Identification of ZFR, an ancient and highly conserved murine chromosome-associated zinc finger protein

In a screen for RNA binding proteins expressed during murine spermatogenesis, we cloned a novel, ancient zinc finger protein possessing a region common to a small class of RNA binding proteins. Zfr (zinc finger RNA binding) encodes a protein of 1052 amino acids with three widely spaced Cys2His2 zinc fingers. Outside of the zinc fingers, ZFR shares a region that is highly conserved between several RNA binding proteins containing copies of the double-stranded RNA binding motif. By northern blotting, Zfr is expressed at highest levels within the testis, ovary and brain. Immunohistochemistry and confocal microscopy were used to show that ZFR is highly expressed during meiosis I in males and females and is chromosome associated. Zfr is also expressed in Sertoli cells in the testis and granulosa cells in the ovary where it is localized to the nucleus. Using fluorescent in situ hybridization we mapped Zfr to chromosome 15 region A. ZFR appears to be an ancient protein, as apparent homologs exist in invertebrates (D. melanogaster) nematodes (C. elegans) and humans (H. sapiens).     

Characterization of TFG in mus musculus and Caenorhabditis elegans

TFG was discovered as a fusion partner of NTRK1 in human papillary thyroid carcinoma. We assembled the mouse TFG cDNA from EST sequences and 5' end RACE product, identified full coding length TFG EST clones in pig (c17b07) and Schistosoma mansoni (SMNAS62), and analyzed the genomic structure of TFG in Caenorhabditis elegans (Y63D3A). The protein sequences of mouse, pig, and S. mansoni TFG are highly homologous to human TFG. The C. elegans sequence has diverged, but its predicted secondary structure is remarkably conserved. Human, mouse, and C. elegans TFG contain a putative trimeric N-terminal coiled-coil domain, glycosylation, myristylation, and phosphorylation sites, and SH2- and SH3-binding motifs. The SH2-binding motif is absent in C. elegans TFG. The expression of TFG does not vary among 7, 11, 15, and 19 day mouse embryonal stages. In situ hybridization with a TFG probe in 10, 5-day whole mouse embryos showed preferential staining of the limb buds, branchial arches, nasal processes, and brain, and weak staining of the primitive spinal cord and dorsal root ganglia.     

Lamin-dependent localization of UNC-84, a protein required for nuclear migration in Caenorhabditis elegans

Mutations in the Caenorhabditis elegans unc-84 gene cause defects in nuclear migration and anchoring. We show that endogenous UNC-84 protein colocalizes with Ce-lamin at the nuclear envelope and that the envelope localization of UNC-84 requires Ce-lamin. We also show that during mitosis, UNC-84 remains at the nuclear periphery until late anaphase, similar to known inner nuclear membrane proteins. UNC-84 protein is first detected at the 26-cell stage and thereafter is present in most cells during development and in adults. UNC-84 is properly expressed in unc-83 and anc-1 lines, which have phenotypes similar to unc-84, suggesting that neither the expression nor nuclear envelope localization of UNC-84 depends on UNC-83 or ANC-1 proteins. The envelope localization of Ce-lamin, Ce-emerin, Ce-MAN1, and nucleoporins are unaffected by the loss of UNC-84. UNC-84 is not required for centrosome attachment to the nucleus because centrosomes are localized normally in unc-84 hyp7 cells despite a nuclear migration defect. Models for UNC-84 localization are discussed.     

