The glial actin cytoskeleton regulates neuronal ciliogenesis.
The glial actin cytoskeleton regulates neuronal ciliogenesis.
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神经胶质肌动蛋白细胞骨架调节神经元纤毛发生。
DOI：
10.1038/cr.2016.131
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发表时间：
2016
期刊：
影响因子：
44.1
通讯作者：
Ou Guangshuo
中科院分区：
文献类型：
--
作者：
Zhu Hao;Chen Lianwan;Yang Yihong;Zhu Zhiwen;Zhang Xianliang;Li Wei;Miao Long;Zhang Yan;Ou Guangshuo
纤毛是以微管(MT)为基础的细胞器，对细胞运动、感觉和信号转导是必不可少的[1]。纤毛缺陷与多种人类疾病有关[1]。虽然棒状纤毛形态和以9对偶MTS为特征的轴丝结构早在几十年前就已被描述[2，3]，但纤毛形态和轴丝结构的分子调控仍然不清楚。纤毛的形成和维持需要双向鞭毛内运输(IFT)，这种运输由基于MT的Kinesin-2和IFT-dynein马达蛋白提供动力[1，4]。尽管在识别纤毛成分和描述IFT的调控事件方面取得了重要进展[1，4]，但其他细胞骨架元素(如肌动蛋白)如何促进纤毛组装以及环境线索如何指导后生动物的纤毛发生仍然令人费解。新出现的证据表明，肌动球蛋白有助于将IFT材料募集到衣藻的基底体[5]。相反，在哺乳动物细胞培养中，基于肌动蛋白的膜转运被认为是通过从基底上去除纤毛前体来抑制纤毛的形成[6，7]，这提出了不同细胞类型的纤毛发生是否依赖于不同的肌动蛋白贡献的问题。重要的是，人们对肌动蛋白在动物纤毛发生中的作用知之甚少。线虫的化学感受器，即双节虫和节肢动物，由感觉神经元的纤毛树突和两个被称为套细胞和鞘细胞的成鞘神经胶质细胞组成，这两个细胞在感觉纤毛周围形成一个圆柱形通道(图1A-1B和补充资料，图S1a)[3]。为了确定肌动蛋白细胞骨架在线虫神经元感觉纤毛形成中的潜在功能，我们测定了含有肌动蛋白调节器缺陷的线虫突变体的染料填充(Dyf)表型。以前的研究已经证实，感觉神经元不能通过化学感觉器官的开口吸收染料DiI(Dyf缺陷)与纤毛结构或感觉间隔的异常相关[3]。通过这一分析，我们发现肌动蛋白成核促进因子Wiskott-Aldrich综合征蛋白(WASP)同系物WSP-1A参与线虫纤毛的形成(补充信息，图S1B)。虽然先前的研究表明WSP-1a(Gm324)突变逆转了修补相关基因daf-6突变体中感觉通道的扩大，但本研究没有在WSP-1a(Gm324)单个突变体中检测到Dyf缺陷，表明WSP-1a(Gm324)不是线虫纤毛发生所必需的[8]。然而，纤毛结构和IFT尚未被仔细研究。我们证实WSP-1a(Gm324)突变体的两栖感觉神经元确实摄取了DII。然而，55%的相核神经元不能吸收DiI(n=390；补充信息，图S1B)。两端和两相之间的差异可能是由于不完全穿透WSP-1a(Gm324)突变体的Dyf缺陷所致，如果八个两端神经元中没有一个或两个相体中没有一个充满DiI，则通常会对Dyf缺陷进行评分。为了更好地评估WSP-1a突变体的纤毛结构和IFT，我们从基因上将这些动物引入OSM-6/IFT52：：mCherry来标记全长纤毛。缺乏WASP功能的动物要么失去了纤毛远端节段，要么形成了弯曲的纤毛，其余纤毛节段的肌动记录仪偶尔才能检测到IFT(图1C-1E)。接下来，我们对…进行了连续切片的透射电子显微镜和聚焦离子束扫描电子显微镜
Cilia are remarkable microtubule (MT)-based organelles that are essential for cell motility, sensory perception and signal transduction [1]. Ciliary defects have been implicated in various human diseases [1]. Although the rod-shaped cilium morphology and the axonemal structure characterized by nine-doublet MTs were described decades ago [2, 3], the molecular regulations of cilium morphology and axonemal structure are still poorly defined. The formation and maintenance of cilia require bidirectional intraflagellar transport (IFT) that is powered by MT-based kinesin-2 and IFT-dynein motor proteins [1, 4]. Despite important progresses in identifying ciliary components and delineating the regulatory events of IFT [1, 4], it remains elusive how other cytoskeletal elements such as actin contribute to cilium assembly and how environmental cues direct ciliogenesis in metazoans. Emerging evidence shows that actomyosin facilitates the recruitment of IFT materials to basal bodies in Chlamydomonas [5]. In contrast, actin-based membrane trafficking was postulated to inhibit cilium formation by removing ciliary precursors from basal bodies in mammalian cell cultures [6, 7], raising the question of whether ciliogenesis in different cell types may rely on distinct actin contributions. Importantly, little is known about the role of actin in ciliogenesis in animals. The C. elegans chemosensory organs, the amphids and phasmids, comprise ciliated dendrites of sensory neurons plus two ensheathing glial cells called socket and sheath cells, which form a cylindrical channel surrounding the sensory cilia (Figure 1A-1B and Supplementary information, Figure S1A)[3]. To identify the potential functions of the actin cytoskeleton in the formation of sensory cilia in C. elegans neurons, we determined the dye-filling (Dyf) phenotype of C. elegans mutants with defective actin regulators. Previous studies have established that the inability of sensory neurons to take up the dye DiI (Dyf defect) through the openings of chemosensory organs correlates with abnormalities in the ciliary structure or the sensory compartments [3]. Through this analysis, we uncovered that an actin nucleation-promoting factor Wiskott-Aldrich syndrome protein (WASP) homologue, WSP-1A, is involved in cilium formation in C. elegans (Supplementary information, Figure S1B). While a previous study showed that the wsp-1a (gm324) mutation reversed the enlargement of sensory channel in a Patched-related gene daf-6 mutant, this study did not detect the Dyf defect in the wsp-1a (gm324) single mutant and suggested that WSP-1A is not required for ciliogenesis in C. elegans [8]. However, cilium structure and IFT have not been examined closely. We confirmed that the amphid sensory neurons of wsp-1a (gm324) mutants indeed took up DiI. However, 55% of the phasmid neurons failed to take up DiI (n= 390; Supplementary information, Figure S1B). The discrepancy between the amphids and phasmids likely resulted from incomplete penetration of the Dyf defect of wsp-1a (gm324) mutants, and the Dyf defect is usually scored if none of the eight amphid neurons or none of the two phasmid neurons are filled with DiI. To better assess the cilium structure and IFT in wsp-1a mutants, we genetically introduced these animals with OSM-6/IFT52:: mCherry to label the fulllength cilium. The animals lacking WASP function either lost their ciliary distal segments or formed curved cilia, and IFT was only occasionally detected by kymographs in the remaining ciliary segments (Figure 1C-1E). Next, we performed serial-section transmission electron microscopy (ss-TEM) and focused ion beam scanning electron microscopy (FIB-SEM) of the …