ZHANG Yu,SHI Xingjuan.Study of cellular localization of Cep70 and its role in microtubule polymerization[J].Journal of Nanjing Agricultural University,2017,40(6):1094-1099.[doi:10.7685/jnau.201707012]





Study of cellular localization of Cep70 and its role in microtubule polymerization
张俞 史兴娟
东南大学生命科学研究院/发育与疾病相关基因教育部重点实验室, 江苏 南京 210096
ZHANG Yu SHI Xingjuan
Institute of Life Sciences/Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Southeast University, Nanjing 210096, China
Cep70antibodyimmunofluorescencemicrotubule polymerization
[目的] 本试验旨在研究Cep70的细胞定位及其在微管聚合中的调节作用。[方法] 利用原核表达质粒pMALp2T-Cep70,在细菌中诱导表达融合蛋白MBP-Cep70。用融合蛋白免疫小鼠,获得特异性的抗Cep70抗体。首先用该抗体对细胞进行免疫荧光显微分析,检测Cep70在细胞内的定位,再利用荧光标记的微管体外组装试剂盒研究Cep70在体外微管聚合中对微管形态的调节,最后利用微管混浊度试验,验证Cep70对微管聚合的功能影响。[结果] 通过从细菌中诱导纯化蛋白,获得了大量高纯度的融合蛋白MBP-Cep70。纯化蛋白免疫小鼠后得到了靶向Cep70的抗体。免疫荧光显微分析发现Cep70定位在微管组织中心(MTOC),即中心体上。通过对荧光标记的微管蛋白进行体外组装分析,发现Cep70增加了体外聚合微管的长度。进一步通过微管浑浊度试验,发现Cep70促进了体外微管的聚合。[结论] Cep70在细胞内定位在微管组织中心上,且在体外微管聚合上具有一定促进作用。
[Objectives] The paper aims to study cellular localization of Cep70 and its role in microtubule polymerization.[Methods] Prokaryotic expression plasmid of pMALp2T-Cep70 was constructed and transfected into BL21 strain of Escherichia coli. MBP-Cep70 fusion protein was induced and purified from bacteria. Anti-Cep70 antibody was obtained by immunizing mice with MBP-Cep70 fusion protein. We first analyzed the cellular localization of Cep70 with anti-Cep70 antibody by immunofluorescence microscopy. We then analyzed the role of Cep70 in microtubule polymerization with in vitro tubulin polymerization assay by examining the morphology of rhodamine-labeled tubulin. Finally, the role of Cep70 in microtubule assembly was further verified by measuring the absorbance of microtubule at a 350 nm wavelength at 37℃.[Results] We harvested large quantity of MBP-Cep70 fusion protein by purification from bacteria, and obtained anti-Cep70 antibody by immunizing mice with fusion protein. The cellular localization of Cep70 was examined with anti-Cep70 antibody by immunofluorescence microscopy analysis. We found that Cep70 protein localized at the microtubule organizing center. In vitro microtubule polymerization assay demonstrated that Cep70 increased the length of polymerized microtubule. By measuring the absorbance of microtubule at 350 nm wavelength, we also found that Cep70 promoted in vitro microtubule polymerization.[Conclusions] Our results demonstrated that Cep70 localized at the microtubule organizing center, and promoted in vitro microtubule assembly.


