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1.
EBioMedicine ; 45: 379-392, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31231018

RESUMO

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.

2.
G3 (Bethesda) ; 8(7): 2277-2290, 2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29720391

RESUMO

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.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas dos Microfilamentos/genética , Morfogênese/genética , Fosfoproteínas Fosfatases/genética , Processamento Alternativo , Animais , Animais Geneticamente Modificados , Embrião não Mamífero , Epiderme/embriologia , Epiderme/metabolismo , Especificidade de Órgãos/genética , Fenótipo
3.
G3 (Bethesda) ; 8(5): 1425-1437, 2018 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-29593072

RESUMO

The ELT-2 GATA factor normally functions in differentiation of the C. elegans endoderm, downstream of endoderm specification. We have previously shown that, if ELT-2 is expressed sufficiently early, it is also able to specify the endoderm and to replace all other members of the core GATA-factor transcriptional cascade (END-1, END-3, ELT-7). However, such rescue requires multiple copies (and presumably overexpression) of the end-1p::elt-2 cDNA transgene; a single copy of the transgene does not rescue. We have made this observation the basis of a genetic screen to search for genetic modifiers that allow a single copy of the end-1p::elt-2 cDNA transgene to rescue the lethality of the end-1 end-3 double mutant. We performed this screen on a strain that has a single copy insertion of the transgene in an end-1 end-3 background. These animals are kept alive by virtue of an extrachromosomal array containing multiple copies of the rescuing transgene; the extrachromosomal array also contains a toxin under heat shock control to counterselect for mutagenized survivors that have been able to lose the rescuing array. A screen of ∼14,000 mutagenized haploid genomes produced 17 independent surviving strains. Whole genome sequencing was performed to identify genes that incurred independent mutations in more than one surviving strain. The C. elegans gene tasp-1 was mutated in four independent strains. tasp-1 encodes the C. elegans homolog of Taspase, a threonine-aspartic acid protease that has been found, in both mammals and insects, to cleave several proteins involved in transcription, in particular MLL1/trithorax and TFIIA. A second gene, pqn-82, was mutated in two independent strains and encodes a glutamine-asparagine rich protein. tasp-1 and pqn-82 were verified as loss-of-function modifiers of the end-1p::elt-2 transgene by RNAi and by CRISPR/Cas9-induced mutations. In both cases, gene loss leads to modest increases in the level of ELT-2 protein in the early endoderm although ELT-2 levels do not strictly correlate with rescue. We suggest that tasp-1 and pqn-82 represent a class of genes acting in the early embryo to modulate levels of critical transcription factors or to modulate the responsiveness of critical target genes. The screen's design, rescuing lethality with an extrachromosomal transgene followed by counterselection, has a background survival rate of <10-4 without mutagenesis and should be readily adapted to the general problem of identifying suppressors of C. elegans lethal mutations.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Diferenciação Celular , Endoderma/metabolismo , Fatores de Transcrição GATA/genética , Genes Modificadores , Intestinos/citologia , Mutação/genética , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/embriologia , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Diferenciação Celular/genética , Embrião não Mamífero/metabolismo , Fatores de Transcrição GATA/química , Fatores de Transcrição GATA/metabolismo , Testes Genéticos , Genótipo , Reprodutibilidade dos Testes , Análise de Sobrevida , Sequenciamento Completo do Genoma , Zigoto/metabolismo
4.
Mol Genet Metab ; 122(4): 160-171, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29153845

