Kinesin-3 KLP-6 regulates intraflagellar transport in male-specific cilia of Caenorhabditis elegans

Cilia are cellular sensory organelles whose integrity of structure and function are important to human health. All cilia are assembled and maintained by kinesin-2 motors in a process termed intraflagellar transport (IFT), but they exhibit great variety of morphology and function. This diversity is proposed to be conferred by cell-specific modulation of the core IFT by additional factors, but examples of such IFT modulators are limited. Here we demonstrate that the cell-specific kinesin-3 KLP-6 acts as a modulator of both IFT dynamics and length in the cephalic male (CEM) cilia of Caenorhabditis elegans. Live imaging of GFP-tagged kinesins in CEM cilia shows partial uncoupling of the IFT motors of the kinesin-2 family, kinesin-II and OSM-3/KIF17, with a portion of OSM-3 moving independently of the IFT complex. KLP-6 moves independently of the kinesin-2 motors and acts to reduce the velocity of OSM-3 and IFT. Additionally, kinesin-II mutants display a novel CEM cilia elongation phenotype that is partially dependent on OSM-3 and KLP-6. Our observations illustrate modulation of the general kinesin-2-driven IFT process by a cell-specific kinesin-3 in cilia of C. elegans male neurons.     

Functional modulation of IFT kinesins extends the sensory repertoire of ciliated neurons in Caenorhabditis elegans

The diversity of sensory cilia on Caenorhabditis elegans neurons allows the animal to detect a variety of sensory stimuli. Sensory cilia are assembled by intraflagellar transport (IFT) kinesins, which transport ciliary precursors, bound to IFT particles, along the ciliary axoneme for incorporation into ciliary structures. Using fluorescence microscopy of living animals and serial section electron microscopy of high pressure-frozen, freeze-substituted IFT motor mutants, we found that two IFT kinesins, homodimeric OSM-3 kinesin and heterotrimeric kinesin II, function in a partially redundant manner to build full-length amphid channel cilia but are completely redundant for building full-length amphid wing (AWC) cilia. This difference reflects cilia-specific differences in OSM-3 activity, which serves to extend distal singlets in channel cilia but not in AWC cilia, which lack such singlets. Moreover, AWC-specific chemotaxis assays reveal novel sensory functions for kinesin II in these wing cilia. We propose that kinesin II is a "canonical" IFT motor, whereas OSM-3 is an "accessory" IFT motor, and that subtle changes in the deployment or actions of these IFT kinesins can contribute to differences in cilia morphology, cilia function, and sensory perception.     

The KLP-6 kinesin is required for male mating behaviors and polycystin localization in Caenorhabditis elegans

BACKGROUND: Male mating behavior of the nematode Caenorhabditis elegans offers an intriguing model to study the genetics of sensory behavior, cilia function, and autosomal dominant polycystic kidney disease (ADPKD). The C. elegans polycystins LOV-1 and PKD-2 act in male-specific sensory cilia required for response and vulva-location mating behaviors. RESULTS: Here, we identify and characterize a new mating mutant, sy511. sy511 behavioral phenotypes were mapped to a mutation in the klp-6 locus, a gene encoding a member of the kinesin-3 family (previously known as the UNC-104/Kif1A family). KLP-6 has a single homolog of unknown function in vertebrate genomes, including fish, chicken, mouse, rat, and human. We show that KLP-6 expresses exclusively in sensory neurons with exposed ciliated endings and colocalizes with the polycystins in cilia of male-specific neurons. Cilia of klp-6 mutants appear normal, suggesting a defect in sensory neuron function but not development. KLP-6 structure-function analysis reveals that the putative cargo binding domain directs the motor to cilia. Consistent with a motor-cargo association between KLP-6 and the polycystins, klp-6 is required for PKD-2 localization and function within cilia. Genetically, we find klp-6 regulates behavior through polycystin-dependent and -independent pathways. CONCLUSION: Multiple ciliary transport pathways dependent on kinesin-II, OSM-3, and KLP-6 may act sequentially to build cilia and localize sensory ciliary membrane proteins such as the polycystins. We propose that KLP-6 and the polycystins function as an evolutionarily conserved ciliary unit. KLP-6 promises new routes to understanding cilia function, behavior, and ADPKD.     

