Interactions of Caenorhabditis elegans ß-tubulins with the microtubule inhibitor and anthelmintic drug albendazole.

Parasitic nematodes are major human and agricultural pests, and benzimidazoles are amongst the most important broad-spectrum anthelmintic drug class used for their control. Benzimidazole resistance is now widespread in many species of parasitic nematodes in livestock globally and an emerging concern for the sustainable control of human soil-transmitted helminths. ß-tubulin is the major benzimidazole target, although other genes may influence resistance. Among the 6 Caenorhabditis elegans ß-tubulin genes, loss of ben-1 causes resistance without other apparent defects. Here, we explored the genetics of C. elegans ß-tubulin genes in relation to the response to the benzimidazole derivative albendazole. The most highly expressed ß-tubulin isotypes, encoded by tbb-1 and tbb-2, were known to be redundant with each other for viability, and their products are predicted not to bind benzimidazoles. We found that tbb-2 mutants, and to a lesser extent tbb-1 mutants, were hypersensitive to albendazole. The double mutant tbb-2 ben-1 is uncoordinated and short, resembling the wild type exposed to albendazole, but the tbb-1 ben-1 double mutant did not show the same phenotypes. These results suggest that tbb-2 is a modifier of albendazole sensitivity. To better understand how BEN-1 mutates to cause benzimidazole resistance, we isolated mutants resistant to albendazole and found that 15 of 16 mutations occurred in the ben-1 coding region. Mutations ranged from likely nulls to hypomorphs, and several corresponded to residues that cause resistance in other organisms. Null alleles of ben-1 are albendazole-resistant and BEN-1 shows high sequence identity with tubulins from other organisms, suggesting that many amino acid changes could cause resistance. However, our results suggest that missense mutations conferring resistance are not evenly distributed across all possible conserved sites. Independent of their roles in benzimidazole resistance, tbb-1 and tbb-2 may have specialized functions as null mutants of tbb-1 or tbb-2 were cold or heat sensitive, respectively.

STRIPAK regulation of katanin microtubule severing in the Caenorhabditis elegans embryo.

Microtubule severing plays important role in cell structure and cell division. The microtubule severing protein katanin, composed of the MEI-1/MEI-2 subunits in Caenorhabditis elegans, is required for oocyte meiotic spindle formation; however, it must be inactivated for mitosis to proceed as continued katanin expression is lethal. Katanin activity is regulated by 2 ubiquitin-based protein degradation pathways. Another ubiquitin ligase, HECD-1, the homolog of human HECTD1/HECT domain E3 ubiquitin protein ligase 1, regulates katanin activity without affecting katanin levels. In other organisms, HECD-1 is a component of the striatin-interacting kinase phosphatase complex, which affects cell proliferation and a variety of signaling pathways. Here we conducted a systematic screen of how mutations in striatin-interacting kinase phosphatase components affect katanin function in C. elegans. Striatin-interacting kinase phosphatase core components (FARL-11, CASH-1, LET-92, and GCK-1) were katanin inhibitors in mitosis and activators in meiosis, much like HECD-1. By contrast, variable components (SLMP-1, OTUB-2) functioned as activators of katanin activity in mitosis, indicating they may function to alter striatin-interacting kinase phosphatase core function. The core component CCM-3 acted as an inhibitor at both divisions, while other components (MOB-4, C49H3.6) showed weak interactions with katanin mutants. Additional experiments indicate that katanin may be involved with the centralspindlin complex and a tubulin chaperone. HECD-1 shows ubiquitous expression in the cytoplasm throughout meiosis and early development. The differing functions of the different subunits could contribute to the diverse functions of the striatin-interacting kinase phosphatase complex in C. elegans and other organisms.

Reduction of Derlin activity suppresses Notch-dependent tumours in the C. elegans germ line.