Characterization of STIP, a multi-domain nuclear protein, highly conserved in metazoans, and essential for embryogenesis in Caenorhabditis elegans

We report here the identification and characterization of STIP, a multi-domain nuclear protein that contains a G-patch, a coiled-coil, and several short tryptophan-tryptophan repeats highly conserved in metazoan species. To analyze their functional role in vivo, we cloned nematode stip-1 genes and determined the spatiotemporal pattern of Caenorhabditis elegans STIP-1 protein. RNA analyses and Western blots revealed that stip-1 mRNA was produced via trans-splicing and translated as a 95-kDa protein. Using reporter constructs, we found STIP-1 to be expressed at all developmental stages and in many tissue/cell types including worm oocyte nuclei. We found that STIP-1 is targeted to the nucleus and forms large polymers with a rod-like shape when expressed in mammalian cells. Using deletion mutants, we mapped the regions of STIP-1 involved in nuclear import and polymer assembly. We further showed that knockdown of C. elegans stip-1 by RNA interference arrested development and resulted in morphologic abnormalities around the 16-cell stage followed by 100% lethality, suggesting its essential role in worm embryogenesis. Importantly, the embryonic lethal phenotype could be faithfully rescued with Drosophila and human genes via transgenic expression. Our data provide the first direct evidence that STIP have a conserved essential nuclear function across metazoans from worms to humans.     

原位杂交检测pal-1 mRNA在秀丽小杆线虫野生型和突变体早期胚胎中的分布

pal-1是秀丽小杆线虫(Caenorhabditis elegans)早期胚胎发育中决定体细胞命运的重要基因,也是转录因子,调控后续基因的表达,凡含有该基因表达的细胞发育成体细胞。本文通过整体原位杂交技术检测pal-1mRNA在C·elegans野生型和par-1、par-2、par-3、par-4突变体、spn-4突变体、mex-5/mex-6突变体早期胚胎中的分布,探讨这些基因在胚胎发育早期对pal-1mRNA的影响。实验结果表明:par-1、par-3、par-4突变使4细胞胚胎pal-1mRNA完全丧失了野生型不对称分布模式,pal-1mRNA分布在所有卵裂球中;par-2对pal-1mRNA的分布影响较小,在par-2突变体4细胞胚胎中pal-1mRNA分布与野生型相同。spn-4、mex-5、mex-6也能影响pal-1mRNA的分布,使其分布丧失不对称性。在par-1、par-4突变的情况下,pal-1mRNA广泛存在,但PAL-1蛋白也不表达,显示对pal-1mRNA的翻译调控是PAL-1蛋白空间和时序不对称分布的主要原因[动物学报51(5):884-891,2005]。     

The Zinc Finger Protein Zfr1p Is Localized Specifically to Conjugation Junction and Required for Sexual Development in Tetrahymena thermophila

Conjugation in Tetrahymena thermophila involves a developmental program consisting of three prezygotic nuclear divisions, pronuclear exchange and fusion, and postzygotic and exconjugant stages. The conjugation junction structure appears during the initiation of conjugation development, and disappears during the exconjugant stage. Many structural and functional proteins are involved in the establishment and maintenance of the junction structure in T. thermophila. In the present study, a zinc finger protein-encoding gene ZFR1 was found to be expressed specifically during conjugation and to localize specifically to the conjugation junction region. Truncated Zfr1p localized at the plasma membrane in ordered arrays and decorated Golgi apparatus located adjacent to basal body. The N-terminal zinc finger and C-terminal hydrophobic domains of Zfr1p were found to be required for its specific conjugation junction localization. Conjugation development of ZFR1 somatic knockout cells was aborted at the pronuclear exchange and fusion conjugation stages. Furthermore, Zfr1p was found to be important for conjugation junction stability during the prezygotic nuclear division stage. Taken together, our data reveal that Zfr1p is required for the stability and integrity of the conjugation junction structure and essential for the sexual life cycle of the Tetrahymena cell.     