Cuticle of Caenorhabditis elegans: its isolation and partial characterization

The adult cuticle of the soil nematode, Caenorhabditis elegans, is a proteinaceous extracellular structure elaborated by the underlying layer of hypodermal cells during the final molt in the animal's life cycle. The cuticle is composed of an outer cortical layer connected by regularly arranged struts to an inner basal layer. The cuticle can be isolated largely intact and free of all cellular material by sonication and treatment with 1% sodium dodecyl sulfate (SDS). Purified cuticles exhibit a negative material in the basal cuticle layer. The cuticle layers differ in their solubility in sulfhydryl reducing agents, susceptibility to various proteolytic enzymes and amino acid composition. The struts, basal layer, and internal cortical layer are composed of collagen proteins that are extensively cross-linked by disulfide bonds. The external cortical layer appears to contain primarily noncollagen proteins that are extensively cross-linked by nonreducible covalent bonds. The collagen proteins extracted from the cuticle with a reducing agent can be separated by SDS-polyacrylamide gel electrophoresis into eight major species differing in apparent molecular weight.     

KLP-18, a Klp2 kinesin,is required for assembly of acentrosomal meiotic spindles in Caenorhabditis elegans

The proper segregation of chromosomes during meiosis or mitosis requires the assembly of well organized spindles. In many organisms, meiotic spindles lack centrosomes. The formation of such acentrosomal spindles seems to involve first assembly or capture of microtubules (MTs) in a random pattern around the meiotic chromosomes and then parallel bundling and bipolar organization by the action of MT motors and other proteins. Here, we describe the structure, distribution, and function of KLP-18, a Caenorhabditis elegans Klp2 kinesin. Previous reports of Klp2 kinesins agree that it concentrates in spindles, but do not provide a clear view of its function. During prometaphase, metaphase, and anaphase, KLP-18 concentrates toward the poles in both meiotic and mitotic spindles. Depletion of KLP-18 by RNA-mediated interference prevents parallel bundling/bipolar organization of the MTs that accumulate around female meiotic chromosomes. Hence, meiotic chromosome segregation fails, leading to haploid or aneuploid embryos. Subsequent assembly and function of centrosomal mitotic spindles is normal except when aberrant maternal chromatin is present. This suggests that although KLP-18 is critical for organizing chromosome-derived MTs into a parallel bipolar spindle, the order inherent in centrosome-derived astral MT arrays greatly reduces or eliminates the need for KLP-18 organizing activity in mitotic spindles.     

Functional characterization of Caenorhabditis elegans DNA topoisomerase IIIalpha

To investigate the function of a DNA topoisomerase III enzyme in Caenorhabditis elegans, the full-length cDNA of C.elegans DNA topoisomerase IIIα was cloned. The deduced amino acid sequence exhibited identities of 48 and 39% with those of human DNA topoisomerase IIIα and Saccharomyces cerevisiae DNA topoisomerase III, respectively. The overexpressed polypeptide showed an optimal activity for removing negative DNA supercoils at a relatively high temperature of 52–57°C, which is similar to the optimum temperatures of other eukaryotic DNA topoisomerase III enzymes. When topoisomerase IIIα expression was interfered with by a cognate double-stranded RNA injection, pleiotropic phenotypes with abnormalities in germ cell proliferation, oogenesis and embryogenesis appeared. These phenotypes were well correlated with mRNA expression localized in the meiotic cells of gonad and early embryonic cells.     

Purification and characterization of recombinant Caenorhabditis elegans metallothionein

The roundworm Caenorhabditis elegans adapted for survival at high concentrations of Cd(II) expresses two isoforms of metallothionein, CeMT-I and CeMT-II. To characterize one of these proteins CeMT-II was prepared as its Cd containing form by expressing its cDNA heterologously in Escherichia coli. The purified 63-amino-acid protein was identified as the desired product by ion-spray mass spectrometry and was found to resemble in most of its chemical and spectroscopic features the metallothioneins of other animal phyla. The recombinant protein contains a total of 18 cysteine residues and, as documented by electrophoresis and mass spectrometry, binds firmly six Cd ions through the cysteine's side chains. The (113)Cd NMR spectrum features six (113)Cd resonances. Their chemical shift positions between 615 and 675 ppm denote the existence of clusters of tetrahedrally coordinated cadmium thiolate complexes. The metal thiolate coordination dominates also the electronic far-UV absorption spectrum. It is characterized by a massive absorption profile with Cd thiolate shoulders at 255 and 235 nm. Upon replacement of Cd by Zn the profile was blue-shifted by 30 nm.     

