Dynein-2 intermediate chains play crucial but distinct roles in primary cilia formation and function.
Dynein-2 intermediate chains play crucial but distinct roles in primary cilia formation and function.
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DOI：
10.7554/elife.39655
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发表时间：
2018-10-16
期刊：
影响因子：
7.7
通讯作者：
Stephens DJ
中科院分区：
文献类型：
--
作者：
Vuolo L;Stevenson NL;Heesom KJ;Stephens DJ
动力蛋白2微管马达是鞭毛内转运的逆行马达。动力蛋白-2组分的突变引起骨骼纤毛病变，特别是Jeune综合征。动力蛋白-2含有两个不相同的中间链WDR 34和WDR 60的异二聚体。在这里，我们使用敲除细胞系来证明每个中间链在纤毛功能中具有不同的作用。使用定量蛋白质组学，我们表明，WDR 34 KO细胞可以组装动力蛋白-2马达复合物，结合IFT蛋白，但未能延长轴丝，表明复杂的功能停滞。相比之下，WDR 60 KO细胞确实延伸轴丝，但显示动力蛋白-2的组装和与IFT蛋白的结合减少。这两种蛋白质都需要维持一个功能性的过渡区和有效的双向鞭毛内运输。我们的研究结果表明，动力蛋白-2复合物内的亚基不对称性与动力蛋白-2中间链之间的功能不对称性相匹配。此外，这项工作表明，动力蛋白-2的功能丧失导致过渡区结构的缺陷，以及鞭毛内运输。人体内几乎所有的细胞都被称为初级纤毛的微小毛发状结构所覆盖。这些结构就像天线一样接收来自细胞外的信号，调节身体的生长和发育。细胞必须将新的蛋白质和其他分子运送到纤毛内的精确位置，以确保它们正常工作。每个纤毛都通过一个称为过渡区的选择性屏障与细胞的其他部分隔开，该屏障控制分子进出细胞其他部分的运动。动力蛋白-2是一种运动蛋白，可在纤毛内移动其他蛋白质和细胞物质。它包括两个亚基，称为WDR 34和WDR 60。产生这些亚基的基因在青少年和短肋多指综合征中发生突变，主要影响骨骼的形成。然而，对单个亚基在马达蛋白中所起的作用知之甚少。Vuolo等人使用了一种名为CRISPR-Cas9的基因编辑技术，从人类细胞中删除了编码动力蛋白-2亚基的一个或两个基因。实验表明，这两种亚基在纤毛中具有非常不同的作用。WDR 34是细胞构建纤毛所必需的，而WDR 60则不是。相反，WDR 60需要在已建立的纤毛内移动蛋白质和其他材料。出乎意料的是，实验表明动力蛋白-2也需要维持过渡区。这项工作为进一步研究动力蛋白2在纤毛结构的构建和维持中的作用奠定了基础。这可能最终有助于开发新的治疗方法，以减少Jeune综合征和其他由纤毛缺陷引起的疾病的症状。
The dynein-2 microtubule motor is the retrograde motor for intraflagellar transport. Mutations in dynein-2 components cause skeletal ciliopathies, notably Jeune syndrome. Dynein-2 contains a heterodimer of two non-identical intermediate chains, WDR34 and WDR60. Here, we use knockout cell lines to demonstrate that each intermediate chain has a distinct role in cilium function. Using quantitative proteomics, we show that WDR34 KO cells can assemble a dynein-2 motor complex that binds IFT proteins yet fails to extend an axoneme, indicating complex function is stalled. In contrast, WDR60 KO cells do extend axonemes but show reduced assembly of dynein-2 and binding to IFT proteins. Both proteins are required to maintain a functional transition zone and for efficient bidirectional intraflagellar transport. Our results indicate that the subunit asymmetry within the dynein-2 complex is matched with a functional asymmetry between the dynein-2 intermediate chains. Furthermore, this work reveals that loss of function of dynein-2 leads to defects in transition zone architecture, as well as intraflagellar transport. Almost all cells in the human body are covered in tiny hair-like structures known as primary cilia. These structures act as antennae to receive signals from outside the cell that regulate how the body grows and develops. The cell has to deliver new proteins and other molecules to precise locations within its cilia to ensure that they work properly. Each cilium is separated from the rest of the cell by a selective barrier known as the transition zone, which controls the movement of molecules to and from the rest of the cell. Dynein-2 is a motor protein that moves other proteins and cell materials within cilia. It includes two subunits known as WDR34 and WDR60. The genes that produce these subunits are mutated in Jeune and short rib polydactyly syndromes that primarily affect how the skeleton forms. However, little is known about the roles the individual subunits play within the motor protein. Vuolo et al. used a gene editing technique called CRISPR-Cas9 to remove one or both of the genes encoding the dynein-2 subunits from human cells. The experiments show that the two subunits have very different roles in cilia. WDR34 is required for cells to build a cilium whereas WDR60 is not. Instead, WDR60 is needed to move proteins and other materials within an established cilium. Unexpectedly, the experiments suggest that dynein-2 is also required to maintain the transition zone. This work provides the foundations for future studies on the role of dynein-2 in building and maintaining the structure of cilia. This could ultimately help to develop new treatments to reduce the symptoms of Jeune syndrome and other diseases caused by defects in cilia.