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

摘要/项目总结 原发性睫状体运动障碍（PCD）是一种遗传异质性疾病， 运动纤毛是由于迄今为止发现的30多个不同基因的突变。PCD的特征是慢性的 肺部疾病、不孕症、偏侧性缺陷，最终导致终末期呼吸衰竭。最常见的 在PCD病例中观察到的运动纤毛异常不存在和/或内部和/或外部动力蛋白缺陷 臂，纤毛运动所必需的蛋白质结构。然而，蛋白质相互作用和机制 对轴丝动力蛋白臂装配所必需的蛋白质在很大程度上是未知的。以前，精子的突变- 相关抗原1（SPAG 1）被发现导致静止纤毛缺失和/或缺陷的内部和 动力蛋白外臂先前在原代人气道上皮细胞（hAEC）培养物中的研究表明， SPAG 1的表达在纤毛发生过程中被诱导，并且SPAG 1主要定位于细胞质和邻近细胞质。 在纤毛轴丝中没有。原代hAEC中SPAG 1的免疫沉淀（IP）研究 裂解物鉴定了两种已知的动力蛋白轴丝组装因子DNAAF 1和DNAAF 2，以及一种潜在的 新的PCD基因PIH 1D 2与SPAG 1共沉淀。因此， SPAG 1可能通过与细胞外基质的相互作用，在轴丝动力蛋白臂的胞质组装中起关键作用。 与其他动力蛋白轴丝组装因子（DNAAFs）。目的1提出表征已鉴定的SPAG 1 与DNAAF 1、DNAAF 2和PIH 1D 2的相互作用。这些鉴定的表达和定位 将通过液滴数字PCR（ddPCR）、westerns和超分辨率显微镜检查相互作用物。到 确定纤毛细胞分化过程中SPAG 1相互作用发生的顺序，时间进程co-IP研究 将被执行。将在分化hAEC中进行邻位连接试验（PLA），以询问是否 DNAAF 1、DNAAF 2或PIH 1D 2与SPAG 1直接相互作用。目标2：确定 动力蛋白臂的同种型和不同的亚基复合物需要SPAG 1进行组装。SPAG 1将是 使用CRISPR/Cas9技术在hAEC培养物中敲除SPAG 1，并确认SPAG 1的这种缺失将 包括测量纤毛搏动频率和测量动力蛋白臂的丰度。错误定位 将研究SPAG 1缺陷细胞中已知的DNAAF和不同动力蛋白臂同种型的动力蛋白链 使用免疫荧光技术。已知DNAAF相互作用和已知动力蛋白的解离 链复合物在动力蛋白臂组装过程中将使用共IP技术在SPAG 1- 缺陷细胞这些研究将使我们更好地理解SPAG 1在细胞质中的作用。 轴丝动力蛋白臂的预组装。这种知识的扩展将导致诊断的改进， 原发性纤毛运动障碍未来治疗药物的基本原理。

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.