the release of transcriptional pausing, and the suppression of premature transcription termination, and our data clearly show the importance of the regulatory region of the adapting gene in this particular act-5/act-3 TA model. Our findings also suggest that RNA fragments can modulate the expression of loci exhibiting limited sequence similarity, possibly a critical observation when designing RNA based therapies. Author summary: Transcriptional adaptation (TA) is a recently identified process that modulates the transcriptome of a cell in response to a mutation in a gene, independent of the mutation's effect on the encoded protein. Depending on the identity of the gene(s) whose expression is modulated, TA can lead to functional compensation or a worse phenotype. TA requires mutant mRNA degradation, and the current hypothesis is that the resulting RNA fragments, or their derivatives, modulate the expression of the so-called adapting genes. A major unresolved question about TA is how the mutant and adapting gene(s) are linked, and it has been proposed that sequence similarity plays an important role. Here, we link a short DNA sequence in the regulatory region of an adapting gene (act-3) to a corresponding sequence in the mutant gene (act-5), and also show that small RNAs containing this act-5 sequence element can lead to higher levels of adapting gene (act-3) mRNA. Because the sequence similarity between these two elements is lower than expected, these results are significant as they suggest that the number of potential adapting genes for a given mutant gene is currently underestimated. [ABSTRACT FROM AUTHOR]

Retroviruses and closely related LTR retrotransposons export full-length, unspliced genomic RNA (gRNA) for packaging into virions and to serve as the mRNA encoding GAG and POL polyproteins. Because gRNA often includes splice acceptor and donor sequences used to splice viral mRNAs, retroelements must overcome host mechanisms that retain intron-containing RNAs in the nucleus. Here we examine gRNA expression in Cer1, an LTR retrotransposon in C. elegans which somehow avoids silencing and is highly expressed in germ cells. Newly exported Cer1 gRNA associates rapidly with the Cer1 GAG protein, which has structural similarity with retroviral GAG proteins. gRNA export requires CERV (C. elegansregulator of viral expression), a novel protein encoded by a spliced Cer1 mRNA. CERV phosphorylation at S214 is essential for gRNA export, and phosphorylated CERV colocalizes with nuclear gRNA at presumptive sites of transcription. By electron microscopy, tagged CERV proteins surround clusters of distinct, linear fibrils that likely represent gRNA molecules. Single fibrils, or groups of aligned fibrils, also localize near nuclear pores. During the C. elegans self-fertile period, when hermaphrodites fertilize oocytes with their own sperm, CERV concentrates in two nuclear foci that are coincident with gRNA. However, as hermaphrodites cease self-fertilization, and can only produce cross-progeny, CERV undergoes a remarkable transition to form giant nuclear rods or cylinders that can be up to 5 microns in length. We propose a novel mechanism of rod formation, in which stage-specific changes in the nucleolus induce CERV to localize to the nucleolar periphery in flattened streaks of protein and gRNA; these streaks then roll up into cylinders. The rods are a widespread feature of Cer1 in wild strains of C. elegans, but their function is not known and might be limited to cross-progeny. We speculate that the adaptive strategy Cer1 uses for the identical self-progeny of a host hermaphrodite might differ for heterozygous cross-progeny sired by males. For example, mating introduces male chromosomes which can have different, or no, Cer1 elements. Author summary: LTR retrotransposons are closely related to retroviruses and are enormously abundant in animals and plants. Cer1 is the most prevalent LTR retrotransposon in the nematode C. elegans, where it is expressed at high levels in adult germ cells. Cer1 produces typical retroviral proteins except for a novel protein called CERV. CERV appears to allow unspliced Cer1 genomic RNA to be exported from the nucleus, escaping host mechanisms that normally retain unspliced RNA. The nuclear export of gRNA is regulated by multiple ON/OFF switches controlled by sex, developmental stage, and environment. CERV is a diffuse nucleoplasmic protein in the OFF states, but in the ON states CERV colocalizes with nuclear gRNA. By transmission electron microscopy, CERV is associated with unusual and distinct linear fibrils that appear to represent gRNA molecules. In older germ cells, CERV undergoes a remarkable transition to form giant cylindrical rods of unknown function that can equal the nuclear diameter in length. Rod formation occurs when C. elegans hermaphrodites can no longer fertilize their own oocytes, and instead require mating with males. Thus, the rods might be part of adaptive strategies Cer1 uses to distinguish potentially heterozygous cross-progeny from homozygous self-progeny. [ABSTRACT FROM AUTHOR]

