Publications by authors named "Asako Sugimoto"

41 Publications

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

Cell Struct Funct 2021 Jun 8;46(1):51-64. Epub 2021 May 8.

Laboratory of Developmental Dynamics, Graduate School of Life Sciences, Tohoku University.

Most organisms have multiple α- and β-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 β-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 β-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.
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http://dx.doi.org/10.1247/csf.21022DOI Listing
June 2021

Fluorescence-labeled neopeltolide derivatives for subcellular localization imaging.

Org Biomol Chem 2019 07;17(28):6771-6776

Department of Applied Chemistry, Faculty of Science and Engineering, Chuo University, 1-13-27 Kasuga, Bunkyo-ku, Tokyo 112-8551, Japan.

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.
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http://dx.doi.org/10.1039/c9ob01276aDOI Listing
July 2019

The Role of Tissue Inhibitors of Metalloproteinases in Organ Development and Regulation of ADAMTS Family Metalloproteinases in .

Genetics 2019 06 16;212(2):523-535. Epub 2019 Apr 16.

Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan.

Remodeling of the extracellular matrix supports tissue and organ development, by regulating cellular morphology and tissue integrity. However, proper extracellular matrix remodeling requires spatiotemporal regulation of extracellular metalloproteinase activity. Members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family, including MIG-17 and GON-1, are evolutionarily conserved, secreted, zinc-requiring metalloproteinases. Although these proteases are required for extracellular matrix remodeling during gonadogenesis in , their regulatory mechanisms remain to be delineated. Therefore, we focused on the tissue inhibitors of metalloproteinases (TIMPs), TIMP-1 and CRI-2 Analysis of the transcription and translation products for GFP/Venus fusions, with TIMP-1 or CRI-2, indicated that these inhibitors were secreted and localized to the basement membrane of gonads and the plasma membrane of germ cells. A deletion mutant exhibited gonadal growth defects and sterility, and the phenotypes of this mutant were fully rescued by a TIMP-1::Venus construct, but not by a TIMP-1(C21S)::Venus mutant construct, in which the inhibitor coding sequence had been mutated. Moreover, genetic data suggested that TIMP-1 negatively regulates proteolysis of the α1 chain of type IV collagen. We also found that the loss-of-function observed for the mutants and involves a partial suppression of gonadal defects found for the mutants and , and that this suppression was canceled upon overexpression of or , respectively. Based on these results, we propose that both TIMP-1 and CRI-2 act as inhibitors of MIG-17 and GON-1 ADAMTSs to regulate gonad development in a noncell-autonomous manner.
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http://dx.doi.org/10.1534/genetics.119.301795DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6553827PMC
June 2019

Biology and genome of a newly discovered sibling species of Caenorhabditis elegans.

Nat Commun 2018 08 10;9(1):3216. Epub 2018 Aug 10.

Faculty of Medicine, University of Miyazaki, Miyazaki, 889-1692, Japan.

A 'sibling' species of the model organism Caenorhabditis elegans has long been sought for use in comparative analyses that would enable deep evolutionary interpretations of biological phenomena. Here, we describe the first sibling species of C. elegans, C. inopinata n. sp., isolated from fig syconia in Okinawa, Japan. We investigate the morphology, developmental processes and behaviour of C. inopinata, which differ significantly from those of C. elegans. The 123-Mb C. inopinata genome was sequenced and assembled into six nuclear chromosomes, allowing delineation of Caenorhabditis genome evolution and revealing unique characteristics, such as highly expanded transposable elements that might have contributed to the genome evolution of C. inopinata. In addition, C. inopinata exhibits massive gene losses in chemoreceptor gene families, which could be correlated with its limited habitat area. We have developed genetic and molecular techniques for C. inopinata; thus C. inopinata provides an exciting new platform for comparative evolutionary studies.
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http://dx.doi.org/10.1038/s41467-018-05712-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6086898PMC
August 2018

Streptothricin acetyl transferase 2 (Sat2): A dominant selection marker for Caenorhabditis elegans genome editing.

