A role for septins in cellular and axonal migration in C. elegans

Caenorhabditis elegans has two genes, unc-59 and unc-61, encoding septin-family GTPases. Mutations in the septin genes cause defects in locomotory behavior that have been previously attributed to cytokinesis failures in postembryonic neuroblasts. We find that mutations in either septin gene frequently cause uncoordination in newly hatched larvae in the absence of cytokinesis failures. The septins exhibit developmentally regulated expression, including expression in various neurons at times when processes are extending and synapses are forming. Motor neurons in the mutant larvae display defects in multiple aspects of axonal migration and guidance that are likely to be responsible for the locomotory behavior defects. The septins are also expressed in migrating distal tip cells, which are leaders for gonad arm extension. Septin mutants affect morphology of the distal tip cells, as well as their migration and guidance during gonadogenesis. These results suggest that septins may be generally required for developmental migrations and pathfinding.     

Sensitized genetic backgrounds reveal a role for C. elegans FGF EGL-17 as a repellent for migrating CAN neurons

Although many molecules are necessary for neuronal cell migrations in C. elegans, no guidance cues are known to be essential for any of these cells to migrate along the anteroposterior (AP) axis. We demonstrate that the fibroblast growth factor (FGF) EGL-17, an attractant for the migrating sex myoblasts (SMs), repels the CANs, a pair of neurons that migrate posteriorly from the head to the center of the embryo. Although mutations in genes encoding EGL-17/FGF and a specific isoform of its receptor EGL-15/FGFR had little effect on CAN migration, they enhanced the CAN migration defects caused by mutations in other genes. Two cells at the anterior end of the embryo express EGL-17/FGF, raising the possibility that EGL-17/FGF functions as a repellent for migrating CANs. Consistent with this hypothesis, ectopic expression of EGL-17/FGF shifted the final CAN cell positions away from these novel sites of expression. Cell-specific rescue experiments demonstrated that EGL-15/FGFR acts in the CANs to promote their migration. We also found that the tyrosine phosphatase receptor CLR-1 regulates CAN migration by inhibiting EGL-15/FGFR signaling, and that the FGFR adaptor protein SEM-5/GRB2 may mediate EGL-15/FGFR signaling in CAN migration. Thus, EGL-17/FGF signaling through an EGL-15/FGFR isoform and possibly SEM-5/GRB2 mediates both attraction of the SMs and repulsion of the CANs. This study also raises the possibility that several guidance cues regulate cell migrations along the C. elegans AP axis, and their role in these migrations may only be revealed in sensitized genetic backgrounds.     

Efficient chaperone-mediated tubulin biogenesis is essential for cell division and cell migration in C. elegans

The efficient folding of actin and tubulin in vitro and in Saccharomyces cerevisiae is known to require the molecular chaperones prefoldin and CCT, yet little is known about the functions of these chaperones in multicellular organisms. Whereas none of the six prefoldin genes are essential in yeast, where prefoldin-independent folding of actin and tubulin is sufficient for viability, we demonstrate that reducing prefoldin function by RNAi in Caenorhabditis elegans causes defects in cell division that result in embryonic lethality. Our analyses suggest that these defects result mainly from a decrease in α-tubulin levels and a subsequent reduction in the microtubule growth rate. Prefoldin subunit 1 (pfd-1) mutant animals with maternally contributed PFD-1 develop to the L4 larval stage with gonadogenesis defects that include aberrant distal tip cell migration. Importantly, RNAi knockdown of prefoldin, CCT or tubulin in developing animals phenocopy the pfd-1 cell migration phenotype. Furthermore, reducing CCT function causes more severe phenotypes (compared with prefoldin knockdown) in the embryo and developing gonad, consistent with a broader role for CCT in protein folding. Overall, our results suggest that efficient chaperone-mediated tubulin biogenesis is essential in C. elegans, owing to the critical role of the microtubule cytoskeleton in metazoan development.     

A primary culture system for functional analysis of C. elegans neurons and muscle cells

C. elegans has provided important insights into neuromuscular system function and development. However, the animal's small size limits access to individual neurons and muscle cells for physiological, biochemical, and molecular study. We describe here primary culture methods that allow C. elegans embryonic cells to differentiate into neurons and muscle cells in vitro. Morphological, electrophysiological, and GFP reporter studies demonstrate that the differentiation and functional properties of cultured cells are similar to those observed in vivo. Enriched populations of cells expressing specific GFP reporters can be generated by fluorescence-activated cell sorting. Addition of double-stranded RNA to the culture medium induces dramatic knockdown of targeted gene expression. Primary nematode cell culture provides a new foundation for a wide variety of experimental opportunities heretofore unavailable in the field.     

