Single-particle imaging reveals intraflagellar transport-independent transport and accumulation of EB1 in Chlamydomonas flagella.
Single-particle imaging reveals intraflagellar transport-independent transport and accumulation of EB1 in Chlamydomonas flagella.
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DOI：
10.1091/mbc.e15-08-0608
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发表时间：
2016-01-15
影响因子：
3.3
通讯作者：
Lechtreck KF
中科院分区：
文献类型：
--
作者：
Harris JA;Liu Y;Yang P;Kner P;Lechtreck KF
微管末端跟踪蛋白EB1进入鞭毛并在鞭毛末端独立地积累。EB1在尖端停留数秒，表明EB1结合位点稳定。模拟表明，扩散捕获是在纤毛中积累蛋白质的另一种机制。微管（MT）正端跟踪蛋白EB1存在于纤毛和鞭毛的尖端；末端结合蛋白1 （EB1）在鞭毛缩短期间和鞭毛中主要的蛋白质运输系统鞭毛内运输（IFT）缺失的情况下留在鞭毛尖端。为了研究EB1是如何在鞭毛尖端积累的，我们在莱茵衣藻中使用了荧光蛋白标记的EB1 （EB1- fp）的体内成像。光漂白后，鞭毛尖端的EB1信号在几分钟内恢复，表明与未漂白的EB1从细胞体进入鞭毛进行了交换。EB1的运动不依赖于IFT序列，EB1- fp的恢复不需要IFT通路。单粒子成像显示EB1-FP沿鞭毛轴高度移动，在鞭毛尖端附近明显降低迁移率。单个EB1- fp颗粒在鞭毛尖端附近停留了几秒钟，表明存在稳定的EB1结合位点。在模拟中，EB1流动的两个不同阶段足以解释其在尖端的积累。我们认为像EB1这样均匀分布在细胞质中的蛋白质通过扩散和捕获在局部积累；相比之下，IFT可能需要将蛋白质输送到纤毛中以对抗细胞浓度梯度。
The microtubule plus-end tracking protein EB1 moves into flagella and accumulates at the tip independently of intraflagellar transport. EB1 dwell for seconds at the tip, indicating stable EB1-binding sites. Simulations show that diffusion to capture is an alternative mechanism to accumulate proteins in cilia. The microtubule (MT) plus-end tracking protein EB1 is present at the tips of cilia and flagella; end-binding protein 1 (EB1) remains at the tip during flagellar shortening and in the absence of intraflagellar transport (IFT), the predominant protein transport system in flagella. To investigate how EB1 accumulates at the flagellar tip, we used in vivo imaging of fluorescent protein–tagged EB1 (EB1-FP) in Chlamydomonas reinhardtii. After photobleaching, the EB1 signal at the flagellar tip recovered within minutes, indicating an exchange with unbleached EB1 entering the flagella from the cell body. EB1 moved independent of IFT trains, and EB1-FP recovery did not require the IFT pathway. Single-particle imaging showed that EB1-FP is highly mobile along the flagellar shaft and displays a markedly reduced mobility near the flagellar tip. Individual EB1-FP particles dwelled for several seconds near the flagellar tip, suggesting the presence of stable EB1 binding sites. In simulations, the two distinct phases of EB1 mobility are sufficient to explain its accumulation at the tip. We propose that proteins uniformly distributed throughout the cytoplasm like EB1 accumulate locally by diffusion and capture; IFT, in contrast, might be required to transport proteins against cellular concentration gradients into or out of cilia.