Brown C R, Doxsey S J, Hong-Brown L Q, Martin R L, Welch W J
Department of Medicine, University of California, San Francisco 94143-0854, USA.
J Biol Chem. 1996 Jan 12;271(2):824-32. doi: 10.1074/jbc.271.2.824.
Molecular chaperones play an important role in facilitating the proper maturation of many newly synthesized proteins. Here we provide evidence that molecular chaperones also participate in regulating the assembly of the microtubule cytoskeleton. Via indirect immunofluorescence analysis, both hsp 73 and TCP-1 localized within the centrosome in interphase and mitotic cells. These proteins, along with the centrosome-specific protein, pericentrin, were also present within an enriched preparation of centrosomes. Because the centrosome serves as an initiation site for microtubule growth, we examined the ability of cells to regrow their microtubule network in the presence of hsp 73 or TCP-1 specific antibodies. Purified tubulin and GTP were added to cells following the depolymerization and extraction of cellular microtubules. Microtubules were observed to nucleate off the centrosome using this system, even in the presence of anti-hsp 73 antibodies. Incubation with anti-TCP-1 antibodies, however, blocked microtubule regrowth off the centrosome. Similarly, anti-TCP-1 antibodies microinjected into living cells first treated with nocodazole also inhibited the regrowth of the microtubule network following removal of the microtubule poison. Our results complement earlier genetic studies in yeast implicating a role for TCP-1 in microtubule mediated processes, and may help to explain the previously reported mitotic and meiotic abnormalities associated with TCP-1 mutations.
分子伴侣在促进许多新合成蛋白质的正确成熟过程中发挥着重要作用。在此，我们提供证据表明分子伴侣也参与调节微管细胞骨架的组装。通过间接免疫荧光分析，热休克蛋白73（hsp 73）和TCP-1在间期和有丝分裂细胞的中心体中均有定位。这些蛋白质，连同中心体特异性蛋白质中心体蛋白，也存在于富集的中心体制剂中。由于中心体是微管生长的起始位点，我们检测了在存在hsp 73或TCP-1特异性抗体的情况下细胞重新生长其微管网络的能力。在细胞微管解聚和提取后，向细胞中加入纯化的微管蛋白和鸟苷三磷酸（GTP）。即使在存在抗hsp 73抗体的情况下，使用该系统也观察到微管从中心体上成核。然而，用抗TCP-1抗体孵育会阻断微管从中心体上的重新生长。同样，将抗TCP-1抗体显微注射到先用诺考达唑处理的活细胞中，在去除微管毒物后也会抑制微管网络的重新生长。我们的结果补充了早期在酵母中的遗传学研究，这些研究表明TCP-1在微管介导的过程中发挥作用，并且可能有助于解释先前报道的与TCP-1突变相关的有丝分裂和减数分裂异常。
