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为了研究LINC(Linker of nucleoskeleton and cytoskeleton)复合体相关基因KASH(Klarsicht/Anc/Syne-1)和SUN(Sad1/UNC84)在海鞘(Ciona robusta)细胞核位置决定中的角色和作用，本研究鉴定并分别克隆了海鞘LINC复合体中编码2种Cr＿KASH蛋白和2种Cr＿SUN蛋白的基因。荧光定量PCR方法分析了上述4个基因在海鞘胚胎各个时期的表达量，发现在胚胎发育的尾芽延伸期，几种基因的表达量均有不同程度的提高。原位杂交方法揭示Cr＿KASH1、Cr＿SUN1和Cr＿SUN2特异表达在海鞘胚胎的脊索组织中，而Cr＿KASH2则特异表达在肌肉组织。在NIH/3T3细胞中的亚细胞定位分析证实，上述LINC复合体蛋白与不同的细胞骨架交联。通过破坏细胞骨架微丝和微管蛋白的聚合发现会影响KASH和SUN在细胞中的定位和结构，暗示海鞘LINC复合体成分和细胞质骨架蛋白协同作用调控细胞核的形态和位置。本文研究结果表明，在海鞘不同胚胎发育阶段和不同组织中，细胞核骨架或许通过不同LINC蛋白组分与细胞骨架结合影响细胞核的行为。
Abstract：In this study, we identified and cloned genes encoded two KASH proteins and two SUN proteins in Ciona genome. Quantitative real-time PCR results showed that expression levels of these genes were increased to different degrees during the embryo development. In situ hybridization revealed that Cr＿KASH1, Cr＿SUN1, and Cr＿SUN2 were specifically expressed in the notochord tissue, while Cr＿KASH2 was specifically expressed in muscle tissue in Ciona embryos, suggesting that these proteins were involved in the regulation of nuclear location in different tissues and developmental stages. Subcellular localization of these proteins in the NIH/3T3 cell line showed that these LINC complex component proteins cross-linked to the cytoplasmic cytoskeleton components. Disruption of the polymerization of actin or microtubule filaments affected the localization and structure of Cr＿KASH and Cr＿SUN in the cells, suggesting that the components of the LINC complex and cytoplasmic skeletal proteins are cooperated in the regulation of the morphology and location of the nucleus. The results of this study indicate that the nuclear skeleton in different Ciona tissues binds to the cytoskeleton through different linker proteins, and they interact and synergistically affect the behavior of the nucleus.
[1] Patterson K,Molofsky A B,Robinson C,et al.The functions of Klarsicht and nuclear lamin in developmentally regulated nuclear migrations of photoreceptor cells in the Drosophila eye[J].Molecular Biology of the Cell,2004,15(2):600-610.
[2] Tomlinson A,Ready D F.Sevenless:A cell-specific homeotic mutation of the drosophila eye[J].Science,1986,231(4736):400-402.
[3] Gomes E R,Jani S,Gundersen G G.Nuclear movement regulated by Cdc42,MRCK,myosin,and actin flow establishes MTOC polarization in migrating cells[J].Cell,2005,121(3):451-463.
[4] Gundersen G G,Worman H J.Nuclear Positioning[J].Cell,2013,152(6):1376-1389.
[5] Muchir A,Wu W,Worman H J.Reduced expression of A-type lamins and emerin activates extracellular signal-regulated kinase in cultured cells[J].Biochimica et Biophysica Acta,2009,1792(1):75-81.
[6] Hale C M,Shrestha A L,Khatau S B,et al.Dysfunctional connections between the nucleus and the actin and microtubule networks in laminopathic models[J].Biophysical Journal,2008,95(11):5462-5475.
[7] Muchir A,Pavlidis P,Decostre V,et al.Activation of MAPK pathways links LMNA mutations to cardiomyopathy in emery-dreifuss muscular dystrophy[J].Journal of Clinical Investigtion,2007,117(5):1282-1293.
[8] Folker E S,?stlund C,Luxton G W G,et al.Lamin A variants that cause striated muscle disease are defective in anchoring transmembrane actin-associated nuclear lines for nuclear movement[J].Proceedings of the National Academy of Sciences,2011,108(1):131-136.
[9] Goetz S C,Anderson K V.The primary cilium:A signalling centre during vertebrate development[J].Nature Review Genetics,2010,11(5):331-344.
[10] Del Bene F,Wehman A M,Link B A,et al.Regulation of neurogenesis by interkinetic nuclear migration through an apical-basal notch gradient[J].Cell,2008,134(6):1055-1065.
