標(biāo) 題: Science:Colorada大學(xué)韓珉實(shí)驗(yàn)室：ANC-1參與把細(xì)胞核拉牢到肌動蛋白骨架發(fā)信站: 生命玄機(jī)站 (Fri Oct 11 05:26:54 2002) , 轉(zhuǎn)信 Science, Vol. 298, Issue 5592, 406-409, October 11, 2002 Role of ANC-1 in Tethering Nuclei to the Actin Cytoskeleton Daniel A. Starr, Min Han* Mutations in anc-1 (nuclear anchorage defective) disrupt the positioning of nucl ei and mitochondria in Caenorhabditis elegans. ANC-1 is shown to consist of most ly coiled regions with a nuclear envelope localization domain (called the KASH d omain) and an actin-binding domain; this structure was conserved with the Drosop hila protein Msp-300 and the mammalian Syne proteins. Antibodies against ANC-1 l ocalized cytoplasmically and were enriched at the nuclear periphery in an UNC-84 -dependent manner. Overexpression of the KASH domain or the actin-binding domain caused a dominant negative anchorage defect. Thus, ANC-1 may connect nuclei to the cytoskeleton by interacting with UNC-84 at the nuclear envelope and with act in in the cytoplasm. Howard Hughes Medical Institute and Department of Molecular, Cellular and Develo pmental Biology, University of Colorado, Boulder, CO 80309, USA. * To whom correspondence should be addressed. E-mail: [email protected] -------------------------------------------------------------------------------- A wide variety of organisms have syncytia, formed either when multiple nuclear d ivisions occur without cell divisions or when cells fuse together. Normally, syn cytial nuclei are located in specific regions or are evenly spaced throughout th e cytoplasm. Nuclear positioning is also essential to a variety of singlely nucl eated polar cells and even in single-celled organisms (1). Microtubules and asso ciated dynein and kinesin motors play a central role in the positioning of nucle i (2). Less is known about the role of actin in the process of nuclear positioni ng. However, a defect in the actin cytoskeleton of nurse cells of Drosophila ooc ytes disrupts nuclear anchorage during cytoplasmic dumping (3). Actin is also re quired for plant nuclear migrations (4). We used the large syncytial cells of C. elegans as a model to study the mechanism of nuclear anchorage. Most of the bod y of an adult worm is covered by four large syncytial hypodermal cells that cont ain more than 100 nuclei (5). Normally, nuclei are evenly spaced throughout the syncytia. However, mutations in either anc-1 or unc-84 cause an Anc phenotype, i n which nuclei float freely within the cytoplasm of syncytial cells and often gr oup together (6, 7). The Anc phenotype is observed in all somatic, postembryonic syncytial cells, even in binucleated intestinal cells (7). We determined the molecular identity of anc-1 (8). RNA interference (RNAi) exper iments and the identification of a molecular lesion in the predicted open readin g frame of anc-1(e1873) confirmed that we had cloned anc-1 (8) (Fig. 1, A to D, and fig. S1). The full-length cDNA of anc-1 was predicted to be 25,639 base pair s (bp) encoding an 8546-residue protein. The bulk of ANC-1 (the anc-1 gene produ ct) consisted of mostly predicted coiled regions, including six repeats of 903 r esidues that are essentially identical at the nucleotide level (supporting onlin e text). The length of the repeat region may be maintained because of a selectiv e advantage of keeping ANC-1 large. This could allow a single ANC-1 molecule to extend more than 0.5 µm, long enough to stretch between the nucleus and th e actin cytoskeleton. The COOH-terminal 60 residues of ANC-1 were found to be co nserved with the COOH-termini of Drosophila Klarsicht (9, 10) and of mammalian S yne-1 and Syne-2 (supporting online text, Fig. 1E, and fig. S2) (11-14). We call this the KASH domain (for Klarsicht/ANC-1/Syne-1 homology). We showed by revers e transcription-polymerase chain reaction (RT-PCR) that the Drosophila CK00024 s equence, located nearly 45 kbp downstream of the previously known 3' end of the msp-300 transcript (15, 16), is part of the msp-300 gene. Syne-1 and Syne-2 (syn aptic nuclei expressed, also known as Myne, Nesprin, and NUANCE) localize to the nuclear envelope of mouse and human muscle cells throughout development (11-14) . The KASH domains of Syne-1 and Syne-2 are sufficient for nuclear envelope targ eting in tissue culture cells (13, 14). Syne-1 