[1] Woodruff J B,Gomes B F,Widlund P O,et al. The centrosome is a selective condensate that nucleates microtubules by concentrating tubulin[J]. Cell,2017,169(6):1066-1077.
[2] Sulimenko V,Hájková Z,Klebanovych A,et al. Regulation of microtubule nucleation mediated by γ-tubulin complexes[J]. Protoplasma,2017,254(3):1187-1199.
[3] Meraldi P. Centrosomes in spindle organization and chromosome segregation:a mechanistic view[J]. Chromosome Research,2016,24(1):19-34.
[4] Rivera-Rivera Y,Saavedra H. Centrosome-a promising anti-cancer target[J]. Biologics,2016,10:167-176.
[5] Chavali P L,Pütz M,Gergely F. Small organelle,big responsibility:the role of centrosomes in development and disease[J]. Philosophical Transactions of The Royal Society B-Biological Sciences,2014,369. DOI:10.1098/rstb.20130468.
[6] Shaheen R,Shamseldin H E,Loucks C M,et al. Mutations in CSPP1,encoding a core centrosomalp rotein,cause a range of ciliopathy phenotypes in humans[J]. The American Journal of Human Genetics,2014,94(1):73-79.
[7] Andersen J S,Wilkinson C J,Mayor T,et al. Proteomic characterization of the human centrosome by protein correlation profiling[J]. Nature,2003,426:570-574.
[8] Wilkinson C J,Carl M,Harris W A. Cep70 and Cep131 contribute to ciliogenesis in zebrafish embryos[J]. BMC Cell Biology,2009,10:17.
[9] Shiratsuchi G,Kamiya R,Hirono M. Scaffolding function of the Chlamydomonas procentriole protein CRC70,a member of the conserved Cep70 family[J]. Journal of Cell Science,2011,124:2964-2975.
[10] Shi X,Sun X,Liu M,et al. CEP70 protein interacts with γ-tubulin to localize at the centrosome and is critical for mitotic spindle assembly[J]. Journal of Biological Chemistry,2011,286(38):33401-33408.
[11] Shi X,Liu M,Li D,et al. Cep70 contributes to angiogenesis by modulating microtubule rearrangement and stimulating cell polarization and migration[J]. Cell Cycle,2012,11(8):1554-1563.
[12] Shi X,Li D,Wang Y,et al. Discovery of centrosomal protein 70 as an important player in the development and progression of breast cancer[J]. The American Journal of Pathology,2017,187(3):679-688.
[13] Shi X,Yao Y,Wang Y,et al. Cep70 regulates microtubule stability byinteracting with HDAC6[J]. FEBS Letters,2015,589(15):1771-1777.
[14] Gao J,Huo L,Sun X,et al. The tumor suppressor CYLD regulates microtubule dynamics and plays a role in cell migration[J]. Journal of Biological Chemistry,2008,283(14):8802-8809.
[15] Sun X,Shi X,Liu M,et al. Mdp3 is a novel microtubule-binding protein that regulates microtubule assembly and stability[J]. Cell Cycle,2011,10(22):3929-3937.
[16] Sun L,Gao J,Dong X,et al. EB1 promotes Aurora-B kinase activity through blocking its inactivation by protein phosphatase 2A[J]. Proc Natl Acad Sci USA,2008,105(20):7153-7158.
[17] Huo L,Li D,Sun L,et al. Tat acetylation regulates its actions on microtubule dynamics and apoptosis in T lymphocytes[J]. Journal of Pathology,2010,223(1):28-36.
[18] Sun L,Gao J,Huo L,et al. Tumour suppressor CYLD is a negative regulator of the mitotic kinase Aurora-B[J]. Journal of Pathology,2010,221(4):425-432.
[19] Huo L,Li D,Sun X,et al. Regulation of Tat acetylation and transactivation activity by the microtubule-associated deacetylase HDAC6[J]. Journal of Biological Chemistry,2011,286(11):9280-9286.
[20] Delgehyr N,Sillibourne J,Bornens M. Microtubule nucleation and anchoring at the centrosome are independent processes linked by ninein function[J]. Journal of Cell Science,2005,118:1565-1575.
[21] Yang Y,Ran J,Liu M,et al. CYLD mediates ciliogenesis in multiple organs by deubiquitinating Cep70 and inactivating HDAC6[J]. Cell Research,2014,24(11):1342-1353.
[22] Xie S,Qin J,Liu S,et al. Cep70 overexpression stimulates pancreatic cancer by inducing centrosome abnormality and microtubule disorganization[J]. Scientific Reports,2016,6:21263.
[23] Tala,Xie S,Sun X,et al. Microtubule-associated protein Mdp3 promotes breast cancer growth and metastasis[J]. Theranostics,2014,4(10):1052-1061.
[24] Delgehyr N,Sillibourne J,Bornens M. Microtubule nucleation and anchoring at the centrosome are independent processes linked by ninein function[J]. Journal of Cell Science,2005,118(8):1565-1575.
[25] Wade R H. On and around microtubules:an overview[J]. Molecular Biotechnology,2009,43(2):177-191.
[26] Tala,Sun X,Chen J,et al. Microtubule stabilization by Mdp3 is partially attributed to its modulation of HDAC6 in addition to its association with tubulin and microtubules[J]. PLoS ONE,2014,9(3):e90932.
[27] 杨笑柳,剧世强,崔盼盼,等. Aurora-A抑制对猪卵母细胞MⅠ期纺锤体组装的影响[J]. 南京农业大学学报,2016,39(4):656-660. DOI:10.7685/jnau.201512009. Yang X L,Ju S Q,Cui P P,et al. Effects of Aurora-A inhibitor on MⅠ-stage spindle configuration of porcine oocytes[J]. Journal of Nanjing Argricultural University,2016,39(4):656-660(in Chinese with English abstract).
[28] Schiebel E. γ-tubulin complexes:binding to the centrosome,regulation and microtubule nucleation[J]. Current Opinion in Cell Biology,2000,12(1):113-118.
[29] van der Vaart B,Akhmanova A,Straube A. Regulation of microtubule dynamic instability[J]. Biochemical Society Transactions,2009,37:1007-1013.
[30] Shi X,Wang Y,Sun X,et al. Centrosomal protein 70 is a mediator of paclitaxel sensitivity[J]. International Journal of Molecular Sciences,2017,18(6):1267.
[31] Shi X,Wang J,Yang Y,et al. Cep70 promotes microtubule assembly in vitro by increasing microtubule elongation[J]. Acta Biochimica et Biophysica Sinica,2012,44(5):450-454.


 WANG Ying,ZHENG Hao,HE Cheng-hua,et al.Characterization of fumonisin B1artificial antigen and production of the antibody[J].Journal of Nanjing Agricultural University,2011,34(6):93.[doi:10.7685/j.issn.1000-2030.2011.05.017]


更新日期/Last Update: 1900-01-01