PLoS One 2018 9;13(5):e0197128. Epub 2018 May 9.

Division of Developmental Dynamics, Graduate School of Life Science Tohoku University, Aoba-ku, Sendai, Japan.

Studies on Caenorhabditis elegans would benefit from the introduction of new selectable markers to allow more complex types of experiments to be conducted with this model animal. We established a new antibiotic selection marker for C. elegans transformation based on nourseothricin (NTC) and its resistance-encoding gene, streptothricin-acetyl transferase 2 (Sat2). NTC was able to efficiently prevent worm development at very low concentrations, and the worms expressing Sat2 were able to survive on the selection plates without any developmental defects. Using CRISPR/Cas9 and NTC selection, we were able to easily insert a 13-kb expression cassette into a defined locus in C. elegans. The structure and spectrum of NTC differs from other antibiotics like hygromycin B and geneticin, making it possible to use NTC alongside them. Indeed, we confirmed NTC-sat2 selection could work with the hygromycin B selection system simultaneously. Thus, the new NTC-Sat2 system can act as a useful dominant marker for gene transfer and genome editing in C. elegans.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197128PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5942822PMC
August 2018

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

Dev Genes Evol 2018 01 20;228(1):75-82. Epub 2018 Jan 20.

Laboratory of Developmental Dynamics, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai, 980-8577, Japan.

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.
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http://dx.doi.org/10.1007/s00427-018-0605-zDOI Listing
January 2018

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

J Cell Sci 2017 05 16;130(9):1652-1661. Epub 2017 Mar 16.

Laboratory of Developmental Dynamics, Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan

Microtubules (MTs) are polymers composed of α- and β-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 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, β1 and β2) cooperatively confer distinct MT properties in 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 β1 have higher switching frequency between growth and shrinkage compared with MTs composed of β2. Lower concentration of β-tubulins results in slower growth rates, and the two α-tubulins distinctively affect growth rates of MTs composed of β1. 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 .
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http://dx.doi.org/10.1242/jcs.200923DOI Listing
May 2017

Caenorhabditis elegans Aurora A kinase is required for the formation of spindle microtubules in female meiosis.

Mol Biol Cell 2015 Nov 16;26(23):4187-96. Epub 2015 Sep 16.

Laboratory of Developmental Dynamics, Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan

In many animals, female meiotic spindles are assembled in the absence of centrosomes, the major microtubule (MT)-organizing centers. How MTs are formed and organized into meiotic spindles is poorly understood. Here we report that, in Caenorhabditis elegans, Aurora A kinase/AIR-1 is required for the formation of spindle microtubules during female meiosis. When AIR-1 was depleted or its kinase activity was inhibited in C. elegans oocytes, although MTs were formed around chromosomes at germinal vesicle breakdown (GVBD), they were decreased during meiotic prometaphase and failed to form a bipolar spindle, and chromosomes were not separated into two masses. Whereas AIR-1 protein was detected on and around meiotic spindles, its kinase-active form was concentrated on chromosomes at prometaphase and on interchromosomal MTs during late anaphase and telophase. We also found that AIR-1 is involved in the assembly of short, dynamic MTs in the meiotic cytoplasm, and these short MTs were actively incorporated into meiotic spindles. Collectively our results suggest that, after GVBD, the kinase activity of AIR-1 is continuously required for the assembly and/or stabilization of female meiotic spindle MTs.
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http://dx.doi.org/10.1091/mbc.E15-05-0258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4642853PMC
November 2015

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

PLoS Genet 2014 Oct 23;10(10):e1004638. Epub 2014 Oct 23.

Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Kyoto, Japan.

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.
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http://dx.doi.org/10.1371/journal.pgen.1004638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4207613PMC
October 2014

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

Dev Biol 2014 Jul 8;391(1):43-53. Epub 2014 Apr 8.

Laboratory of Developmental Dynamics, Graduate School of Life Sciences, Tohoku University, Sendai 980-8577, Japan; Laboratory for Developmental Genomics, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ydbio.2014.04.002DOI Listing
July 2014

The UBXN-2/p37/p47 adaptors of CDC-48/p97 regulate mitosis by limiting the centrosomal recruitment of Aurora A.

