Sanchez I, Twersky L H, Cohen W D
Department of Biological Sciences, Hunter College, New York, NY 10021.
Eur J Cell Biol. 1990 Aug;52(2):349-58.
We have developed a new, detergent-based method for the isolation of marginal bands (MBs) of microtubules from non-mammalian vertebrate erythrocytes. The critical step in MB isolation is selective removal of the "membrane skeleton" network (MS), within which the MB is enclosed. To test potential MS solubilizing agents systematically, we prepared dogfish (Mustelus canis) erythrocyte cytoskeletons in the presence of protease inhibitors and stored them at -20 degrees C in medium containing 50% glycerol and 10 microM taxol to stabilize the MB. Using this as a standard starting material, we found that low concentrations of sodium dodecyl sulfate (SDS) (0.025-0.1%) in the presence of Triton X-100 (0.1-0.4%) released both MBs and nuclei intact from cytoskeletons. Either detergent alone was ineffective. MB release from cytoskeletons was blocked by excess Triton X-100 and slowed by glycerol, and this was useful for stopping the release reaction during quantitative time-course studies. Most MBs (greater than 90%) were liberated from cytoskeletons in 5 to 30 min, depending upon detergent concentrations and other conditions, and they were sufficiently stable for mass isolation by differential centrifugation. Added standard proteins were not proteolyzed during MB release, nor was release blocked by protease inhibitors, indicating that endogenous proteases were not involved. As observed in thin sections and negatively stained whole mounts (transmission electron microscopy) and in critical-point dried preparations (scanning electron microscopy), the isolated MBs consisted principally of bundled microtubules, with some additional adhering material. SDS-polyacrylamide gel electrophoresis showed the isolated MBs to be composed primarily of four tubulin region polypeptides, with the same stoichiometry as in the whole cytoskeleton. As determined by immunofluorescence microscopy, isolated MBs bound antibody to both chicken brain and erythrocyte tau, in addition to anti-tubulin. Thus, proteins of the tau family may be involved in bundling of MB microtubules. Unlike previous MB isolation methods, that employed here is applicable to erythrocytes of diverse species, including the marine toad (Bufo marinus) and the chicken (Gallus domestica), both of which should be of value for comparative studies.
我们开发了一种基于去污剂的新方法，用于从非哺乳动物脊椎动物红细胞中分离微管边缘带（MBs）。MB分离的关键步骤是选择性去除包裹着MB的“膜骨架”网络（MS）。为了系统地测试潜在的MS溶解剂，我们在蛋白酶抑制剂存在的情况下制备了角鲨（皱唇鲨）红细胞细胞骨架，并将其保存在含有50%甘油和10 microM紫杉醇的培养基中于-20℃下，以稳定MB。以这个作为标准起始材料，我们发现，在Triton X-100（0.1 - 0.4%）存在的情况下，低浓度的十二烷基硫酸钠（SDS）（0.025 - 0.1%）能完整地从细胞骨架中释放出MB和细胞核。单独使用任何一种去污剂都无效。过量的Triton X-100会阻止MB从细胞骨架中释放，甘油会减缓释放，这在定量时间进程研究中用于停止释放反应很有用。根据去污剂浓度和其他条件，大多数MB（超过90%）在5到30分钟内从细胞骨架中释放出来，并且它们足够稳定，可以通过差速离心进行大量分离。在MB释放过程中，添加的标准蛋白质没有被蛋白酶水解，蛋白酶抑制剂也没有阻止释放，这表明内源性蛋白酶没有参与。正如在超薄切片、负染整装标本（透射电子显微镜）以及临界点干燥制剂（扫描电子显微镜）中观察到的那样，分离出的MB主要由成束的微管组成，还有一些额外的附着物质。SDS-聚丙烯酰胺凝胶电泳显示，分离出的MB主要由四种微管蛋白区域多肽组成，其化学计量与整个细胞骨架中的相同。通过免疫荧光显微镜测定，除了抗微管蛋白抗体外，分离出的MB还能结合针对鸡脑和红细胞tau的抗体。因此，tau家族的蛋白质可能参与了MB微管的成束。与以前的MB分离方法不同，这里采用的方法适用于多种物种的红细胞，包括海蟾蜍（美洲蟾蜍）和鸡（家鸡），这两种物种对于比较研究都应该是有价值的。
