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

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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