J Cell Biol 2013 May 6;201(4):559-75. Epub 2013 May 6.

Department of Physiology and Metabolism, Faculty of Medicine, University of Geneva, 1211 Geneva, Switzerland.

Coordination of cell cycle events in space and time is crucial to achieve a successful cell division. Here, we demonstrate that UBXN-2, a substrate adaptor of the AAA ATPase Cdc48/p97, is required to coordinate centrosome maturation timing with mitosis. In UBXN-2-depleted Caenorhabditis elegans embryos, centrosomes recruited more AIR-1 (Aurora A), matured precociously, and alignment of the mitotic spindle with the axis of polarity was impaired. UBXN-2 and CDC-48 coimmunoprecipitated with AIR-1 and the spindle alignment defect was partially rescued by co-depleting AIR-1, indicating that UBXN-2 controls these processes via AIR-1. Similarly, depletion in human cells of the UBXN-2 orthologues p37/p47 resulted in an accumulation of Aurora A at centrosomes and a delay in centrosome separation. The latter defect was also rescued by inhibiting Aurora A. We therefore postulate that the role of this adaptor in cell cycle regulation is conserved.
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http://dx.doi.org/10.1083/jcb.201209107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3653362PMC
May 2013

The nucleoporin Nup205/NPP-3 is lost near centrosomes at mitotic onset and can modulate the timing of this process in Caenorhabditis elegans embryos.

Mol Biol Cell 2012 Aug 27;23(16):3111-21. Epub 2012 Jun 27.

Swiss Institute for Experimental Cancer Research, School of Life Sciences, Swiss Federal Institute of Technology (EPFL), Lausanne, Switzerland.

Regulation of mitosis in time and space is critical for proper cell division. We conducted an RNA interference-based modifier screen to identify novel regulators of mitosis in Caenorhabditis elegans embryos. Of particular interest, this screen revealed that the Nup205 nucleoporin NPP-3 can negatively modulate the timing of mitotic onset. Furthermore, we discovered that NPP-3 and nucleoporins that are associated with it are lost from the nuclear envelope (NE) in the vicinity of centrosomes at the onset of mitosis. We demonstrate that centrosomes are both necessary and sufficient for NPP-3 local loss, which also requires the activity of the Aurora-A kinase AIR-1. Our findings taken together support a model in which centrosomes and AIR-1 promote timely onset of mitosis by locally removing NPP-3 and associated nucleoporins from the NE.
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http://dx.doi.org/10.1091/mbc.E12-03-0204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3418306PMC
August 2012

Cell polarity: centrosomes release signals for polarization.

Curr Biol 2012 Apr;22(8):R281-3

Graduate School of Life Sciences, Tohoku University, Sendai, 980-8577, Japan.

New findings reveal that, in Caenorhabditis elegans embryos, the centrosome provides signals that induce cell polarization, independently of its function as the microtubule-organizing center.
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http://dx.doi.org/10.1016/j.cub.2012.03.009DOI Listing
April 2012

Tissue architecture in the Caenorhabditis elegans gonad depends on interactions among fibulin-1, type IV collagen and the ADAMTS extracellular protease.

Genetics 2012 Apr 31;190(4):1379-88. Epub 2012 Jan 31.

Department of Bioscience, Kwansei Gakuin University, Sanda, Hyogo 669-1337, Japan.