is enriched at the nuclear envelo pe of myonuclei clustered at the neuromuscular junction, which suggests a role i n nuclear positioning at the synapse (11). Msp-300, Syne-1, and Syne-2 have larg e central domains of multiple spectrin repeats that could function analogously t o the long coiled domains of ANC-1 (17). The NH2-terminus of ANC-1 is also simil ar to that of Msp-300 and the Syne proteins, containing two approximately 100-am ino-acid stretches of homology to the calponin domains of human alpha-actinin (s upporting online text, Fig. 1E, and fig. S2). Calponin domains are actin-binding motifs (18), suggesting that the NH2-terminal domain of ANC-1 interacts with th e actin cytoskeleton. -------------------------------------------------------------------------------- Fig. 1. Nomarski photographs of the lateral syncytial hypodermis of L2 hermaphr odites. Selected nuclei are marked with arrows. (A) N2 animal. (B) anc-1(e1873) animal. (C) RNAi-treated animal from a mother injected with double-stranded RNA (dsRNA) against exons 13 to 18 of anc-1. dsRNA against a full repeat of anc-1 or the five exons at the 3' end of anc-1 gave similar results. Scale bar, 10 &micr o;m. (D) The predicted intron/exon structure of anc-1. The site of the anc-1(e18 73) molecular lesion is marked by an arrow. Sequenced cDNAs that were identified either by expressed sequence tags or as RT-PCR products (8) are depicted below the structure (E) A depiction of the predicted domains of ANC-1 and related prot eins. All shades of blue represent regions highly predicted to be helical, with interspersed regions of predicted coiled-coil domains. The dark blue regions of the Syne proteins and of Msp-300 represent spectrin repeats. The mid-shade blue in ANC-1 represents repetitive regions. The conserved KASH domain at the COOH-te rminus is yellow. Black arrowheads mark predicted transmembrane domains. The NH2 -terminal regions with homology to calponin-type actin-binding domains are red. Some of the Syne protein sequences were deduced from genome projects (supporting online text). Human Syne-2 corresponds to NUANCE (14). [View Larger Version of this Image (62K GIF file)] -------------------------------------------------------------------------------- Antibodies were raised against the tandem repeat region of ANC-1; the specificit y of the affinity-purified antibody was shown by immunoblot analysis (Fig. 2A) a nd by the fact that purified antibodies in anc-1 mutant tissues failed to locali ze. ANC-1 was first detected in L1 larvae and was observed through adult stages where antibodies localized to the cytoplasm of all postembryonic somatic cells ( Fig. 2, B and C). Peripheral nuclear localization of ANC-1 was observed in a var iety of cells, including uterine cells. The nuclear envelope component UNC-84 is required for nuclear migration and anchorage (6, 19, 20). We tested whether ANC -1 localized properly in a collection of unc-84 mutant backgrounds. ANC-1 failed to localize to the nuclear periphery in the null unc-84 allele and in alleles t hat have missense mutations in or near the conserved SUN (for Sad1p, UNC-84 homo logy) domain of UNC-84 and that disrupt both nuclear migration and anchorage (6) (Fig. 2E). In contrast, ANC-1 was detected at the nuclear envelope in unc-84 al leles that have missense mutations or small deletions in the NH2-terminus of UNC -84 and that disrupt only migration (6) (Fig. 2F). ANC-1 was still detected at n ormal levels in the cytoplasm in all unc-84 mutants. Thus the UNC-84 SUN domain is required for ANC-1 localization to the nuclear envelope but not for overall A NC-1 levels. UNC-83, a component required for nuclear migration, also localizes to the nuclear envelope in an UNC-84-dependent manner (21). The predicted transm embrane domains in both ANC-1 and UNC-83 may play important roles in localizatio n or maintenance of the proteins at the nuclear envelope, as appears to be the c ase for Syne-1 (13). -------------------------------------------------------------------------------- Fig. 2. (A) Affinity-purified antibodies to ANC-1 were used to probe an immunob lot from a 5% acrylamide gel. Antibodies recognized multiple bands that migrate more slowly than the 175-kD marker. (B and C) ANC-1 antibody localization is pse udocolored red and 4',6'- diamidino-2-phenylindole (DAPI)-stained nuclei are blu e. (B) The lateral surface of the mid-body of an L4 N2 worm. ANC-1 antibodies lo calized to the cytoplasm and were enriched at the nuclear periphery. (C) The ext ruded gut of an N2 adult. (D) The molecular lesions in UNC-84 that were tested f or their ability to disrupt ANC-1 localization. The conserved SUN domain of UNC- 84 is shaded. (E and F) Samples shown are of extruded uterine tissue from (E) un c-84(n399) or (F) unc-84(e1411) adult hermaphrodites. Arrows point to examples o f uterine nuclei. Scale bars, 10 µm. ANC-1 localization was tested in 11 m utant strains (supporting online text). [View Larger Version of this Image (40K GIF file)] -------------------------------------------------------------------------------- There may be a limited number of ANC-1 docking sites at the nuclear envelope. To test this possibility, we bred animals that overexpressed the COOH-terminal 346 residues of ANC-1, including the KASH domain, using a heat shock promoter. Heat shock of these transgenic animals for 2 hours caused a strong nuclear anchorage defect in 100% of larval animals (Fig. 3C). In addition, staining with hemagglu tinin (HA) epitope antibodies showed that the COOH-terminal domain of ANC-1 loca lized to the nuclear envelope (Fig. 3, H to J). As a control, overexpression of an 1887-amino acid fragment in the middle of ANC-1 did not cause any detectable mutant phenotype (Fig. 3B). Thus, multiple ANC-1 molecules are required to bind to a limited number of docking sites at the nuclear envelope, suggesting that th e overexpressed domain blocks endogenous ANC-1 from docking at the nuclear envel ope. This is likely to occur by disrupting the interaction between UNC-84 and AN C-1. However, we were unable to detect a direct physical interaction between the SUN domain of UNC-84 and the COOH-terminal domain of ANC-1 using the two-hybrid system or by glutathione S-transferase pull-down assays. Thus, UNC-84 may funct ion through other proteins to recruit or maintain ANC-1 at the nuclear envelope. -------------------------------------------------------------------------------- Fig. 3. (A to C) Hypodermal nuclei (green) of L4 animals were identified by the coinjection marker protein SUR-5::GFP. Schematic figures of the constructs are shown at the bottom of the panels. Overexpression of (A) the NH2-terminus or (C) the COOH-terminus, but not (B) a middle region, of ANC-1 caused a nuclear ancho rage defect. (D) An autoradiograph is shown detecting [35S]methionine-labeled en d domains of ANC-1. Lanes with equally loaded pellets (P) and supernatants (S) a re coupled. The presence of actin filaments (F actin) is noted above. The left-h and lanes show that the NH2-terminal 700 residues of ANC-1 bound to filamentous actin. The right-hand lanes show that the COOH-terminal 346 residues of ANC-1 di d not bind to actin filaments. (E to G) An adult body wall muscle is shown in a worm expressing the NH2-terminal 566 residues of ANC-1 fused to GFP, expressed f rom a heat shock promoter after 2 hours at 33°C. (E) GFP fluorescence and (F) a ctin filaments stained with phalloidin colored red. (G) Colocalization is shown in yellow. (H to J) An embryo overexpressing the COOH-terminal domain of ANC-1 s tained with (H) antibodies against the HA epitope, pseudocolored red, or (I) DAP I, colored blue, to show nuclei. (J) The merged image. Scale bars, 10 µm. [View Larger Version of this Image (42K GIF file)] -------------------------------------------------------------------------------- To determine whether ANC-1 could bind directly to actin, an in vitro F-actin bin ding assay was performed. The NH2-terminal domain of ANC-1 was shown to bind to filamentous actin but not to monomeric actin (Fig. 3D). The NH2-domains of MSP-3 00 and Syne-2 also bind actin in vitro (14, 15). The conserved nature of the NH2 -terminus of ANC-1 suggests that ANC-1 binds actin in vivo. To test this, the NH 2-terminal 566 residues of ANC-1 were fused to green fluorescent protein (GFP) a nd expressed in worms from a heat shock promoter. When subjected to a short heat shock at 33°C for 2 hours, the NH2-terminal ANC-1::GFP completely colocalized with actin in body wall muscles (Fig. 3, E to G), suggesting that the in vitro a ctin-binding activity is functional in the