Pfister K K, Wagner M C, Stenoien D L, Brady S T, Bloom G S
Department of Cell Biology and Anatomy, University of Texas Southwestern Medical Center, Dallas 75235.
J Cell Biol. 1989 Apr;108(4):1453-63. doi: 10.1083/jcb.108.4.1453.
Kinesin, a microtubule-activated ATPase and putative motor protein for the transport of membrane-bounded organelles along microtubules, was purified from bovine brain and used as an immunogen for the production of murine monoclonal antibodies. Hybridoma lines that secreted five distinct antikinesin IgGs were cloned. Three of the antibodies reacted on immunoblots with the 124-kD heavy chain of kinesin, while the other two antibodies recognized the 64-kD light chain. When used for immunofluorescence microscopy, the antibodies stained punctate, cytoplasmic structures in a variety of cultured mammalian cell types. Consistent with the identification of these structures as membrane-bounded organelles was the observation that cells which had been extracted with Triton X-100 before fixation contained little or no immunoreactive material. Staining of microtubules in the interphase cytoplasm or mitotic spindle was never observed, nor were associated structures, such as centrosomes and primary cilia, labeled by any of the antibodies. Nevertheless, in double-labeling experiments using antibodies to kinesin and tubulin, kinesin-containing particles were most abundant in regions where microtubules were most highly concentrated and the particles often appeared to be aligned on microtubules. These results constitute the first direct evidence for the association of kinesin with membrane-bounded organelles, and suggest a molecular mechanism for organelle motility based on transient interactions of organelle-bound kinesin with the microtubule surface.
驱动蛋白是一种微管激活的ATP酶，被认为是沿微管运输膜结合细胞器的驱动蛋白，它从牛脑中纯化出来，并用作免疫原以产生鼠单克隆抗体。分泌五种不同抗驱动蛋白IgG的杂交瘤细胞系被克隆。其中三种抗体在免疫印迹上与驱动蛋白的124-kD重链发生反应，而另外两种抗体识别64-kD轻链。当用于免疫荧光显微镜检查时，这些抗体在多种培养的哺乳动物细胞类型中对点状细胞质结构进行染色。与这些结构被鉴定为膜结合细胞器相一致的是，在固定前用Triton X-100提取的细胞几乎不含或不含免疫反应性物质。从未观察到间期细胞质或有丝分裂纺锤体中的微管染色，也没有任何抗体标记中心体和初级纤毛等相关结构。然而，在使用抗驱动蛋白和微管蛋白抗体的双重标记实验中，含驱动蛋白的颗粒在微管高度集中的区域最为丰富，并且颗粒常常似乎排列在微管上。这些结果构成了驱动蛋白与膜结合细胞器关联的首个直接证据，并提出了一种基于细胞器结合的驱动蛋白与微管表面短暂相互作用的细胞器运动分子机制。