Regulating the balance between self-renewal (proliferation) and differentiation is key to the long-term functioning of all stem cell pools. In the Caenorhabditis elegans germline, the primary signal controlling this balance is the conserved Notch signaling pathway. Gain-of-function mutations in the GLP-1/Notch receptor cause increased stem cell self-renewal, resulting in a tumour of proliferating germline stem cells. Notch gain-of-function mutations activate the receptor, even in the presence of little or no ligand, and have been associated with many human diseases, including cancers. We demonstrate that reduction in CUP-2 and DER-2 function, which are Derlin family proteins that function in endoplasmic reticulum-associated degradation (ERAD), suppresses the C. elegans germline over-proliferation phenotype associated with glp-1(gain-of-function) mutations. We further demonstrate that their reduction does not suppress other mutations that cause over-proliferation, suggesting that over-proliferation suppression due to loss of Derlin activity is specific to glp-1/Notch (gain-of-function) mutations. Reduction of CUP-2 Derlin activity reduces the expression of a read-out of GLP-1/Notch signaling, suggesting that the suppression of over-proliferation in Derlin loss-of-function mutants is due to a reduction in the activity of the mutated GLP-1/Notch(GF) receptor. Over-proliferation suppression in cup-2 mutants is only seen when the Unfolded Protein Response (UPR) is functioning properly, suggesting that the suppression, and reduction in GLP-1/Notch signaling levels, observed in Derlin mutants may be the result of activation of the UPR. Chemically inducing ER stress also suppress glp-1(gf) over-proliferation but not other mutations that cause over-proliferation. Therefore, ER stress and activation of the UPR may help correct for increased GLP-1/Notch signaling levels, and associated over-proliferation, in the C. elegans germline.

Tissue-specific regulation of epidermal contraction during Caenorhabditis elegans embryonic morphogenesis.

Actin and myosin mediate the epidermal cell contractions that elongate the Caenorhabditis elegans embryo from an ovoid to a tubular-shaped worm. Contraction occurs mainly in the lateral epidermal cells, while the dorsoventral epidermis plays a more passive role. Two parallel pathways trigger actinomyosin contraction, one mediated by LET-502/Rho kinase and the other by PAK-1/p21 activated kinase. A number of genes mediating morphogenesis have been shown to be sufficient when expressed either laterally or dorsoventrally. Additional genes show either lateral or dorsoventral phenotypes. This led us to a model where contractile genes have discrete functions in one or the other cell type. We tested this by examining several genes for either lateral or dorsoventral sufficiency. LET-502 expression in the lateral cells was sufficient to drive elongation. MEL-11/Myosin phosphatase, which antagonizes contraction, and PAK-1 were expected to function dorsoventrally, but we could not detect tissue-specific sufficiency. Double mutants of lethal alleles predicted to decrease lateral contraction with those thought to increase dorsoventral force were previously shown to be viable. We hypothesized that these mutant combinations shifted the contractile force from the lateral to the dorsoventral cells and so the embryos would elongate with less lateral cell contraction. This was tested by examining 10 single and double mutant strains. In most cases, elongation proceeded without a noticeable alteration in lateral contraction. We suggest that many embryonic elongation genes likely act in both lateral and dorsoventral cells, even though they may have their primary focus in one or the other cell type.

The R941L mutation in MYH14 disrupts mitochondrial fission and associates with peripheral neuropathy.

BACKGROUND: Peripheral neuropathies are often caused by disruption of genes responsible for myelination or axonal transport. In particular, impairment in mitochondrial fission and fusion are known causes of peripheral neuropathies. However, the causal mechanisms for peripheral neuropathy gene mutations are not always known. While loss of function mutations in MYH14 typically cause non-syndromic hearing loss, the recently described R941L mutation in MYH14, encoding the non-muscle myosin protein isoform NMIIC, leads to a complex clinical presentation with an unexplained peripheral neuropathy phenotype. METHODS: Confocal microscopy was used to examine mitochondrial dynamics in MYH14 patient fibroblast cells, as well as U2OS and M17 cells overexpressing NMIIC. The consequence of the R941L mutation on myosin activity was modeled in C. elegans. FINDINGS: We describe the third family carrying the R941L mutation in MYH14, and demonstrate that the R941L mutation impairs non-muscle myosin protein function. To better understand the molecular basis of the peripheral neuropathy phenotype associated with the R941L mutation, which has been hindered by the fact that NMIIC is largely uncharacterized, we have established a previously unrecognized biological role for NMIIC in mediating mitochondrial fission in human cells. Notably, the R941L mutation acts in a dominant-negative fashion to inhibit mitochondrial fission, especially in the cell periphery. In addition, we observed alterations to the organization of the mitochondrial genome. INTERPRETATION: As impairments in mitochondrial fission cause peripheral neuropathy, this insight into the function of NMIIC likely explains the peripheral neuropathy phenotype associated with the R941L mutation. FUND: This study was supported by the Alberta Children's Hospital Research Institute, the Canadian Institutes of Health Research and the Care4Rare Canada Consortium.

Tissue-Specific Functions of fem-2/PP2c Phosphatase and fhod-1/formin During Caenorhabditis elegans Embryonic Morphogenesis.