A role for Caenorhabditis elegans importin IMA-2 in germ line and embryonic mitosis

The importin alpha family of nuclear-cytoplasmic transport factors mediates the nuclear localization of proteins containing classical nuclear localization signals. Metazoan animals express multiple importin alpha proteins, suggesting their possible roles in cell differentiation and development. Adult Caenorhabditis elegans hermaphrodites express three importin alpha proteins, IMA-1, IMA-2, and IMA-3, each with a distinct expression and localization pattern. IMA-2 was expressed exclusively in germ line cells from the early embryonic through adult stages. The protein has a dynamic pattern of localization dependent on the stage of the cell cycle. In interphase germ cells and embryonic cells, IMA-2 is cytoplasmic and nuclear envelope associated, whereas in developing oocytes, the protein is cytoplasmic and intranuclear. During mitosis in germ line cells and embryos, IMA-2 surrounded the condensed chromosomes but was not directly associated with the mitotic spindle. The timing of IMA-2 nuclear localization suggested that the protein surrounded the chromosomes after fenestration of the nuclear envelope in prometaphase. Depletion of IMA-2 by RNA-mediated gene interference (RNAi) resulted in embryonic lethality and a terminal aneuploid phenotype. ima-2(RNAi) embryos have severe defects in nuclear envelope formation, accumulating nucleoporins and lamin in the cytoplasm. We conclude that IMA-2 is required for proper chromosome dynamics in germ line and early embryonic mitosis and is involved in nuclear envelope assembly at the conclusion of mitosis.     

Transmission electron microscope studies of the nuclear envelope in Caenorhabditis elegans embryos

Nuclear membranes and nuclear pore complexes (NPCs) are conserved in both animals and plants. However, the lamina composition and the dimensions of NPCs vary between plants, yeast, and vertebrates. In this study, we established a protocol that preserves the structure of Caenorhabditis elegans embryonic cells for high-resolution studies with thin-section transmission electron microscopy (TEM). We show that the NPCs are bigger in C. elegans embryos than in yeast, with dimensions similar to those in higher eukaryotes. We also localized the C. elegans nuclear envelope proteins Ce-lamin and Ce-emerin by pre-embedding gold labeling immunoelectron microscopy. Both proteins are present at or near the inner nuclear membrane. A fraction of Ce-lamin, but not Ce-emerin, is present in the nuclear interior. Removing the nuclear membranes leaves both Ce-lamin and Ce-emerin associated with the chromatin. Eliminating the single lamin protein caused cell death as visualized by characteristic changes in nuclear architecture including condensation of chromatin, clustering of NPCs, membrane blebbing, and the presence of vesicles inside the nucleus. Taken together, these results show evolutionarily conserved protein localization, interactions, and functions of the C. elegans nuclear envelope.     

Caenorhabditis elegans WASP-interacting protein homologue WIP-1 is involved in morphogenesis through maintenance of WSP-1 protein levels

Mammalian WASP and N-WASP are involved in reorganization of the actin cytoskeleton through activation of the Arp2/3 complex and in regulation of cell motility or cell shape changes. In the present study, we identified WASP-interacting protein homologue (WIP)-1 in Caenorhabditis elegans. WIP-1 contains the domains and sequences conserved among mammalian WIP family proteins. Yeast two-hybrid analysis detected a physical interaction between WIP-1 and WSP-1, the sole homologue of WASP/N-WASP in C. elegans. Western analysis of embryo lysates showed that RNA interference (RNAi) treatment for wip-1 decreased levels of WSP-1 protein, and wsp-1(RNAi) treatment decreased levels of WIP-1 protein. However, wsp-1 mRNA levels were not decreased in wip-1(RNAi)-treated embryos, and wip-1 mRNA levels were not decreased in wsp-1(RNAi)-treated embryos. Furthermore, disruption of WIP-1 by RNAi resulted in embryonic lethality with morphologic defects in hypodermal cell migration, a process known as ventral enclosure. This phenotype was similar to that observed in RNAi experiments for wsp-1. Immunostaining showed that WIP-1 was expressed by migrating hypodermal cells, as was WSP-1. This expression during ventral enclosure was reduced in wip-1(RNAi)-treated embryos and wsp-1(RNAi)-treated embryos. Our results suggest that C. elegans WIP-1 may function in hypodermal cell migration during ventral enclosure by maintaining levels of WSP-1.     