RESUMO

Vitamin B12 (cobalamin, Cbl) is a micronutrient essential to human health. Cbl is not utilized as is but must go through complex subcellular and metabolic processing to generate two cofactor forms: methyl-Cbl for methionine synthase, a cytosolic enzyme; and adenosyl-Cbl for methylmalonyl-CoA mutase, a mitochondrial enzyme. Some 10-12 human genes have been identified responsible for the intracellular conversion of Cbl to cofactor forms, including genes that code for ATP-binding cassette (ABC) transporters acting at the lysosomal and plasma membranes. However, the gene for mitochondrial uptake is not known. We hypothesized that ABC transporters should be candidates for other uptake and efflux functions, including mitochondrial transport, and set out to screen ABC transporter mutants for blocks in Cbl utilization using the nematode roundworm Caenorhabditis elegans. Thirty-seven mutant ABC transporters were screened for the excretion of methylmalonic acid (MMA), which should result from loss of Cbl transport into the mitochondria. One mutant, wht-6, showed elevated MMA excretion and reduced [14C]-propionate incorporation, pointing to a functional block in methylmalonyl-CoA mutase. In contrast, the wht-6 mutant appeared to have a normal cytosolic pathway based on analysis of cystathionine excretion, suggesting that cytosolic methionine synthase was functioning properly. Further, the MMA excretion in wht-6 could be partially reversed by including vitamin B12 in the assay medium. The human ortholog of wht-6 is a member of the G family of ABC transporters. We propose wht-6 as a candidate for the transport of Cbl into mitochondria and suggest that a member of the corresponding ABCG family of ABC transporters has this role in humans. Our ABC transporter screen also revealed that mrp-1 and mrp-2 mutants excreted lower MMA than wild type, suggesting they were concentrating intracellular Cbl, consistent with the cellular efflux defect proposed for the mammalian MRP1 ABC transporter.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Caenorhabditis elegans/metabolismo , Vitamina B 12/metabolismo , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Animais , Transporte Biológico , Caenorhabditis elegans/genética , Citosol/enzimologia , Citosol/metabolismo , Humanos , Lisossomos/metabolismo , Espectrometria de Massas , Ácido Metilmalônico/metabolismo , Metilmalonil-CoA Mutase/metabolismo , Mitocôndrias/enzimologia , Mutação , Propionatos/metabolismo
5.
G3 (Bethesda) ; 6(10): 3257-3268, 2016 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-27527792

RESUMO

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.


Assuntos
Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Animais , Caenorhabditis elegans/embriologia , Ciclo Celular/genética , Epistasia Genética , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Katanina , Meiose/genética , Microtúbulos , Mitose/genética , Complexos Multiproteicos/metabolismo , Ligação Proteica , Interferência de RNA , Transdução de Sinais
6.
Dev Biol ; 405(2): 250-9, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26188247

RESUMO

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.


Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Quinases Associadas a rho/fisiologia , Alelos , Animais , Tamanho Corporal , Citocinese , Epiderme/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Microscopia de Fluorescência , Morfogênese , Mutação , Reação em Cadeia da Polimerase , Interferência de RNA , Temperatura Ambiente , Proteínas rho de Ligação ao GTP/metabolismo
7.
Elife ; 4: e06056, 2015 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-25848744

RESUMO

Trisomy, the presence of a third copy of one chromosome, is deleterious and results in inviable or defective progeny if passed through the germ line. Random segregation of an extra chromosome is predicted to result in a high frequency of trisomic offspring from a trisomic parent. Caenorhabditis elegans with trisomy of the X chromosome, however, have far fewer trisomic offspring than expected. We found that the extra X chromosome was preferentially eliminated during anaphase I of female meiosis. We utilized a mutant with a specific defect in pairing of the X chromosome as a model to investigate the apparent bias against univalent inheritance. First, univalents lagged during anaphase I and their movement was biased toward the cortex and future polar body. Second, late-lagging univalents were frequently captured by the ingressing polar body contractile ring. The asymmetry of female meiosis can thus partially correct pre-existing trisomy.


Assuntos
Divisão Celular Assimétrica/genética , Caenorhabditis elegans/genética , Padrões de Herança , Trissomia , Cromossomo X/química , Anáfase , Animais , Segregação de Cromossomos , Feminino , Oócitos/metabolismo , Oócitos/ultraestrutura , Cromossomo X/ultraestrutura
8.
Mol Biol Cell ; 25(8): 1298-311, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24554763

