Stepanova Tatiana, Slemmer Jenny, Hoogenraad Casper C, Lansbergen Gideon, Dortland Bjorn, De Zeeuw Chris I, Grosveld Frank, van Cappellen Gert, Akhmanova Anna, Galjart Niels
Medical Genetics Center Department of Cell Biology and Genetics, Erasmus University, 3000 DR Rotterdam, The Netherlands.
J Neurosci. 2003 Apr 1;23(7):2655-64. doi: 10.1523/JNEUROSCI.23-07-02655.2003.
Several microtubule binding proteins, including CLIP-170 (cytoplasmic linker protein-170), CLIP-115, and EB1 (end-binding protein 1), have been shown to associate specifically with the ends of growing microtubules in non-neuronal cells, thereby regulating microtubule dynamics and the binding of microtubules to protein complexes, organelles, and membranes. When fused to GFP (green fluorescent protein), these proteins, which collectively are called +TIPs (plus end tracking proteins), also serve as powerful markers for visualizing microtubule growth events. Here we demonstrate that endogenous +TIPs are present at distal ends of microtubules in fixed neurons. Using EB3-GFP as a marker of microtubule growth in live cells, we subsequently analyze microtubule dynamics in neurons. Our results indicate that microtubules grow slower in neurons than in glia and COS-1 cells. The average speed and length of EB3-GFP movements are comparable in cell bodies, dendrites, axons, and growth cones. In the proximal region of differentiated dendrites approximately 65% of EB3-GFP movements are directed toward the distal end, whereas 35% are directed toward the cell body. In more distal dendritic regions and in axons most EB3-GFP dots move toward the growth cone. This difference in directionality of EB3-GFP movements in dendrites and axons reflects the highly specific microtubule organization in neurons. Together, these results suggest that local microtubule polymerization contributes to the formation of the microtubule network in all neuronal compartments. We propose that similar mechanisms underlie the specific association of CLIPs and EB1-related proteins with the ends of growing microtubules in non-neuronal and neuronal cells.
包括CLIP-170（细胞质连接蛋白-170）、CLIP-115和EB1（末端结合蛋白1）在内的几种微管结合蛋白已被证明在非神经元细胞中与正在生长的微管末端特异性结合，从而调节微管动力学以及微管与蛋白质复合物、细胞器和膜的结合。当与绿色荧光蛋白（GFP）融合时，这些蛋白统称为+TIPs（正端追踪蛋白），它们也是可视化微管生长事件的有力标记物。在这里，我们证明内源性+TIPs存在于固定神经元微管的远端。利用EB3-GFP作为活细胞中微管生长的标记物，我们随后分析了神经元中的微管动力学。我们的结果表明，神经元中的微管生长比神经胶质细胞和COS-1细胞中的微管生长慢。EB3-GFP运动的平均速度和长度在细胞体、树突、轴突和生长锥中相当。在分化树突的近端区域，约65%的EB3-GFP运动指向远端，而35%指向细胞体。在更远端的树突区域和轴突中，大多数EB3-GFP点向生长锥移动。树突和轴突中EB3-GFP运动方向性的这种差异反映了神经元中高度特异性的微管组织。总之，这些结果表明局部微管聚合有助于所有神经元区室中微管网络的形成。我们提出，类似的机制是CLIPs和EB1相关蛋白在非神经元和神经元细胞中与正在生长的微管末端特异性结合的基础。
