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1.
2.
Autoinhibition regulates the motility of the C. elegans intraflagellar transport motor OSM-3
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OSM-3 is a Kinesin-2 family member from Caenorhabditis elegans that is involved in intraflagellar transport (IFT), a process essential for the construction and maintenance of sensory cilia. In this study, using a single-molecule fluorescence assay, we show that bacterially expressed OSM-3 in solution does not move processively (multiple steps along a microtubule without dissociation) and displays low microtubule-stimulated adenosine triphosphatase (ATPase) activity. However, a point mutation (G444E) in a predicted hinge region of OSM-3's coiled-coil stalk as well as a deletion of that hinge activate ATPase activity and induce robust processive movement. These hinge mutations also cause a conformational change in OSM-3, causing it to adopt a more extended conformation. The motility of wild-type OSM-3 also can be activated by attaching the motor to beads in an optical trap, a situation that may mimic attachment to IFT cargo. Our results suggest that OSM-3 motility is repressed by an intramolecular interaction that involves folding about a central hinge and that IFT cargo binding relieves this autoinhibition in vivo. Interestingly, the G444E allele in C. elegans produces similar ciliary defects to an osm-3-null mutation, suggesting that autoinhibition is important for OSM-3's biological function. 相似文献
3.
Robin G DeBonis S Dornier A Cappello G Ebel C Wade RH Thierry-Mieg D Kozielski F 《Biochemistry》2005,44(17):6526-6536
Kinesins form a superfamily of molecular motors involved in cell division and intracellular transport. Twenty kinesins have been found in the Caenorhabditis elegans genome, and four of these belong to the kinesin-14 subfamily, i.e., kinesins with C-terminal motor domains. Three of these kinesin-14s, KLP-15, KLP-16, and KLP-17, form a distinct subgroup in which KLP-15 and KLP-16 are more than 90% identical and appear to be related by a relatively recent gene duplication. They are essential for meiotic spindle organization and chromosome segregation, and are mostly expressed in the germline. With 587 amino acids each, they are among the smallest kinesins known. Using bacterially expressed KLP-15 constructs with different length extensions preceding the motor domain, we have determined in vitro the following characteristic properties: ATPase activity, microtubule binding, oligomeric state, microtubule gliding activity, and direction of movement. The constructs exhibit a monomer-dimer equilibrium that depends on the length of the predicted alpha-helical coiled-coil region preceding the motor domain. The longest construct with the complete coiled-coil domain is a stable dimer, and the shortest construct with only seven amino acids preceding the motor domain is a monomer. In microtubule gliding assays, the monomer is immobile whereas the fully dimeric KLP-15 construct supports gliding at 2.3 microm/min and moves toward microtubule minus ends, like other members of the kinesin-14 subfamily studied to date. 相似文献
4.
Caenorhabditis elegans DYF-2, an orthologue of human WDR19, is a component of the intraflagellar transport machinery in sensory cilia
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Efimenko E Blacque OE Ou G Haycraft CJ Yoder BK Scholey JM Leroux MR Swoboda P 《Molecular biology of the cell》2006,17(11):4801-4811
The intraflagellar transport (IFT) machinery required to build functional cilia consists of a multisubunit complex whose molecular composition, organization, and function are poorly understood. Here, we describe a novel tryptophan-aspartic acid (WD) repeat (WDR) containing IFT protein from Caenorhabditis elegans, DYF-2, that plays a critical role in maintaining the structural and functional integrity of the IFT machinery. We determined the identity of the dyf-2 gene by transgenic rescue of mutant phenotypes and by sequencing of mutant alleles. Loss of DYF-2 function selectively affects the assembly and motility of different IFT components and leads to defects in cilia structure and chemosensation in the nematode. Based on these observations, and the analysis of DYF-2 movement in a Bardet-Biedl syndrome mutant with partially disrupted IFT particles, we conclude that DYF-2 can associate with IFT particle complex B. At the same time, mutations in dyf-2 can interfere with the function of complex A components, suggesting an important role of this protein in the assembly of the IFT particle as a whole. Importantly, the mouse orthologue of DYF-2, WDR19, also localizes to cilia, pointing to an important evolutionarily conserved role for this WDR protein in cilia development and function. 相似文献
5.