The conserved ATPase, PCH-2/TRIP13, is required during both the spindle checkpoint and meiotic prophase. However, its specific role in regulating meiotic homolog pairing, synapsis and recombination has been enigmatic. Here, we report that this enzyme is required to proofread meiotic homolog interactions. We generated a mutant version of PCH-2 in C. elegans that binds ATP but cannot hydrolyze it: pch-2E253Q. In vitro, this mutant can bind a known substrate but is unable to remodel it. This mutation results in some non-homologous synapsis and impaired crossover assurance. Surprisingly, worms with a null mutation in PCH-2s adapter protein, CMT-1, the ortholog of p31comet, localize PCH-2 to meiotic chromosomes, exhibit non-homologous synapsis and lose crossover assurance. The similarity in phenotypes between cmt-1 and pch-2E253Q mutants suggest that PCH-2 can bind its meiotic substrates in the absence of CMT-1, in contrast to its role during the spindle checkpoint, but requires its adapter to hydrolyze ATP and remodel them.

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.

Human NIMA-related kinases have primarily been studied for their roles in cell cycle progression (NEK1/2/6/7/9), checkpoint–DNA-damage control (NEK1/2/4/5/10/11), and ciliogenesis (NEK1/4/8). We previously showed that Caenorhabditis elegans NEKL-2 (NEK8/9 homolog) and NEKL-3 (NEK6/7 homolog) regulate apical clathrin-mediated endocytosis (CME) in the worm epidermis and are essential for molting. Here we show that NEKL-2 and NEKL-3 also have distinct roles in controlling endosome function and morphology. Specifically, loss of NEKL-2 led to enlarged early endosomes with long tubular extensions but showed minimal effects on other compartments. In contrast, NEKL-3 depletion caused pronounced defects in early, late, and recycling endosomes. Consistently, NEKL-2 was strongly localized to early endosomes, whereas NEKL-3 was localized to multiple endosomal compartments. Loss of NEKLs also led to variable defects in the recycling of two resident cargoes of the trans-Golgi network (TGN), MIG-14/Wntless and TGN-38/TGN38, which were missorted to lysosomes after NEKL depletion. In addition, defects were observed in the uptake of clathrin-dependent (SMA-6/Type I BMP receptor) and independent cargoes (DAF-4/Type II BMP receptor) from the basolateral surface of epidermal cells after NEKL-2 or NEKL-3 depletion. Complementary studies in human cell lines further showed that siRNA knockdown of the NEKL-3 orthologs NEK6 and NEK7 led to missorting of the mannose 6-phosphate receptor from endosomes. Moreover, in multiple human cell types, depletion of NEK6 or NEK7 disrupted both early and recycling endosomal compartments, including the presence of excess tubulation within recycling endosomes, a defect also observed after NEKL-3 depletion in worms. Thus, NIMA family kinases carry out multiple functions during endocytosis in both worms and humans, consistent with our previous observation that human NEKL-3 orthologs can rescue molting and trafficking defects in C. elegans nekl-3 mutants. Our findings suggest that trafficking defects could underlie some of the proposed roles for NEK kinases in human disease. Author summary: Intracellular trafficking is an evolutionary conserved process whereby cargoes, which include proteins, lipids, and other macromolecules, are internalized by cells, packaged into vesicles, and distributed to their proper places within the cell. This study demonstrated that two conserved NIMA-related kinases, NEKL-2 and NEKL-3, are required for the transport of multiple cargoes in the epidermis of C. elegans. NEKL-2 and NEKL-3 function at organelles, called endosomes, to regulate their morphology and control the sorting of cargoes between different intracellular compartments. In the absence of NEKL activities, various cargoes, including components of the BMP and Wnt signaling pathways, were misregulated. Our studies are further supported by results showing that the human counterparts of NEKL-3, NEK6 and NEK7, were also required for maintaining endosome morphologies and for the proper sorting of cargo in human cells. Notably, NIMA-kinases are well studied for their roles in cell cycle regulation, and overexpression of these kinases is linked to cancer formation and poor prognosis. Our study suggests their role in cancer progression could be partly due to the abnormal intracellular trafficking of conserved signaling components with known roles in cancer formation. [ABSTRACT FROM AUTHOR]

