A germline-specific role for unconventional components of the γ-tubulin complex in Caenorhabditis elegans.

The γ-tubulin complex (γTuC) is a widely conserved microtubule nucleator, but some of its components, namely GCP4, GCP5 and GCP6 (also known as TUBGCP4, TUBGCP5 and TUBGCP6, respectively), have not been detected in Caenorhabditis elegans. Here, we identified two γTuC-associated proteins in C. elegans, GTAP-1 and GTAP-2, for which apparent orthologs were detected only in the genus Caenorhabditis. GTAP-1 and GTAP-2 were found to localize at centrosomes and the plasma membrane of the germline, and their centrosomal localization was interdependent. In early C. elegans embryos, whereas the conserved γTuC component MZT-1 (also known as MOZART1 and MZT1) was essential for the localization of centrosomal γ-tubulin, depletion of GTAP-1 and/or GTAP-2 caused up to 50% reduction of centrosomal γ-tubulin and precocious disassembly of spindle poles during mitotic telophase. In the adult germline, GTAP-1 and GTAP-2 contributed to efficient recruitment of the γTuC to the plasma membrane. Depletion of GTAP-1, but not of GTAP-2, severely disrupted both the microtubule array and the honeycomb-like structure of the adult germline. We propose that GTAP-1 and GTAP-2 are unconventional components of the γTuC that contribute to the organization of both centrosomal and non-centrosomal microtubules by targeting the γTuC to specific subcellular sites in a tissue-specific manner.

Centrosome maturation requires phosphorylation-mediated sequential domain interactions of SPD-5.

Centrosomes consist of two centrioles and the surrounding pericentriolar material (PCM). The PCM expands during mitosis in a process called centrosome maturation, in which PCM scaffold proteins play pivotal roles to recruit other centrosomal proteins. In Caenorhabditis elegans, the scaffold protein SPD-5 forms a PCM scaffold in a polo-like kinase 1 (PLK-1) phosphorylation-dependent manner. However, how phosphorylation of SPD-5 promotes PCM scaffold assembly is unclear. Here, we identified three functional domains of SPD-5 through in vivo domain analyses, and propose that sequential domain interactions of SPD-5 are required for mitotic PCM scaffold assembly. Firstly, SPD-5 is targeted to centrioles through a direct interaction between its centriole localization (CL) domain and the centriolar protein PCMD-1. Then, intramolecular and intermolecular interactions between the SPD-5 phospho-regulated multimerization (PReM) domain and the PReM association (PA) domain are enhanced by phosphorylation by PLK-1, which leads to PCM scaffold expansion. Our findings suggest that the sequential domain interactions of scaffold proteins mediated by PLK-1 phosphorylation is an evolutionarily conserved mechanism of PCM scaffold assembly. This article has an associated First Person interview with the first author of the paper.

The PAF1 complex cell autonomously promotes oogenesis in Caenorhabditis elegans.

The RNA polymerase II-associated factor 1 complex (PAF1C) is a protein complex that consists of LEO1, RTF1, PAF1, CDC73, and CTR9, and has been shown to be involved in RNA polymerase II-mediated transcriptional and chromatin regulation. Although it has been shown to regulate a variety of biological processes, the precise role of the PAF1C during germ line development has not been clarified. In this study, we found that reduction in the function of the PAF1C components, LEO-1, RTFO-1, PAFO-1, CDC-73, and CTR-9, in Caenorhabditis elegans affects oogenesis. Defects in oogenesis were also confirmed using an oocyte maturation marker, OMA-1::GFP. While four to five OMA-1::GFP-positive oocytes were observed in wild-type animals, their numbers were significantly decreased in pafo-1 mutant and leo-1(RNAi), pafo-1(RNAi), and cdc-73(RNAi) animals. Expression of a functional PAFO-1::mCherry transgene in the germline significantly rescued the oogenesis-defective phenotype of the pafo-1 mutants, suggesting that expression of the PAF1C in germ cells is required for oogenesis. Notably, overexpression of OMA-1::GFP partially rescued the oogenesis defect in the pafo-1 mutants. Based on our findings, we propose that the PAF1C promotes oogenesis in a cell-autonomous manner by positively regulating the expression of genes involved in oocyte maturation.

Expression Patterns and Levels of All Tubulin Isotypes Analyzed in GFP Knock-In C. elegans Strains.

