DETECTION OF SINGLE FLUORESCENT MICROTUBULES AND METHODS FOR DETERMINING THEIR DYNAMICS IN LIVING CELLS
DETECTION OF SINGLE FLUORESCENT MICROTUBULES AND METHODS FOR DETERMINING THEIR DYNAMICS IN LIVING CELLS
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DOI：
10.1002/cm.970100128
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发表时间：
1988-01-01
影响因子：
--
通讯作者：
BORISY, GG
中科院分区：
文献类型：
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作者：
SAMMAK, PJ;BORISY, GG
荧光标记生物分子并检测其在活细胞中的分布的能力促进了对细胞质组织的研究，特别是微管动力学。我们选择和开发的技术允许在单个微管水平上测定活成纤维细胞中微管网络的空间和时间变化。我们采用了两种一般的方法来确定模式的变化：直接视频显微镜和光漂白和随后的观察。通过高清视频显微镜直接观察荧光微管在几个时间点提供了良好的空间分辨率，但仅限于细胞的较不拥挤和较薄的周边。这种方法是通过一种相对明亮的光稳定报告基因xrhodamine‐tubulin实现的，并表明微管从其末端经历了多轮的组装和拆卸。溶解细胞的漂白和随后的观察通过提取可溶性发色团改善了信噪比，并允许在拥挤区域进行观察，但仅限于单个时间间隔。这种方法表明，微管结构域被一个接一个地替换，并且在细胞周边的周转最快。非漂白发色团的特异性抗体可用于进一步增强信噪比或通过使用免疫电子显微镜来扩展空间分辨率。直接视频显微镜和光漂白是研究动态的两种方法，它们具有互补的优势，并广泛应用于活细胞的生物学。
The ability to tag biological molecules fluorescently and to detect their distribution in living cells has promoted the study of cytoplasmic organization in general and microtubule dynamics in particular. The techniques that we have selected and developed allowed the determination of spatial and temporal changes of the microtubule network in living fibroblasts at the level of individual microtubules. We have employed two general approaches for determining pattern changes: direct video microscopy and photobleaching and subsequent observation. Direct observation of fluorescent microtubules by high‐definition video microscopy provided good spatial resolution at several time points, but was limited to the less congested and thinner periphery of the cell. This approach was made possible by a relatively bright, photostable reporter, xrhodamine‐tubulin, and showed that microtubules underwent rounds of assembly and disassembly from their ends. Bleaching and subsequent observation of lysed cells improved the signal to noise ratio by extracting soluble chromophore and permitted observations in congested areas, but was limited to a single time interval. This approach demonstrated that microtubule domains were replaced one by one and that turnover was most rapid at the cell periphery. Antibodies specific for nonbleached chromophore can be used to enhance the signal to noise ratio further or to extend spatial resolution by the use of immunoelectron microscopy. Direct video microscopy and photo‐bleaching are two approaches to the study of dynamics that have complementary strengths and wide application to the biology of living cells.