Wolff P, Abreu P A, Espreafico E M, Costa M C, Larson R E, Ho P L
Centro de Biotecnologia, Instituto Butantan, São Paulo, SP, 05503-900, Brazil.
Biochem Biophys Res Commun. 1999 Aug 19;262(1):98-102. doi: 10.1006/bbrc.1999.1171.
PC12 cell line is a cellular model to study neurite outgrowth and neurotransmitter release mechanisms. Molecular motors may be involved in these responses and myosin V could be a candidate to mediate these effects. Overlay experiments using [(125)I]-calmodulin showed that PC12 cells possess several calmodulin-binding proteins, some of them around 190-210 kDa. Western blots using affinity purified polyclonal antibodies raised against chicken brain myosin V revealed a component of 190 kDa, a molecular mass typical of myosin V. Furthermore, Northern blots using a myosin V probe also detected a transcript of around 12 kbp. Immunofluorescence cytochemistry demonstrated the localization of myosin V throughout the cytoplasm, in the neurites, growth cone tips, and with an intense asymmetrical perinuclear labeling. Western blot analyses of PC12 cellular extracts after FGF-2 and/or dibutyryl cAMP treatment revealed variations between myosin V and myosin II expression during neuronal differentiation. These results demonstrated the presence of myosin V in PC12 cells and also suggest a role for this motor molecule in the neuronal differentiation response in PC12 cells.
PC12细胞系是一种用于研究神经突生长和神经递质释放机制的细胞模型。分子马达可能参与这些反应，肌球蛋白V可能是介导这些效应的候选分子。使用[（125）I]-钙调蛋白的覆盖实验表明，PC12细胞拥有几种钙调蛋白结合蛋白，其中一些分子量约为190-210 kDa。使用针对鸡脑肌球蛋白V产生的亲和纯化多克隆抗体进行的蛋白质印迹分析显示出一条190 kDa的条带，这是肌球蛋白V的典型分子量。此外，使用肌球蛋白V探针进行的Northern印迹分析也检测到一条约12 kbp的转录本。免疫荧光细胞化学显示肌球蛋白V在整个细胞质、神经突、生长锥尖端均有定位，并且在核周有强烈的不对称标记。对FGF-2和/或二丁酰cAMP处理后的PC12细胞提取物进行的蛋白质印迹分析显示，在神经元分化过程中，肌球蛋白V和肌球蛋白II的表达存在差异。这些结果证明了PC12细胞中存在肌球蛋白V，也表明这种马达分子在PC12细胞的神经元分化反应中发挥作用。