RESUMO

In many animals, including vertebrates, oocyte meiotic spindles are bipolar but assemble in the absence of centrosomes. Although meiotic spindle positioning in oocytes has been investigated extensively, much less is known about their assembly. In Caenorhabditis elegans, three genes previously shown to contribute to oocyte meiotic spindle assembly are the calponin homology domain protein encoded by aspm-1, the katanin family member mei-1, and the kinesin-12 family member klp-18. We isolated temperature-sensitive alleles of all three and investigated their requirements using live-cell imaging to reveal previously undocumented requirements for aspm-1 and mei-1. Our results indicate that bipolar but abnormal oocyte meiotic spindles assemble in aspm-1(-) embryos, whereas klp-18(-) and mei-1(-) mutants assemble monopolar and apolar spindles, respectively. Furthermore, two MEI-1 functions--ASPM-1 recruitment to the spindle and microtubule severing--both contribute to monopolar spindle assembly in klp-18(-) mutants. We conclude that microtubule severing and ASPM-1 both promote meiotic spindle pole assembly in C. elegans oocytes, whereas the kinesin 12 family member KLP-18 promotes spindle bipolarity.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriologia , Fuso Acromático/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Proteínas de Caenorhabditis elegans/genética , Cinesina/genética , Cinesina/metabolismo , Meiose , Microtúbulos/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/genética , Mutação , Oócitos/enzimologia , Interferência de RNA , RNA Interferente Pequeno , Fuso Acromático/metabolismo , Temperatura Ambiente
9.
Mol Biol Cell ; 25(7): 1037-49, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24501424

RESUMO

Assembly of Caenorhabditis elegans female meiotic spindles requires both MEI-1 and MEI-2 subunits of the microtubule-severing ATPase katanin. Strong loss-of-function mutants assemble apolar intersecting microtubule arrays, whereas weaker mutants assemble bipolar meiotic spindles that are longer than wild type. To determine whether katanin is also required for spindle maintenance, we monitored metaphase I spindles after a fast-acting mei-1(ts) mutant was shifted to a nonpermissive temperature. Within 4 min of temperature shift, bivalents moved off the metaphase plate, and microtubule bundles within the spindle lengthened and developed a high degree of curvature. Spindles eventually lost bipolar structure. Immunofluorescence of embryos fixed at increasing temperature indicated that MEI-1 was lost from spindle microtubules before loss of ASPM-1, indicating that MEI-1 and ASPM-1 act independently at spindle poles. We quantified the microtubule-severing activity of purified MEI-1/MEI-2 complexes corresponding to six different point mutations and found a linear relationship between microtubule disassembly rate and meiotic spindle length. Previous work showed that katanin is required for severing at points where two microtubules intersect in vivo. We show that purified MEI-1/MEI-2 complexes preferentially sever at intersections between two microtubules and directly bundle microtubules in vitro. These activities could promote parallel/antiparallel microtubule organization in meiotic spindles.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Cromossomos/metabolismo , Meiose , Metáfase , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Alelos , Animais , Caenorhabditis elegans/metabolismo , Feminino , Katanina , Modelos Biológicos , Mutação Puntual , Ligação Proteica , Transporte Proteico , Temperatura Ambiente
10.
G3 (Bethesda) ; 3(11): 2015-29, 2013 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-24048649

RESUMO

The guidance of axons to their correct targets is a critical step in development. The C. elegans pharynx presents an attractive system to study neuronal pathfinding in the context of a developing organ. The worm pharynx contains relatively few cells and cell types, but each cell has a known lineage and stereotyped developmental patterns. We found that extension of the M1 pharyngeal axon, which spans the entire length of the pharynx, occurs in two distinct phases. The first proximal phase does not require genes that function in axon extension (unc-34, unc-51, unc-115, and unc-119), whereas the second distal phase does use these genes and is guided in part by the adjacent g1P gland cell projection. unc-34, unc-51, and unc-115 had incompletely penetrant defects and appeared to act in conjunction with the g1P cell for distal outgrowth. Only unc-119 showed fully penetrant defects for the distal phase. Mutations affecting classical neuronal guidance cues (Netrin, Semaphorin, Slit/Robo, Ephrin) or adhesion molecules (cadherin, IgCAM) had, at best, weak effects on the M1 axon. None of the mutations we tested affected the proximal phase of M1 elongation. In a forward genetic screen, we isolated nine mutations in five genes, three of which are novel, showing defects in M1, including axon overextension, truncation, or ectopic branching. One of these mutations appeared to affect the generation or differentiation of the M1 neuron. We conclude that M1 axon extension is a robust process that is not completely dependent on any single guidance mechanism.


