Regulation of Cilium Length

项目总结/摘要 纤毛是围绕一束特化微管（轴丝）构建的细胞突起， 基本的运动和感觉功能。纤毛推动细胞，产生细胞外液流，支持 信号转导和释放细胞外囊泡。纤毛的长度是一个重要的参数。 运动纤毛太短或太长不能产生或响应流体动力。在 在哺乳动物中，hedgehog信号被改变纤毛长度的突变所损害。遗传 研究发现了几种高度保守的激酶，它们负调节纤毛长度 包括MAK/ICK、LF 4/MOK、LF 2/CCRK/DYF-18、CNK/NEK/NRK和LF 5/CDKL 5。突变 这些激酶引起包括发育障碍（内分泌-脑- 骨发育不良综合征、短肋多指综合征）、色素性视网膜炎、多囊肾 青少年癫痫病纤毛长度激酶的活性是如何调节的，以及它们是如何起作用的 调节纤毛长度的方法知之甚少。稳态纤毛长度依赖于一种平衡 纤毛组装速率（由鞭毛内运输驱动，递送前体）和 去除轴丝亚单位的纤毛分解途径。传感器的身份， 测量纤毛长度是未知的。纤毛长度激酶的活性如何影响执行 调节纤毛长度的途径（鞭毛内运输和分解）知之甚少。 纤毛长度激酶的磷酸化底物的鉴定将有助于揭示纤毛长度激酶的磷酸化作用。 纤毛长度感测和调节的潜在机械原理。纤毛长度激酶 从纤毛虫原生生物到人类都是保守的。我们将使用一个具有高密度纤毛的单细胞模型， 嗜热四膜虫是一种优良的遗传和生化模型， 基于保守纤毛长度激酶中功能突变获得， LF 4/MOK，导致纤毛缩短和细胞麻痹。我们将使用这个屏幕和一个新的 用于比较全基因组测序的管道，以鉴定LF 4/MOK的基因外支持物 功能的增益。该筛选具有鉴定新型LF 4相互作用物的强大潜力，包括其 磷酸化底物、反向磷酸酶和上游激活剂。LF 4的性质 相互作用者将决定对纤毛长度的感知和执行机制的功能研究。

PROJECT SUMMARY/ABSTRACT Cilia are cell projections built around a bundle of specialized microtubules (the axoneme) that mediate essential motile and sensory functions. Cilia propel cells, generate extracellular fluid flows, support signal transduction and release extracellular vesicles. The length of cilium is an important parameter. Motile cilia that are either too short or too long fail to generate or respond to hydrodynamic forces. In mammals, hedgehog signaling is compromised by mutations that alter the cilium length. Genetic studies led to the discovery of several highly conserved kinases that negatively regulate cilium length including: MAK/ICK, LF4/MOK, LF2/CCRK/DYF-18, CNK/NEK/NRK and LF5/CDKL5. Mutations in these kinases cause diseases (ciliopathies) including developmental disorders (endocrine-cerebro- osteodysplasia syndrome, short rib polydactyly syndrome), retinitis pigmentosa, polycystic kidney disease and juvenile epilepsy. How the activity of cilium length kinases is regulated and how they act to adjust cilium length is poorly understood. The steady-state cilium length is dependent on a balance between the rates of ciliary assembly (driven by intraflagellar transport that delivers precursors) and ciliary disassembly pathway that removes axoneme subunits. The identity of the sensor that measures cilium length is unknown. How the activity of cilium length kinases affects the executive pathways that adjust cilium length (intraflagellar transport and disassembly) is poorly understood. Identification of phosphorylation substrates of cilium length kinases will help in revealing the underlying mechanistic principle of cilium length sensing and adjustment. The cilium length kinases are conserved from ciliated protists to man. We will use a unicellular model with a high density of cilia, Tetrahymena thermophila, an excellent genetic and biochemical model, for a novel genetic suppressor screen, based on a gain of function mutation in a conserved cilium length kinase, LF4/MOK, that causes shortening of cilia and cell paralysis. We will use this screen and a new pipeline for comparative whole genome sequencing, to identify extragenic suppressions of LF4/MOK gain of function. This screen has strong potential for identification of novel LF4 interactors including its phosphorylation substrates, opposing phosphatases and upstream activators. The nature of LF4 interactors will dictate functional studies into the mechanism of sensing and execution of cilium length.