Most organisms have multiple α- and ß-tubulin isotypes that likely contribute to the diversity of microtubule (MT) functions. To understand the functional differences of tubulin isotypes in Caenorhabditis elegans, which has nine α-tubulin isotypes and six ß-tubulin isotypes, we systematically constructed null mutants and GFP-fusion strains for all tubulin isotypes with the CRISPR/Cas9 system and analyzed their expression patterns and levels in adult hermaphrodites. Four isotypes-α-tubulins TBA-1 and TBA-2 and ß-tubulins TBB-1 and TBB-2-were expressed in virtually all tissues, with a distinct tissue-specific spectrum. Other isotypes were expressed in specific tissues or cell types at significantly lower levels than the broadly expressed isotypes. Four isotypes (TBA-5, TBA-6, TBA-9, and TBB-4) were expressed in different subsets of ciliated sensory neurons, and TBB-4 was inefficiently incorporated into mitotic spindle MTs. Taken together, we propose that MTs in C. elegans are mainly composed of four broadly expressed tubulin isotypes and that incorporation of a small amount of tissue-specific isotypes may contribute to tissue-specific MT properties. These newly constructed strains will be useful for further elucidating the distinct roles of tubulin isotypes.Key words: tubulin isotypes, microtubules, C. elegans.

Tubulin isotype substitution revealed that isotype combination modulates microtubule dynamics in C. elegans embryos.

Microtubules (MTs) are polymers composed of α- and ß-tubulin heterodimers that are generally encoded by genes at multiple loci. Despite implications of distinct properties depending on the isotype, how these heterodimers contribute to the diverse MT dynamics in vivo remains unclear. Here, by using genome editing and depletion of tubulin isotypes following RNAi, we demonstrate that four tubulin isotypes (hereafter referred to as α1, α2, ß1 and ß2) cooperatively confer distinct MT properties in Caenorhabditis elegans early embryos. GFP insertion into each isotype locus reveals their distinct expression levels and MT incorporation rates. Substitution of isotype coding regions demonstrates that, under the same isotype concentration, MTs composed of ß1 have higher switching frequency between growth and shrinkage compared with MTs composed of ß2. Lower concentration of ß-tubulins results in slower growth rates, and the two α-tubulins distinctively affect growth rates of MTs composed of ß1. Alteration of ratio and concentration of isotypes distinctively modulates both growth rate and switching frequency, and affects the amplitude of mitotic spindle oscillation. Collectively, our findings demonstrate that MT dynamics are modulated by the combination (ratio and concentration) of tubulin isotypes with distinct properties, which contributes to create diverse MT behaviors in vivo.

Fluorescence-labeled neopeltolide derivatives for subcellular localization imaging.

Design, synthesis and functional analysis of fluorescent derivatives of neopeltolide, an antiproliferative marine macrolide, are reported herein. Live cell imaging using the fluorescent derivatives showed rapid cellular uptake and localization within the endoplasmic reticulum as well as the mitochondria.

Transgenesis by microparticle bombardment for live imaging of fluorescent proteins in Pristionchus pacificus germline and early embryos.

Pristionchus pacificus is a free-living nematode used as a model organism for evolutionary developmental and ecological biology. Although a transgenic technique to form complex arrays by microinjection has been established in P. pacificus, transgene expression from the array in the germline and early embryos tends to be silenced. Here, we established a method to integrate transgenes into the genome of P. pacificus using microparticle bombardment with hygromycin B selection. Additionally, we isolated a mutant exhibiting significantly lower autofluorescence in the germline and early embryos, facilitating visualization of transgene-derived fluorescent proteins for live imaging. Transgenic lines constructed using these tools successfully expressed GFP-tagged proteins in the germline and early embryos and enabled live imaging of chromosomes, microtubules, and centrosomes.

Protein phosphatase 4 promotes chromosome pairing and synapsis, and contributes to maintaining crossover competence with increasing age.

