Establishment of the early cilia preassembly protein complex during motile ciliogenesis
Establishment of the early cilia preassembly protein complex during motile ciliogenesis
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DOI：
10.1073/pnas.1715915115
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发表时间：
2018-02-06
影响因子：
11.1
通讯作者：
Brody, Steven L.
中科院分区：
文献类型：
--
作者：
Horani, Amjad;Ustione, Alessandro;Brody, Steven L.
运动纤毛的特征在于动力蛋白运动单位，其在运输到纤毛之前在细胞质中预组装。动力蛋白预组装所需的蛋白质是通过发现导致纤毛马达缺失的人类突变而发现的，但对其表达、功能或相互作用知之甚少。通过监测原代气道上皮细胞和MCIDAS调节的诱导多能干细胞中的纤毛发生，我们发现了预组装蛋白表达的两个阶段。由HEATR 2、SPAG 1和DNAAF 2组成的早期阶段先于其他预组装蛋白，并且独立于MCIDAS调节。早期的预组装蛋白共定位在核周病灶，也包含动力蛋白臂蛋白。这些蛋白质也相互作用的基础上免疫沉淀和福斯特共振能量转移（FRET）的研究。HEAT结构域缺失和人类突变的FRET分析表明，HEATR 2与自身和SPAG 1在多个HEAT结构域相互作用，而DNAAF 2与SPAG 1相互作用。HEATR 2中的人类突变并不影响这种相互作用，但触发了含有早期预组装蛋白的p62/Sequestosome-1阳性聚集体的形成，这表明早期预组装复合物的降解是疾病的原因，并指向HEATR 2支架稳定性所需的关键区域。我们推测HEATR 2是运动纤毛中动力蛋白复合物组装起始的早期支架。
Motile cilia are characterized by dynein motor units, which preassemble in the cytoplasm before trafficking into the cilia. Proteins required for dynein preassembly were discovered by finding human mutations that result in absent ciliary motors, but little is known about their expression, function, or interactions. By monitoring ciliogenesis in primary airway epithelial cells and MCIDAS-regulated induced pluripotent stem cells, we uncovered two phases of expression of preassembly proteins. An early phase, composed of HEATR2, SPAG1, and DNAAF2, preceded other preassembly proteins and was independent of MCIDAS regulation. The early preassembly proteins colocalized within perinuclear foci that also contained dynein arm proteins. These proteins also interacted based on immunoprecipitation and Forster resonance energy transfer (FRET) studies. FRET analysis of HEAT domain deletions and human mutations showed that HEATR2 interacted with itself and SPAG1 at multiple HEAT domains, while DNAAF2 interacted with SPAG1. Human mutations in HEATR2 did not affect this interaction, but triggered the formation of p62/Sequestosome-1-positive aggregates containing the early preassembly proteins, suggesting that degradation of an early preassembly complex is responsible for disease and pointing to key regions required for HEATR2 scaffold stability. We speculate that HEATR2 is an early scaffold for the initiation of dynein complex assembly in motile cilia.