Overexpression of neurofilament H disrupts normal cell structure and function.
Overexpression of neurofilament H disrupts normal cell structure and function.
复制标题
神经丝 H 的过度表达会破坏正常的细胞结构和功能。
DOI：
10.1002/jnr.10212
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发表时间：
2002
期刊：
影响因子：
--
通讯作者：
Brady,ScottT
中科院分区：
文献类型：
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作者：
Szebenyi,Gyorgyi;Smith,GeorgeM;Li,Ping;Brady,ScottT
研究活细胞中外源表达的标记蛋白已成为评估蛋白质分布和功能的标准技术。通常，实验引入的蛋白质的表达水平不受调节，高水平通常是为了便于检测。然而，许多蛋白质的过表达会导致定位错误和病理。因此，对于规范性研究，中等水平的表达可能更合适。为了更好地了解健康活细胞中中间纤维形成、运输和稳定性的动力学，我们将神经丝重链(NFH) -绿色荧光蛋白（GFP）融合构建物插入四环素（tet）调控启动子的腺病毒载体中。该系统允许以剂量依赖的方式在选定的时间内开启或关闭NFH - GFP的合成。我们使用该诱导系统对与NFH - GFP表达增加相关的丝结构和细胞形状、运动和运输的变化进行活细胞成像。低至中等水平的NFH - GFP细胞在结构和功能上与邻近的非表达细胞相似。相反，过表达导致纤维组织和细胞功能的病理改变。©2002 Wiley‐Liss, Inc。
Studying exogenously expressed tagged proteins in live cells has become a standard technique for evaluating protein distribution and function. Typically, expression levels of experimentally introduced proteins are not regulated, and high levels are often preferred to facilitate detection. However, overexpression of many proteins leads to mislocalization and pathologies. Therefore, for normative studies, moderate levels of expression may be more suitable. To understand better the dynamics of intermediate filament formation, transport, and stability in a healthy, living cell, we inserted neurofilament heavy chain (NFH)‐green fluorescent protein (GFP) fusion constructs in adenoviral vectors with tetracycline (tet)‐regulated promoters. This system allows for turning on or off the synthesis of NFH‐GFP at a selected time, for a defined period, in a dose‐dependent manner. We used this inducible system for live cell imaging of changes in filament structure and cell shape, motility, and transport associated with increasing NFH‐GFP expression. Cells with low to intermediate levels of NFH‐GFP were structurally and functionally similar to neighboring, nonexpressing cells. In contrast, overexpression led to pathological alterations in both filament organization and cell function. © 2002 Wiley‐Liss, Inc.