UNC-84 localizes to the nuclear envelope and is required for nuclear migration and anchoring during C. elegans development.

Nuclear migrations are essential for metazoan development. Two nuclear migrations that occur during C. elegans development require the function of the unc-84 gene. unc-84 mutants are also defective in the anchoring of nuclei within the hypodermal syncytium and in the migrations of the two distal tip cells of the gonad. Complementation analyses of 17 unc-84 alleles defined two genetically separable functions. Both functions are required for nuclear and distal tip cell migrations, but only one is required for nuclear anchorage. The DNA lesions associated with these 17 mutations indicate that the two genetically defined functions correspond to two distinct regions of the UNC-84 protein. The UNC-84 protein has a predicted transmembrane domain and a C-terminal region with similarity to the S. pombe spindle pole body protein Sad1 and to two predicted mammalian proteins. Analysis of a green fluorescent protein reporter indicated that UNC-84 is widely expressed and localized to the nuclear envelope. We propose that UNC-84 functions to facilitate a nuclear-centrosomal interaction required for nuclear migration and anchorage.     

Global cell sorting in the C. elegans embryo defines a new mechanism for pattern formation

4D microscopic observations of Caenorhabditis elegans development show that the nematode uses an unprecedented strategy for development. The embryo achieves pattern formation by sorting cells, through far-ranging movements, into coherent regions before morphogenesis is initiated. This sorting of cells is coupled to their particular fate. If cell identity is altered by experiment, cells are rerouted to positions appropriate to their new fates even across the whole embryo. This cell behavior defines a new mechanism of pattern formation, a mechanism that is also found in other animals. We call this new mechanism "cell focusing". When the fate of cells is changed, they move to new positions which also affect the shape of the body. Thus, this process is also important for morphogenesis.     

The C. elegans Frizzled CFZ-2 is required for cell migration and interacts with multiple Wnt signaling pathways

Members of the Frizzled family of integral membrane proteins are implicated in many developmental events, including specifying cell fate, orienting cell and planar polarity, and directing cell migration. Frizzleds function as cell surface receptors for secreted Wnt proteins. We report here the isolation of a mutation in cfz-2, a Caenorhabditis elegans Frizzled gene. Mutation of cfz-2 causes defective cell migration, disorganization of head neurons, and can cause ectopic axon outgrowth. Analysis of mosaic animals shows that CFZ-2 functions cell nonautonomously, but does not rule out an autonomous role. CFZ-2 is expressed primarily in the anterior of embryos and in several cells in the head of adults. Our analysis of interactions between CFZ-2 and other Wnt pathways reveals that three Wnts, CWN-1, CWN-2 and EGL-20, and a Frizzled, MOM-5, function redundantly with one another and with CFZ-2 for specific cell migrations. In contrast, CWN-1, CWN-2, EGL-20, CFZ-2, and MOM-5 antagonize one another for other migrations. Therefore, CFZ-2 functions by collaborating with and/or antagonizing other Wnt signaling pathways to regulate specific cell migrations.     

Control of the basement membrane and cell migration by ADAMTS proteinases: Lessons from C. elegans genetics

The members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of secreted proteins, MIG-17 and GON-1, play essential roles in Caenorhabditis elegans gonadogenesis. The genetic and molecular analyses of these proteinases uncovered novel molecular interactions regulating the basement membrane (BM) during the migration of the gonadal leader cells. MIG-17, which is localized to the gonadal BM recruits or activates fibulin-1 and type IV collagen, which then recruits nidogen, thereby inducing the remodeling of the BM that is required for directional control of leader cell migration. GON-1 acts antagonistically with fibulin-1 to regulate the levels of type IV collagen accumulation in the gonadal BM, which facilitates active migration of the leader cells. The cooperative action of MIG-17 and GON-1 represents an excellent model for understanding the mechanisms of organogenesis mediated by ADAMTS proteinases.     