The molecular identities of the Caenorhabditis elegans intraflagellar transport genes dyf-6, daf-10 and osm-1
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The Caenorhabditis elegans genes dyf-6, daf-10, and osm-1 are among the set of genes that affect chemotaxis and the ability of certain sensory neurons to take up fluorescent dyes from the environment. Some genes in this category are known to be required for intraflagellar transport (IFT), which is the bidirectional movement of raft-like particles along the axonemes of cilia and flagella. The cloning of dyf-6, daf-10, and osm-1 are described here. The daf-10 and osm-1 gene products resemble each other and contain WD and WAA repeats. DYF-6, the product of a complex locus, lacks known motifs, but orthologs are present in flies and mammals. Phenotypic analysis of dyf-6 mutants expressing an OSM-6::GFP reporter indicates that the cilia of the amphid and phasmid dendritic endings are foreshortened. Consistent with genetic mosaic analysis, which indicates that dyf-6 functions in neurons of the amphid sensilla, DYF-6::GFP is expressed in amphid and phasmid neurons. Movement of DYF-6::GFP within the ciliated endings of the neurons indicates that DYF-6 is involved in IFT. In addition, IFT can be observed in dauer larvae. 相似文献
6.
Brown JM Fine NA Pandiyan G Thazhath R Gaertig J 《Molecular biology of the cell》2003,14(8):3192-3207
We cloned a Tetrahymena thermophila gene, IFT52, encoding a homolog of the Chlamydomonas intraflagellar transport protein, IFT52. Disruption of IFT52 led to loss of cilia and incomplete cytokinesis, a phenotype indistinguishable from that of mutants lacking kinesin-II, a known ciliary assembly transporter. The cytokinesis failures seem to result from lack of cell movement rather than from direct involvement of ciliary assembly pathway components in cytokinesis. Spontaneous partial suppressors of the IFT52 null mutants occurred, which assembled cilia at high cell density and resorbed cilia at low cell density. The stimulating effect of high cell density on cilia formation is based on the creation of pericellular hypoxia. Thus, at least under certain conditions, ciliary assembly is affected by an extracellular signal and the Ift52p function may be integrated into signaling pathways that regulate ciliogenesis. 相似文献
7.
Snow JJ Ou G Gunnarson AL Walker MR Zhou HM Brust-Mascher I Scholey JM 《Nature cell biology》2004,6(11):1109-1113
Cilia have diverse roles in motility and sensory reception and their dysfunction contributes to cilia-related diseases. Assembly and maintenance of cilia depends on the intraflagellar transport (IFT) of axoneme, membrane, matrix and signalling proteins to appropriate destinations within the organelle. In the current model, these diverse cargo proteins bind to multiple sites on macromolecular IFT particles, which are moved by a single anterograde IFT motor, kinesin-II, from the ciliary base to its distal tip, where cargo-unloading occurs. Here, we describe the observation of fluorescent IFT motors and IFT particles moving along distinct domains within sensory cilia of wild-type and IFT-motor-mutant Caenorhabditis elegans. We show that two anterograde IFT motor holoenzymes, kinesin-II and Osm-3-kinesin, cooperate in a surprising way to control two pathways of IFT that build distinct parts of cilia. Instead of each motor independently moving its own specific cargo to a distinct destination, the two motors function redundantly to transport IFT particles along doublet microtubules adjacent to the transition zone to form the axoneme middle segment. Next, Osm-3-kinesin alone transports IFT particles along the distal singlet microtubules to stabilize the distal segment. Thus, the subtle coordinate activity of these IFT motors creates two sequential transport pathways. 相似文献
8.
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. 相似文献
9.
MI Monteiro S Ahlawat JR Kowalski E Malkin SP Koushika P Juo 《Molecular biology of the cell》2012,23(18):3647-3662
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. 相似文献
10.
