Active transport and diffusion barriers restrict Joubert Syndrome-associated ARL13B/ARL-13 to an Inv-like ciliary membrane subdomain.
Active transport and diffusion barriers restrict Joubert Syndrome-associated ARL13B/ARL-13 to an Inv-like ciliary membrane subdomain.
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主动运输和扩散屏障将 Joubert 综合征相关的 ARL13B/ARL-13 限制在类似 Inv 的睫状膜子域。
DOI：
10.1371/journal.pgen.1003977
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发表时间：
2013
期刊：
影响因子：
4.5
通讯作者：
Blacque OE
中科院分区：
文献类型：
--
作者：
Cevik S;Sanders AA;Van Wijk E;Boldt K;Clarke L;van Reeuwijk J;Hori Y;Horn N;Hetterschijt L;Wdowicz A;Mullins A;Kida K;Kaplan OI;van Beersum SE;Man Wu K;Letteboer SJ;Mans DA;Katada T;Kontani K;Ueffing M;Roepman R;Kremer H;Blacque OE
纤毛是基于微管的细胞附属物，具有运动性、化学/机械/光感觉和发育信号传导功能。纤毛由不同的结构和功能亚区域组成，包括基体、过渡区 (TZ) 和反相 (Inv) 区室，该细胞器的缺陷与一系列遗传性疾病相关，包括巴代-比德尔综合征 (BBS)、梅克尔-格鲁伯综合征 (MKS)、朱伯特综合征 (JS) 和肾痨 (NPHP)。尽管在理解纤毛运输途径（例如鞭毛内运输（IFT））方面取得了重大进展，但蛋白质如何运输到纤毛下膜仍然知之甚少。使用秀丽隐杆线虫和哺乳动物细胞，我们研究了 JS 相关 ARL13B/ARL-13 区室化的运输机制，我们之前发现这种机制仅限于近端睫状膜。我们现在证明，在许多秀丽隐杆线虫纤毛神经元和哺乳动物纤毛亚型的子集中，ARL13B/ARL-13 定位到类似 Inv 的纤毛下膜区室（不包括 TZ）的进化保守性。线虫 ARL-13 的区室化需要 C 端 RVVP 基序和膜锚定，以分别防止远端纤毛和核靶向。超过 20 个突变体的定量成像揭示了 IFT 和纤毛病模块在定义 ARL-13 区室中的不同贡献； IFT-A/B、IFT-动力蛋白和 BBS 基因可防止 ARL-13 在纤毛周膜上积聚，而 MKS/NPHP 模块还可抑制 ARL-13 与 TZ 膜的结合。此外，体内 FRAP 分析揭示了 IFT 和 MKS/NPHP 基因在调节 TZ 屏障对 ARL-13 扩散和纤毛内 ARL-13 扩散方面的不同作用。最后，线虫 ARL-13 对人类 ARL13B 进行类 IFT 运动和定量蛋白质复合物分析，确定与 IFT-B 复合物的功能关联，映射到 IFT46 和 IFT74 相互作用。总之，这些发现揭示了在定义 ARL-13 睫状膜区室时对序列基序、IFT 和纤毛病模块的独特要求。我们得出的结论是，MKS/NPHP 模块构成了 ARL-13 扩散的 TZ 屏障，而 IFT 基因主要通过主动转运机制促进 ARL-13 纤毛进入和/或保留。从大多数细胞表面突出的是一根毛发状的延伸部分，称为初级纤毛。该细胞器充当细胞天线，接收物理和化学信号，例如光、气味和协调细胞生长、分化和迁移的分子。纤毛缺陷是一系列称为纤毛病的疾病的基础，其特征是囊性肾、失明和骨骼异常等广泛的症状，这凸显了它们的重要性。一个关键问题是纤毛蛋白如何靶向并保留在纤毛内。最了解的系统是鞭毛内运输（IFT），被认为在纤毛基部和尖端之间运送蛋白质。此外，纤毛病蛋白模块在纤毛基部过渡区（TZ）组织蛋白质扩散屏障。尽管取得了重大进展，但人们对蛋白质如何靶向纤毛，特别是纤毛膜子域仍然知之甚少。在这里，我们研究了 Joubert 综合征相关的 ARL13B/ARL-13 如何在睫状膜下区分开。使用秀丽隐杆线虫线虫和哺乳动物细胞实验系统，我们发现了序列基序、IFT 和纤毛病模块在调节 ARL-13 纤毛限制、活动性和区室长度方面的差异要求。此外，我们还提供了有关 IFT 和纤毛病相关蛋白复合物和模块如何影响纤毛膜蛋白转运、跨 TZ 扩散、纤毛膜完整性和纤毛下蛋白组成的重要见解。
Cilia are microtubule-based cell appendages, serving motility, chemo-/mechano-/photo- sensation, and developmental signaling functions. Cilia are comprised of distinct structural and functional subregions including the basal body, transition zone (TZ) and inversin (Inv) compartments, and defects in this organelle are associated with an expanding spectrum of inherited disorders including Bardet-Biedl syndrome (BBS), Meckel-Gruber Syndrome (MKS), Joubert Syndrome (JS) and Nephronophthisis (NPHP). Despite major advances in understanding ciliary trafficking pathways such as intraflagellar transport (IFT), how proteins are transported to subciliary membranes remains poorly understood. Using Caenorhabditis elegans and mammalian cells, we investigated the transport mechanisms underlying compartmentalization of JS-associated ARL13B/ARL-13, which we previously found is restricted at proximal ciliary membranes. We now show evolutionary