Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110;
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110.
Proc Natl Acad Sci U S A. 2018 Feb 6;115(6):E1221-E1228. doi: 10.1073/pnas.1715915115. Epub 2018 Jan 22.
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 Förster 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.
纤毛的运动特征是包含动力蛋白单元，这些单元在运输到纤毛之前会在细胞质中预先组装。通过发现导致纤毛动力蛋白缺失的人类突变，发现了用于动力蛋白预组装的蛋白质，但人们对它们的表达、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，我们揭示了预组装蛋白表达的两个阶段。一个早期阶段，由 HEATR2、SPAG1 和 DNAAF2 组成，先于其他预组装蛋白，并独立于 MCIDAS 调节。早期预组装蛋白在核周焦点内共定位，该焦点还包含动力蛋白臂蛋白。这些蛋白质还通过免疫沉淀和Förster 共振能量转移（FRET）研究相互作用。基于 FRET 分析 HEAT 结构域缺失和人类突变，表明 HEATR2 在多个 HEAT 结构域上与自身和 SPAG1 相互作用，而 DNAAF2 与 SPAG1 相互作用。HEATR2 中的人类突变不会影响这种相互作用，但会触发包含早期预组装蛋白的 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导致疾病的原因，并指出了 HEATR2 支架稳定性所需的关键区域。我们推测 HEATR2 是纤毛中动力蛋白复合物组装的早期支架。
