Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan.
J Cell Biol. 2011 Jul 25;194(2):245-55. doi: 10.1083/jcb.201104034. Epub 2011 Jul 18.
Polarized transport in neurons is fundamental for the formation of neuronal circuitry. A motor domain-containing truncated KIF5 (a kinesin-1) recognizes axonal microtubules, which are enriched in EB1 binding sites, and selectively accumulates at the tips of axons. However, it remains unknown what cue KIF5 recognizes to result in this selective accumulation. We found that axonal microtubules were preferentially stained by the anti-GTP-tubulin antibody hMB11. Super-resolution microscopy combined with EM immunocytochemistry revealed that hMB11 was localized at KIF5 attachment sites. In addition, EB1, which binds preferentially to guanylyl-methylene-diphosphate (GMPCPP) microtubules in vitro, recognized hMB11 binding sites on axonal microtubules. Further, expression of hMB11 antibody in neurons disrupted the selective accumulation of truncated KIF5 in the axon tips. In vitro studies revealed approximately threefold stronger binding of KIF5 motor head to GMPCPP microtubules than to GDP microtubules. Collectively, these data suggest that the abundance of GTP-tubulin in axonal microtubules may underlie selective KIF5 localization and polarized axonal vesicular transport.
神经元中的极化运输对于神经元回路的形成至关重要。一种含有运动结构域的截断型 KIF5（驱动蛋白-1）识别富含 EB1 结合位点的轴突微管，并选择性地积累在轴突的末端。然而，目前尚不清楚 KIF5 识别什么线索导致这种选择性积累。我们发现抗 GTP-微管蛋白抗体 hMB11 优先染色轴突微管。超分辨率显微镜结合 EM 免疫细胞化学显示 hMB11 定位于 KIF5 附着部位。此外，体外优先结合鸟苷酰亚甲基二磷酸（GMPCPP）微管的 EB1 识别轴突微管上的 hMB11 结合位点。进一步，神经元中 hMB11 抗体的表达破坏了截断型 KIF5 在轴突末端的选择性积累。体外研究表明，KIF5 马达头部与 GMPCPP 微管的结合强度约是 GDP 微管的三倍。综上所述，这些数据表明轴突微管中 GTP-微管蛋白的丰度可能是 KIF5 选择性定位和极化轴突囊泡运输的基础。
