Department of Medicine IV, Faculty of Medicine, Medical Center-University of Freiburg, 79106, Freiburg, Germany.
Bio Imaging Core Light Microscopy (BiMiC), Medical Faculty-Institute for Disease Modeling and Targeted Medicine (IMITATE), 79106, Freiburg, Germany.
Sci Rep. 2023 Oct 17;13(1):17647. doi: 10.1038/s41598-023-44235-y.
CLIC5 belongs to a family of ion channels with six members reported so far. In vertebrates, the CLIC5 gene encodes two different isoforms, CLIC5A and CLIC5B. In addition to its ion channel activity, there is evidence for further functions of CLIC5A, such as the remodeling of the actin cytoskeleton during the formation of a functional glomerulus in the vertebrate kidney. However, its specific role is still incompletely understood and a specific functional role for CLIC5B has not been described yet. Here we report our findings on the differential expression and functions of Clic5a and Clic5b during zebrafish kidney development. Whole-mount in situ hybridization studies revealed specific expression of clic5a in the eye and pronephric glomerulus, and clic5b is expressed in the gut, liver and the pronephric tubules. Clic5 immunostainings revealed that Clic5b is localized in the cilia. Whereas knockdown of Clic5a resulted in leakiness of the glomerular filtration barrier, Clic5b deficient embryos displayed defective ciliogenesis, leading to ciliopathy-associated phenotypes such as ventral body curvature, otolith deposition defects, altered left-right asymmetry and formation of hydrocephalus and pronephric cysts. In addition, Clic5 deficiency resulted in dysregulation of cilia-dependent Wnt signalling pathway components. Mechanistically, we identified a Clic5-dependent activation of the membrane-cytoskeletal linker proteins Ezrin/Radixin/Moesin (ERM) in the pronephric tubules of zebrafish. In conclusion, our in vivo data demonstrates a novel role for Clic5 in regulating essential ciliary functions and identified Clic5 as a positive regulator of ERM phosphorylation.
CLIC5 属于目前已报道的六成员离子通道家族。在脊椎动物中，CLIC5 基因编码两种不同的异构体，CLIC5A 和 CLIC5B。除了其离子通道活性外，还有证据表明 CLIC5A 具有进一步的功能，例如在脊椎动物肾脏中功能性肾小球形成过程中重塑肌动蛋白细胞骨架。然而，其具体作用仍不完全清楚，并且尚未描述 CLIC5B 的特定功能作用。在这里，我们报告了我们在斑马鱼肾脏发育过程中对 Clic5a 和 Clic5b 差异表达和功能的研究结果。整体原位杂交研究表明 clic5a 在眼睛和肾小囊肾小球中特异性表达，而 clic5b 在肠道、肝脏和肾小囊小管中表达。Clic5 免疫染色显示 Clic5b 定位于纤毛。尽管 Clic5a 的敲低导致肾小球滤过屏障的渗漏，但 Clic5b 缺陷胚胎显示出纤毛发生缺陷，导致纤毛病相关表型，如腹侧身体弯曲、耳石沉积缺陷、左右不对称改变以及脑积水和肾小囊囊肿的形成。此外，Clic5 缺乏导致依赖纤毛的 Wnt 信号通路成分的失调。在机制上，我们在斑马鱼的肾小囊小管中鉴定了 Clic5 依赖性膜细胞骨架连接蛋白 Ezrin/Radixin/Moesin (ERM) 的激活。总之，我们的体内数据表明 Clic5 在调节基本纤毛功能方面具有新的作用，并确定 Clic5 是 ERM 磷酸化的正调节剂。