[11] Gurusaran M,Davies O R.A molecular mechanism for LINC complex branching by structurally diverse SUN-KASH 6:6 assemblies[J].Elife,2021,10:e60175.
[12] Carley E,Stewart R M,Zieman A,et al.The LINC complex transmits integrin-dependent tension to the nuclear lamina and represses epidermal differentiation[J].Elife,2021,10:e58541.
[13] Stroud M J,Banerjee I,Lowe J,et al.LINC complex proteins in cardiac structure,function,and disease[J].Circulation Research,2014,114(3):538.
[14] Horn H F.LINC complex proteins in development and disease[J].Current Topics in Developmental Biology,2014,109(109):287.
[15] Razafsky D,Hodzic D.Bringing KASH under the SUN:The many faces of nucleo-cytoskeletal connections[J].Journal of Cell Biology,2009,186(4):461-472.
[16] Fridolfsson H N,Starr D A.Kinesin-1 and dynein at the nuclear envelope mediate the bidirectional migrations of nuclei[J].Journal of Cell Biology,2010,191(1):115-128.
[17] Bouzid T,Kim E,Riehl B D,et al.The LINC complex,mechanotransduction,and mesenchymal stem cell function and fate[J].Journal of Biological Engineering,2019,13(1):68.
[18] Hao H,Starr D A.SUN/KASH interactions facilitate force transmission across the nuclear envelope[J].Nucleus (Austin,Tex),2019,10(1):73-80.
[19] D'Angelo M A,Hetzer M W.The role of the nuclear envelope in cellular organization[J].Cellular and Molecular Life Sciences,2006,63(3):316-332.
[20] Yin L M,Schnoor M,Jun C D.Structural characteristics,binding partners and related diseases of the calponin homology (CH) domain[J].Frontiers in Cell and Developmental Biology,2020,8:342.
[21] Dejardin T,Carollo P S,Sipieter F,et al.Nesprins are mechanotransducers that discriminate epithelial-mesenchymal transition programs[J].Journal of Cell Biology,2020,219(10):e201908036.
[22] Starr D A,Han M.ANChors away:An actin based mechanism of nuclear positioning[J].Journal of Cell Science,2003,116(2):211-216.
[23] Adames,N.R.Microtubule interactions with the cell cortex causing nuclear movements in Saccharomyces cerevisiae[J].Journal of Cell Biology,2000,149(4):863-874.
[24] Dong B,Horie T,Denker E,et al.Tube formation by complex cellular processes in Ciona intestinalis notochord[J].Developmental Biology,2009,330(2):237-249.
[25] Jiang D,Smith W C.Ascidian notochord morphogenesis[J].Developmental Dynamics,2007,236(7):1748-1757.
[26] Jiang D,Munro E M,Smith W C.Ascidian prickle regulates both mediolateral and anterior-posterior cell polarity of notochord cells[J].Current Biology,2005,15(1):79-85.
[27] Zhou X,Graumann K,Evans D E,et al.Novel plant SUN-KASH bridges are involved in RanGAP anchoring and nuclear shape determination[J].Journal of Cell Biology,2012,196(2):203-211.
[28] Dardaillon J,Dauga D,Simion P,et al.ANISEED 2019:4D exploration of genetic data for an extended range of tunicates[J].Nucleic Acids Research,2020,48(1):668-675.
[29] Brozovic M,Dantec C,Dardaillon J,et al.ANISEED 2017:Extending the integrated ascidian database to the exploration and evolutionary comparison of genome-scale datasets[J].Nucleic Acids Research,2018,46(1):718-725.
[30] Christiaen L,Wagner E,Shi W,et al.Whole-mount in situ hybridization on sea squirt (Ciona intestinalis) embryos[J].Cold Spring Harbor Perspectives in Biology,2009,2009(12):PDB.PROT5348.
[31] Bhattachan P,Rae J,Yu H,et al.Ascidian caveolin induces membrane curvature and protects tissue integrity and morphology during embryogenesis[J].Faseb Journal,2020,34(1):1345-1361.
[32] Rosenberghasson Y,Renertpasca M,Volk T.A Drosophila dystrophin-related protein,MSP-300,is required for embryonic muscle morphogenesis[J].Mechanisms of Development,1996,60(1):83-94.
[33] Starr D A.A nuclear-envelope bridge positions nuclei and moves chromosomes[J].Journal of Cell Science,2009,122(5):577-586.
