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尹家奇,何燕,吴俊林,生欣.2022.纤毛病相关蛋白CEP43和CCDC13在艾美游仆虫中的细胞与亚细胞定位.动物学杂志,57(3):412-421.
纤毛病相关蛋白CEP43和CCDC13在艾美游仆虫中的细胞与亚细胞定位
Cellular and Subcellular Localization of Ciliopathy-associated Proteins CEP43 and CCDC13 in Euplotes amieti
投稿时间:2021-07-25  修订日期:2022-04-11
DOI:10.13859/j.cjz.202203010
中文关键词:  艾美游仆虫  纤毛病蛋白  CEP43  CCDC13  免疫荧光  免疫电镜
英文关键词:Euplotes amieti  Ciliopathy-associated protein  CEP43  CCDC13  Immunofluorescence  Immuno- electron microscopy
基金项目:国家自然科学基金项目(No. 31760616)
作者单位E-mail
尹家奇 遵义医科大学生物化学与分子生物学教研室 遵义 563000 1154195642@qq.com 
何燕 遵义医科大学 生物化学与分子生物学教研室 遵义 563000 1458820307@qq.com 
吴俊林 遵义医科大学 生物化学与分子生物学教研室 遵义 563000 1690942115@qq.com 
生欣 遵义医科大学 生物化学与分子生物学教研室 遵义 563000 xshengbio@163.com 
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中文摘要:
      艾美游仆虫(Euplotes amieti)含有几乎所有已知的纤毛病基因,但绝大多数基因及其表达产物的细胞定位和功能未知。为明确中心粒蛋白43(CEP43)和卷曲螺旋域蛋白13(CCDC13)在艾美游仆虫中的细胞以及亚细胞定位,本研究采用免疫荧光和免疫电镜技术对其进行显微与超微结构观察。免疫荧光结果显示,CEP43主要定位于艾美游仆虫的细胞核、腹面纤毛器(口围带、尾棘毛、额腹横棘毛)的基体及其附属微管,CCDC13主要定位于腹面纤毛器杆部和基体以及银线系统,附属微管及大核未见其定位。CEP43与γ-微管蛋白定位相同,CCDC13与γ-微管蛋白仅在腹面定位相同。2种蛋白在免疫电镜下显示与荧光标记定位相同,而且CCDC13在额腹棘毛基部的胶体金数量远多于CEP43。结合已有研究推测,纤毛形成后多余的CEP43受γ-微管蛋白复合体调控且被募集于细胞核,CCDC13参与形成银线系统,但为增加生长期艾美游仆虫的微管再生能力,附属微管结构不需要CCDC13的参与。本结果为进一步研究上述蛋白在腹毛类纤毛虫中调节和维持皮层微管类细胞骨架的装配和稳定性中的作用和机制提供资料。
英文摘要:
      [Objectives] Recently, many ciliopathy-associated proteins are found in the basal body of cilia, and these proteins are also located in ciliates, Euplotes amieti. Ciliates have thousands of basal bodies of cilia and a complicated microtubular cytoskeleton, therefore, it is an ideal material for studying the interaction between the basal body and the microtubular cytoskeleton. [Methods] In this experiment, immunofluorescence and immuno-electron microscopy were used to observe the cellular and subcellular localization of Centrosomal protein 43 (CEP43) and Coiled-coil domain-containing 13 protein (CCDC13) in Euplotes amieti. [Results] The results showed that the cilia microtubular organelles of Euplotes amieti were composed of the adoral zone of membranelles, caudal cirrus, marginal cirrus, frontoventral cirrus, transverse cirrus, radiating microtubule, and dorsum bristle (Fig. 2). Immunofluorescence showed that CCDC13 was mainly located in the basal body and rod of cilia microtubular organelles and the silverline system (Fig. 3a, b), while CEP43 was mainly located in the basal body of cilia microtubular organelles, and cirrus accessory microtubules and nucleus (Fig. 3c). CEP43 and γ-tubulin were co-localized in the regions mentioned above (Fig. 4), while CCDC13 and γ-tubulin were only located in the same regions on the ventral side of Euplotes amieti (Fig. 5a, b). On the dorsum, CCDC13 was located in the silverline system (Fig. 5d), while γ-tubulin was located in the basal body of the dorsal cirri (Fig. 5e). During the cell division, CEP43 was not located in the nucleus, but in the basal body and the adoral zone of membranelles (Fig. 6a, b), while CCDC13 was observed in the adoral zone of membranelles (Fig. 6c, d). Further, the immuno-electron microscopy showed that CEP43 and CCDC13 were located in the basal bodies of the cilia, the adoral zone of membranelles, the base of the frontoventral cirrus, and the base of the transverse cirrus (Fig. 7), and the amount of colloidal gold at the base of frontoventral cirrus of CCDC13 was much more than that of CEP43 (Fig. 7h), while CCDC13 was not observed in the nucleus (Fig. 7j). [Conclusion] 1) CCDC13 and CEP43 are involved in the formation of cilia and basal body; 2) CEP43 can form complexes with CAP350 to provide attachment points for microtubule extension; 3) the excess CEP43 will be regulated and recruited to the nucleus after the formation of cilia; 4) the CCDC13 participates in the formation of the silverline system, in addition, it will affect the rate of microtubule regeneration. However, in order to increase the regeneration off the anterior longitudinal microtubules of Euplotes amieti during the growth period, CCDC13 is not required. The results provide basic data for further research on the roles and mechanisms of the above proteins in regulating and maintaining the assembly and stability of the cortical microtubule cytoskeleton in hypotrichous ciliate.
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