Mutant sensory cilia in the nematode Caenorhabditis elegans 总被引:13,自引:0,他引:13
Eight classes of chemosensory neurons in C. elegans fill with fluorescein when living animals are placed in a dye solution. Fluorescein enters the neurons through their exposed sensory cilia. Mutations in 14 genes prevent dye uptake and disrupt chemosensory behaviors. Each of these genes affects the ultrastructure of the chemosensory cilia or their accessory cells. In each case, the cilia are shorter or less exposed than normal, suggesting that dye contact is the principal factor under selection. Ten genes affect many or all of the sensory cilia in the head. The daf-19 (m86) mutation eliminates all cilia, leaving only occasional centrioles in the dendrites. The cilia in che-13 (e1805), osm-1 (p808), osm-5 (p813), and osm-6 (p811) mutants have normal transition zones and severely shortened axonemes. Doublet-microtubules, attached to the membrane by Y links, assemble ectopically proximal to the cilia in these mutants. The amphid cilia in che-11 (e1810) are irregular in diameter and contain dark ground material in the middle of the axonemes. Certain mechanocilia are also affected. The amphid cilia in che-10 (e1809) apparently degenerate, leaving dendrites with bulb-shaped endings filled with dark ground material. The mechanocilia lack striated rootlets. Cilia defects have also been found in che-2, che-3, and daf-10 mutants. The osm-3 (p802) mutation specifically eliminates the distal segment of the amphid cilia. Mutations in three genes affect sensillar support cells. The che-12 (e1812) mutation eliminates matrix material normally secreted by the amphid sheath cell. The che-14 (e1960) mutation disrupts the joining of the amphid sheath and socket cells to form the receptor channel. A similar defect has been observed in daf-6 mutants. Four additional genes affect specific classes of ciliated sensory neurons. The mec-1 and mec-8 (e398) mutations disrupt the fasciculation of the amphid cilia. The cat-6 (e1861) mutation disrupts the tubular bodies of the CEP mechanocilia. A cryophilic thermotaxis mutant, ttx-1 (p767), lacks fingers on the AFD dendrite, suggesting this neuron is thermosensory. 相似文献
11.
Marie Lopez Christo P Christov María Dolores Sequedo Mareike Herzog Anna E Herlihy Maxime Bodak Roxani Gatsi Howard A Baylis 《EMBO reports》2015,16(3):341-350
RNA interference (RNAi) is a widespread and widely exploited phenomenon. Here, we show that changing inositol 1,4,5‐trisphosphate (IP3) signalling alters RNAi sensitivity in Caenorhabditis elegans. Reducing IP3 signalling enhances sensitivity to RNAi in a broad range of genes and tissues. Conversely up‐regulating IP3 signalling decreases sensitivity. Tissue‐specific rescue experiments suggest IP3 functions in the intestine. We also exploit IP3 signalling mutants to further enhance the sensitivity of RNAi hypersensitive strains. These results demonstrate that conserved cell signalling pathways can modify RNAi responses, implying that RNAi responses may be influenced by an animal's physiology or environment. 相似文献
12.
Krishna Kumar Vasudevan Yu-Yang Jiang Karl F. Lechtreck Yasuharu Kushida Lea M. Alford Winfield S. Sale Todd Hennessey Jacek Gaertig 《Molecular biology of the cell》2015,26(3):478-494
Kinesin-13, an end depolymerizer of cytoplasmic and spindle microtubules, also affects the length of cilia. However, in different models, depletion of kinesin-13 either lengthens or shortens cilia, and therefore the exact function of kinesin-13 in cilia remains unclear. We generated null mutations of all kinesin-13 paralogues in the ciliate Tetrahymena. One of the paralogues, Kin13Ap, localizes to the nuclei and is essential for nuclear divisions. The remaining two paralogues, Kin13Bp and Kin13Cp, localize to the cell body and inside assembling cilia. Loss of both Kin13Bp and Kin13Cp resulted in slow cell multiplication and motility, overgrowth of cell body microtubules, shortening of cilia, and synthetic lethality with either paclitaxel or a deletion of MEC-17/ATAT1, the α-tubulin acetyltransferase. The mutant cilia assembled slowly and contained abnormal tubulin, characterized by altered posttranslational modifications and hypersensitivity to paclitaxel. The mutant cilia beat slowly and axonemes showed reduced velocity of microtubule sliding. Thus kinesin-13 positively regulates the axoneme length, influences the properties of ciliary tubulin, and likely indirectly, through its effects on the axonemal microtubules, affects the ciliary dynein-dependent motility. 相似文献
13.