Molecules in the extracellular matrix (ECM) regulate cellular behavior in both development and pathology. Fibulin-1 is a conserved ECM protein. The Caenorhabditis elegans ortholog, FBL-1, regulates gonad-arm elongation and expansion by acting antagonistically to GON-1, an ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family protease. The elongation of gonad arms is directed by gonadal distal tip cells (DTCs). Here we report that a dominant mutation in the EMB-9/type IV collagen α1 subunit can compensate for loss of FBL-1 activity in gonadogenesis. A specific amino acid substitution in the noncollagenous 1 (NC1) domain of EMB-9 suppressed the fbl-1 null mutant. FBL-1 was required to maintain wild-type EMB-9 in the basement membrane (BM), whereas mutant EMB-9 was retained in the absence of FBL-1. EMB-9 (either wild type or mutant) localization in the BM enhanced PAT-3/β-integrin expression in DTCs. In addition, overexpression of PAT-3 partially rescued the DTC migration defects in fbl-1 mutants, suggesting that EMB-9 acts in part through PAT-3 to control DTC migration. In contrast to the suppression of fbl-1(tk45), mutant EMB-9 enhanced the gonadal defects of gon-1(e1254), suggesting that it gained a function similar to that of wild-type FBL-1, which promotes DTC migration by inhibiting GON-1. We propose that FBL-1 and GON-1 control EMB-9 accumulation in the BM and promote PAT-3 expression to control DTC migration.
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http://dx.doi.org/10.1534/genetics.111.133173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3316650PMC
April 2012

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

Authors:
Asako Sugimoto

Genes Cells 2011 Jul 16;16(7):745-7. Epub 2011 Jun 16.

Graduate School of Life Sciences, Tohoku University, Katahira, Aoba-ku, Sendai, Japan.

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?
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http://dx.doi.org/10.1111/j.1365-2443.2011.01531.xDOI Listing
July 2011

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

Dev Biol 2011 Jul 6;355(2):302-12. Epub 2011 May 6.

Genome Biology Laboratory, National Institute of Genetics, 1111 Yata, Mishima, 411-8540, Japan.

The Wnt and Src pathways are widely used signal transduction pathways in development. β-catenin is utilized in both pathways, as a signal transducer and a component of the cadherin cell adhesion complex, respectively. A C. elegans β-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.
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http://dx.doi.org/10.1016/j.ydbio.2011.04.034DOI Listing
July 2011

A kinase-independent role for Aurora A in the assembly of mitotic spindle microtubules in Caenorhabditis elegans embryos.

Nat Cell Biol 2011 Jun 15;13(6):708-14. Epub 2011 May 15.

Laboratory for Developmental Genomics, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan.

The assembly of a functional mitotic spindle is crucial for achieving successful mitosis. Aurora A kinase is one of the key regulators of mitotic events, including mitotic entry, centrosome maturation and spindle bipolarity. Caenorhabditis elegans Aurora A (AIR-1) is responsible for the assembly of γ-tubulin-independent microtubules in early embryos; however, the mechanism by which AIR-1 contributes to microtubule assembly during mitosis has been unclear. Here we show by live-cell imaging and RNA-mediated interference (RNAi)-based modulation of gene activity that AIR-1 has a crucial role in the assembly of chromatin-stimulated microtubules that is independent of the γ-tubulin complex. Surprisingly, the kinase activity of AIR-1 is dispensable for this process. Although the kinase-inactive form of AIR-1 was detected along the microtubules as well as on centrosomes, the kinase-active form of AIR-1 was restricted to centrosomes. Thus, we propose that AIR-1 has a kinase-dependent role at centrosomes and a kinase-independent role for stabilizing spindle microtubules and that coordination of these two roles is crucial for the assembly of mitotic spindles.
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http://dx.doi.org/10.1038/ncb2242DOI Listing
June 2011

PGL proteins self associate and bind RNPs to mediate germ granule assembly in C. elegans.

J Cell Biol 2011 Mar 14;192(6):929-37. Epub 2011 Mar 14.

Laboratory for Developmental Genomics, RIKEN Center for Developmental Biology, Kobe, Hyogo, 650-0047 Japan.