Purification and characterization of Caenorhabditis elegans NTH,a homolog of human endonuclease III: Essential role of N-terminal region

Oxidatively damaged bases in DNA cause many types of deleterious effects. The main enzyme that removes such lesions is DNA glycosylase, and accordingly, DNA glycosylase plays an important role in genome stability. Recently, a relationship between DNA glycosylases and aging has been suggested, but it remains controversial. Here, we investigated DNA glycosylases of C. elegans, which is a useful model organism for studying aging. We firstly identified a C. elegans homolog of endonuclease III (NTH), which is a well-conserved DNA glycosylase for oxidatively damaged pyrimidine bases, based on the activity and homology. Blast searching of the Wormbase database retrieved a sequence R10E4.5, highly homologous to the human NTH1. However, the R10E4.5-encoded protein did not have NTH activity, and this was considered to be due to lack of the N-terminal region crucial for the activity. Therefore, we purified the protein encoded by the sequence containing both R10E4.5 and the 117-bp region upstream from it, and found that the protein had the NTH activity. The endogenous CeNTH in the extract of C. elegans showed the same DNA glycosylase activity. Therefore, we concluded that the genuine C. elegans NTH gene is not the R10E4.5 but the sequence containing both R10E4.5 and the 117-bp upstream region. NTH-deficient C. elegans showed no difference from the wild-type in lifespan and was not more sensitive to two oxidizing agents, H₂O₂ and methyl viologen. This suggests that C. elegans has an alternative DNA glycosylase that repairs pyrimidine bases damaged by these agents. Indeed, DNA glycosylase activity that cleaved thymine glycol containing oligonucleotides was detected in the extract of the NTH-deficient C. elegans.     

Functional characterization of Caenorhabditis elegans heteromeric amino acid transporters

Mammalian heteromeric amino acid transporters (HATs) are composed of a multi-transmembrane spanning catalytic protein covalently associated with a type II glycoprotein (e.g. 4F2hc, rBAT) through a disulfide bond. Caenorhabditis elegans has nine genes encoding close homologues of the HAT catalytic proteins. Three of these genes (designated AAT-1 to AAT-3) have a much higher degree of similarity to the mammalian homologues than the other six, including the presence of a cysteine residue at the position known to form a disulfide bridge to the glycoprotein partner in mammalian HATs. C. elegans also has two genes encoding homologues of the heteromeric amino acid transporter type II glycoprotein subunits (designated ATG-1 and ATG-2). Both ATG, and/or AAT-1, -2, -3 proteins were expressed in Xenopus oocytes and tested for amino acid transport function. This screen revealed that AAT-1 and AAT-3 facilitate amino acid transport when expressed together with ATG-2 but not with ATG-1 or the mammalian type II glycoproteins 4F2hc and rBAT. AAT-1 and AAT-3 covalently bind to both C. elegans ATG glycoproteins, but only the pairs with ATG-2 traffic to the oocyte surface. Both of these functional, surface-expressed C. elegans HATs transport most neutral amino acids and display the highest transport rate for l-Ala and l-Ser (apparent K(m) 100 microm range). Similar to their mammalian counterparts, the C. elegans HATs function as (near) obligatory amino acid exchangers. Taken together, this study demonstrates that the heteromeric structure and the amino acid exchange function of HATs have been conserved throughout the evolution of nematodes to mammals.     

Purification and characterization of beta-galactoside-binding proteins from Caenorhabditis elegans.

Two carbohydrate-binding proteins (subunit molecular masses, 32 and 16 kDa, respectively) were isolated for the first time from a nematode, Caenorhabditis elegans. They were specifically extracted with lactose and adsorbed on asialofetuin-Sepharose in the absence of a metal ion. Although these two proteins were co-eluted from a gel filtration column at a position corresponding to an apparent molecular size of 30 kDa under non-denaturing conditions, they could be separated by reversed-phase chromatography. The 32 kDa protein, the main component, was further characterized. Together with its solubility, saccharide specificity and metal independence, some other structural properties, including its amino acid composition, UV spectrum, and partial amino acid sequence, strongly suggested that the 32 kDa protein is a member of a class of soluble beta-galactoside-binding lectins which had previously been only found in vertebrates.     