Phospholipase Cepsilon (PLCepsilon) is a novel class of phosphoinositide-specific PLC with unknown physiological functions. Here, we present the first genetic analysis of PLCepsilon in an intact organism, the nematode Caenorhabditis elegans. Ovulation in C. elegans is dependent on an inositol 1,4,5-trisphosphate (IP(3)) signaling pathway activated by the receptor tyrosine kinase LET-23. We generated deletion mutants of the gene, plc-1, encoding C. elegans PLCepsilon. We observed a novel ovulation phenotype whereby oocytes are trapped in the spermatheca due to delayed dilation of the spermatheca-uterine valve. The expression of plc-1 in the adult spermatheca is consistent with its involvement in regulation of ovulation. On the other hand, we failed to observe genetic interaction of plc-1 with let-23-mediated IP(3) signaling pathway genes, suggesting a complex mechanism for control of ovulation. 相似文献
14.
van Swinderen B Metz LB Shebester LD Mendel JE Sternberg PW Crowder CM 《Genetics》2001,158(2):643-655
To identify genes controlling volatile anesthetic (VA) action, we have screened through existing Caenorhabditis elegans mutants and found that strains with a reduction in Go signaling are VA resistant. Loss-of-function mutants of the gene goa-1, which codes for the alpha-subunit of Go, have EC(50)s for the VA isoflurane of 1.7- to 2.4-fold that of wild type. Strains overexpressing egl-10, which codes for an RGS protein negatively regulating goa-1, are also isoflurane resistant. However, sensitivity to halothane, a structurally distinct VA, is differentially affected by Go pathway mutants. The RGS overexpressing strains, a goa-1 missense mutant found to carry a novel mutation near the GTP-binding domain, and eat-16(rf) mutants, which suppress goa-1(gf) mutations, are all halothane resistant; goa-1(null) mutants have wild-type sensitivities. Double mutant strains carrying mutations in both goa-1 and unc-64, which codes for a neuronal syntaxin previously found to regulate VA sensitivity, show that the syntaxin mutant phenotypes depend in part on goa-1 expression. Pharmacological assays using the cholinesterase inhibitor aldicarb suggest that VAs and GOA-1 similarly downregulate cholinergic neurotransmitter release in C. elegans. Thus, the mechanism of action of VAs in C. elegans is regulated by Goalpha, and presynaptic Goalpha-effectors are candidate VA molecular targets. 相似文献
15.
Mating behavior of animals is regulated by the sensory stimuli provided by the other sex. Sexually receptive females emit mating signals that can be inhibited by male ejaculate. The genetic mechanisms controlling the release of mating signals and encoding behavioral responses remain enigmatic. Here we present evidence of a Caenorhabditis elegans hermaphrodite-derived cue that stimulates male mating-response behavior and is dynamically regulated by her reproductive status. Wild-type males preferentially mated with older hermaphrodites. Increased sex appeal of older hermaphrodites was potent enough to stimulate robust response from mating-deficient pkd-2 and lov-1 polycystin mutant males. This enhanced response of pkd-2 males toward older hermaphrodites was independent of short-chain ascaroside pheromones, but was contingent on the absence of active sperm in the hermaphrodites. The improved pkd-2 male response toward spermless hermaphrodites was blocked by prior insemination or by genetic ablation of the ceh-18-dependent sperm-sensing pathway of the hermaphrodite somatic gonad. Our work suggests an interaction between sperm and the soma that has a negative but reversible effect on a hermaphrodite-derived mating cue that regulates male mating response, a phenomenon to date attributed to gonochoristic species only. 相似文献
16.
Migration of cells within epithelial sheets is an important feature of embryogenesis and other biological processes. Previous work has demonstrated a role for inositol 1,4,5-trisphosphate (IP(3))-mediated calcium signalling in the rearrangement of epidermal cells (also known as hypodermal cells) during embryonic morphogenesis in Caenorhabditis elegans. However the mechanism by which IP(3) production is stimulated is unknown. IP(3) is produced by the action of phospholipase C (PLC). We therefore surveyed the PLC family of C. elegans using RNAi and mutant strains, and found that depletion of PLC-1/PLC-epsilon produced substantial embryonic lethality. We used the epithelial cell marker ajm-1::gfp to follow the behaviour of epidermal cells and found that 96% of the arrested embryos have morphogenetic defects. These defects include defective ventral enclosure and aberrant dorsal intercalation. Using time-lapse confocal microscopy we show that the migration of the ventral epidermal cells, especially of the leading cells, is slower and often fails in plc-1(tm753) embryos. As a consequence plc-1 loss of function results in ruptured embryos with a Gex phenotype (gut on exterior) and lumpy larvae. Thus PLC-1 is involved in the regulation of morphogenesis. Genetic studies using gain- and loss-of-function alleles of itr-1, the gene encoding the IP(3) receptor in C. elegans, demonstrate that PLC-1 acts through ITR-1. Using RNAi and double mutants to deplete the other PLCs in a plc-1 background, we show that PLC-3/PLC-gamma and EGL-8/PLC-beta can compensate for reduced PLC-1 activity. Our work places PLC-epsilon into a pathway controlling epidermal cell migration, thus establishing a novel role for PLC-epsilon. 相似文献
17.
