Application of Atomic Force Microscopy to the Study of Expressed Molecules in or on a Single Living Cell

Abstract

One of the most useful ways to study the physical and biochemical properties of expressed molecules in or on living cells is to directly manipulate the molecules in question. In this chapter, we describe howthe atomic forcemicroscope (AFM)was used as a nanomanipulator, and membrane receptors or expressed messenger RNA (mRNAs) were touched, pulled and picked up to study the expressed information and biophysical properties of these molecules. First, the chemical and physical modifications of the AFM tip are introduced. For the study of membrane receptors over a large area of cell surface using an AFM, the colloidal probe method is useful. Details of probe preparation, chemical modification of colloids and data analysis are introduced. Furthermore, a useful method to measure the interaction force between a single molecular pair on a soft material is also introduced. Second, some examples of expressed receptor mapping are given. Ligand molecules were immobilized on a microbead attached to the AFM tip and force measurements were performed one by one over a large area of living cell surface. Distributions and amounts of expressed receptors on the cell surface were studied by measuring ligand–receptor interactions. Third, extractions of both membrane receptors and mRNAs are introduced. For the extraction of membrane receptors, the AFM tip was treated with cross-linkers. The tip was moved close to the cell surface and covalent bonds were formed between the tip and the receptors through the cross-linkers. Extraction of membrane proteins was confirmed by monitoring the force in each assay. For the extraction of mRNAs, the AFM tip was inserted into the cell body, and incubated for a few minutes. After incubation, the tip was withdrawn and inserted into a PCR tube. Collection of mRNAs was then confirmed with PCR analysis. By application of this method, the time course expression of specific mRNAs was successfully measured. The methods introduced in this chapter represent new and useful applications of the AFM to the study of the properties of single cells.