Assuntos
Axônios/metabolismo , Caenorhabditis elegans/genética , Neurônios/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Genoma , Genótipo , Faringe/fisiologia , Fenótipo , Polimorfismo de Nucleotídeo Único , Transdução de Sinais/genética
11.
J Cell Biol ; 202(3): 431-9, 2013 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-23918937

RESUMO

Katanin is an evolutionarily conserved microtubule (MT)-severing complex implicated in multiple aspects of MT dynamics. In Caenorhabditis elegans, the katanin homologue MEI-1 is required for meiosis, but must be inactivated before mitosis. Here we show that PPFR-1, a regulatory subunit of a trimeric protein phosphatase 4 complex, enhanced katanin MT-severing activity during C. elegans meiosis. Loss of ppfr-1, similarly to the inactivation of MT severing, caused a specific defect in meiosis II spindle disassembly. We show that a fraction of PPFR-1 was degraded after meiosis, contributing to katanin inactivation. PPFR-1 interacted with MEL-26, the substrate recognition subunit of the CUL-3 RING E3 ligase (CRL3(MEL-26)), which also targeted MEI-1 for post-meiotic degradation. Reversible protein phosphorylation of MEI-1 may ensure temporal activation of the katanin complex during meiosis, whereas CRL3(MEL-26)-mediated degradation of both MEI-1 and its activator PPFR-1 ensure efficient katanin inactivation in the transition to mitosis.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Microtúbulos/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Animais , Caenorhabditis elegans/genética , Katanina , Complexos Multiproteicos/metabolismo , Fosforilação
12.
Worm ; 2(3): e25040, 2013 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24778933

RESUMO

During the second half of embryogenesis, the ellipsoidal Caenorhabditis elegans embryo elongates into a long, thin worm. This elongation requires a highly organized cytoskeleton composed of actin microfilaments, microtubules and intermediate filaments throughout the epidermis of the embryo. This architecture allows the embryonic epidermal cells to undergo a smooth muscle-like actin/myosin-based contraction that is redundantly controlled by LET- 502/Rho kinase and MEL-11/myosin phosphatase in one pathway and FEM-2/PP2c phosphatase and PAK-1/p21-activated kinase in a parallel pathway(s). Although actin microfilaments surround the embryo, the force for contraction is generated mainly in the lateral (seam) epidermal cells whose actin microfilaments appear qualitatively different from those in their dorsal/ventral neighbors. We have identified FHOD-1, a formin family actin nucleator, which acts in the lateral epidermis. fhod-1 mutants show microfilament defects in the embryonic lateral epidermal cells and FHOD-1 protein is detected only in those cells. fhod-1 genetic interactions with let-502, mel-11, fem-2 and pak-1 indicate that fhod-1 preferentially regulates those microfilaments acting with let-502 and mel-11, and in parallel to fem-2 and pak-1. Thus, FHOD-1 may contribute to the qualitative differences in microfilaments found in the contractile lateral epidermal cells and their non-contractile dorsal and ventral neighbors. Different microfilament populations may be involved in the different contractile pathways.

13.
Mol Biol Cell ; 23(14): 2623-34, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22621901

RESUMO

The ubiquitin proteasome system is involved in degradation of old or damaged sarcomeric proteins. Most E3 ubiquitin ligases are associated with cullins, which function as scaffolds for assembly of the protein degradation machinery. Cullin 3 uses an adaptor to link to substrates; in Caenorhabditis elegans, one of these adaptors is the BTB-domain protein MEL-26 (maternal effect lethal). Here we show that MEL-26 interacts with the giant sarcomeric protein UNC-89 (obscurin). MEL-26 and UNC-89 partially colocalize at sarcomeric M-lines. Loss of function or gain of function of mel-26 results in disorganization of myosin thick filaments similar to that found in unc-89 mutants. It had been reported that in early C. elegans embryos, a target of the CUL-3/MEL-26 ubiquitylation complex is the microtubule-severing enzyme katanin (MEI-1). Loss of function or gain of function of mei-1 also results in disorganization of thick filaments similar to unc-89 mutants. Genetic data indicate that at least some of the mel-26 loss-of-function phenotype in muscle can be attributed to increased microtubule-severing activity of MEI-1. The level of MEI-1 protein is reduced in an unc-89 mutant, suggesting that the normal role of UNC-89 is to inhibit the CUL-3/MEL-26 complex toward MEI-1.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas Musculares/metabolismo , Músculo Estriado/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Adenosina Trifosfatases/genética , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas Culina/metabolismo , Proteínas Musculares/genética , Miosinas/metabolismo , Proteólise , Interferência de RNA , RNA Interferente Pequeno , Sarcômeros/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
14.
Dev Biol ; 330(2): 349-57, 2009 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-19361490