Evolutionary plasticity in the requirement for force exerted by ligand endocytosis to activate C. elegans Notch proteins

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Conserved and divergent processing of neuroligin and neurexin genes: from the nematode C. elegans to human

Neuroligins are cell-adhesion proteins that interact with neurexins at the synapse. This interaction may contribute to differentiation, plasticity and specificity of synapses. In humans, single mutations in neuroligin-encoding genes are implicated in autism spectrum disorder and/or mental retardation. Moreover, some copy number variations and point mutations in neurexin-encoding genes have been linked to neurodevelopmental disorders including autism. Neurexins are subject to extensive alternative splicing, highly regulated in mammals, with a great physiological importance. In addition, neuroligins and neurexins are subjected to proteolytic processes that regulate synaptic transmission modifying pre- and postsynaptic activities and may also regulate the remodelling of spines at specific synapses. Four neuroligin genes exist in mice and five in human, whilst in the nematode Caenorhabditis elegans, there is only one orthologous gene. In a similar manner, in mammals, there are three neurexin genes, each of them encoding two major isoforms named α and β, respectively. In contrast, there is one neurexin gene in C. elegans that also generates two isoforms like mammals. The complexity of the genetic organization of neurexins is due to extensive processing resulting in hundreds of isoforms. In this review, a wide comparison is made between the genes in the nematode and human with a view to better understanding the conservation of processing in these synaptic proteins in C. elegans, which may serve as a genetic model to decipher the synaptopathies underpinning neurodevelopmental disorders such as autism.     

Novel gain-of-function alleles demonstrate a role for the heterochronic gene lin-41 in C. elegans male tail tip morphogenesis

To gain an understanding of the genes and mechanisms that govern morphogenesis and its evolution, we have analyzed mutations that disrupt this process in a simple model structure, the male tail tip of the rhabditid nematode C. elegans. During the evolution of rhabditid male tails, there have been several independent changes from tails with rounded tips ("peloderan", as in C. elegans) to those with pointed tips ("leptoderan"). Mutations which produce leptoderan (Lep) tails in C. elegans thus identify candidate genes and pathways in which evolutionary changes could have produced leptoderan tails from peloderan ancestors. Here we report that two novel, gain-of-function (gf) alleles of lin-41 have lesions predicted to affect the N-terminus of the RBCC-domain LIN-41 protein. Both gf alleles cause the tail tip of adult males to retain the pointed shape of the juvenile tails, producing a Lep phenotype that looks like the tails of leptoderan species. Consistent with its role in the heterochronic pathway, we find that lin-41 governs the timing and extent of male tail tip morphogenesis in a dose-dependent manner. Specifically, the Lep phenotype results from a heterochronic delay in the retraction and fusion of the tail tip cells during L4 morphogenesis, such that retraction is not completed before the adult molt. Conversely, we find that tail tip morphogenesis and cell fusions begin precociously at the L3 stage in the reduced-function lin-41 mutant, ma104, resulting in over-retracted male tails in the adult. Because modulated anti-LIN-41 RNAi knockdowns in the gf mutants restore wild-type phenotype, we suggest that the leptoderan phenotype of the gf alleles is due to a higher activity of otherwise normal LIN-41. Additionally, the gf allele is suppressed by the wild-type allele, suggesting that LIN-41 normally regulates itself, possibly by autoubiquitination. We speculate that small changes affecting LIN-41 could have been significant for male tail evolution.     

Function of the her-1 gene is required for maintenance of male differentiation in adult tissues of C. elegans

Function of the sex-determining gene her-1 is required in XO embryos cf C. elegans to specify male development. Using a temperature-sensitive mutant of her-1, we show that when XO males reared at a permissive temperature are shifted as adults to a nonpermissive temperature, they initiate vitellogenin synthesis in the intestine and oocyte production in the germline. A similar shift has no effect on her-1(+) males. We conclude that sexual differentiation of the intestine and germline is plastic, requiring her-1 expression throughout adulthood for maintenance of the male state. © 1994 Wiley-Liss, Inc.     

Evidence for N----O acetyl migration as the mechanism for O acetylation of peptidoglycan in Proteus mirabilis.

