Constable Sandii, Ott Carolyn M, Lemire Andrew L, White Kevin, Xun Yu, Lim Amin, Lippincott-Schwartz Jennifer, Mukhopadhyay Saikat
Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390.
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147.
Proc Natl Acad Sci U S A. 2024 Dec 24;121(52):e2408083121. doi: 10.1073/pnas.2408083121. Epub 2024 Dec 20.
Brain neurons utilize the primary cilium as a privileged compartment to detect and respond to extracellular ligands such as Sonic hedgehog (SHH). However, cilia in cerebellar granule cell (GC) neurons disassemble during differentiation through ultrastructurally unique intermediates, a process we refer to as cilia deconstruction. In addition, mature neurons do not reciliate despite having docked centrioles. Here, we identify molecular changes that accompany cilia deconstruction and centriole docking in GC neurons. We used single cell transcriptomic and immunocytological analyses to compare the transcript levels and subcellular localization of proteins between progenitor, differentiating, and mature GCs. Differentiating GCs lacked transcripts for key activators of premitotic cilia resorption, indicating that cilia disassembly in differentiating cells is distinct from premitotic cilia resorption. Instead, during differentiation, transcripts of many genes required for cilia maintenance-specifically those encoding components of intraflagellar transport, pericentrosomal material, and centriolar satellites-decreased. The abundance of several corresponding proteins in and around cilia and centrosomes also decreased. These changes coincided with downregulation of SHH signaling prior to differentiation, even in a mutant with excessive SHH activation. Finally, mother centrioles in maturing granule neurons recruited the cap complex protein, CEP97. These data suggest that a global, developmentally programmed decrease in cilium maintenance in differentiating GCs mediates cilia deconstruction, while capping of docked mother centrioles prevents cilia regrowth and dysregulated SHH signaling. Our study provides mechanistic insights expanding our understanding of permanent cilia loss in multiple tissue-specific contexts.
脑神经元利用初级纤毛作为一个特殊的区室来检测并响应细胞外配体,如音猬因子(SHH)。然而,小脑颗粒细胞(GC)神经元中的纤毛在分化过程中会通过超微结构独特的中间体进行拆解,我们将这一过程称为纤毛解构。此外,成熟神经元尽管有停靠的中心粒,但不会重新长出纤毛。在这里,我们确定了GC神经元中纤毛解构和中心粒停靠过程中伴随的分子变化。我们使用单细胞转录组学和免疫细胞分析来比较祖细胞、分化中的细胞和成熟GC之间的转录水平和蛋白质的亚细胞定位。分化中的GC缺乏有丝分裂前纤毛吸收关键激活因子的转录本,这表明分化细胞中的纤毛拆解不同于有丝分裂前的纤毛吸收。相反,在分化过程中,纤毛维持所需的许多基因的转录本——特别是那些编码鞭毛内运输、中心体周围物质和中心粒卫星成分的基因——减少了。纤毛和中心体内部及周围几种相应蛋白质的丰度也降低了。这些变化与分化前SHH信号通路的下调同时发生,即使在SHH激活过度的突变体中也是如此。最后,成熟颗粒神经元中的母中心粒招募了帽复合体蛋白CEP97。这些数据表明,分化中的GC中纤毛维持的全局性、发育程序性减少介导了纤毛解构,而停靠的母中心粒的加帽则阻止了纤毛再生和SHH信号通路失调。我们的研究提供了机制上的见解,扩展了我们对多种组织特异性情况下永久性纤毛丧失的理解。