Prior to the meiotic divisions, dynamic chromosome reorganizations including pairing, synapsis, and recombination of maternal and paternal chromosome pairs must occur in a highly regulated fashion during meiotic prophase. How chromosomes identify each other's homology and exclusively pair and synapse with their homologous partners, while rejecting illegitimate synapsis with non-homologous chromosomes, remains obscure. In addition, how the levels of recombination initiation and crossover formation are regulated so that sufficient, but not deleterious, levels of DNA breaks are made and processed into crossovers is not understood well. We show that in Caenorhabditis elegans, the highly conserved Serine/Threonine protein phosphatase PP4 homolog, PPH-4.1, is required independently to carry out four separate functions involving meiotic chromosome dynamics: (1) synapsis-independent chromosome pairing, (2) restriction of synapsis to homologous chromosomes, (3) programmed DNA double-strand break initiation, and (4) crossover formation. Using quantitative imaging of mutant strains, including super-resolution (3D-SIM) microscopy of chromosomes and the synaptonemal complex, we show that independently-arising defects in each of these processes in the absence of PPH-4.1 activity ultimately lead to meiotic nondisjunction and embryonic lethality. Interestingly, we find that defects in double-strand break initiation and crossover formation, but not pairing or synapsis, become even more severe in the germlines of older mutant animals, indicating an increased dependence on PPH-4.1 with increasing maternal age. Our results demonstrate that PPH-4.1 plays multiple, independent roles in meiotic prophase chromosome dynamics and maintaining meiotic competence in aging germlines. PP4's high degree of conservation suggests it may be a universal regulator of meiotic prophase chromosome dynamics.

The PAF1 complex is involved in embryonic epidermal morphogenesis in Caenorhabditis elegans.

The PAF1 complex (PAF1C) is an evolutionarily conserved protein complex involved in transcriptional regulation and chromatin remodeling. How the PAF1C is involved in animal development is still not well understood. Here, we report that, in the nematode Caenorhabditis elegans, the PAF1C is involved in epidermal morphogenesis in late embryogenesis. From an RNAi screen we identified the C. elegans ortholog of a component of the PAF1C, CTR-9, as a gene whose depletion caused various defects during embryonic epidermal morphogenesis, including epidermal cell positioning, ventral enclosure and epidermal elongation. RNAi of orthologs of other four components of the PAF1C (PAFO-1, LEO-1, CDC-73 and RTFO-1) caused similar epidermal defects. In these embryos, whereas the number and cell fate determination of epidermal cells were apparently unaffected, their position and shape were severely disorganized. PAFO-1::mCherry, mCherry::LEO-1 and GFP::RTFO-1 driven by the authentic promoters were detected in the nuclei of a wide range of cells. Nuclear localization of GFP::RTFO-1 was independent of other PAF1C components, while PAFO-1::mCherry and mCherry::LEO-1 dependent on other components except RTFO-1. Epidermis-specific expression of mCherry::LEO-1 rescued embryonic lethality of the leo-1 deletion mutant. Thus, although the PAF1C is universally expressed in C. elegans embryos, its epidermal function is crucial for the viability of this animal.

The impact of differential transposition activities of autonomous and nonautonomous hAT transposable elements on genome architecture and gene expression in Caenorhabditis inopinata.

Transposable elements are DNA sequences capable of moving within genomes and significantly influence genomic evolution. The nematode Caenorhabditis inopinata exhibits a much higher transposable element copy number than its sister species, Caenorhabditis elegans. In this study, we identified a novel autonomous transposable element belonging to the hAT superfamily from a spontaneous transposable element-insertion mutant in C. inopinata and named this transposon Ci-hAT1. Further bioinformatic analyses uncovered 3 additional autonomous hAT elements-Ci-hAT2, Ci-hAT3, and Ci-hAT4-along with over 1,000 copies of 2 nonautonomous miniature inverted-repeat transposable elements, mCi-hAT1 and mCi-hAT4, likely derived from Ci-hAT1 and Ci-hAT4 through internal deletion. We tracked at least 3 sequential transpositions of Ci-hAT1 over several years. However, the transposition rates of the other 3 autonomous hAT elements were lower, suggesting varying activity levels. Notably, the distribution patterns of the 2 miniature inverted-repeat transposable element families differed significantly: mCi-hAT1 was primarily located in the chromosome arms, a pattern observed in the transposable elements of other Caenorhabditis species, whereas mCi-hAT4 was more evenly distributed across chromosomes. Additionally, interspecific transcriptome analysis indicated that C. inopinata genes with upstream or intronic these miniature inverted-repeat transposable element insertions tend to be more highly expressed than their orthologous genes in C. elegans. These findings highlight the significant role of de-silenced transposable elements in driving the evolution of genomes and transcriptomes, leading to species-specific genetic diversity.

