The zebrafish fleer gene encodes an essential regulator of cilia tubulin polyglutamylation

Cilia and basal bodies are essential organelles for a broad spectrum of functions, including the development of left-right asymmetry, kidney function, cerebrospinal fluid transport, generation of photoreceptor outer segments, and hedgehog signaling. Zebrafish fleer (flr) mutants exhibit kidney cysts, randomized left-right asymmetry, hydrocephalus, and rod outer segment defects, suggesting a pleiotropic defect in ciliogenesis. Positional cloning flr identified a tetratricopeptide repeat protein homologous to the Caenorhabditis elegans protein DYF1 that was highly expressed in ciliated cells. flr pronephric cilia were shortened and showed a reduced beat amplitude, and olfactory cilia were absent in mutants. flr cilia exhibited ultrastructural defects in microtubule 13-tubules, similar to axonemes that lack tubulin posttranslational modifications (polyglutamylation or polyglycylation). flr cilia showed a dramatic reduction in cilia polyglutamylated tubulin, indicating that flr encodes a novel modulator of tubulin polyglutamylation. We also found that the C. elegans flr homologue, dyf-1, is also required for tubulin polyglutamylation in sensory neuron cilia. Knockdown of zebrafish Tt116, a tubulin polyglutamylase, specifically eliminated tubulin polyglutamylation and cilia formation in olfactory placodes, similar to flr mutants. These results are the first in vivo evidence that tubulin polyglutamylation is required for vertebrate cilia motility and structure, and, when compromised, results in failed ciliogenesis.

纤毛和基体是具有广泛功能的重要细胞器，包括左右不对称的发育、肾功能、脑脊液运输、光感受器外段的产生以及刺猬信号传导。斑马鱼fleer（flr）突变体表现出肾囊肿、左右不对称随机化、脑积水以及视杆外段缺陷，这表明在纤毛发生方面存在多效性缺陷。通过定位克隆flr，鉴定出一种与秀丽隐杆线虫蛋白DYF1同源的四肽重复蛋白，该蛋白在纤毛细胞中高度表达。flr前肾纤毛缩短，摆动幅度减小，并且突变体中嗅觉纤毛缺失。flr纤毛在微管13 - 微管中表现出超微结构缺陷，类似于缺乏微管蛋白翻译后修饰（多聚谷氨酰化或多聚甘氨酰化）的轴丝。flr纤毛中纤毛多聚谷氨酰化微管蛋白显著减少，这表明flr编码一种新型的微管蛋白多聚谷氨酰化调节剂。我们还发现秀丽隐杆线虫的flr同源物dyf - 1也是感觉神经元纤毛中微管蛋白多聚谷氨酰化所必需的。斑马鱼Tt116（一种微管蛋白多聚谷氨酰化酶）的敲低特异性地消除了嗅基板中的微管蛋白多聚谷氨酰化和纤毛形成，这与flr突变体相似。这些结果是体内的首个证据，表明微管蛋白多聚谷氨酰化对于脊椎动物纤毛的运动和结构是必需的，并且当受损时会导致纤毛发生失败。