[34] Grady R M,Starr D A,Ackerman G L,et al.Syne proteins anchor muscle nuclei at the neuromuscular junction[J].Proceedings of the National Academy of Sciences of the United States of America,2005,102(12):4359-4364.
[35] Zhang X,Lei K,Yuan X,et al.SUN1/2 and Syne/Nesprin-1/2 complexes connect centrosome to the nucleus during neurogenesis and neuronal migration in mice[J].Neuron,2009,64(2):173-187.
[36] Horn H F,Brownstein Z,Lenz D R,et al.The LINC complex is essential for hearing[J].Journal of Clinical Investigtion,2013,123(2):740-750.
[37] Roux K J,Crisp M L,Liu Q,et al.Nesprin 4 is an outer nuclear membrane protein that can induce kinesin-mediated cell polarization[J].Proceedings of the National Academy of Sciences,2009,106(7):2194-2199.
[38] Starr D A,Hermann G J,Malone C J,et al.unc-83 encodes a novel component of the nuclear envelope and is essential for proper nuclear migration[J].Development,2001,128(24):5039-5050.
[39] Starr D A,Han M.Role of ANC-1 in tethering nuclei to the actin cytoskeleton[J].Science,2002,298(5592):406-409.
[40] Horvitz H R,Sulston J E.Isolation and genetic characterization of cell-lineage mutants of the nematode Caenorhabditis elegans[J].Genetics,1980,96(2):435-454.
[41] Malone C J,Misner L,Bot N L,et al.The C.elegans hook protein,ZYG-12,mediates the essential attachment between the centrosome and nucleus[J].Cell,2004,115(7):825-836.
[42] Mosley-Bishop K L,Qinghong L,Patterson K,et al.Molecular analysis of the klarsicht gene and its role in nuclear migration within differentiating cells of the Drosophila eye[J].Current Biology,1999,9(21):1211-1220.
[43] Fischer J A.Drosophila Klarsicht has distinct subcellular localization domains for nuclear envelope and microtubule localization in the eye[J].Genetics,2004,168(3):1385-1393.
[44] Crisp M.Coupling of the nucleus and cytoplasm:Role of the LINC complex[J].Journal of Cell Biology,2006,172(1):41-53.
[45] Haque F,Lloyd D J,Smallwood D T,et al.SUN1 interacts with nuclear lamin A and cytoplasmic nesprins to provide a physical connection between the nuclear lamina and the cytoskeleton[J].Molecular Biology of the Cell,2006,26(10):3738-3751.
[46] Padmakumar V C,Libotte T,Lu W,et al.The inner nuclear membrane protein Sun1 mediates the anchorage of Nesprin-2 to the nuclear envelope[J].Journal of Cell Science,2005,118(15):3419-3430.
[47] Hodzic D M,Yeater D B,Bengtsson L,et al.Sun2 is a novel mammalian inner nuclear membrane protein[J].Journal of Biological Chemistry,2004,279(24):25805-25812.
[48] Malone C J,Fixsen W D,Horvitz H R,et al.UNC-84 localizes to the nuclear envelope and is required for nuclear migration and anchoring during C.elegans development[J].Development,1999,126(14):3171-3181.
[49] Kracklauer M P,Banks S M,Xie X,et al.Drosophila klaroid encodes a SUN domain protein required for Klarsicht localization to the nuclear envelope and nuclear migration in the eye[J].Fly (Austin),2007,1(2):75-85.
[50] Yu J,Lei K,Zhou M,et al.KASH protein Syne-2/Nesprin-2 and SUN proteins SUN1/2 mediate nuclear migration during mammalian retinal development[J].Human Molecular Genetics,2011,20(6):1061-1073.
[51] Tsujikawa M,Omori Y,Biyanwila J,et al.Mechanism of positioning the cell nucleus in vertebrate photoreceptors[J].Proceedings of the National Academy of Sciences of the United States of America,2007,104(37):14819-14824.
[52] Peng H,Qiao R,Dong B.Polarity establishment and maintenance in ascidian notochord[J].Frontiers in Cell and Developmental Biology,2020,8:597446.
基本信息:
DOI：10.16441/j.cnki.hdxb.20210419
中图分类号:Q78
引用信息:
[1]乔润雨,彭鸿哲,董波.海鞘细胞核骨架与细胞质骨架连接复合体相关基因的克隆及表达分析[J],2023,53(05):89-100.DOI:10.16441/j.cnki.hdxb.20210419.
基金信息:
国家重点研究发展计划项目(2019YFE0190900);; 山东省泰山学者建设工程专项经费资助~~
2021-11-14
2021
2021-11-26
2022-01-03
2022
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