Germ granules are germ lineage-specific ribonucleoprotein (RNP) complexes, but how they are assembled and specifically segregated to germ lineage cells remains unclear. Here, we show that the PGL proteins PGL-1 and PGL-3 serve as the scaffold for germ granule formation in Caenorhabditis elegans. Using cultured mammalian cells, we found that PGL proteins have the ability to self-associate and recruit RNPs. Depletion of PGL proteins from early C. elegans embryos caused dispersal of other germ granule components in the cytoplasm, suggesting that PGL proteins are essential for the architecture of germ granules. Using a structure-function analysis in vivo, we found that two functional domains of PGL proteins contribute to germ granule assembly: an RGG box for recruiting RNA and RNA-binding proteins and a self-association domain for formation of globular granules. We propose that self-association of scaffold proteins that can bind to RNPs is a general mechanism by which large RNP granules are formed.
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http://dx.doi.org/10.1083/jcb.201010106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3063142PMC
March 2011

[Germ granule formation and distribution in C. elegans].

Tanpakushitsu Kakusan Koso 2009 Dec;54(16 Suppl):2147-52

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December 2009

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.

Genes Cells 2010 Nov 21;15(11):1145-57. Epub 2010 Oct 21.

Laboratory for Developmental Genomics, RIKEN Center for Developmental Biology, Kobe, Hyogo, Japan.

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.
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http://dx.doi.org/10.1111/j.1365-2443.2010.01451.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3036819PMC
November 2010

Imaging of mitotic spindle dynamics in Caenorhabditis elegans embryos.

Methods Cell Biol 2010 ;97:359-72

Laboratory for Developmental Genomics, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan.

Development of the nematode Caenorhabditis elegans is highly reproducible, and the cell division patterns are virtually invariant. Transparency of the eggshell and cells enables the observation of intracellular events with a high temporal and spatial resolution. These unique features, along with the sophisticated genetic techniques, make this organism one of the most attractive model systems for dissecting regulatory mechanisms of dynamic cellular behaviors, such as mitosis, at an organismal level. In this chapter, we describe immunofluorescence and live imaging methods for analyzing mitotic spindle regulation. In particular, we present the use of double- or triple-labeled fluorescent strains for high-resolution two-dimensional and three-dimensional live imaging to analyze dynamic behaviors of mitotic spindles.
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http://dx.doi.org/10.1016/S0091-679X(10)97019-2DOI Listing
December 2010

Clearance of germ granules in the soma.

Authors:
Asako Sugimoto

F1000 Biol Rep 2009 Jun 29;1:49. Epub 2009 Jun 29.

Laboratory for Developmental Genomics, RIKEN Center for Developmental Biology 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047 Japan.

Germ granules are ribonucleoprotein complexes specifically segregated into germ cell lineages in diverse organisms. Recent studies indicate that multiple mechanisms are involved in the clearance of germ granules and their components in somatic cells in Caenorhabditis elegans embryos.
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http://dx.doi.org/10.3410/B1-49DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2924698PMC
June 2009

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

Genetics 2009 Mar 15;181(3):933-43. Epub 2008 Dec 15.

Genes and Development Research Group, Department of Biochemistry and Molecular Biology, University of Calgary, Alberta, Canada.

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.
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http://dx.doi.org/10.1534/genetics.108.096016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2651065PMC
March 2009

A new mechanism controlling kinetochore-microtubule interactions revealed by comparison of two dynein-targeting components: SPDL-1 and the Rod/Zwilch/Zw10 complex.

Genes Dev 2008 Sep;22(17):2385-99

Ludwig Institute for Cancer Research/Dept of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California 92093, USA.

Chromosome segregation requires stable bipolar attachments of spindle microtubules to kinetochores. The dynein/dynactin motor complex localizes transiently to kinetochores and is implicated in chromosome segregation, but its role remains poorly understood. Here, we use the Caenorhabditis elegans embryo to investigate the function of kinetochore dynein by analyzing the Rod/Zwilch/Zw10 (RZZ) complex and the associated coiled-coil protein SPDL-1. Both components are essential for Mad2 targeting to kinetochores and spindle checkpoint activation. RZZ complex inhibition, which abolishes both SPDL-1 and dynein/dynactin targeting to kinetochores, slows but does not prevent the formation of load-bearing kinetochore-microtubule attachments and reduces the fidelity of chromosome segregation. Surprisingly, inhibition of SPDL-1, which abolishes dynein/dynactin targeting to kinetochores without perturbing RZZ complex localization, prevents the formation of load-bearing attachments during most of prometaphase and results in extensive chromosome missegregation. Coinhibition of SPDL-1 along with the RZZ complex reduces the phenotypic severity to that observed following RZZ complex inhibition alone. We propose that the RZZ complex can inhibit the formation of load-bearing attachments and that this activity of the RZZ complex is normally controlled by dynein/dynactin localized via SPDL-1. This mechanism could coordinate the hand-off from initial weak dynein-mediated lateral attachments, which help orient kinetochores and enhance their ability to capture microtubules, to strong end-coupled attachments that drive chromosome segregation.
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http://dx.doi.org/10.1101/gad.1687508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2532926PMC
September 2008

