LaserLaB and Department of Physics and Astronomy, Vrije Universiteit Amsterdam, De Boelelaan 1081, 1081 HV, Amsterdam, the Netherlands.
LaserLaB and Department of Physics and Astronomy, Vrije Universiteit Amsterdam, De Boelelaan 1081, 1081 HV, Amsterdam, the Netherlands.
Cell Rep. 2018 Nov 13;25(7):1701-1707.e2. doi: 10.1016/j.celrep.2018.10.050.
Cilia are microtubule-based sensing hubs that rely on intraflagellar transport (IFT) for their development, maintenance, and function. Kinesin-2 motors transport IFT trains, consisting of IFT proteins and cargo, from ciliary base to tip. There, trains turn around and are transported back by IFT dynein. The mechanism of tip turnaround has remained elusive. Here, we employ single-molecule fluorescence microscopy of IFT components in the tips of phasmid cilia of living C. elegans. Analysis of the trajectories reveals that while motor proteins and IFT-A particle component CHE-11 mostly turn around immediately, the IFT-B particle component OSM-6 pauses for several seconds. Our data indicate that IFT trains disassemble into at least IFT-A, IFT-B, IFT-dynein, and OSM-3 complexes at the tip, where OSM-6 is temporarily retained or undergoes modification, prior to train reassembly and retrograde transport. The single-molecule approach used here is a valuable tool to study how directional switches occur in microtubule-based transport processes.
纤毛是基于微管的感应中心，其发育、维持和功能依赖于鞭毛内运输（IFT）。驱动蛋白-2 马达将包含 IFT 蛋白和货物的 IFT 列车从纤毛基部运送到顶端。在那里，列车掉头并由 IFT 动力蛋白反向运输。然而，目前仍然难以确定纤毛顶端的列车掉头机制。在这里，我们利用活体秀丽隐杆线虫（C. elegans）的翼状线虫纤毛顶端的 IFT 成分的单分子荧光显微镜技术。对轨迹的分析表明，虽然马达蛋白和 IFT-A 颗粒成分 CHE-11 主要立即掉头，但 IFT-B 颗粒成分 OSM-6 会暂停几秒钟。我们的数据表明，IFT 列车在顶端至少会分解为 IFT-A、IFT-B、IFT-动力蛋白和 OSM-3 复合物，在此期间，OSM-6 会被暂时保留或发生修饰，然后再重新组装和逆行运输。这里使用的单分子方法是研究微管基运输过程中如何发生定向转换的有价值的工具。
