i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal.
i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal; ICBAS - Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal.
Cell Rep. 2023 Nov 28;42(11):113337. doi: 10.1016/j.celrep.2023.113337. Epub 2023 Oct 25.
Intraflagellar transport (IFT) trains, built around IFT-A and IFT-B complexes, are carried by opposing motors to import and export ciliary cargo. While transported by kinesin-2 on anterograde IFT trains, the dynein-2 motor adopts an autoinhibitory conformation until it needs to be activated at the ciliary tip to power retrograde IFT. Growing evidence has linked the IFT-A complex to retrograde IFT; however, its roles in this process remain unknown. Here, we use CRISPR-Cas9-mediated genome editing to disable the dynein-2 autoinhibition mechanism in Caenorhabditis elegans and assess its impact on IFT with high-resolution live imaging and photobleaching analyses. Remarkably, this dynein-2 "hot-wiring" approach reignites retrograde motility inside IFT-A-deficient cilia without triggering tug-of-war events. In addition to providing functional evidence that multiple mechanisms maintain dynein-2 inhibited during anterograde IFT, our data establish key roles for IFT-A in mediating motor-train coupling during IFT turnaround, promoting retrograde IFT initiation, and modulating dynein-2 retrograde motility.
鞭毛内运输（IFT）列车，围绕着 IFT-A 和 IFT-B 复合物构建，由相反的马达携带，以导入和导出纤毛货物。当正向 IFT 列车由 kinesin-2 运输时，dynein-2 马达采用自动抑制构象，直到需要在纤毛尖端被激活以提供逆行 IFT 的动力。越来越多的证据将 IFT-A 复合物与逆行 IFT 联系起来；然而，其在这个过程中的作用仍然未知。在这里，我们使用 CRISPR-Cas9 介导的基因组编辑来使秀丽隐杆线虫中的 dynein-2 自动抑制机制失活，并使用高分辨率活细胞成像和光漂白分析来评估其对 IFT 的影响。值得注意的是，这种 dynein-2“热接线”方法重新点燃了 IFT-A 缺陷纤毛内的逆行运动，而不会引发拔河事件。除了提供功能证据表明多个机制在正向 IFT 期间维持 dynein-2 抑制外，我们的数据还确立了 IFT-A 在介导 IFT 转换过程中的马达列车耦合、促进逆行 IFT 起始和调节 dynein-2 逆行运动方面的关键作用。