Functional characterization of SR and SR-related genes in Caenorhabditis elegans

The SR proteins constitute a family of nuclear phosphoproteins, which are required for constitutive splicing and also influence alternative splicing regulation. Initially, it was suggested that SR proteins were functionally redundant in constitutive splicing. However, differences have been observed in alternative splicing regulation, suggesting unique functions for individual SR proteins. Homology searches of the Caenorhabditis elegans genome identified seven genes encoding putative orthologues of the human factors SF2/ASF, SRp20, SC35, SRp40, SRp75 and p54, and also several SR-related genes. To address the issue of functional redundancy, we used dsRNA interference (RNAi) to inhibit specific SR protein function during C.elegans development. RNAi with CeSF2/ASF caused late embryonic lethality, suggesting that this gene has an essential function during C.elegans development. RNAi with other SR genes resulted in no obvious phenotype, which is indicative of gene redundancy. Simultaneous interference of two or more SR proteins in certain combinations caused lethality or other developmental defects. RNAi with CeSRPK, an SR protein kinase, resulted in early embryonic lethality, suggesting an essential role for SR protein phosphorylation during development.     

Molecular characterization of the Caenorhabditis elegans Rho GDP-dissociation inhibitor.

GDP-dissociation inhibitors (GDIs) form one of the classes of regulatory proteins that modulate the cycling of the Ras superfamily of GTPases between active GTP-bound and inactive GDP-bound states. We report here the characterization of the Caenorhabditis elegans RhoGDI (CeRhoGDI) as part of our investigations into Rho-GTPase signalling pathways that are involved in nematode development. CeRhoGDI is a 23-kDa protein that is localized predominantly in the cytosol. CeRhoGDI interacts only with the lipid-modified forms of C. elegans Rho-GTPases, CeRhoA, CeRac1 and Cdc42Ce, in vitro and is able to solubilize the membrane-bound forms of these GTPases. CeRhoGDI recognizes the GTPases in both GTP- and GDP-bound forms; hence it inhibits both the guanine-nucleotide dissociation and GTP-hydrolysis activities. The inhibitory activity towards the GTP-bound GTPases is weak compared with that towards GDP-bound GTPases. CeRhoGDI is expressed throughout development and is highly expressed in marginal and vulval epithelial cells, in sperm cells and spicules. Taken together, our results suggest that CeRhoGDI may be involved in specific morphogenetic events mediated by the C. elegans Rho-GTPases.     

Essential kinase-independent role of a Fer-like non-receptor tyrosine kinase in Caenorhabditis elegans morphogenesis

Morphogenesis requires coordination of cell surface activity and cytoskeletal architecture. During the initial stage of morphogenesis in Caenorhabditis elegans, the concerted movement of surface epithelial cells results in enclosure of the embryo by the epidermis. We report that Fer-related kinase-1 (FRK-1), an ortholog of the mammalian non-receptor tyrosine kinase Fer, is necessary for embryonic enclosure and morphogenesis in C. elegans. Expression of FRK-1 in epidermal cells is sufficient to rescue a chromosomal deficiency that removes the frk-1 locus, demonstrating its autonomous requirement in the epidermis. The essential function of FRK-1 is independent of its kinase domain, suggesting a non-enzymatic role in morphogenesis. Localization of FRK-1 to the plasma membrane requires beta-catenin, but not cadherin or alpha-catenin, and muscle-expressed beta-integrin is non-autonomously required for this localization; in the absence of these components FRK-1 becomes nuclear. Mouse FerT rescues the morphogenetic defects of frk-1 mutants and expression of FRK-1 in mammalian cells results in loss of adhesion, implying a conserved function for FRK-1/FerT in cell adhesion and morphogenesis. Thus, FRK-1 performs a kinase-independent function in differentiation and morphogenesis of the C. elegans epidermis during embryogenesis.     

