Sanchez I, Cohen W D
Department of Biological Sciences, Hunter College of CUNY, New York, New York.
Cell Motil Cytoskeleton. 1994;29(1):57-71. doi: 10.1002/cm.970290106.
Microtubule protein extracted from dogfish erythrocyte cytoskeletons by disassembly of marginal bands at low temperature formed linear microtubule (MT) bundles upon reassembly at 22 degrees C. The bundles, which were readily visible by video-enhanced phase contrast or DIC microscopy, increased in length and thickness with time. At steady state after 1 hour, most bundles were 6-11 microns in length and 2-5 MTs in thickness. No inter-MT cross-bridges were visible by negative staining. The bundles exhibited mechanical stability in flow as well as flexibility, in this respect resembling native marginal bands. As analyzed by SDS-PAGE and immunoblotting, our standard extraction conditions yielded MT protein preparations and bundles containing tau protein but not high molecular weight MAPs such as MAP-2 or syncolin. In addition, late fractions of MT protein obtained by gel filtration were devoid of high molecular weight proteins but still produced MT bundles. The marginal band tau was salt-extractable and heat-stable, bound antibodies to mammalian brain tau, and formed aggregates upon desalting. Antibodies to tau blocked MT assembly, but both assembly and bundling occurred in the presence of antibodies to actin or syncolin. The MTs were "unbundled" by subtilisin or by high salt (0.5-1 M KCl or NaCl), consistent with tau involvement in bundling. High salt extracts retained bundling activity, and salt-induced unbundling was reversible with desalting. However, reversibility was observed only after salt-induced MT disassembly had occurred. Reconstitution experiments showed that addition of marginal band tau to preassembled MTs did not produce bundles, whereas tau presence during MT reassembly did yield bundles. Thus, in this system, tau appears to play a role in both MT assembly and bundling, serving in the latter function as a coassembly factor.
通过在低温下拆解边缘带从狗鱼红细胞细胞骨架中提取的微管蛋白，在22摄氏度重新组装时形成线性微管束。这些微管束通过视频增强相差显微镜或微分干涉对比显微镜很容易观察到，其长度和厚度随时间增加。在1小时后的稳态下，大多数微管束长度为6 - 11微米，厚度为2 - 5根微管。负染法未观察到微管间的交叉桥。这些微管束在流动中表现出机械稳定性以及柔韧性，在这方面类似于天然边缘带。通过SDS - PAGE和免疫印迹分析，我们的标准提取条件得到的微管蛋白制剂和微管束含有tau蛋白，但不含有高分子量微管相关蛋白，如MAP - 2或syncolin。此外，通过凝胶过滤获得的微管蛋白后期组分不含高分子量蛋白质，但仍能产生微管束。边缘带tau可被盐提取且热稳定，能与抗哺乳动物脑tau的抗体结合，并在脱盐时形成聚集体。抗tau抗体可阻断微管组装，但在存在抗肌动蛋白或抗syncolin抗体时，组装和束集均会发生。枯草杆菌蛋白酶或高盐（0.5 - 1 M KCl或NaCl）可使微管束“解束”，这与tau参与束集作用一致。高盐提取物保留束集活性，盐诱导的解束通过脱盐是可逆的。然而，只有在盐诱导微管解聚发生后才观察到可逆性。重组实验表明，将边缘带tau添加到预先组装的微管中不会产生微管束，而在微管重新组装过程中存在tau则会产生微管束。因此，在这个系统中，tau似乎在微管组装和束集中都起作用，在后者的功能中作为共组装因子。
