Bearer E L, DeGiorgis J A, Bodner R A, Kao A W, Reese T S
Division of Biology and Medicine, Brown University, Providence, RI 02912.
Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11252-6. doi: 10.1073/pnas.90.23.11252.
Squid axoplasm has proved a rich source for the identification of motors involved in organelle transport. Recently, squid axoplasmic organelles have been shown to move on invisible tracks that are sensitive to cytochalasin, suggesting that these tracks are actin filaments. Here, an assay is described that permits observation of organelles moving on unipolar actin bundles. This assay is used to demonstrate that axoplasmic organelles move on actin filaments in the barbed-end direction, suggesting the presence of a myosin motor on axoplasmic organelles. Indeed, axoplasm contains actin-dependent ATPase activity, and a pan-myosin antibody recognized at least four bands in Western blots of axoplasm. An approximately 235-kDa band copurified in sucrose gradients with KI-extracted axoplasmic organelles, and the myosin antibody stained the organelle surfaces by immunogold electron microscopy. The myosin is present on the surface of at least some axoplasmic organelles and thus may be involved in their transport through the axoplasm, their movement through the cortical actin in the synapse, or some other aspect of axonal function.
鱿鱼轴浆已被证明是鉴定参与细胞器运输的分子马达的丰富来源。最近，已表明鱿鱼轴浆细胞器在对细胞松弛素敏感的不可见轨道上移动，这表明这些轨道是肌动蛋白丝。在此，描述了一种可观察细胞器在单极肌动蛋白束上移动的检测方法。该检测方法用于证明轴浆细胞器沿肌动蛋白丝的带刺端方向移动，这表明轴浆细胞器上存在肌球蛋白分子马达。实际上，轴浆含有肌动蛋白依赖性ATP酶活性，并且泛肌球蛋白抗体在轴浆的蛋白质免疫印迹中识别出至少四条带。一条约235 kDa的带在蔗糖梯度中与碘化钾提取的轴浆细胞器共纯化，并且肌球蛋白抗体通过免疫金电子显微镜对细胞器表面进行了染色。肌球蛋白存在于至少一些轴浆细胞器的表面，因此可能参与它们通过轴浆的运输、它们通过突触中皮质肌动蛋白的移动或轴突功能的其他方面。
