Time lapse nano-analysis of single cell components : Development of Harvesting, Identification and Injection Methods of Functional Molecules

利用原子力显微镜技术开发了纳米级、纳米牛顿级的活细胞和生物大分子（如蛋白质和DNA）操作技术。这项开发工作的目的是“细胞手术”，这意味着我们将获得工具和手段来监测活细胞内部生物化学的时间依赖性过程，并通过将功能性生物大分子插入细胞来改变细胞的特性。为此，我们做了以下实验，得到了如下所述的结果。蛋白质从活细胞表面拉出的力曲线分析。利用改良的AFM探针从活细胞表面提取膜蛋白。在这种拉伸实验中获得的力曲线显示，细胞膜的拉伸很可能与脂质系链的形成相对应，然后最终提取目标蛋白。力曲线的特征可分为：1)拉不与细胞骨架和其他细胞内结构连接的膜蛋白，2)拉与细胞骨架连接的膜蛋白。以前者为例对糖蛋白A的特异性凝集素和后者为例对Band 3的特异性凝集素在红细胞上进行模型实验。在前一种情况下，与细胞骨架没有联系，力曲线的特征是平台力的延伸约为70 pN，以一步跳跃到零力水平结束。后一种情况下，受力曲线表现为在平台力延伸前后出现多个大于100 pN的力峰。我们提出这两种类型的力曲线可以用来监测膜蛋白与胞内细胞骨架结构的连接状态。mRNA的提取和质粒DNA的插入。利用AFM探针从活细胞细胞质中提取mRNA和其他功能大分子。将探针推入细胞的目标位点，作为RT-PCR和PCR扩增探针上收获的mRNA的起始材料。mRNA定位在活细胞的一个非常有限的位置可以常规分析其身份和定量。这种方法对活细胞不同部位特定蛋白质的生产的时间依赖性监测是有用的。通过吸附质粒DNA的AFM探针，我们发现以绿色荧光蛋白基因为标记的质粒DNA可以转染许多细胞。少

Atomic force microscopy was used to develop the technology of nano-meter, nano-newton level manipulation of live cells and biological macromolecules such as proteins and DNA. The aim of this development effort is in "cell surgery" meaning that we would acquire the tools and means to monitor the time dependent process of the biochemistry of the interior of a living cell and to change the properties of the cell by inserting functional biomacromolecules into the cell. For this purpose we have done the following experiments and obtained the results as explained.1.Analysis of force curves of protein pull-out from the surface of a living cell.The modified AFM probes were used to pull out the membrane protein from the surface of live cells. Force curves obtained in such pulling experiments showed a long stretch of the cell membrane most likely corresponding to the lipid tether formation followed by eventual extraction of targeted protein. The characteristics of force curves were classified in … More to, 1) pulling membrane proteins without linkage with the cytoskeleton and other intracellular structures, and 2) pulling those with linkage to the cytoskeletons. The model experiments were performed on the red blood cell using specific lectins towards glycophorin A as the example of the former type, and to Band 3 an the example of the latter type of membrane proteins. In the former case of no linkage to cytoskeleton, force curves were characterized by the extension of a plateau force of about 70 pN ending with a single step jump to zero force level. Whereas, in the latter case, force curves were characterized by the appearance of multiple force peaks greater than 100 pN before and after the extension of plateau force. We proposed that the two types of force curves could be used to monitor the linkage status of membrane protein with the intracellular cytoskeletal structure.2.Extraction of mRNA and insertion of plasmid DNA.AFM probes were used to extract mRNA and other functional macromolecules from the cytoplasm of living cells. A probe that was pushed into a targeted locus of the cell was used as the starting material for RT-PCR and PCR amplification of the harvested mRNA on the probe. mRNA localization in a very limited position of a live cell can be routinely analyzed for its identity and quantification. This method will be useful for the time dependent monitoring of the production of specific proteins in different parts of a living cell. By using an AFM probe with adsorbed plasmid DNA, we showed that a number of cells could be transfected with the plasmid DNA having the gene of green fluorescent protein as a marker. Less