Bulinski J C, Gruber D, Faire K, Prasad P, Chang W
Department of Anatomy & Cell Biology, Integrated Program in Cell, Molecular, & Biophysical Studies, Columbia University, College of Physicians & Surgeons, 630 W. 168th St., New York, NY 10032-3702, USA.
Cell Struct Funct. 1999 Oct;24(5):313-20. doi: 10.1247/csf.24.313.
E-MAP-115 (ensconsin) is a microtubule-associated protein (MAP) abundant in carcinoma and other epithelia-derived cells. We expressed chimeras of green fluorescent protein (GFP) conjugated to ensconsin's N-terminal MT-binding domain (EMTB), to study distribution, dynamics, and function of the MAP in living cells. We tested the hypothesis that behavior of expressed GFP-EMTB accurately matched behavior of endogenous ensconsin. Like endogenous MAP, GFP-EMTB was associated with microtubules in living or fixed cells, and microtubule association of either molecule was impervious to extraction with nonionic detergents. In cell lysates both GFP-EMTB and endogenous ensconsin were dissociated from microtubules by identical salt extraction conditions, and both molecules remained bound to a calcium-stable subset of Taxol-stabilized microtubules. These data show that microtubule association of ensconsin was affected neither by the absence of domains other than its microtubule-binding domain, nor by the presence of appended GFP. We took advantage of this finding to generate constructs in which additional GFP moieties were attached to EMTB, to obtain a more intensely fluorescent reporter of in vivo MAP binding. We show here that expression of chimeric proteins consisting of five GFP molecules attached to a single EMTB molecule produces brightly labeled microtubules without compromising the behavior of the MAP or the microtubules to which it is attached. Thus, we have demonstrated the utility of chimeric proteins containing GFP multimers as authentic reporters of ensconsin distribution and dynamics; expression of these GFP-EMTB chimeric molecules also provides a non-perturbing label of the microtubule system in living cells.
E-MAP-115（ensin）是一种在癌组织和其他上皮来源细胞中大量存在的微管相关蛋白（MAP）。我们表达了与ensin的N端微管结合结构域（EMTB）偶联的绿色荧光蛋白（GFP）嵌合体，以研究该MAP在活细胞中的分布、动态变化及功能。我们验证了一个假设，即表达的GFP-EMTB的行为与内源性ensin的行为精确匹配。与内源性MAP一样，GFP-EMTB在活细胞或固定细胞中均与微管相关，并且这两种分子与微管的结合不受非离子去污剂提取的影响。在细胞裂解物中，GFP-EMTB和内源性ensin在相同的盐提取条件下均从微管上解离，并且这两种分子都保持与紫杉醇稳定的微管的钙稳定亚组结合。这些数据表明，ensin与微管的结合既不受其微管结合结构域以外其他结构域缺失的影响，也不受附加GFP的影响。我们利用这一发现构建了在EMTB上连接额外GFP部分的构建体，以获得体内MAP结合的更强荧光报告物。我们在此表明，由五个GFP分子连接到单个EMTB分子组成的嵌合蛋白的表达产生了明亮标记的微管，而不会损害MAP或其附着的微管的行为。因此，我们证明了含有GFP多聚体的嵌合蛋白作为ensin分布和动态变化的真实报告物的实用性；这些GFP-EMTB嵌合分子的表达还为活细胞中的微管系统提供了一种无干扰的标记。
