Abstract：
Motile cilia in a vertebrate are important to sustaining activities of life. Fluid flow on the apical surface of several tissues, including bronchial epithelium, ependymal epithelium, and fallopian tubules is generated by the ciliary beating of motile cilia. Multi-ciliated cells in ependymal tissue are responsible for the circulation of cerebrospinal fluid (CSF), which is essential for the development and homeostasis of the central nervous system, and airway tissues are protected from external contaminants by cilia-driven mucosal flow over the top of the airway epithelium.
A previous study reported that reduction of Ribc2 protein leads to disruption of ciliary beating in multi-ciliated cells. However, knowledge regarding the molecular function of Ribc2 is limited, thus currently available information is also limited. Therefore, we evaluated the importance of proteins involved in the interaction with Ribc2 in the process of ciliary beating.
Immunoprecipitation and mass spectrometry analysis was performed for the discovery of proteins involved in the interaction with Ribc2. Expression of the target gene was inhibited by injection of antisense morpholinos and measurement of the fluid flow on the embryonic epidermis of Xenopus was performed using fluorescent beads for examination of the ciliary beating of multi cilia. In addition, the flag-tagged protein was expressed by injection of mRNA and the changes in protein localization in the cilia were measured by immunostaining and western blot analysis for analysis of the molecular interaction between Ribc2 and Ribc2 binding proteins in multi-cilia.
The IP/MS analysis identified Ckb and Ybx2 as Ribc2 binding proteins and our results showed that localization of both Ckb and Ybx2 occurs at the axoneme of multi-cilia on the embryonic epithelium of Xenopus laevis. In addition, our findings confirmed that knock-down of Ckb or Ybx2 resulted in abnormal ciliary beating and reduction of cilia-driven fluid flow on multi-cilia of Xenopus laevis. In addition, significantly decreased localization of Ckb or Ybx2 in the ciliary axoneme was observed in Ribc2-depleted multi-cilia.
Ckb and Ybx2 are involved in the interaction with Ribc2 and are necessary for the ciliary beating of multi-cilia.
A previous study reported that reduction of Ribc2 protein leads to disruption of ciliary beating in multi-ciliated cells. However, knowledge regarding the molecular function of Ribc2 is limited, thus currently available information is also limited. Therefore, we evaluated the importance of proteins involved in the interaction with Ribc2 in the process of ciliary beating.
Immunoprecipitation and mass spectrometry analysis was performed for the discovery of proteins involved in the interaction with Ribc2. Expression of the target gene was inhibited by injection of antisense morpholinos and measurement of the fluid flow on the embryonic epidermis of Xenopus was performed using fluorescent beads for examination of the ciliary beating of multi cilia. In addition, the flag-tagged protein was expressed by injection of mRNA and the changes in protein localization in the cilia were measured by immunostaining and western blot analysis for analysis of the molecular interaction between Ribc2 and Ribc2 binding proteins in multi-cilia.
The IP/MS analysis identified Ckb and Ybx2 as Ribc2 binding proteins and our results showed that localization of both Ckb and Ybx2 occurs at the axoneme of multi-cilia on the embryonic epithelium of Xenopus laevis. In addition, our findings confirmed that knock-down of Ckb or Ybx2 resulted in abnormal ciliary beating and reduction of cilia-driven fluid flow on multi-cilia of Xenopus laevis. In addition, significantly decreased localization of Ckb or Ybx2 in the ciliary axoneme was observed in Ribc2-depleted multi-cilia.
Ckb and Ybx2 are involved in the interaction with Ribc2 and are necessary for the ciliary beating of multi-cilia.
摘要：
背景：脊椎动物的运动性纤毛对维持生命活动很重要。流体在几个组织的顶端表面流动，包括支气管上皮,室管膜上皮,输卵管是由能动纤毛的纤毛跳动产生的。室管膜组织中的多纤毛细胞负责脑脊液（CSF）的循环，这对中枢神经系统的发育和稳态至关重要，气道上皮顶部的纤毛驱动的粘膜流保护气道组织免受外部污染物的影响。
目的：之前的一项研究报道，Ribc2蛋白的减少导致多纤毛细胞纤毛搏动的破坏。然而,关于Ribc2分子功能的知识是有限的，因此，目前可用的信息也是有限的。因此,我们评估了参与与Ribc2相互作用的蛋白质在纤毛搏动过程中的重要性。
方法：进行免疫沉淀和质谱分析，以发现与Ribc2相互作用有关的蛋白质。通过注射反义吗啉代抑制靶基因的表达，并使用荧光珠测量非洲爪的胚胎表皮上的流体流量，以检查多纤毛的纤毛跳动。此外,通过注射mRNA表达标记的蛋白质，并通过免疫染色和蛋白质印迹分析测量纤毛中蛋白质定位的变化，以分析多纤毛中Ribc2和Ribc2结合蛋白之间的分子相互作用。
结果：IP/MS分析将Ckb和Ybx2鉴定为Ribc2结合蛋白，我们的结果表明，Ckb和Ybx2的定位都发生在非洲爪的胚胎上皮上多纤毛的轴突处。此外,我们的发现证实，敲除Ckb或Ybx2会导致异常的纤毛搏动和纤毛驱动的流体流量减少。此外,在Ribc2耗尽的多纤毛中观察到Ckb或Ybx2在睫状轴突中的定位显着降低。
结论：Ckb和Ybx2参与与Ribc2的相互作用，并且是多纤毛纤毛搏动所必需的。
目的：之前的一项研究报道，Ribc2蛋白的减少导致多纤毛细胞纤毛搏动的破坏。然而,关于Ribc2分子功能的知识是有限的，因此，目前可用的信息也是有限的。因此,我们评估了参与与Ribc2相互作用的蛋白质在纤毛搏动过程中的重要性。
方法：进行免疫沉淀和质谱分析，以发现与Ribc2相互作用有关的蛋白质。通过注射反义吗啉代抑制靶基因的表达，并使用荧光珠测量非洲爪的胚胎表皮上的流体流量，以检查多纤毛的纤毛跳动。此外,通过注射mRNA表达标记的蛋白质，并通过免疫染色和蛋白质印迹分析测量纤毛中蛋白质定位的变化，以分析多纤毛中Ribc2和Ribc2结合蛋白之间的分子相互作用。
结果：IP/MS分析将Ckb和Ybx2鉴定为Ribc2结合蛋白，我们的结果表明，Ckb和Ybx2的定位都发生在非洲爪的胚胎上皮上多纤毛的轴突处。此外,我们的发现证实，敲除Ckb或Ybx2会导致异常的纤毛搏动和纤毛驱动的流体流量减少。此外,在Ribc2耗尽的多纤毛中观察到Ckb或Ybx2在睫状轴突中的定位显着降低。
结论：Ckb和Ybx2参与与Ribc2的相互作用，并且是多纤毛纤毛搏动所必需的。
