初级纤毛是通过基于微管的轴突的延伸产生的。中心体蛋白104(CEP104)定位于伸长的轴突的尖端,CEP104突变与纤毛病有关,Joubert综合征.因此,CEP104与纤毛发生有关。然而,CEP104调节纤毛生成的机制仍然难以捉摸。我们在此报告,CEP104对纤毛伸长至关重要,但对启动纤毛生成至关重要。我们还证明了CEP104的肿瘤过表达基因(TOG)结构域具有微管聚合活性,并且该活性对于CEP104的纤毛延伸活性至关重要。击倒/挽救实验表明,N末端果冻卷(JR)折叠部分有助于CEP104的纤毛延伸活性,但该活性既不需要锌指区域也不需要SXIP基序。CEP104与中心蛋白结合,CP110,通过锌指区域和微管加端结合蛋白,EB1,通过SXIP图案,表明CP110和EB1的结合对于CEP104的纤毛延伸活性是不必要的。此外,CEP104耗尽不影响从母体中心粒中去除CP110,这表明CEP104在移除CP110后起作用。最后,我们还表明,在Hedgehog信号激活后,平滑的纤毛进入和GPR161的输出需要CEP104,并且TOG域在该活性中起关键作用。我们的结果定义了CEP104单个结构域在纤毛伸长和Hedgehog信号传导中的作用,并应增强我们对CEP104突变相关纤毛病潜在机制的理解。
Primary cilia are generated through the extension of the microtubule-based axoneme. Centrosomal protein 104 (
CEP104) localizes to the tip of the elongating axoneme, and
CEP104 mutations are linked to a ciliopathy, Joubert syndrome. Thus,
CEP104 has been implicated in ciliogenesis. However, the mechanism by which
CEP104 regulates ciliogenesis remains elusive. We report here that CEP104 is critical for cilium elongation but not for initiating ciliogenesis. We also demonstrated that the tumor-overexpressed gene (TOG) domain of CEP104 exhibits microtubule-polymerizing activity and that this activity is essential for the cilium-elongating activity of
CEP104. Knockdown/rescue experiments showed that the N-terminal jelly-roll (JR) fold partially contributes to cilium-elongating activity of CEP104, but neither the zinc-finger region nor the SXIP motif is required for this activity. CEP104 binds to a centriole-capping protein, CP110, through the zinc-finger region and to a microtubule plus-end-binding protein, EB1, through the SXIP motif, indicating that the binding of CP110 and EB1 is dispensable for the cilium-elongating activity of CEP104. Moreover, CEP104 depletion does not affect CP110 removal from the mother centriole, which suggests that
CEP104 functions after the removal of CP110. Last, we also showed that CEP104 is required for the ciliary entry of Smoothened and export of GPR161 upon Hedgehog signal activation and that the TOG domain plays a critical role in this activity. Our results define the roles of the individual domains of CEP104 in its functions in cilium elongation and Hedgehog signaling and should enhance our understanding of the mechanism underlying CEP104 mutation-associated ciliopathies.