RESUMO

The MEI-1/MEI-2 microtubule-severing complex, katanin, is required for oocyte meiotic spindle formation and function in C. elegans, but the microtubule-severing activity must be quickly downregulated so that it does not interfere with formation of the first mitotic spindle. Post-meiotic MEI-1 inactivation is accomplished by two parallel protein degradation pathways, one of which requires MEL-26, the substrate-specific adaptor that recruits MEI-1 to a CUL-3 based ubiquitin ligase. Here we address the question of how MEL-26 mediated MEI-1 degradation is triggered only after the completion of MEI-1's meiotic function. We find that MEL-26 is present only at low levels until the completion of meiosis, after which protein levels increase substantially, likely increasing the post-meiotic degradation of MEI-1. During meiosis, MEL-26 levels are kept low by the action of another type of ubiquitin ligase, which contains CUL-2. However, we find that the low levels of meiotic MEL-26 have a subtle function, acting to moderate MEI-1 activity during meiosis. We also show that MEI-1 is the only essential target for MEL-26, and possibly for the E3 ubiquitin ligase CUL-3, but the upstream ubiquitin ligase activating enzyme RFL-1 has additional essential targets.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas Culina/metabolismo , Meiose , Microtúbulos , Mitose , Animais , Caenorhabditis elegans/citologia , Katanina
15.
Genetics ; 181(2): 473-82, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19015549

RESUMO

The inhibitor of growth (ING) family of type II tumor suppressors are encoded by five genes in mammals and by three genes in Caenorhabditis elegans. All ING proteins contain a highly conserved plant homeodomain (PHD) zinc finger. ING proteins are activated by stresses, including ionizing radiation, leading to the activation of p53. ING proteins in mammals and yeast have recently been shown to read the histone code in a methylation-sensitive manner to regulate gene expression. Here we identify and characterize ing-3, the C. elegans gene with the highest sequence identity to the human ING3 gene. ING-3 colocalizes with chromatin in embryos, the germline, and somatic cells. The ing-3 gene is part of an operon but is also transcribed from its own promoter. Both ing-3(RNAi) and ing-3 mutant strains demonstrate that the gene likely functions in concert with the C. elegans p53 homolog, cep-1, to induce germ-cell apoptosis in response to ionizing radiation. Somatically, the ing-3 mutant has a weak kinker uncoordinated (kinker Unc) phenotype, indicating a possible neuronal function.


Assuntos
Apoptose/genética , Caenorhabditis elegans/genética , Genes de Helmintos , Genes Supressores de Tumor , Animais , Animais Geneticamente Modificados , Apoptose/efeitos da radiação , Sequência de Bases , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/efeitos da radiação , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Primers do DNA/genética , DNA de Helmintos/genética , Expressão Gênica , Células Germinativas/citologia , Células Germinativas/efeitos da radiação , Proteínas de Homeodomínio/genética , Humanos , Imuno-Histoquímica , Mutação , Interferência de RNA , Especificidade da Espécie , Distribuição Tecidual , Transativadores/genética , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
16.
Genetics ; 181(3): 933-43, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19087961