Sequential functioning of the ECT-2 RhoGEF, RHO-1 and CDC-42 establishes cell polarity in Caenorhabditis elegans embryos.

During development, the establishment of cell polarity is important for cells to undergo asymmetric cell divisions that give rise to diverse cell types. In C. elegans embryos, cues from the centrosome trigger the cortical flow of an actomyosin network, leading to the formation of anterior-posterior polarity. However, its precise mechanism is poorly understood. Here, we show that small GTPases have sequential and crucial functions in this process. ECT-2, a potential guanine nucleotide-exchange factor (GEF) for RHO-1, was uniformly distributed at the cortex before polarization, but was excluded from the posterior cortex by the polarity cue from the centrosomes. This local exclusion of ECT-2 led to an asymmetric RHO-1 distribution, which generated a cortical flow of the actomyosin that translocated PAR proteins and CDC-42 (Refs 4, 5) to the anterior cortex. Polarized CDC-42 was, in turn, involved in maintaining the established anterior-cortical domains. Our results suggest that a local change in the function of ECT-2 and RHO-1 links the centrosomal polarity cue with the polarization of the cell cortex.

C. elegans ATG-5 mutants associated with ataxia.

Intercellular cleaning via autophagy is crucial for maintaining cellular homeostasis, and impaired autophagy has been associated with the accumulation of protein aggregates that can contribute to neurological diseases. Specifically, the loss-of-function mutation in the human autophagy-related gene 5 (ATG5) at E122D has been linked to the pathogenesis of spinocerebellar ataxia in humans. In this study, we generated two homozygous C. elegans strains with mutations (E121D and E121A) at positions corresponding to the human ATG5 ataxia mutation to investigate the effects of ATG5 mutations on autophagy and motility. Our results showed that both mutants exhibited a reduction in autophagy activity and impaired motility, suggesting that the conserved mechanism of autophagy-mediated regulation of motility extends from C. elegans to humans.

Differentially Expressed Genes Associated with Body Size Changes and Transposable Element Insertions between Caenorhabditis elegans and Its Sister Species, Caenorhabditis inopinata.

Why the recently discovered nematode Caenorhabditis inopinata differs so greatly from its sibling species Caenorhabditis elegans remains unknown. A previous study showed that C. inopinata has more transposable elements (TEs), sequences that replicate and move autonomously throughout the genome, potentially altering the expression of neighboring genes. In this study, we focused on how the body size of this species has evolved and whether TEs could affect the expression of genes related to species-specific traits such as body size. First, we compared gene expression levels between C. inopinata and C. elegans in the L4 larval and young adult stages-when growth rates differ most prominently between these species-to identify candidate genes contributing to their differences. The results showed that the expression levels of collagen genes were consistently higher in C. inopinata than in C. elegans and that some genes related to cell size were differentially expressed between the species. Then, we examined whether genes with TE insertions are differentially expressed between species. Indeed, the genes featuring C. inopinata-specific TE insertions had higher expression levels in C. inopinata than in C. elegans. These upregulated genes included those related to body size, suggesting that these genes could be candidates for artificial TE insertion to examine the role of TEs in the body size evolution of C. inopinata.

The ß-catenin HMP-2 functions downstream of Src in parallel with the Wnt pathway in early embryogenesis of C. elegans.

The Wnt and Src pathways are widely used signal transduction pathways in development. ß-catenin is utilized in both pathways, as a signal transducer and a component of the cadherin cell adhesion complex, respectively. A C. elegans ß-catenin HMP-2 is involved in cell adhesion, but its signaling role has been unknown. Here, we report that in early embryogenesis HMP-2 acts as a signaling molecule in the Src signal. During early embryogenesis in C. elegans, the Wnt and Src pathways are redundantly involved in endoderm induction at the four-cell stage and spindle orientation in an ABar blastomere. RNAi experiments demonstrated that HMP-2 functions in the Src pathway, but in parallel with the Wnt pathway in these processes. HMP-2 localized at the cell boundaries and nuclei, and its localization at cell boundaries was negatively regulated by SRC-1. In addition, HMP-2 was Tyr-phosphorylated in a SRC-1-dependent manner in vivo. Taken together, we propose that HMP-2 functions downstream of the Src signaling pathway and contribute to endoderm induction and ABar spindle orientation, in parallel with the Wnt signaling pathway.

Toward the second stage of recovery from the 3.11 Tohoku Earthquake.

