Wilson R, Brophy P J
Division of Cell Biology and Biochemistry, School of Molecular and Biological Sciences, University of Stirling, Scotland.
J Neurosci Res. 1989 Apr;22(4):439-48. doi: 10.1002/jnr.490220409.
Enriched cultures of rat brain oligodendrocytes were extracted with a buffer that separated the cells into a Triton X-100-soluble fraction and an insoluble cytoskeleton (CSK) residue. The buffer was optimised so that intact microtubules were preserved in the CSK residue. The partition of four myelin proteins between the soluble and the CSK fractions was determined by immunoblotting and immunofluorescence. Immunoblotting showed that two integral membrane proteins of myelin, the proteolipid protein (PLP) and the DM-20 protein, were completely extracted under these conditions. By contrast, a substantial amount of myelin basic protein (MBP) and to a lesser extent 2,3-cyclic nucleotide-3-phosphohydrolase (CNP) remained associated with the CSK residue. The association of these proteins with the CSK was confirmed by immunofluorescence. A remarkable difference in the distribution of microfilaments and microtubules was observed in oligodendrocytes. Immature cells possessed many fine processes that were rich in microfilaments. The cell body of these oligodendrocytes was devoid of microfilaments but did contain microtubules. Furthermore, a close association between CNP and microfilaments and between MBP and microtubules was revealed after detergent lysis. The strong interaction between CNP and filamentous actin was underlined by their concomitant disappearance from the extremities of the cell at a later stage of development when extensive membrane sheets had formed. Mature cells had fewer, thicker processes than younger cells and their processes contained microtubules, not microfilaments. MBP was present throughout the thick processes and the membrane sheets. These observations suggest roles for CNP and MBP at distinct stages of myelin process formation and support a directive role for the oligodendrocyte's CSK in the formation of myelin.
用一种缓冲液提取大鼠脑少突胶质细胞的富集培养物，该缓冲液将细胞分离为Triton X - 100可溶性部分和不溶性细胞骨架（CSK）残余物。对缓冲液进行了优化，以便完整的微管保留在CSK残余物中。通过免疫印迹和免疫荧光法测定了四种髓磷脂蛋白在可溶性部分和CSK部分之间的分配情况。免疫印迹显示，在这些条件下，髓磷脂的两种整合膜蛋白，即蛋白脂蛋白（PLP）和DM - 20蛋白被完全提取出来。相比之下，大量的髓磷脂碱性蛋白（MBP）以及少量的2,3 - 环核苷酸 - 3 - 磷酸水解酶（CNP）仍与CSK残余物相关联。这些蛋白与CSK的关联通过免疫荧光得到证实。在少突胶质细胞中观察到微丝和微管分布存在显著差异。未成熟细胞具有许多富含微丝的细突起。这些少突胶质细胞的细胞体没有微丝，但确实含有微管。此外，去污剂裂解后发现CNP与微丝之间以及MBP与微管之间存在密切关联。在发育后期形成广泛的膜片时，CNP和丝状肌动蛋白从细胞末端同时消失，这突出了它们之间的强烈相互作用。成熟细胞的突起比年轻细胞少且更粗，其突起含有微管而非微丝。MBP存在于整个粗突起和膜片中。这些观察结果表明CNP和MBP在髓鞘形成的不同阶段发挥作用，并支持少突胶质细胞的CSK在髓鞘形成中起指导作用。
