Brady S T, Pfister K K, Bloom G S
Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas 75235-9039.
Proc Natl Acad Sci U S A. 1990 Feb;87(3):1061-5. doi: 10.1073/pnas.87.3.1061.
One of our monoclonal antibodies against the heavy chain of bovine kinesin (H2) also recognized the heavy chain of squid kinesin. The immunofluorescence pattern of H2 in axoplasm was similar to that seen in mammalian cells with antibodies specific for kinesin light and heavy chains, indicating that squid kinesin is also concentrated on membrane-bounded organelles. Although kinesin is assumed to be a motor for translocation of membrane-bounded organelles in fast axonal transport, direct evidence has been lacking. Perfusion of axoplasm with purified H2 at 0.1-0.4 mg/ml resulted in a profound inhibition of both the rates and number of organelles moving in anterograde and retrograde directions in the interior of the axoplasm, and comparable inhibition was noted in bidirectional movement along individual microtubules at the periphery. Maximal inhibition developed over 30-60 min. Perfusion with higher concentrations of H2 (greater than 1 mg of IgG per ml) were less effective, whereas perfusion with 0.04 mg of H2 per ml resulted in minimal inhibition. Movement of membrane-bounded organelles after perfusion with comparable levels of irrelevant mouse IgG (0.04 to greater than 1 mg/ml) were not distinguishable from perfusion with buffer controls. Inhibition of fast axonal transport by an antibody specific for kinesin provides direct evidence that kinesin is involved in the translocation of membrane-bounded organelles in axons. Moreover, the inhibition of bidirectional axonal transport by H2 raises the possibility that kinesin may play some role in both anterograde and retrograde axonal transport.
我们的一种抗牛驱动蛋白重链的单克隆抗体（H2）也能识别鱿鱼驱动蛋白的重链。H2在轴浆中的免疫荧光模式与用驱动蛋白轻链和重链特异性抗体处理的哺乳动物细胞中的模式相似，这表明鱿鱼驱动蛋白也集中在膜结合细胞器上。尽管驱动蛋白被认为是快速轴突运输中膜结合细胞器转运的动力，但一直缺乏直接证据。用0.1 - 0.4 mg/ml的纯化H2灌注轴浆，导致轴浆内部顺行和逆行方向移动的细胞器的速度和数量都受到显著抑制，并且在周边沿单个微管的双向移动中也观察到类似的抑制。最大抑制在30 - 60分钟内出现。用更高浓度的H2（每毫升大于1 mg IgG）灌注效果较差，而每毫升用0.04 mg H2灌注导致的抑制最小。用相当水平的无关小鼠IgG（0.04至大于1 mg/ml）灌注后，膜结合细胞器的移动与用缓冲液对照灌注没有区别。驱动蛋白特异性抗体对快速轴突运输的抑制提供了直接证据，表明驱动蛋白参与轴突中膜结合细胞器的转运。此外，H2对轴突双向运输的抑制增加了驱动蛋白可能在顺行和逆行轴突运输中都发挥某种作用的可能性。
