A dominant mutation in mec-7/β-tubulin affects axon development and regeneration in Caenorhabditis elegans neurons

Microtubules have been known for decades to be basic elements of the cytoskeleton. They form long, dynamic, rope-like structures within the cell that are essential for mitosis, maintenance of cell shape, and intracellular transport. More recently, in vitro studies have implicated microtubules as signaling molecules that, through changes in their stability, have the potential to trigger growth of axons and dendrites in developing neurons. In this study, we show that specific mutations in the Caenorhabditis elegans mec-7/β-tubulin gene cause ectopic axon formation in mechanosensory neurons in vivo. In mec-7 mutants, the ALM mechanosensory neuron forms a long ectopic neurite that extends posteriorly, a phenotype that can be mimicked in wild-type worms with a microtubule-stabilizing drug (paclitaxel), and suppressed by mutations in unc-33/CRMP2 and the kinesin-related gene, vab-8. Our results also reveal that these ectopic neurites contain RAB-3, a marker for presynaptic loci, suggesting that they have axon-like properties. Interestingly, in contrast with the excessive axonal growth observed during development, mec-7 mutants are inhibited in axonal regrowth and remodeling following axonal injury. Together our results suggest that MEC-7/β-tubulin integrity is necessary for the correct number of neurites a neuron generates in vivo and for the capacity of an axon to regenerate.     

Evidence for dynamic alteration in histone gene clusters of Caenorhabditis elegans: a topoisomerase II connection?

Chromatin integrity is maintained throughout the cell cycle through repair mechanisms and intrinsically by the ordered packaging of DNA in association with histone proteins; however, aberrant rearrangements within and between chromosomes do occur. The role of the nuclear matrix protein topoisomerase II (TopoII) in generating chromosome breakpoints has been a focus of recent investigations. TopoII preferentially binds in vitro to scaffold-associated regions (SARs) and is involved in many DNA processing activities that require chromosome untangling. SARs, biochemically defined DNA elements rich in A + T, have been proposed to serve as structural boundaries for chromatin loops and to delineate functional domains. In our investigation of gene compartmentalization in a eukaryotic genome, SAR-associated nucleotide motifs from Drosophila were mapped in the regions of three histone gene clusters in an in silico analysis of the genome of Caenorhabditis elegans. Sites with similarity to the 15 bp consensus for TopoII cleavage were found predominantly in A + T enriched intergenic regions. Reiteration of sites matching the TopoII core consensus led to the identification of a novel core histone gene on chromosome IV and provided evidence for duplication and inversion in each of the three histone gene clusters. Breakpoint analysis of DNA flanking reiterated regions revealed potential sites for TopoII cleavage and a base composition phenomenon suggestive of a trigger for inversion events.     

PPM-1, a PP2Cα/β phosphatase, regulates axon termination and synapse formation in Caenorhabditis elegans

The PHR (Pam/Highwire/RPM-1) proteins are evolutionarily conserved ubiquitin ligases that regulate axon guidance and synapse formation in Caenorhabditis elegans, Drosophila, zebrafish, and mice. In C. elegans, RPM-1 (Regulator of Presynaptic Morphology-1) functions in synapse formation, axon guidance, axon termination, and postsynaptic GLR-1 trafficking. Acting as an E3 ubiquitin ligase, RPM-1 negatively regulates a MAP kinase pathway that includes: dlk-1, mkk-4, and the p38 MAPK, pmk-3. Here we provide evidence that ppm-1, a serine/threonine phosphatase homologous to human PP2Cα(PPM1A) and PP2Cβ(PPM1B) acts as a second negative regulatory mechanism to control the dlk-1 pathway. We show that ppm-1 functions through its phosphatase activity in a parallel genetic pathway with glo-4 and fsn-1 to regulate both synapse formation in the GABAergic motorneurons and axon termination in the mechanosensory neurons. Our transgenic analysis shows that ppm-1 acts downstream of rpm-1 to negatively regulate the DLK-1 pathway, with PPM-1 most likely acting at the level of pmk-3. Our study provides insight into the negative regulatory mechanisms that control the dlk-1 pathway in neurons and demonstrates a new role for the PP2C/PPM phosphatases as regulators of neuronal development.     

SUN-1 and ZYG-12, Mediators of Centrosome–Nucleus Attachment,Are a Functional SUN/KASH Pair in Caenorhabditis elegans

Klarsicht/ANC-1/Syne/homology (KASH)/Sad-1/UNC-84 (SUN) protein pairs can act as connectors between cytoplasmic organelles and the nucleoskeleton. Caenorhabditis elegans ZYG-12 and SUN-1 are essential for centrosome–nucleus attachment. Although SUN-1 has a canonical SUN domain, ZYG-12 has a divergent KASH domain. Here, we establish that the ZYG-12 mini KASH domain is functional and, in combination with a portion of coiled-coil domain, is sufficient for nuclear envelope localization. ZYG-12 and SUN-1 are hypothesized to be outer and inner nuclear membrane proteins, respectively, and to interact, but neither their topologies nor their physical interaction has been directly investigated. We show that ZYG-12 is a type II outer nuclear membrane (ONM) protein and that SUN-1 is a type II inner nuclear membrane protein. The proteins interact in the luminal space of the nuclear envelope via the ZYG-12 mini KASH domain and a region of SUN-1 that does not include the SUN domain. SUN-1 is hypothesized to restrict ZYG-12 to the ONM, preventing diffusion through the endoplasmic reticulum. We establish that ZYG-12 is indeed immobile at the ONM by using fluorescence recovery after photobleaching and show that SUN-1 is sufficient to localize ZYG-12 in cells. This work supports current models of KASH/SUN pairs and highlights the diversity in sequence elements defining KASH domains.     