cell. The analogous construct of huma n Syne-2 also binds actin in vitro and colocalizes with actin in tissue culture (14). Long-term overexpression of NH2-terminal ANC-1::GFP led to a paralyzed and arrested elongation at twofold (Pat) phenotype, in which embryos failed to elon gate because of blocked muscle development (supporting online text and fig. S3). Overexpression of the NH2-terminus of ANC-1 by a stronger heat shock caused a w eak Anc phenotype; clumps of at least three hypodermal nuclei were observed in 2 3% of larval animals (n = 77), whereas 4% of worms had a severe Anc phenotype (F ig. 3A). The lack of a more severe dominant Anc phenotype in the syncytial hypod ermis may be due to the lower expression level of the transgene in hypodermal ce ll lineages, as judged by GFP expression, or due to an abundance of actin-bindin g sites available for ANC-1 in these cells. Mutations in anc-1 disrupt the positioning and shape of mitochondria (7). We exa mined this phenotype in live animals using a GFP construct targeted to the mitoc hondria of body wall muscles (22). In wild-type animals, mitochondria appeared l ong and string-like (Fig. 4A). The mitochondria also remained anchored and sprea d throughout the cell as the worm moved. In contrast, mitochondria in anc-1(e187 3) animals were spherically shaped, often clustered together, and were pushed ar ound within the cytoplasm as the animal moved (Fig. 4B). Mitochondria were not s haped or positioned properly in an unc-60(r398) mutant background (Fig. 4D). A p artial loss-of-function allele in the C. elegans cofilin homolog, unc-60(r398), disrupts actin filaments in the body wall muscle of adult hermaphrodites (23). T herefore, actin filaments are required for proper positioning of mitochondria. T he anchorage of mitochondria in unc-84(n369) was normal (Fig. 4C), suggesting th at ANC-1 does not require UNC-84 to anchor mitochondria as it does for nuclear a nchorage. -------------------------------------------------------------------------------- Fig. 4. GFP-labeled mitochondria are shown in the body wall muscle cells of L4 animals of the following genotypes: (A) wild-type N2, (B) anc-1(e1873), (C) unc- 84(n369), and (D) cofilin unc-60(r398). The severely abnormal mitochondria in an c-1(e1873) are not anchored. In live animals, they were seen moving throughout t he muscle as the animal moved. Scale bar, 10 µm. [View Larger Version of t his Image (47K GIF file)] -------------------------------------------------------------------------------- Our model (fig. S4) suggests that ANC-1 functions to anchor nuclei by tethering the nucleus to the actin cytoskeleton and predicts that the KASH domain of ANC-1 is localized to the outer nuclear envelope by UNC-84. Digitonin extraction expe riments show that human Syne-2 localizes to the outer nuclear envelope (14). ANC -1 would then extend away from the nucleus, where its NH2-terminus binds to the stable actin cytoskeleton. As a result, ANC-1 molecules function to directly att ach the actin cytoskeleton to the nuclear envelope. Before a nucleus can migrate through the cytoplasm of the cell, the nuclear anchor must be released. The SUN domain of UNC-84 is likely to be intimately involved with this switch in nuclea r behavior, because it is required for both ANC-1 and UNC-83 localization at the nuclear envelope (21) (Fig. 2). UNC-83 is required for normal nuclear migration but not for nuclear anchorage (21). It is not known whether ANC-1 and UNC-83 ca n interact with UNC-84 simultaneously, although both antigens are detected at th e nuclear envelope of adult hypodermal cells. Overexpression of UNC-83 did not c ause any obvious anchorage phenotype, eliminating a competition model. Dystrophin and the associated dystrophin-glycoprotein complex function to connec t the actin cytoskeleton to the extracellular matrix; mutations in these compone nts lead to Duchenne or Becker muscular dystrophies (24). Although ANC-1 and Syn e connect the actin cytoskeleton to the nuclear matrix whereas dystrophin connec ts actin to the extracellular matrix, there are some similarities between these two mechanisms. ANC-1 and associated proteins, including UNC-84 and lamin A/C (1 2), are likely to create a bridge across the nuclear envelope. 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