Ahmad Fridoon J, He Yan, Myers Kenneth A, Hasaka Thomas P, Francis Franto, Black Mark M, Baas Peter W
Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, PA, USA.
Traffic. 2006 May;7(5):524-37. doi: 10.1111/j.1600-0854.2006.00403.x.
We investigated potential roles of cytoplasmic dynein in organizing axonal microtubules either by depleting dynein heavy chain from cultured neurons or by experimentally disrupting dynactin. The former was accomplished by siRNA while the latter was accomplished by overexpressing P50-dynamitin. Both methods resulted in a persistent reduction in the frequency of transport of short microtubules. To determine if the long microtubules in the axon also undergo dynein-dependent transport, we ascertained the rates of EGFP-EB3 "comets" observed at the tips of microtubules during assembly. The rates of the comets, in theory, should reflect a combination of the assembly rate and any potential transport of the microtubule. Comets were initially slowed during P50-dynamitin overexpression, but this effect did not persist beyond the first day and was never observed in dynein-depleted axons. In fact, the rates of the comets were slightly faster in dynein-depleted axons. We conclude that the transient effect of P50-dynamitin overexpression reflects a reduction in microtubule polymerization rates. Interestingly, after prolonged dynein depletion, the long microtubules were noticeably misaligned in the distal regions of axons and failed to enter the filopodia of growth cones. These results suggest that the forces generated by cytoplasmic dynein do not transport long microtubules, but may serve to align them with one another and also permit them to invade filopodia.
我们通过从培养的神经元中去除动力蛋白重链或通过实验破坏动力蛋白激活蛋白来研究细胞质动力蛋白在组织轴突微管中的潜在作用。前者通过小干扰RNA（siRNA）实现，而后者通过过表达P50-动力蛋白抑制因子来实现。两种方法都导致短微管运输频率持续降低。为了确定轴突中的长微管是否也经历动力蛋白依赖性运输，我们确定了在组装过程中在微管尖端观察到的增强绿色荧光蛋白（EGFP）-EB3“彗星”的速率。理论上，彗星的速率应反映微管组装速率和任何潜在运输的综合情况。在过表达P50-动力蛋白抑制因子期间，彗星最初速度减慢，但这种效应在第一天之后并未持续，并且在动力蛋白缺失的轴突中从未观察到。事实上，在动力蛋白缺失的轴突中，彗星的速率略快。我们得出结论，过表达P50-动力蛋白抑制因子的短暂效应反映了微管聚合速率的降低。有趣的是，在长期去除动力蛋白后，长微管在轴突的远端区域明显排列不齐，并且无法进入生长锥的丝状伪足。这些结果表明，细胞质动力蛋白产生的力不会运输长微管，但可能有助于使它们彼此对齐，并允许它们侵入丝状伪足。