RESUMO

MEI-1, the catalytic subunit of the Caenorhabditis elegans "katanin" microtubule-severing complex, is required for meiotic spindle formation. However, MEI-1 must be inactivated after the completion of meiosis to allow formation of the first mitotic spindle. Recent work demonstrated that post-meiotic MEI-1 undergoes ubiquitin-dependent degradation mediated by two independent pathways. Here we describe another level of MEI-1 regulation involving the protein phosphatase 4 (PP4) complex. The PP4 R1 regulatory subunit protein phosphatase four regulatory subunit 1 (ppfr-1) was identified in an RNA interference (RNAi) screen for suppressors of a mei-1(gf) allele that is refractory to post-meiotic degradation. RNAi to the PP4 catalytic subunit PPH-4.1 or to the alpha4 regulatory PPFR-4 also suppressed lethality of ectopic MEI-1. These results suggest that PP4(+) activates MEI-1, and therefore loss of PP4 decreases ectopic MEI-1(gf) activity. PPH-4.1 and MEI-1 co-immunoprecipitate with one another, indicating that the PP4 complex likely regulates MEI-1 activity directly rather than through an intermediate. The ppfr-1 mutant has subtle meiotic defects indicating that PPFR-1 also regulates MEI-1 during meiosis. MBK-2 is the only kinase known to phosphorylate MEI-1 and triggers post-meiotic MEI-1 degradation. However, genetic interactions between PP4 and mbk-2 were not consistent with an antagonistic relationship between the phosphatase and kinase. Additionally, reducing PP4 in mei-1(gf) did not change the level or localization of post-meiotic MEI-1. Thus, by making use of a genetic background where MEI-1 is ectopically expressed, we have uncovered a third mechanism of MEI-1 regulation, one based on phosphorylation but independent of degradation. The redundant regulatory pathways likely contribute in different ways to the rapid and precise post-meiotic inactivation of MEI-1 microtubule-severing activity.


Assuntos
Caenorhabditis elegans/citologia , Caenorhabditis elegans/enzimologia , Microtúbulos/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Técnicas de Silenciamento de Genes , Meiose , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/genética , Fosforilação , Subunidades Proteicas/deficiência , Subunidades Proteicas/genética
17.
Dev Biol ; 302(2): 438-47, 2007 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-17069791

RESUMO

The C. elegans embryo supports both meiotic and mitotic spindles, requiring careful regulation of components specific to each spindle type. The MEI-1/katanin microtubule-severing complex is required for meiosis but must be inactivated prior to mitosis. Downregulation of MEI-1 depends on MEL-26, which binds MEI-1, targeting it for degradation by the CUL-3 E3 ubiquitin ligase complex. Here we report that other protein degradation pathways, involving the anaphase promoting complex (APC) and the MBK-2/DYRK kinase, act in parallel to MEL-26 to inactivate MEI-1. At 25 degrees all mel-26(null) embryos die due to persistence of MEI-1 into mitosis, but at 15 degrees a significant portion of embryos hatch due to lower levels of ectopic MEI-1, suggesting that a redundant pathway also regulates MEI-1 degradation at 15 degrees. Previously the MBK-2/DYRK kinase was suggested to trigger MEL-26 mediated MEI-1 degradation. However, mbk-2 enhances the incomplete lethality of mel-26(null) at 15 degrees, arguing that MEL-26 acts in parallel to MBK-2. APC mutants behave similarly. In mel-26 embryos, ectopic MEI-1 remains until the onset of gastrulation, but in mbk-2; apc embryos, MEI-1 only persists through the first mitosis. We propose that mbk-2 and apc couple the initial phase of MEI-1 degradation to meiotic exit, after which MEL-26 completes MEI-1 degradation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Adenosina Trifosfatases/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/fisiologia , Meiose/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Tirosina Quinases/fisiologia , Complexos Ubiquitina-Proteína Ligase/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Ciclossomo-Complexo Promotor de Anáfase , Animais , Caenorhabditis elegans/embriologia , Microtúbulos/metabolismo , Mitose , Mutação , Complexos Ubiquitina-Proteína Ligase/genética
18.
Curr Biol ; 15(18): 1605-15, 2005 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-16169482