conservation of ARL13B/ARL-13 localisation to an Inv-like subciliary membrane compartment, excluding the TZ, in many C. elegans ciliated neurons and in a subset of mammalian ciliary subtypes. Compartmentalisation of C. elegans ARL-13 requires a C-terminal RVVP motif and membrane anchoring to prevent distal cilium and nuclear targeting, respectively. Quantitative imaging in more than 20 mutants revealed differential contributions for IFT and ciliopathy modules in defining the ARL-13 compartment; IFT-A/B, IFT-dynein and BBS genes prevent ARL-13 accumulation at periciliary membranes, whereas MKS/NPHP modules additionally inhibit ARL-13 association with TZ membranes. Furthermore, in vivo FRAP analyses revealed distinct roles for IFT and MKS/NPHP genes in regulating a TZ barrier to ARL-13 diffusion, and intraciliary ARL-13 diffusion. Finally, C. elegans ARL-13 undergoes IFT-like motility and quantitative protein complex analysis of human ARL13B identified functional associations with IFT-B complexes, mapped to IFT46 and IFT74 interactions. Together, these findings reveal distinct requirements for sequence motifs, IFT and ciliopathy modules in defining an ARL-13 subciliary membrane compartment. We conclude that MKS/NPHP modules comprise a TZ barrier to ARL-13 diffusion, whereas IFT genes predominantly facilitate ARL-13 ciliary entry and/or retention via active transport mechanisms. Protruding from most cells surfaces is a hair-like extension called the primary cilium. This organelle functions as a cellular antenna, receiving physical and chemical signals such as light, odorants, and molecules that coordinate cell growth, differentiation and migration. Underscoring their importance, cilium defects underlie an expanding spectrum of diseases termed ciliopathies, characterised by wide-ranging symptoms such as cystic kidneys, blindness and bone abnormalities. A key question is how ciliary proteins are targeted to and retained within cilia. The best understood system is intraflagellar transport (IFT), thought to ferry proteins between the ciliary base and tip. Also, ciliopathy protein modules organise protein diffusion barriers at the ciliary base transition zone (TZ). Despite major advances, it remains poorly understood how proteins are targeted to cilia, and ciliary membrane subdomains in particular. Here, we investigated how Joubert syndrome-associated ARL13B/ARL-13 is compartmentalized at subciliary membranes. Using C. elegans nematodes and mammalian cell experimental systems, we uncovered differential requirements for sequence motifs, IFT and ciliopathy modules in regulating ARL-13 ciliary restriction, mobility and compartment length. Also, we provide essential insight into how IFT and ciliopathy-associated protein complexes and modules influence ciliary membrane protein transport, diffusion across the TZ, the integrity of the ciliary membrane, and subciliary protein composition.
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