Oxidative stress response is a fundamental biological process mediated by conserved mechanisms. The identities and functions of some key regulators remain unknown. Here, we report a novel role of C. elegans casein kinase 1 gamma CSNK-1 (also known as CK1γ or CSNK1G) in regulating oxidative stress response and ROS levels. csnk-1 interacted with the bli-3/tsp-15/doxa-1 NADPH dual oxidase genes via genetic nonallelic noncomplementation to affect C. elegans survival in oxidative stress. The genetic interaction was supported by specific biochemical interactions between DOXA-1 and CSNK-1 and potentially between their human orthologs DUOXA2 and CSNK1G2. Consistently, CSNK-1 was required for normal ROS levels in C. elegans. CSNK1G2 and DUOXA2 each can promote ROS levels in human cells, effects that were suppressed by a small molecule casein kinase 1 inhibitor. We also detected genetic interactions between csnk-1 and skn-1 Nrf2 in oxidative stress response. Together, we propose that CSNK-1 CSNK1G defines a novel conserved regulatory mechanism for ROS homeostasis. Author summary: Oxidative stress response is a fundamental life process that regulates the generation and elimination of reactive oxygen species (ROS) and manages ROS-elicited damages. Some key regulatory mechanisms remain to be elucidated. While analyzing mutations promoting the survival of the nematode Caenorhabditis elegans in excess iodide, an oxidative stressor, we identified the casein kinase 1 gamma gene csnk-1. Casein kinase 1 gamma proteins are highly conserved and proposed to have important biomedical functions. However, they are understudied and their effects on oxidative stress response are unknown. We combined genetic, biochemical and cell biology approaches to show that casein kinase 1 gamma members in C. elegans and humans are novel regulators of oxidative stress response and promoters of ROS levels. These activities involve interactions with the NADPH dual oxidase complex, which are likely mediated by physical interactions between casein kinase 1 gamma and the dual oxidase maturation factor. Our findings provide new mechanistic insights into the regulation of ROS homeostasis and uncover an important function of the "dark" casein kinase 1 gamma. [ABSTRACT FROM AUTHOR]

One of the most common cell shape changes driving morphogenesis in diverse animals is the constriction of the apical cell surface. Apical constriction depends on contraction of an actomyosin network in the apical cell cortex, but such actomyosin networks have been shown to undergo continual, conveyor belt-like contractions before the shrinking of an apical surface begins. This finding suggests that apical constriction is not necessarily triggered by the contraction of actomyosin networks, but rather can be triggered by unidentified, temporally-regulated mechanical links between actomyosin and junctions. Here, we used C. elegans gastrulation as a model to seek genes that contribute to such dynamic linkage. We found that α-catenin and β-catenin initially failed to move centripetally with contracting cortical actomyosin networks, suggesting that linkage is regulated between intact cadherin-catenin complexes and actomyosin. We used proteomic and transcriptomic approaches to identify new players, including the candidate linkers AFD-1/afadin and ZYX-1/zyxin, as contributing to C. elegans gastrulation. We found that ZYX-1/zyxin is among a family of LIM domain proteins that have transcripts that become enriched in multiple cells just before they undergo apical constriction. We developed a semi-automated image analysis tool and used it to find that ZYX-1/zyxin contributes to cell-cell junctions' centripetal movement in concert with contracting actomyosin networks. These results identify several new genes that contribute to C. elegans gastrulation, and they identify zyxin as a key protein important for actomyosin networks to effectively pull cell-cell junctions inward during apical constriction. The transcriptional upregulation of ZYX-1/zyxin in specific cells in C. elegans points to one way that developmental patterning spatiotemporally regulates cell biological mechanisms in vivo. Because zyxin and related proteins contribute to membrane-cytoskeleton linkage in other systems, we anticipate that its roles in regulating apical constriction in this manner may be conserved. Author summary: Animals take shape during development in large part by the bending of tissues. Failures in this process are common causes of human birth defects. Such tissue bending is driven primarily by individual cells changing shape: in many examples, one side of a cell shrinks, pulling on junctions that connect the cell to neighboring cells. But the networks that drive one side of a cell to shrink are not always connected to junctions. As a result, focus has turned to understanding how connections between such networks and junctions are dynamically regulated to trigger cell shape change. We sought to identify genes that contribute to these dynamic connections. Here, we describe proteomic and transcriptomic methods that we used to identify proteins that contribute to cell shape change. We developed a new image analysis tool and used it to reveal that loss of one of these genes results in networks moving without efficiently pulling in junctions. Our results identify new molecular players, and they pinpoint a key gene whose products might contribute to dynamically connecting networks to junctions to trigger tissue shape changes in C. elegans and other organisms. [ABSTRACT FROM AUTHOR]