O-acetylated peptidoglycan was purified from Proteus mirabilis grown in the presence of specifically radiolabelled glucosamine derivatives, and the migration of the radiolabel was monitored. Mild-base hydrolysis of the isolated peptidoglycan (to release ester-linked acetate) from cells grown in the presence of 40 microM [acetyl-3H]N-acetyl-D-glucosamine resulted in the release of [3H]acetate, as detected by high-pressure liquid chromatography. The inclusion of either acetate, pyruvate, or acetyl phosphate, each at 1 mM final concentration, did not result in a diminution of mild-base-released [3H]acetate levels. No such release of [3H]acetate was observed with peptidoglycan isolated from either Escherichia coli incubated with the same radiolabel or P. mirabilis grown with [1,6-3H]N-acetyl-D-glucosamine or D-[1-14C]glucosamine. These observations support a hypothesis that O acetylation occurs by N----O acetyl transfer within the sacculus. A decrease in [3H]acetate release by mild-base hydrolysis was observed with the peptidoglycan of P. mirabilis cultures incubated in the presence of antagonists of peptidoglycan biosynthesis, penicillin G and D-cycloserine. The absence of free-amino sugars in the peptidoglycan of P. mirabilis but the detection of glucosamine in spent culture broths implies that N----O transacetylation is intimately associated with peptidoglycan turnover.     

The RNA-binding proteins PUF-5, PUF-6, and PUF-7 reveal multiple systems for maternal mRNA regulation during C. elegans oogenesis

In metazoans, many mRNAs needed for embryogenesis are produced during oogenesis and must be tightly regulated during the complex events of oocyte development. In C. elegans, translation of the Notch receptor GLP-1 is repressed during oogenesis and is then activated specifically in anterior cells of the early embryo. The KH domain protein GLD-1 represses glp-1 translation during early stages of meiosis, but the factors that repress glp-1 during late oogenesis are not known. Here, we provide evidence that the PUF domain protein PUF-5 and two nearly identical PUF proteins PUF-6 and PUF-7 function during a specific period of oocyte differentiation to repress glp-1 and other maternal mRNAs. Depletion of PUF-5 and PUF-6/7 together caused defects in oocyte formation and early embryonic cell divisions. Loss of PUF-5 and PUF-6/7 also caused inappropriate expression of GLP-1 protein in oocytes, but GLP-1 remained repressed in meiotic germ cells. PUF-5 and PUF-6/7 function was required directly or indirectly for translational repression through elements of the glp-1 3' untranslated region. Oogenesis and embryonic defects could not be rescued by loss of GLP-1 activity, suggesting that PUF-5 and PUF-6/7 regulate other mRNAs in addition to glp-1. PUF-5 and PUF-6/7 depletion, however, did not perturb repression of the maternal factors GLD-1 and POS-1, suggesting that subsets of maternal gene products may be regulated by distinct pathways. Interestingly, PUF-5 protein was detected exclusively during mid to late oogenesis but became undetectable prior to completion of oocyte differentiation. These results reveal a previously unknown maternal mRNA control system that is specific to late stages of oogenesis and suggest new functions for PUF family proteins in post-mitotic differentiation. Multiple sets of RNA-binding complexes function in different domains of the C. elegans germ line to maintain silencing of Notch/glp-1 and other mRNAs.     

Genes required for axon pathfinding and extension in the C. elegans nerve ring.

Over half of the neurons in Caenorhabditis elegans send axons to the nerve ring, a large neuropil in the head of the animal. Genetic screens in animals that express the green fluorescent protein in a subset of sensory neurons identified eight new sax genes that affect the morphology of nerve ring axons. sax-3/robo mutations disrupt axon guidance in the nerve ring, while sax-5, sax-9 and unc-44 disrupt both axon guidance and axon extension. Axon extension and guidance proceed normally in sax-1, sax-2, sax-6, sax-7 and sax-8 mutants, but these animals exhibit later defects in the maintenance of nerve ring structure. The functions of existing guidance genes in nerve ring development were also examined, revealing that SAX-3/Robo acts in parallel to the VAB-1/Eph receptor and the UNC-6/netrin, UNC-40/DCC guidance systems for ventral guidance of axons in the amphid commissure, a major route of axon entry into the nerve ring. In addition, SAX-3/Robo and the VAB-1/Eph receptor both function to prevent aberrant axon crossing at the ventral midline. Together, these genes define pathways required for axon growth, guidance and maintenance during nervous system development.     