The kinesin-3 family motor KLP-4 regulates anterograde trafficking of GLR-1 glutamate receptors in the ventral nerve cord of Caenorhabditis elegans

The transport of glutamate receptors from the cell body to synapses is essential during neuronal development and may contribute to the regulation of synaptic strength in the mature nervous system. We previously showed that cyclin-dependent kinase-5 (CDK-5) positively regulates the abundance of GLR-1 glutamate receptors at synapses in the ventral nerve cord (VNC) of Caenorhabditis elegans. Here we identify a kinesin-3 family motor klp-4/KIF13 in a cdk-5 suppressor screen for genes that regulate GLR-1 trafficking. klp-4 mutants have decreased abundance of GLR-1 in the VNC. Genetic analysis of klp-4 and the clathrin adaptin unc-11/AP180 suggests that klp-4 functions before endocytosis in the ventral cord. Time-lapse microscopy indicates that klp-4 mutants exhibit decreased anterograde flux of GLR-1. Genetic analysis of cdk-5 and klp-4 suggests that they function in the same pathway to regulate GLR-1 in the VNC. Interestingly, GLR-1 accumulates in cell bodies of cdk-5 but not klp-4 mutants. However, GLR-1 does accumulate in klp-4-mutant cell bodies if receptor degradation in the multivesicular body/lysosome pathway is blocked. This study identifies kinesin KLP-4 as a novel regulator of anterograde glutamate receptor trafficking and reveals a cellular control mechanism by which receptor cargo is targeted for degradation in the absence of its motor.     

Biochemical characterization of the WRN-1 RecQ helicase of Caenorhabditis elegans

The highly conserved RecQ helicases are essential for the maintenance of genomic stability. Werner syndrome protein, WRN, is one of five human RecQ helicase homologues, and a deficiency of the protein causes a hereditary premature aging disorder that is characterized by genomic instability. A WRN orthologue, wrn-1 lacking the exonuclease domain, has been identified in the nematode Caenorhabditis elegans. wrn-1(RNAi) in C. elegans has a shortened life span, increased sensitivity to DNA damage, and accelerated aging phenotypes. However, little is known about its enzymatic activity. We purified the recombinant C. elegans WRN-1 protein (CeWRN-1) and then investigated its substrate specificity in vitro to improve our understanding of its function in vivo. We found that CeWRN-1 is an ATP-dependent 3'-5' helicase capable of unwinding a variety of DNA structures such as forked duplexes, Holliday junctions, bubble substrates, D-loops, and flap duplexes, and 3'-tailed duplex substrates. Distinctly, CeWRN-1 is able to unwind a long forked duplex compared to human WRN. Furthermore, CeWRN-1 helicase activity on a long DNA duplex is stimulated by C. elegans replication protein A (CeRPA) that is shown to interact with CeWRN-1 by a dot blot. The ability of CeWRN-1 to unwind these DNA structures may improve the access for DNA repair and replication proteins that are important for preventing the accumulation of abnormal structures, contributing to genomic stability.     

Functional characterization of a water channel of the nematode Caenorhabditis elegans

A genome project for the species Caenorhabditis elegans has demonstrated the presence of eight cDNAs belonging to the major intrinsic protein (MIP) family. We previously characterized one of these cDNAs known as C01G6.1. C01G6.1 was confirmed to be a water channel and newly designated as AQP-CE1 [Am. J. Physiol. 275 (1998) C1459-C1464]. In this paper, we examined the function of another MIP protein encoded by F40F9.9. This cDNA encodes a 274-amino acid protein showing a high sequence identity with mammalian aquaporin-8 (AQP8) water channel (35%) and d-TIP (34%), an AQP of Arabidopsis. The expression of F40F9.9 in Xenopus oocytes increased the osmotic water permeability (P(f)) 10.4-fold, and the activation energy for P(f) from Arrhenius plot was 4.7 kcal/mol, suggesting that F40F9.9 is a water channel (AQP-CE2). AQP-CE2 was not permeable to glycerol or urea. Oocyte P(f) was reversibly inhibited by 58% after an incubation with 0.3 mM HgCl(2). To identify the mercury-sensitive site, four individual cysteine residues in AQP-CE2 (at positions 47, 132, 149, 259) were altered to serine by site-directed mutagenesis. Of these mutants, only C132S had a P(f) similar to that of the wild-type together with an acquired mercury resistance, suggesting that Cys-132 is the mercury-sensitive site. Similar results were obtained by the mutation of Cys-132 to alanine (C132A). Replacement of Cys-132 with tryptophan decreased P(f) by 64%, but P(f) was still 2.5 times higher than that of the control. Cys-132 is located in the transmembrane helix 3, close to the transition to the extracellular loop C. These results suggest that the transmembrane helix 3, including Cys-132, might participate in the aqueous pore formation, or, alternatively, that Cys-132 might contribute to the construction of the AQP protein.