Sleep is controlled by neurons that induce behavioral quiescence and physiological restoration. It is not known, however, how sleep neurons link sleep behavior and survival. In Caenorhabditis elegans, the sleep-active RIS neuron induces sleep behavior and is required for survival of starvation and wounding. Sleep-active neurons such as RIS might hypothetically promote survival primarily by causing sleep behavior and associated conservation of energy. Alternatively, RIS might provide a survival benefit that does not depend on behavioral sleep. To probe these hypotheses, we tested how activity of the sleep-active RIS neuron in Caenorhabditis elegans controls sleep behavior and survival during larval starvation. To manipulate the activity of RIS, we expressed constitutively active potassium channel (twk-18gf and egl-23gf) or sodium channel (unc-58gf) mutant alleles in this neuron. Low levels of unc-58gf expression in RIS increased RIS calcium transients and sleep. High levels of unc-58gf expression in RIS elevated baseline calcium activity and inhibited calcium activation transients, thus locking RIS activity at a high but constant level. This manipulation caused a nearly complete loss of sleep behavior but increased survival. Long-term optogenetic activation also caused constantly elevated RIS activity and a small trend towards increased survival. Disturbing sleep by lethal blue-light stimulation also overactivated RIS, which again increased survival. FLP-11 neuropeptides were important for both, induction of sleep behavior and starvation survival, suggesting that FLP-11 might have divergent roles downstream of RIS. These results indicate that promotion of sleep behavior and survival are separable functions of RIS. These two functions may normally be coupled but can be uncoupled during conditions of strong RIS activation or when sleep behavior is impaired. Through this uncoupling, RIS can provide survival benefits under conditions when behavioral sleep is disturbed. Promoting survival in the face of impaired sleep might be a general function of sleep neurons. Author summary: The sleep neuron RIS in Caenorhabditis elegans is required to induce sleep behavior and to promote survival upon stress. Here we show that RIS can provide survival benefits even when sleep behavior is disturbed. [ABSTRACT FROM AUTHOR]

A hallmark of gastrulation is the establishment of germ layers by internalization of cells initially on the exterior. In C. elegans the end of gastrulation is marked by the closure of the ventral cleft, a structure formed as cells internalize during gastrulation, and the subsequent rearrangement of adjacent neuroblasts that remain on the surface. We found that a nonsense allele of srgp-1/srGAP leads to 10–15% cleft closure failure. Deletion of the SRGP-1/srGAP C-terminal domain led to a comparable rate of cleft closure failure, whereas deletion of the N-terminal F-BAR region resulted in milder defects. Loss of the SRGP-1/srGAP C-terminus or F-BAR domain results in defects in rosette formation and defective clustering of HMP-1/⍺-catenin in surface cells during cleft closure. A mutant form of HMP-1/⍺-catenin with an open M domain can suppress cleft closure defects in srgp-1 mutant backgrounds, suggesting that this mutation acts as a gain-of-function allele. Since SRGP-1 binding to HMP-1/⍺-catenin is not favored in this case, we sought another HMP-1 interactor that might be recruited when HMP-1/⍺-catenin is constitutively open. A good candidate is AFD-1/afadin, which genetically interacts with cadherin-based adhesion later during embryonic elongation. AFD-1/afadin is prominently expressed at the vertex of neuroblast rosettes in wildtype, and depletion of AFD-1/afadin increases cleft closure defects in srgp-1/srGAP and hmp-1R551/554A/⍺-catenin backgrounds. We propose that SRGP-1/srGAP promotes nascent junction formation in rosettes; as junctions mature and sustain higher levels of tension, the M domain of HMP-1/⍺-catenin opens, allowing maturing junctions to transition from recruitment of SRGP-1/srGAP to AFD-1/afadin. Our work identifies new roles for ⍺-catenin interactors during a process crucial to metazoan development. Author summary: A key feature of early embryonic development in animals is gastrulation. Gastrulation often involves the movement of cells initially on the surface to the interior, where they serve as founder cells for the formation of key tissues in the embryo. We used the early embryo of the nematode, C. elegans, as a model system to investigate how cells on the surface seal gaps left behind as cells move into the interior. We found that two parts of a protein known as SRGP-1/srGAP are important for the formation of "rosettes" at the end of gastrulation: a part that binds membranes and another that binds a key adhesion protein known as HMP-1/α-catenin. Defects in SRGP-1 function lead to defects in the clustering of ⍺-catenin that are important for strengthening cell-cell adhesion. These defects can be bypassed by activating mutations in ⍺-catenin. We found that a second protein, AFD-1/afadin, is normally prominently expressed at rosettes, but that depletion of AFD-1/afadin frequently leads to sealing defects. SRGP-1 and AFD-1 appear to act largely in parallel during sealing. Our work identifies new roles for ⍺-catenin interactors during a process crucial to animal development. [ABSTRACT FROM AUTHOR]

