Denoulet P, Filliatreau G, de Néchaud B, Gros F, Di Giamberardino L
Collège de France, Paris.
J Cell Biol. 1989 Mar;108(3):965-71. doi: 10.1083/jcb.108.3.965.
The axonal transport of the diverse isotubulins in the motor axons of the rat sciatic nerve was studied by two-dimensional polyacrylamide gel electrophoresis after intraspinal injection of [35S]methionine. 3 wk after injection, the nerve segments carrying the labeled axonal proteins of the slow components a (SCa) and b (SCb) of axonal transport were homogenized in a cytoskeleton-stabilizing buffer and two distinct fractions, cytoskeletal (pellet, insoluble) and soluble (supernatant), were obtained by centrifugation. About two-thirds of the transported-labeled tubulin moved with SCa, the remainder with SCb. In both waves, tubulin was found to be associated mainly with the cytoskeletal fraction. The same isoforms of tubulin were transported with SCa and SCb; however, the level of a neuron-specific beta-tubulin subcomponent, termed beta', composed of two related isotubulins beta'1 and beta'2, was significantly greater in SCb than in SCa, relative to the other tubulin isoforms. In addition, certain specific isotubulins were unequally distributed between the cytoskeletal and the soluble fractions. In SCa as well as in SCb, alpha''-isotubulins were completely soluble in the motor axons. By contrast, alpha''' and beta'2-isotubulins, both posttranslationally modified isoforms, were always recovered in the cytoskeletal fraction and thus may represent isotubulins restricted to microtubule polymers. The different distribution of isotubulins suggests that a recruitment of tubulin isoforms, including specific posttranslational modifications of defined isoforms (such as, at least, phosphorylation of beta' and acetylation of alpha'), might be involved in the assembly of distinct subsets of axonal microtubules displaying differential properties of stability, velocity and perhaps of function.
在大鼠脊髓内注射[35S]蛋氨酸后，通过二维聚丙烯酰胺凝胶电泳研究了大鼠坐骨神经运动轴突中不同微管蛋白的轴突运输。注射3周后，将携带轴突运输慢成分a（SCa）和b（SCb）标记轴突蛋白的神经节段在细胞骨架稳定缓冲液中匀浆，通过离心获得细胞骨架（沉淀，不溶性）和可溶性（上清液）两个不同的组分。约三分之二的运输标记微管蛋白与SCa一起移动，其余的与SCb一起移动。在这两个波中，微管蛋白主要与细胞骨架组分相关。与SCa和SCb一起运输的是相同的微管蛋白亚型；然而，相对于其他微管蛋白亚型，由两种相关的微管蛋白亚型β'1和β'2组成的神经元特异性β-微管蛋白亚组分β'在SCb中的水平明显高于SCa。此外，某些特定的微管蛋白亚型在细胞骨架和可溶性组分之间分布不均。在SCa以及SCb中，α''-微管蛋白在运动轴突中完全可溶。相比之下，α'''和β'2-微管蛋白这两种翻译后修饰的亚型总是在细胞骨架组分中回收，因此可能代表限于微管聚合物的微管蛋白亚型。微管蛋白亚型的不同分布表明，微管蛋白亚型的募集，包括特定亚型的特定翻译后修饰（如至少β'的磷酸化和α'的乙酰化），可能参与了具有不同稳定性、速度和功能特性的轴突微管不同亚群的组装。