原位杂交检测lin-4 mRNA在Caenorhabditis elegans中的表达

lin-4是控制Caenorhabditis elegans(C.elegans)幼虫发育的异时性基因,也是一种小RNA分子(microRNA)。通过整体原位杂交检测小RNA lin-4在野生型和lin-14、lin-28突变体中的区域性表达,探讨lin-4在C.elegans发育时空控制中的作用。结果表明:lin-4 mRNA在胚胎发育的早期和中期表达,胚胎后期至L1期末没有表达,之后又持续表达,成虫中也可以检测到lin-4 mRNA的存在。在lin-14和lin-28突变体中,lin-4的表达基本与野生型一致,不受lin-14、lin-28基因突变的影响,说明lin-14和lin-28是lin-4的下游基因。     

Location of specific messenger RNAs in Caenorhabditis elegans by cytological hybridization

We have developed an autoradiographic technique for detecting specific Caenorhabditis elegans messenger RNA molecules in situ by hybridization of labeled, cloned DNA probes to fixed tissue sections and squashes of embryos and adults. We report analyses with probes of actin and collagen gene sequences from a C. elegans genomic clone library. Hybridization is RNase sensitive and tissue specific. In adults the actin probe, which recognizes cytoplasmic as well as muscle actin mRNA, hybridizes strongly to muscle and distal gonad (ovary), somewhat less strongly to maturing oocytes, and weakly to intestine. The collagen probe hybridizes weakly to distal gonad and intestine and very strongly to subcuticular tissues, in particular to the hypodermal cells and syncytial cytoplasm of the lateral hypodermal ridges, which are the sites of cuticle synthesis. In embryos, hybridization to squashes indicates that actin message is present at fertilization, decreases during early cleavage, and then increases again during morphogenesis. By contrast, collagen message is absent until the 100-cell stage and then increases rapidly during morphogenesis. The number of cells labeled is consistent with the view that the collagen probe hybridizes to hypodermal precursor cells. We estimate that our present methods can detect messages representing about 0.2% or more of the total mRNA population, and increases in this sensitivity should be possible. Therefore, the cytological hybridization technique should be useful for determining temporal and spatial patterns of specific mRNA distributions during development, at least for abundant and moderately abundant messages.     

The Caenorhabditis elegans polarity gene ooc-5 encodes a Torsin-related protein of the AAA ATPase superfamily.

The PAR proteins are required for polarity and asymmetric localization of cell fate determinants in C. elegans embryos. In addition, several of the PAR proteins are conserved and localized asymmetrically in polarized cells in Drosophila, Xenopus and mammals. We have previously shown that ooc-5 and ooc-3 mutations result in defects in spindle orientation and polarity in early C. elegans embryos. In particular, mutations in these genes affect the re-establishment of PAR protein asymmetry in the P(1) cell of two-cell embryos. We now report that ooc-5 encodes a putative ATPase of the Clp/Hsp100 and AAA superfamilies of proteins, with highest sequence similarity to Torsin proteins; the gene for human Torsin A is mutated in individuals with early-onset torsion dystonia, a neuromuscular disease. Although Clp/Hsp100 and AAA family proteins have roles in diverse cellular activities, many are involved in the assembly or disassembly of proteins or protein complexes; thus, OOC-5 may function as a chaperone. OOC-5 protein co-localizes with a marker of the endoplasmic reticulum in all blastomeres of the early C. elegans embryo, in a pattern indistinguishable from that of OOC-3 protein. Furthermore, OOC-5 localization depends on the normal function of the ooc-3 gene. These results suggest that OOC-3 and OOC-5 function in the secretion of proteins required for the localization of PAR proteins in the P(1) cell, and may have implications for the study of torsion dystonia.     

