Determining the role of SPAG1 in the cytoplasmic assembly of axonemal dynein arms

摘要/项目摘要 原发性睫状肌运动障碍(PCD)是一种由功能障碍引起的遗传性异质性疾病 到目前为止发现的30多种不同基因的突变导致的游动纤毛。PCD的特点是慢性 肺部疾病、不孕不育、偏侧缺陷，最终导致终末期呼吸衰竭。最常见的 PCD患者运动纤毛无异常和/或内和/或外动力缺陷 手臂，纤毛运动所必需的蛋白质结构。然而，蛋白质的相互作用和机制 轴丝动力蛋白臂组装所必需的基因在很大程度上是未知的。此前，精子中的突变- 相关抗原1(SPAG1)被发现导致静态纤毛缺失和/或内部和/或缺陷 外动力臂。先前对原代人呼吸道上皮细胞(HAEC)培养的研究表明， SPAG1‘S在纤毛发生过程中被诱导表达，SPAG1主要定位于细胞质和近胞质 基底体，但不在纤毛轴丝。原代hAEC中SPAG1的免疫沉淀研究 裂解物鉴定了两个已知的动力蛋白轴丝组装因子，DNAAF1和DNAAF2，以及一个潜在的 新的PCD基因PIH1D2与SPAG1共沉淀。 推测SPAG1通过相互作用在轴丝动力蛋白臂的胞浆组装中起关键作用 与其他动力蛋白轴丝组装因子(DNAAFs)共同作用。目标1建议对已鉴定的SPAG1进行特征描述 进一步与DNAAF1、DNAAF2和PIH1D2相互作用。这些已鉴定基因的表达和定位 将通过液滴数字聚合酶链式反应(DdPCR)、westerns和超分辨率显微镜对相互作用进行检查。至 确定纤毛细胞分化过程中SPAG1相互作用发生的顺序，时间进程共IP研究 将会被执行。在鉴别hAEC时，将进行邻近连接试验(LA)以询问是否 DNAAF1、DNAAF2或PIH1D2与SPAG1直接相互作用。目标2建议确定哪一个 动力蛋白臂的异构体和不同的亚基复合体需要SPAG1进行组装。SPAG1将是 在使用CRISPR/Cas9技术的hAEC培养中被敲除，并且确认这种SPAG1的丢失将 包括测量纤毛搏动频率和测量动力蛋白臂的丰度。错误的本地化 将研究SPAG1缺陷细胞中不同动力蛋白臂亚型的已知DNAAFs和动力蛋白链 使用免疫荧光技术。已知DNAAF相互作用和已知动力蛋白的分离 将使用共IP技术在SPAG1-1中研究动力蛋白臂组装过程中的链状络合物。 缺乏细胞。这些研究将提供对SPAG1在细胞质中的作用的更好的理解 轴突动力臂的预装配。这种知识的扩展将导致更好的诊断和 未来治疗原发纤毛运动障碍药物的基本原理。

ABSTRACT/PROJECT SUMMARY Primary ciliary dyskinesia (PCD) is a genetically heterogenous disorder resulting from dysfunctional motile cilia due to mutations in over 30 different genes discovered thus far. PCD is characterized by chronic pulmonary disease, infertility, laterality defects, and ultimately end-stage respiratory failure. The most common abnormality observed in motile cilia in cases of PCD are absent and/or defective inner and/or outer dynein arms, the protein structures essential for ciliary movement. However, the protein interactions and mechanisms that are necessary for axonemal dynein arm assembly are largely unknown. Previously, mutations in sperm- associated antigen 1 (SPAG1) were discovered to result in static cilia with missing and/or defective inner and outer dynein arms. Previous studies in primary human airway epithelial cell (hAEC) cultures demonstrates that SPAG1's expression is induced during ciliogenesis and SPAG1 localizes primarily in the cytoplasm and near basal bodies, but not in the ciliary axoneme. Immunoprecipitation (IP) studies for SPAG1 in primary hAEC lysates identified two known dynein axonemal assembly factors, DNAAF1 and DNAAF2, as well as a potential novel PCD gene, PIH1D2, to have co-precipitated with SPAG1.Therefore, the central hypothesis of this proposal is that SPAG1 plays a key role in the cytoplasmic assembly of axonemal dynein arms by interacting with other dynein axonemal assembly factors (DNAAFs). Aim 1 proposes to characterize the identified SPAG1 interactions with DNAAF1, DNAAF2, and PIH1D2 further. The expression and localization of these identified interactors will be examined by droplet digital PCR (ddPCR), westerns, and super-resolution microscopy. To determine the order that SPAG1 interactions occur during ciliated cell differentiation, time course co-IP studies will be performed. Proximity ligation assays (PLA) in differentiating hAEC will be performed to interrogate if DNAAF1, DNAAF2, or PIH1D2 are directly interacting with SPAG1. Aim 2 proposes to determine which isoforms and distinct subunit complexes of dynein arms require SPAG1 for their assembly. SPAG1 will be knocked out in hAEC cultures using CRISPR/Cas9 technology, and confirmation of this loss of SPAG1 will include measuring ciliary beat frequency and measuring the abundance of dynein arms. Mislocalization of known DNAAFs and dynein chains of different dynein arm isoforms in SPAG1-deficent cells will be studied using immunofluorescence techniques. The disassociation of known DNAAF interactions and known dynein chain complexes in the dynein arm assembly process will be studied using co-IP techniques in SPAG1- deficient cells. These studies will provide a greater understanding of the role SPAG1 has in the cytoplasmic pre-assembly of axonemal dynein arms. This expansion of knowledge will lead to improved diagnostics and the rationale for future therapeutic agents for primary ciliary dyskinesia.