The role of Dzip1L in ciliogenesis and Hedgehog signaling

 描述（由申请人提供）：初级纤毛是一种从细胞表面突出的基于微管的孤立细胞器，存在于大多数脊椎动物细胞中。初级纤毛 起源于中心体的母中心粒，并通过鞭毛内运输（IFT）延长和维持。先前的透射电子显微镜（TEM）研究定义了两种不同的纤毛发生途径的初级纤毛。在上皮细胞中，母中心粒似乎直接停靠在顶端质膜上;从那里，轴丝向细胞外环境生长。这被称为细胞外途径。相反，在成纤维细胞和平滑肌细胞中，纤毛似乎首先在细胞体内生长，在母亲中心粒停靠高尔基体衍生的初级纤毛囊泡。轴丝的生长发生在这个囊泡内。这种囊泡与质膜的融合最终允许纤毛进入细胞外环境。这被称为细胞内途径。到目前为止，还不知道这两种途径是如何在分子水平上差异调节。 纤毛功能障碍通过影响Hh信号传导的两种主要调节因子Gli 2和Gli 3转录因子的活性来破坏Hedgehog（Hh）信号传导。迄今为止，大多数已知的纤毛基因突变导致Gli 2激活失败和Gli 3阻遏物水平降低，但其潜在的分子机制仍知之甚少。 我们最近突变了小鼠Dzip 1 L基因，该基因编码中心粒蛋白。Dzip 1 L突变小鼠表现出多指（趾）畸形和增大的前脑和中脑。Dzip 1 L的缺失导致Gli 3阻遏物活性降低，但不影响Gli 2活化。令人惊讶的是，虽然纤毛在Dzip 1 L突变体大脑的神经上皮细胞中是正常的，但在突变体肢芽间充质细胞和胚胎成纤维细胞中纤毛明显缺乏。因此，Dzip 1 L是第一个已知的纤毛相关蛋白，它特异性地调节成纤维细胞中的纤毛发生，但不影响上皮细胞，并且只影响Gli 3的加工，但不影响Gli 2的激活。我们的目标是了解Dzip 1 L突变如何影响Gli 3处理，但不Gli 2激活，以及Dzip 1 L如何使用遗传，分子和细胞生物学方法差异调节两个纤毛发生途径。

 DESCRIPTION (provided by applicant): The primary cilium is a solitary microtubule-based organelle that protrudes from the cell surface and is found on most vertebrate cells. Primary cilia originate in the mother centriole of the centrosome and are elongated and maintained by intraflagellar transport (IFT). Previous transmission electron microscopy (TEM) studies defined two different ciliogenesis pathways for primary cilia. In epithelial cells, the mother centriole appears to dock directly at the apical plasma membrane; from there, the axoneme grows out toward the extracellular environment. This is called the extracellular pathway. In contrast, in fibroblasts and smooth muscle cells, the cilium appears to first grow within the cell body, upon docking of the mother centriole to a Golgi-derived primary ciliary vesicle. The growth of the axoneme takes place within this vesicle. Fusion of this vesicle with the plasma membrane finally allows the cilium to access the extracellular environment. This is called the intracellular pathway. Thus far, nothing is known about how these two pathways are differentially regulated at the molecular levels. Ciliary dysfunction disrupts Hedgehog (Hh) signaling by affecting the activity of the Gli2 and Gli3 transcription factors, two primary regulators of Hh signaling. Most known ciliary gene mutations identified thus far result in failed Gli2 activation and decreased Gli3 repressor levels, but the underlying molecular mechanisms remain poorly understood. We recently mutated the mouse Dzip1L gene, which encodes a centriolar protein. Dzip1L mutant mice exhibit polydactyly and an enlarged forebrain and midbrain. Loss of Dzip1L results in reduced Gli3 repressor activity but does not affect Gli2 activation. Surprisingly, although cilia ae normal in neuroepithelial cells of the Dzip1L mutant brain, there is a pronounced paucity of cilia in mutant limb bud mesenchymal cells and embryonic fibroblasts. Thus, Dzip1L is the first known cilia-related protein that specifically regulates ciliogenesis in fibroblasts, but not epitheial cells, and affects only Gli3 processing but not Gli2 activation. Our objective is to understand how Dzip1L mutation affects Gli3 processing but not Gli2 activation and how Dzip1L differentially regulates the two ciliogenesis pathways using genetic, molecular, and cell biological approaches.