A Caenorhabditis elegans homeobox gene expressed in the male tail, a link between pattern formation and sexual dimorphism?

 ceh-7 is a small Caenorhabditis elegans homeobox gene. We have shown that this gene is transcribed. Examination of the expression pattern of ceh-7 using reporter constructs revealed that it is expressed in a few cells of the male tail, which form a ring around the rectum. The most posterior member of the C. elegans Hox cluster, egl-5, an Abd-B homologue, has previously been shown to be required for the proper development of several blast cells in the male tail. We have examined the expression of ceh-7 in mutant backgrounds of egl-5 and also mab-5, an Antp/Ubx/Abd-A homologue. We find that ceh-7 is not expressed in egl-5 mutants, but is still expressed in mab-5 mutants. Received: 16 March 1998 / Accepted: 4 September 1998     

WDR34 Mutations that Cause Short-Rib Polydactyly Syndrome Type III/Severe Asphyxiating Thoracic Dysplasia Reveal a Role for the NF-κB Pathway in Cilia

Short-rib polydactyly (SRP) syndrome type III, or Verma-Naumoff syndrome, is an autosomal-recessive chondrodysplasia characterized by short ribs, a narrow thorax, short long bones, an abnormal acetabulum, and numerous extraskeletal malformations and is lethal in the perinatal period. Presently, mutations in two genes, IFT80 and DYNC2H1, have been identified as being responsible for SRP type III. Via homozygosity mapping in three affected siblings, a locus for the disease was identified on chromosome 9q34.11, and homozygosity for three missense mutations in WDR34 were found in three independent families, as well as compound heterozygosity for mutations in one family. WDR34 encodes a member of the WD repeat protein family with five WD40 domains, which acts as a TAK1-associated suppressor of the IL-1R/TLR3/TLR4-induced NF-κB activation pathway. We showed, through structural modeling, that two of the three mutations altered specific structural domains of WDR34. We found that primary cilia in WDR34 mutant fibroblasts were significantly shorter than normal and had a bulbous tip. This report expands on the pathogenesis of SRP type III and demonstrates that a regulator of the NF-κB activation pathway is involved in the pathogenesis of the skeletal ciliopathies.     

SMG-ly Knocking Out Gene Expression in Specific Cells: An Educational Primer for Use with “A Novel Strategy for Cell-Autonomous Gene Knockdown in Caenorhabditis elegans Defines a Cell-Specific Function for the G-Protein Subunit GOA-1”

SUMMARY

A recent article by Maher et al. in GENETICS introduces an alternative approach to cell-type-specific gene knockdown in Caenorhabditis elegans, using nonsense-mediated decay. This strategy has the potential to be applicable to other organisms (this strategy requires that animals can survive without nonsense-mediated decay—not all can). This Primer article provides a guide and resource for educators and students by describing different gene knockdown methodologies, by assisting with the technically difficult portions of the Maher et al. article, and by providing conceptual questions relating to the article.Related article in GENETICS: Maher, K. N., A. Swaminathan, P. Patel, and D. L. Chase, 2013 A novel strategy for cell-autonomous gene knockdown in Caenorhabditis elegans defines a cell-specific function for the G-protein subunit GOA-1. Genetics 194: 363–373.     

A Highlights from MBoC Selection: RSBP-1 Is a Membrane-targeting Subunit Required by the Gαq-specific But Not the Gαo-specific R7 Regulator of G protein Signaling in Caenorhabditis elegans

Regulator of G protein signaling (RGS) proteins inhibit G protein signaling by activating Gα GTPase activity, but the mechanisms that regulate RGS activity are not well understood. The mammalian R7 binding protein (R7BP) can interact with all members of the R7 family of RGS proteins, and palmitoylation of R7BP can target R7 RGS proteins to the plasma membrane in cultured cells. However, whether endogenous R7 RGS proteins in neurons require R7BP or membrane localization for function remains unclear. We have identified and knocked out the only apparent R7BP homolog in Caenorhabditis elegans, RSBP-1. Genetic studies show that loss of RSBP-1 phenocopies loss of the R7 RGS protein EAT-16, but does not disrupt function of the related R7 RGS protein EGL-10. Biochemical analyses find that EAT-16 coimmunoprecipitates with RSBP-1 and is predominantly plasma membrane-associated, whereas EGL-10 does not coimmunoprecipitate with RSBP-1 and is not predominantly membrane-associated. Mutating the conserved membrane-targeting sequence in RSBP-1 disrupts both the membrane association and function of EAT-16, demonstrating that membrane targeting by RSBP-1 is essential for EAT-16 activity. Our analysis of endogenous R7 RGS proteins in C. elegans neurons reveals key differences in the functional requirements for membrane targeting between members of this protein family.