Nearly 2 months have passed since the 3.11 Tohoku Earthquake. In central Sendai, where Tohoku University is located, life is slowly returning back to normal, at least on the surface. However, just a few kilometers away, where the tsunami washed away an entire town, wreckage still litters the coast. The second stage of recovering from the disaster has begun. What can we do to revive universities and communities in the affected areas?

The auxin-inducible degron 2 (AID2) system enables controlled protein knockdown during embryogenesis and development in Caenorhabditis elegans.

Targeted protein degradation using the auxin-inducible degron (AID) system is garnering attention in the research field of Caenorhabditis elegans, because of the rapid and efficient target depletion it affords, which can be controlled by treating the animals with the phytohormone auxin. However, the current AID system has drawbacks, i.e., leaky degradation in the absence of auxin and the requirement for high auxin doses. Furthermore, it is challenging to deplete degron-fused proteins in embryos because of their eggshell, which blocks auxin permeability. Here, we apply an improved AID2 system utilizing AtTIR1(F79G) and 5-phenyl-indole-3-acetic acid (5-Ph-IAA) to C. elegans and demonstrated that it confers better degradation control vs the previous system by suppressing leaky degradation and inducing sharp degradation using 1,300-fold lower 5-Ph-IAA doses. We successfully degraded the endogenous histone H2A.Z protein fused to an mAID degron and disclosed its requirement in larval growth and reproduction, regardless of the presence of maternally inherited H2A.Z molecules. Moreover, we developed an eggshell-permeable 5-Ph-IAA analog, 5-Ph-IAA-AM, that affords an enhanced degradation in laid embryos. Our improved system will contribute to the disclosure of the roles of proteins in C. elegans, in particular those that are involved in embryogenesis and development, through temporally controlled protein degradation.

Two phases of astral microtubule activity during cytokinesis in C. elegans embryos.

Microtubules of the mitotic spindle are believed to provide positional cues for the assembly of the actin-based contractile ring and the formation of the subsequent cleavage furrow during cytokinesis. In Caenorhabditis elegans, astral microtubules have been thought to inhibit cortical contraction outside the cleavage furrow. Here, we demonstrate by live imaging and RNA interference (RNAi) that astral microtubules play two distinct roles in initiating cleavage furrow formation. In early anaphase, microtubules are required for contractile ring assembly; in late anaphase, microtubules show different cortical behavior and seem to suppress cortical contraction at the poles, as suggested in previous studies. These two distinct phases of microtubule behavior depend on distinct regulatory pathways, one involving the gamma-tubulin complex and the other requiring aurora-A kinase. We propose that temporal and spatial regulation of two distinct phases of astral microtubule behavior is crucial in specifying the position and timing of furrowing.

Caenorhabditis elegans ortholog of the p24/p22 subunit, DNC-3, is essential for the formation of the dynactin complex by bridging DNC-1/p150(Glued) and DNC-2/dynamitin.

Dynactin is a multisubunit protein complex required for the activity of cytoplasmic dynein. In Caenorhabditis elegans, although 10 of the 11 dynactin subunits were identified based on the sequence similarities to their orthologs, the p24/p22 subunit has not been detected in the genome. Here, we demonstrate that DNC-3 (W10G11.20) is the functional counterpart of the p24/p22 subunit in C. elegans. RNAi phenotypes and subcellular localization of DNC-3 in early C. elegans embryos were nearly identical to those of the known dynactin components. All other dynactin subunits were co-immunoprecipitated with DNC-3, indicating that DNC-3 is a core component of dynactin. Furthermore, the overall secondary structure of DNC-3 resembles to those of the mammalian and yeast p24/p22. We found that DNC-3 is required for the localization of the DNC-1/p150(Glued) and DNC-2/dynamitin, the two components of the projection arm of dynactin, to the nuclear envelope of meiotic nuclei in the adult gonad. Moreover, DNC-3 physically interacted with DNC-1 and DNC-2 and significantly enhanced the binding ability between DNC-1 and DNC-2 in vitro. These results suggest that DNC-3 is essential for the formation of the projection arm subcomplex of dynactin.

EGG-3 regulates cell-surface and cortex rearrangements during egg activation in Caenorhabditis elegans.

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.

The role of protein phosphatase 4 in regulating microtubule severing in the Caenorhabditis elegans embryo.

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.