Tai Hoon Kim Do Hyun Kim Hyung Wook Nam Sang Yoon Park Jaegal Shim Jin Won Cho 《Molecules and cells》2010,29(4):413-418
The sulfation of tyrosine residues is an important post-translational modification involved in the regulation of protein function.
We examined the activity of worm tyrosylprotein sulfotransferase (TPST-1) on a typical cuticle collagen, ROL-6, in C. elegans. We verified that TPST-1 sulfates three tyrosine residues of ROL-6 in vitro. We found that these tyrosine residues are important for the secretion of ROL-6::GFP. Mutant ROL-6::GFP proteins that contain
more than two substitutions of the target tyrosine residues are severely deficient in cuticle localization. Consistently,
knock down of tpst-1 blocked the cuticle localization of ROL-6::GFP. Therefore, the sulfation of ROL-6 by TPST-1 is critical for the proper localization
of ROL-6. We also confirmed that worm TPST-1 is localized to the trans-Golgi network (TGN). Our results indicate that TPST-1 regulates cuticle organization by promoting the transport of ROL-6
from the TGN to the cuticle. 相似文献
18.
The cell cycles in C. elegans are tightly controlled but appear to use the same regulators found in other organisms. Four homologues of the dual-specificity phosphatase Cdc25 are present in the C. elegans genome. In our study, we have characterized a deletion mutant for one of these orthologues. We show that embryonic defects are absent in cdc-25.1 homozygous mutants, presumably because of maternally contributed CDC-25.1 product. These embryos hatch and develop into sterile adults. The adults do not appear to have any somatic defects. The sterility results from inadequate germline proliferation. Germline precursors divide slowly and produce abnormally sized daughter cells. Only three to four rounds of germ-cell division occur before they die during the L3 and L4 larval stages. 相似文献
19.
Fujii M Matsumoto Y Tanaka N Miki K Suzuki T Ishii N Ayusawa D 《The Journal of biological chemistry》2004,279(19):20277-20282
The relationship between oxidative stress and longevity is a matter of concern in various organisms. We isolated mutants resistant to paraquat from nematode Caenorhabditis elegans. One mutant named mev-4 was long-lived and showed cross-resistance to heat and Dyf phenotype (defective in dye filling). Genetic and sequence analysis revealed that mev-4 had a nonsense mutation on the che-11 gene, homologues of which are involved in formation of cilia and flagella in other organisms. The paraquat resistance was commonly observed in various Dyf mutants and did not depend on the daf-16 gene, whereas the extension of life span did depend on it. Expression of antioxidant enzyme genes seemed normal. These results suggest that chemosensory neurons are a target of oxidative stress and influence longevity dependent on the daf-16 signaling in C. elegans. 相似文献
20.
Maruyama R Velarde NV Klancer R Gordon S Kadandale P Parry JM Hang JS Rubin J Stewart-Michaelis A Schweinsberg P Grant BD Piano F Sugimoto A Singson A 《Current biology : CB》2007,17(18):1555-1560
Fertilization triggers egg activation and converts the egg into a developing embryo. The events of this egg-to-embryo transition typically include the resumption of meiosis, the reorganization of the cortical actin cytoskeleton, and the remodeling of the oocyte surface. The factors that regulate sperm-dependent egg-activation events are not well understood. Caenorhabditis elegans EGG-3, a member of the protein tyrosine phosphatase-like (PTPL) family, is essential for regulating cell-surface and cortex rearrangements during egg activation in response to sperm entry. Although fertilization occurred normally in egg-3 mutants, the polarized dispersal of F-actin is altered, a chitin eggshell is not formed, and no polar bodies are produced. EGG-3 is associated with the oocyte plasma membrane in a pattern that is similar to CHS-1 and MBK-2. CHS-1 is required for eggshell deposition, whereas MBK-2 is required for the degradation of maternal proteins during the egg-to-embryo transition. The localization of CHS-1 and EGG-3 are interdependent and both genes were required for the proper localization of MBK-2 in oocytes. Therefore, EGG-3 plays a central role in egg activation by influencing polarized F-actin dynamics and the localization or activity of molecules that are directly involved in executing the egg-to-embryo transition. 相似文献