Molecular dynamics simulations reveal initial structural and dynamic features for the A2AR as a result of ligand binding

G-protein-coupled receptors (GPCRs) are membrane proteins that have a wide variety of physiological roles. Adenosine receptors belong to the GPCR family. Adenosine receptors are implicated in many physiological disorders, such as Parkinson's disease, Huntington's disease, inflammatory and immune's disease and many others. Interestingly, crystal structures of the active and inactive conformations of the A₂-subtype adenosine receptor (A₂AR) have been solved. These two structures could be used to get insights about the conformational changes that occur during the process of activation/inactivation processes of this receptor. Therefore, two ligand-free simulations of the native active (PDB code: 3QAK) and inactive (PDB code: 3EML) conformations of the A₂AR and two halo-simulations were carried out to observe the initial conformational changes induced by coupling adenosine to the inactive conformation and caffeine to the active conformation. Furthermore, we constructed an A₂AR model that contained four thermostabilising mutations, L48A, T65A, Q89A and A54L, which had previously been determined to stabilise the bound conformation of the agonist, and we ran molecular dynamics simulations of this mutant to investigate how these point mutations might affect the inactive conformation of this receptor. This study provides insights about the initial structural and dynamic features that occur as a result of the binding of caffeine and adenosine in the active and inactive A₂AR structures, respectively, as well as the introduction of some mutations on the inactive structure of the A₂AR. Moreover, we provide useful and detailed information regarding structural features such as toggle switch and ionic lock during the activation/inactivation processes of this receptor.     

Functional characterization of the C. elegans nephrocystins NPHP-1 and NPHP-4 and their role in cilia and male sensory behaviors

Autosomal dominant polycystic kidney disease (ADPKD) and nephronophthisis (NPH) share two common features: cystic kidneys and ciliary localized gene products. Mutation in either the PKD1 or PKD2 gene accounts for 95% of all ADPKD cases. Mutation in one of four genes (NPHP1-4) results in nephronophthisis. The NPHP1, NPHP2, PKD1, and PKD2 protein products (nephrocystin-1, nephrocystin-2 or inversin, polycystin-1, and polycystin-2, respectively) localize to primary cilia of renal epithelia. However, the relationship between the nephrocystins and polycystins, if any, is unknown. In the nematode Caenorhabditis elegans, the LOV-1 and PKD-2 polycystins localize to male-specific sensory cilia and are required for male mating behaviors. To test the hypothesis that ADPKD and NPH cysts arise from a common defect in cilia, we characterized the C. elegans homologs of NPHP1 and NPHP4. C. elegans nphp-1 and nphp-4 are expressed in a subset of sensory neurons. GFP-tagged NPHP-1 and NPHP-4 proteins localize to ciliated sensory endings of dendrites and colocalize with PKD-2 in male-specific sensory cilia. The cilia of nphp-1(ok500) and nphp-4(tm925) mutants are intact. nphp-1; nphp-4 double, but not single, mutant males are response defective. We propose that NPHP-1 and NPHP-4 proteins play important and redundant roles in facilitating ciliary sensory signal transduction.     

RNT-1, the C. elegans homologue of mammalian RUNX transcription factors, regulates body size and male tail development

The rnt-1 gene is the only Caenorhabditis elegans homologue of the mammalian RUNX genes. Several lines of molecular biological evidence have demonstrated that the RUNX proteins interact and cooperate with Smads, which are transforming growth factor-beta (TGF-beta) signal mediators. However, the involvement of RUNX in TGF-beta signaling has not yet been supported by any genetic evidence. The Sma/Mab TGF-beta signaling pathway in C. elegans is known to regulate body length and male tail development. The rnt-1(ok351) mutants show the characteristic phenotypes observed in mutants of the Sma/Mab pathway, namely, they have a small body size and ray defects. Moreover, RNT-1 can physically interact with SMA-4 which is one of the Smads in C. elegans, and double mutant animals containing both the rnt-1(ok351) mutation and a mutation in a known Sma/Mab pathway gene displayed synergism in the aberrant phenotypes. In addition, lon-1(e185) mutants was epistatic to rnt-1(ok351) mutants in terms of long phenotype, suggesting that lon-1 is indeed downstream target of rnt-1. Our data reveal that RNT-1 functionally cooperates with the SMA-4 proteins to regulate body size and male tail development in C. elegans.