The cytoskeleton is the basic machinery that drives many morphogenetic events. Elongation of the C. elegans embryo from a spheroid into a long, thin larva initially results from actomyosin contractility, mainly in the lateral epidermal seam cells, while the corresponding dorsal and ventral epidermal cells play a more passive role. This is followed by a later elongation phase involving muscle contraction. Early elongation is mediated by parallel genetic pathways involving LET-502/Rho kinase and MEL-11/MYPT myosin phosphatase in one pathway and FEM-2/PP2c phosphatase and PAK-1/p21 activated kinase in another. While the LET-502/MEL-11 pathway appears to act primarily in the lateral epidermis, here we show that FEM-2 can mediate early elongation when expressed in the dorsal and ventral epidermis. We also investigated the early elongation function of FHOD-1, a member of the formin family of actin nucleators and bundlers. Previous work showed that FHOD-1 acts in the LET-502/MEL-11 branch of the early elongation pathway as well as in muscle for sarcomere organization. Consistent with this, we found that lateral epidermal cell-specific expression of FHOD-1 is sufficient for elongation, and FHOD-1 effects on elongation appear to be independent of its role in muscle. Also, we found that fhod-1 encodes long and short isoforms that differ in the presence of a predicted coiled-coil domain. Based on tissue-specific expression constructions and an isoform-specific CRISPR allele, the two FHOD-1 isoforms show partially specialized epidermal or muscle function. Although fhod-1 shows only impenetrant elongation phenotypes, we were unable to detect redundancy with other C. elegans formin genes.

Regulation of the MEI-1/MEI-2 Microtubule-Severing Katanin Complex in Early Caenorhabditis elegans Development.

After fertilization, rapid changes of the Caenorhabditis elegans cytoskeleton occur in the transition from meiosis to mitosis, requiring precise regulation. The MEI-1/MEI-2 katanin microtubule-severing complex is essential for meiotic spindle formation but must be quickly inactivated to allow for proper formation of the mitotic spindle. MEI-1/MEI-2 inactivation is dependent on multiple redundant pathways. The primary pathway employs the MEL-26 substrate adaptor for the CUL-3/cullin-based E3 ubiquitin ligase, which targets MEI-1 for proteosomal degradation. Here, we used quantitative antibody staining to measure MEI-1 levels to determine how other genes implicated in MEI-1 regulation act relative to CUL-3/MEL-26 The anaphase-promoting complex/cyclosome, APC/C, the DYRK (Dual-specificity tyrosine-regulated kinase), MBK-2, and the CUL-2-based E3 ubiquitin ligase act together to degrade MEI-1, in parallel to MEL-26/CUL-3 CUL-2 is known to keep MEL-26 low during meiosis, so CUL-2 apparently changes its target from MEL-26 in meiosis to MEI-1 in mitosis. RFL-1, an activator of cullin E3 ubiquitin ligases, activates CUL-2 but not CUL-3 for MEI-1 elimination. HECD-1 (HECT/Homologous to the E6AP carboxyl terminus domain) E3 ligase acts as a MEI-1 activator in meiosis but functions as an inhibitor during mitosis, without affecting levels of MEI-1 or MEI-2 Our results highlight the multiple layers of MEI-1 regulation that are required during the switch from the meiotic to mitotic modes of cell division.

The Rho guanine exchange factor RHGF-2 acts through the Rho-binding kinase LET-502 to mediate embryonic elongation in C. elegans.

Morphogenesis allows an organism to develop its final body shape. In Caenorhabditis elegans, a smooth muscle-like contraction of an actin/myosin network in the epidermis mediates the elongation of the worm embryo from a ball of cells into a long, thin worm. This process is controlled by two redundant pathways, one involving the small GTPase RHO-1 and its downstream effectors LET-502/Rho-binding kinase and MEL-11/myosin phosphatase, and another involving PAK-1/p21 activated kinase and FEM-2/PP2c phosphatase. Contraction occurs primarily in the lateral epidermal cells during elongation while the dorsal and ventral epidermal cells have a more passive role, and localized activity of a Rho GEF (guanine exchange factor) could contribute to this asymmetry. We found that loss of the C. elegans Rho GEF encoded by rhgf-2 results in arrest during early elongation. Genetically, rhgf-2 acts as an activator of let-502/Rho-binding kinase, in parallel to fem-2/PP2c phosphatase. Although expressed throughout the embryo, lateral cell-specific RHGF-2 expression can mediate elongation. The Rho GTPase activating protein (GAP) RGA-2 is known to inhibit contraction in the dorsal and ventral epidermis. Although rhgf-2 and rga-2 are individually lethal, the double mutant is viable with elongation still occurring in a let-502 dependent fashion. This indicates that LET-502/Rho-binding kinase has activity independent of the GEF and GAP. Finally, maternal LET-502 and MEL-11 are known to regulate the rate of cleavage furrow ingression in the early embryo and we show that maternal RHGF-2 also influences cleavage but RGA-2 does not. Thus while the LET-502/MEL-11 pathway is employed multiple times during embryogenesis, regulation by GEFs and GAPs differs at different points of the life cycle and fine tunes contractile function.