RESUMO

BACKGROUND: The initiation of a cleavage furrow is essential to separate cells during cytokinesis, but little is known about the mechanisms controlling this actin-driven process. Previous studies in C. elegans embryos revealed that inactivation of the CUL-3-based E3 ligase activator rfl-1 results in an aberrant microtubule network, ectopic furrowing during pronuclear migration, and defects during cytokinesis. RESULTS: Here, we show that MEL-26, a substrate-specific adaptor of the CUL-3-based E3 ligase, is required for efficient cell separation and cleavage furrow ingression during the C. elegans early mitotic divisions. Loss of MEL-26 function leads to delayed onset and slow ingression of cytokinesis furrows that frequently regress. Conversely, increased levels of MEL-26 in cul-3(RNAi) and rfl-1 mutant embryos cause a hypercontractile cortex, with several simultaneously ingressing furrows during pronuclear migration. MEL-26 accumulates at cleavage furrows and binds the actin-interacting protein POD-1. Importantly, POD-1 is not a substrate of the MEL-26/CUL-3 ligase but is required to localize MEL-26 to the cortex. CONCLUSIONS: Our results suggest that MEL-26 not only acts as a substrate-specific adaptor within the MEL-26/CUL-3 complex, but also promotes cytokinesis by a CUL-3- and microtubule-independent mechanism.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Citocinese/fisiologia , Animais , Caenorhabditis elegans/fisiologia , Proteínas Culina/genética , Escherichia coli , Vetores Genéticos , Glutationa Transferase , Immunoblotting , Imuno-Histoquímica , Proteínas dos Microfilamentos/metabolismo , Interferência de RNA , Saccharomyces cerevisiae
19.
J Cell Biol ; 169(3): 447-57, 2005 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-15883196

RESUMO

In animals, female meiotic spindles are attached to the egg cortex in a perpendicular orientation at anaphase to allow the selective disposal of three haploid chromosome sets into polar bodies. We have identified a complex of interacting Caenorhabditis elegans proteins that are involved in the earliest step in asymmetric positioning of anastral meiotic spindles, translocation to the cortex. This complex is composed of the kinesin-1 heavy chain orthologue, UNC-116, the kinesin light chain orthologues, KLC-1 and -2, and a novel cargo adaptor, KCA-1. Depletion of any of these subunits by RNA interference resulted in meiosis I metaphase spindles that remained stationary at a position several micrometers from the cell cortex during the time when wild-type spindles translocated to the cortex. After this prolonged stationary period, unc-116(RNAi) spindles moved to the cortex through a partially redundant mechanism that is dependent on the anaphase-promoting complex. This study thus reveals two sequential mechanisms for translocating anastral spindles to the oocyte cortex.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cinesina/metabolismo , Meiose/fisiologia , Oócitos/metabolismo , Fuso Acromático/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Caenorhabditis elegans/citologia , Proteínas de Caenorhabditis elegans/genética , Feminino , Metáfase/fisiologia , Microscopia de Vídeo , Oócitos/citologia , Subunidades Proteicas/metabolismo , Transporte Proteico/fisiologia , RNA Interferente Pequeno/metabolismo , Fuso Acromático/ultraestrutura
20.
Genetics ; 170(1): 115-26, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15781712

RESUMO

The C. elegans zygote supports both meiosis and mitosis within a common cytoplasm. The meiotic spindle is small and is located anteriorly, whereas the first mitotic spindle fills the zygote. The C. elegans microtubule-severing complex, katanin, is encoded by the mei-1 and mei-2 genes and is solely required for oocyte meiotic spindle formation; ectopic mitotic katanin activity disrupts mitotic spindles. Here we characterize two mutations that rescue the lethality caused by ectopic MEI-1/MEI-2. Both mutations are gain-of-function alleles of tba-2 alpha-tubulin. These tba-2 alleles do not prevent MEI-1/MEI-2 microtubule localization but do interfere with its activity. TBA-1 and TBA-2 are redundant for viability, but when katanin activity is limiting, TBA-2 is preferred over TBA-1 by katanin. This is similar to what we previously reported for the beta-tubulins. Removing both preferred alpha- and beta-isoforms results in normal development, suggesting that the katanin isoform preferences are not absolute. We conclude that while the C. elegans embryo expresses redundant alpha- and beta-tubulin isoforms, they nevertheless have subtle functional specializations. Finally, we identified a dominant tba-2 allele that disrupts both meiotic and mitotic spindle formation independently of MEI-1/MEI-2 activity. Genetic studies suggest that this tba-2 mutation has a "poisonous" effect on microtubule function.


Assuntos
Adenosina Trifosfatases/fisiologia , Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/genética , Fase de Clivagem do Zigoto/metabolismo , Tubulina (Proteína)/genética , Adenosina Trifosfatases/biossíntese , Adenosina Trifosfatases/genética , Alelos , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/biossíntese , Proteínas de Caenorhabditis elegans/genética , Katanina , Meiose/genética , Microtúbulos/genética , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Alinhamento de Sequência , Fuso Acromático/genética , Tubulina (Proteína)/fisiologia
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