Nuclear pore protein gp210 is essential for viability in HeLa cells and Caenorhabditis elegans

Gp210 is an evolutionarily conserved membrane protein of the nuclear pore complex (NPC). We studied the phenotypes produced by RNAi-induced downregulation of gp210 in both human (HeLa) cells and Caenorhabditis elegans embryos. HeLa cell viability requires Gp210 activity. The dying cells accumulated clustered NPCs and aberrant membrane structures at the nuclear envelope, suggesting that gp210 is required directly or indirectly for nuclear pore formation and dilation as well as the anchoring or structural integrity of mature NPCs. Essential roles for gp210 were confirmed in C. elegans, where RNAi-induced reduction of gp210 caused embryonic lethality. The nuclear envelopes of embryos with reduced gp210 also had aberrant nuclear membrane structures and clustered NPCs, confirming that gp210 plays critical roles at the nuclear membrane through mechanisms that are conserved from nematodes to humans.     

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

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

Molecular cloning and three-dimensional structure prediction of a novel alpha-tubulin in Caenorhabditis elegans

This paper reports on the isolation of a cDNA clone ( tba-6 ) encoded by a novel a-tubulin gene in the nematode C. elegans . The tba-6 gene is located on chromosome I, that encode a protein of 460 amino acids, as well as the expression of the gene during the development. Here we discuss the structure of the coding region and the regulatory sequences in the promoter region. The comparison of the amino acid sequence of TBA6 with other α-tubulin isotypes of C. elegans , suggests that these proteins are highly conserved in most of the N-terminal and intermediate sequence, but they have highly divergent C-terminal sequences. TBA6 has also high homology with other α-tubulin families (e.g. human, mouse, Drosophila melangaster ). The in situ experiment results suggest that the tba-6 α-tubulin gene is required during the entire embryonic development, therefore it is required during the early cell division stages. Further, we determined the 3D structure of C. elegans TBA6 α-tubulin by altering (computationally) the crystal structure of the α-tubulin (TBA_pig) from porcine α-β tubulin dimer. We discuss structural conservation and changes in the pattern of interactions between secondary structure elements of TBA_pig and TBA6, respectively.     

The polo-like kinase PLK-1 is required for nuclear envelope breakdown and the completion of meiosis in Caenorhabditis elegans

The Polo-like kinases are key regulatory molecules required during the cell cycle for the successful completion of mitosis. We have cloned a C. elegans homolog of the Drosophila melanogaster polo gene (designated plk-1 for C. elegans polo-like kinase-1) and present the subcellular localization of the PLK-1 protein during the meiotic and mitotic cell cycles in C. elegans oocytes and embryos, respectively. Disruption of PLK-1 expression by RNA-mediated interference (RNAi) disrupts normal oocyte and embryonic development. Inspection of oocytes revealed a defect in nuclear envelope breakdown (NEBD) before ovulation. This defect in NEBD was also observed in oocytes that were depleted of the cyclin-dependent kinase NCC-1 (C. elegans homolog of Cdc2). The plk-1 RNAi oocytes were fertilized; however the resulting embryos were unable to separate their meiotic chromosomes or form and extrude polar bodies. These defects led to embryonic arrest as single cells. genesis 26:26-41, 2000. Published 2000 Wiley-Liss, Inc.     

线虫(Caenorhabditis elegans)受精卵中细胞极性的建立

早期线虫胚胎提供了一个研究发育过程的极佳模型。线虫胚胎的第一次分裂是不对称的,产生的两个子细胞在尺度的大小和发育命运上均有不同,而这些不同是由第一次有丝分裂周期中胞质决定子的不均匀分布造成的。通常相信,在受精过程中,精子所携带的中心体介导了对极性建成至关重要的胞质流动的产生。同时,细胞骨架成分被认为参与了胞质成分的定位事件。关于par基因的研究目前进展迅速,大多数par基因的突变都导致了线虫早期胚胎分裂不对称性的丧失。