The Caenorhabditis elegans DDX-23, a homolog of yeast splicing factor PRP28, is required for the sperm-oocyte switch and differentiation of various cell types.

Dev Dyn 2008 Sep;237(9):2367-77

Laboratory for Developmental Genomics, RIKEN Center for Developmental Biology, Chuo-ku, Kobe, Japan.

DEAD/H-box proteins are involved in various aspects of RNA metabolism. Here we report the developmental function of a DEAD-box protein, DDX-23, in Caenorhabditis elegans, which has significant homology with the yeast splicing factor PRP28. We found by RNAi and mutant analyses that DDX-23 is essential for both embryonic and post-embryonic development, and required for differentiation of the majority of somatic tissues. When the germline function of ddx-23 was inhibited, hermaphrodite animals showed a reduced number of germ cells and failed to switch from spermatogenesis to oogenesis. These phenotypes were similar to those of the mutants of the three DEAH-box proteins (MOG-1, MOG-4, and MOG-5) whose yeast orthologs are involved in the pre-mRNA splicing pathway. We speculate that DDX-23 functions with the three MOG proteins in the same pathway to regulate tissue differentiation, robust germline proliferation, and the sperm/oocyte switch through modulations of ribonucleoprotein complexes.
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http://dx.doi.org/10.1002/dvdy.21649DOI Listing
September 2008

Efficient production of monoclonal antibodies recognizing specific structures in Caenorhabditis elegans embryos using an antigen subtraction method.

Genes Cells 2008 Jul 21;13(7):653-65. Epub 2008 May 21.

Laboratory for Developmental Genomics, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan.

Monoclonal antibodies (mAbs) have been widely used to probe molecular components of specific cell types or cellular structures. We have developed a method to enrich antigens of low abundance in heterogeneous molecule mixtures by subtracting abundant antigens. The subtracted immunogen mixture is then used for immunization, which significantly increases the production of mAbs that exhibit specific staining patterns. By applying this "antigen subtraction" method to the embryonic extract of Caenorhabditis elegans, we have successfully isolated 35 mAbs that recognize specific structures, including P granules, muscles, the pharynx, and subsets of hypodermal cells; some of the mAbs revealed previously unreported cellular structures. This antigen subtraction approach can be used in various applications to produce mAbs against relatively scarce antigens in complex molecular mixtures. The mAbs will be useful tools for developmental and cell biological studies.
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http://dx.doi.org/10.1111/j.1365-2443.2008.01195.xDOI Listing
July 2008

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

Curr Biol 2007 Sep;17(18):1555-60

Waksman Institute, Department of Genetics, Rutgers University, Piscataway, New Jersey 08854, USA.

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.
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http://dx.doi.org/10.1016/j.cub.2007.08.011DOI Listing
September 2007

[Function of microtubules at the onset of cytokinesis].

Tanpakushitsu Kakusan Koso 2006 Sep;51(11):1590-5

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September 2006

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

Nat Cell Biol 2006 Sep 20;8(9):978-85. Epub 2006 Aug 20.

Laboratory for Developmental Genomics, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Japan.

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.
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http://dx.doi.org/10.1038/ncb1459DOI Listing
September 2006

[Cell polarization: lessons from C. elegans asymmetric cell division].

Tanpakushitsu Kakusan Koso 2006 May;51(6 Suppl):776-81

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May 2006
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