Transcriptional regulation of HLH-6-independent and subtype-specific genes expressed in the Caenorhabditis elegans pharyngeal glands.

The Caenorhabditis elegans pharyngeal glands represent one of five cell types in the pharynx. We have previously shown that the bHLH transcription factor, HLH-6, is required for gland development and for expression of many, but not all, gland genes (Smit et al., 2008). Here, we have identified additional gland-expressed genes and find that transcriptional regulatory inputs other than HLH-6 are necessary for their regulation. We demonstrate that at least two hlh-6 independent gland genes, nas-12 and Y8A9A.2, require a cis-acting motif (HRL3- Hlh-6 Regulatory eLement 3), previously described based on its requirement for hlh-6 expression (Ghai and Gaudet, 2008). We also show that expression of the gland-expressed genes, ZK596.1, scl-3, wrt-3, and Y76B12C.3, rely on cis-elements and trans-acting factor(s) other than HLH-6 and HRL3. In addition, we show that negative regulatory mechanisms are employed to refine the spatial expression of some genes, resulting in expression in only a subset of the five gland cells. We show that one of these genes, Y8A9A.2, is negatively regulated by the NHR transcription factor encoded by nhr-48, which represses Y8A9A.2 expression in the g1A cells. We also show that another gene expressed in the reciprocal subset of gland cells, phat-5, is negatively regulated in the g1P and g2 cells by an unknown factor acting through a conserved cis-element in the phat-5 promoter. Overall, this work reveals levels of regulation of gene expression in a single cell type beyond that previously known, and suggests mechanisms by which the different gland sub-types are distinguished.

The C. elegans Snail homolog CES-1 can activate gene expression in vivo and share targets with bHLH transcription factors.

Snail-type transcription factors (TFs) are found in numerous metazoan organisms and function in a plethora of cellular and developmental processes including mesoderm and neuronal development, apoptosis and cancer. So far, Snail-type TFs are exclusively known as transcriptional repressors. They repress gene expression by recruiting transcriptional co-repressors and/or by preventing DNA binding of activators from the basic helix-loop-helix (bHLH) family of TFs to CAGGTG E-box sequences. Here we report that the Caenorhabditis elegans Snail-type TF CES-1 can activate transcription in vivo. Moreover, we provide results that suggest that CES-1 can share its binding site with bHLH TFs, in different tissues, rather than only occluding bHLH DNA binding. Together, our data indicate that there are at least two types of CES-1 target genes and, therefore, that the molecular function of Snail-type TFs is more plastic than previously appreciated.

The HLH-6 transcription factor regulates C. elegans pharyngeal gland development and function.

The Caenorhabditis elegans pharynx (or foregut) functions as a pump that draws in food (bacteria) from the environment. While the "organ identity factor" PHA-4 is critical for formation of the C. elegans pharynx as a whole, little is known about the specification of distinct cell types within the pharynx. Here, we use a combination of bioinformatics, molecular biology, and genetics to identify a helix-loop-helix transcription factor (HLH-6) as a critical regulator of pharyngeal gland development. HLH-6 is required for expression of a number of gland-specific genes, acting through a discrete cis-regulatory element named PGM1 (Pharyngeal Gland Motif 1). hlh-6 mutants exhibit a frequent loss of a subset of glands, while the remaining glands have impaired activity, indicating a role for hlh-6 in both gland development and function. Interestingly, hlh-6 mutants are also feeding defective, ascribing a biological function for the glands. Pharyngeal pumping in hlh-6 mutants is normal, but hlh-6 mutants lack expression of a class of mucin-related proteins that are normally secreted by pharyngeal glands and line the pharyngeal cuticle. An interesting possibility is that one function of pharyngeal glands is to secrete a pharyngeal lining that ensures efficient transport of food along the pharyngeal lumen.