Some epigenetic information can be transmitted between generations without changes in the underlying DNA sequence. Changes in epigenetic regulators, termed epimutations, can occur spontaneously and be propagated in populations in a manner reminiscent of DNA mutations. Small RNA-based epimutations occur in C. elegans and persist for around 3–5 generations on average. Here, we explored whether chromatin states also undergo spontaneous change and whether this could be a potential alternative mechanism for transgenerational inheritance of gene expression changes. We compared the chromatin and gene expression profiles at matched time points from three independent lineages of C. elegans propagated at minimal population size. Spontaneous changes in chromatin occurred in around 1% of regulatory regions each generation. Some were heritable epimutations and were significantly enriched for heritable changes in expression of nearby protein-coding genes. Most chromatin-based epimutations were short-lived but a subset had longer duration. Genes subject to long-lived epimutations were enriched for multiple components of xenobiotic response pathways. This points to a possible role for epimutations in adaptation to environmental stressors. Author summary: Evolution is known to occur because of changes in DNA sequence which are inherited between generations. Recently, though, it has been discovered that information beyond the DNA sequence can be transmitted between generations. This information, known as epigenetic, can control how the DNA sequence is used. Epigenetic information that is transmitted between generations could drive evolutionary processes in populations, but this is yet to be tested. We used a simple nematode worm to investigate the contribution of different types of epigenetic information to evolution. We evolved populations of worms in the laboratory and investigated epigenetic differences that emerged in different lineages. Most epigenetic differences were very short-lived, so unlikely to be able to contribute to long-term evolutionary processes. However, we identified some changes that lasted much longer. Intriguingly, genes that control the worms' responses to external threats such as bacterial infections or noxious chemicals were most likely to undergo long-term epigenetic changes, despite the fact that the environment of the worms was stable and did not contain these stresses. We think that epigenetic processes might be able to create a fast-acting form of variation that could help in situations where organisms need to adapt to dangerous environments. [ABSTRACT FROM AUTHOR]

Chromosome movements and licensing of synapsis must be tightly regulated during early meiosis to ensure accurate chromosome segregation and avoid aneuploidy, although how these steps are coordinated is not fully understood. Here we show that GRAS-1, the worm homolog of mammalian GRASP/Tamalin and CYTIP, coordinates early meiotic events with cytoskeletal forces outside the nucleus. GRAS-1 localizes close to the nuclear envelope (NE) in early prophase I and interacts with NE and cytoskeleton proteins. Delayed homologous chromosome pairing, synaptonemal complex (SC) assembly, and DNA double-strand break repair progression are partially rescued by the expression of human CYTIP in gras-1 mutants, supporting functional conservation. However, Tamalin, Cytip double knockout mice do not exhibit obvious fertility or meiotic defects, suggesting evolutionary differences between mammals. gras-1 mutants show accelerated chromosome movement during early prophase I, implicating GRAS-1 in regulating chromosome dynamics. GRAS-1-mediated regulation of chromosome movement is DHC-1-dependent, placing it acting within the LINC-controlled pathway, and depends on GRAS-1 phosphorylation at a C-terminal S/T cluster. We propose that GRAS-1 coordinates the early steps of homology search and licensing of SC assembly by regulating the pace of chromosome movement in early prophase I. Author summary: Successful sexual reproduction depends on the formation of gametes (i.e., eggs and sperm) carrying a correct number of chromosomes. This involves a series of well-choreographed steps during meiosis, the specialized cell division program resulting in the formation of eggs and sperm. Some of these steps include pairing between homologous chromosomes, assembly of a scaffold (the synaptonemal complex) between homologs, and inter-homolog recombination. These steps must be properly coordinated to ensure normal meiotic progression. Here, we show that the GRAS-1 protein in the nematode C. elegans, which shares sequence conservation with the mammalian proteins GRASP/Tamalin and CYTIP, contributes to normal meiotic progression by coordinating early events in meiosis. GRAS-1 is required for the normal speed of chromosome movements early in prophase I in a manner dependent of its phosphorylation and on dynein heavy chain. Delayed homologous chromosome pairing, synaptonemal complex assembly, and DNA double-strand break repair progression are partially rescued by the expression of human CYTIP in gras-1 mutants, supporting functional conservation. We identified a new factor coordinating the early steps of pairing and synapsis by regulating the pace of chromosome movement in early prophase I. [ABSTRACT FROM AUTHOR]