Dunn Sarah, Morrison Ewan E, Liverpool Tanniemola B, Molina-París Carmen, Cross Robert A, Alonso Maria C, Peckham Michelle
Institute for Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK.
J Cell Sci. 2008 Apr 1;121(Pt 7):1085-95. doi: 10.1242/jcs.026492. Epub 2008 Mar 11.
Kinesin-1 is a molecular transporter that trafficks along microtubules. There is some evidence that kinesin-1 targets specific cellular sites, but it is unclear how this spatial regulation is achieved. To investigate this process, we used a combination of in vivo imaging of kinesin heavy-chain Kif5c (an isoform of kinesin-1) fused to GFP, in vitro analyses and mathematical modelling. GFP-Kif5c fluorescent puncta localised to a subset of microtubules in live cells. These puncta moved at speeds of up to 1 microm second(-1) and exchanged into cortically labelled clusters at microtubule ends. This behaviour depended on the presence of a functional motor domain, because a rigor-mutant GFP-Kif5c bound to microtubules but did not move along them. Further analysis indicated that the microtubule subset decorated by GFP-Kif5c was highly stable and primarily composed of detyrosinated tubulin. In vitro motility assays showed that the motor domain of Kif5c moved detyrosinated microtubules at significantly lower velocities than tyrosinated (unmodified) microtubules. Mathematical modelling predicted that a small increase in detyrosination would bias kinesin-1 occupancy towards detyrosinated microtubules. These data suggest that kinesin-1 preferentially binds to and trafficks on detyrosinated microtubules in vivo, providing a potential basis for the spatial targeting of kinesin-1-based cargo transport.
驱动蛋白-1是一种沿微管运输的分子转运体。有证据表明驱动蛋白-1靶向特定的细胞位点，但尚不清楚这种空间调控是如何实现的。为了研究这一过程，我们结合了对与绿色荧光蛋白（GFP）融合的驱动蛋白重链Kif5c（驱动蛋白-1的一种同工型）进行体内成像、体外分析和数学建模。GFP-Kif5c荧光斑点定位于活细胞中微管的一个子集。这些斑点以高达1微米每秒（-1）的速度移动，并在微管末端交换到皮质标记的簇中。这种行为取决于功能性运动结构域的存在，因为一种僵直突变体GFP-Kif5c与微管结合但不沿微管移动。进一步分析表明，由GFP-Kif5c修饰的微管子集高度稳定，主要由去酪氨酸化微管蛋白组成。体外运动分析表明，Kif5c的运动结构域移动去酪氨酸化微管的速度明显低于酪氨酸化（未修饰）微管。数学建模预测，去酪氨酸化的小幅增加会使驱动蛋白-1对去酪氨酸化微管的占有率产生偏差。这些数据表明，驱动蛋白-1在体内优先结合并在去酪氨酸化微管上运输，为基于驱动蛋白-1的货物运输的空间靶向提供了潜在基础。
