As an extension of the previous work on the pH-dependent reorganization of artificial lipid vesicles by poly(2-ethylacrylic acid) (PEAA), the effect of PEAA on living cells are examined by utilizing the acidification process of the epidermal growth factor (EGF) endocytic pathway in A431 cells. Fluoresceinated EGF (f-EGF) was used in conjunction with two-wavelength frame transfer fluorescence imaging techniques to measure the pH's of endocytic vesicles of individual cells. Preliminary results, including feasibility study and PEAA toxicity to A431 cells, are presented in chapter 3. Two projects branched out of this initial study; one involves the recording of PEAA channel activity using single-channel recording techniques (chapter 2), and another the measurement of binding kinetics of f-EGF to A431 cells at the single cell level (chapter 1). The synthetic anionic polyelectrolyte PEAA has been shown to reorganize lipid vesicles under acidic conditions. Single-channel recording techniques are used to further characterize the interactions between PEAA and artificial phospholipid bilayers. PEAA is shown to induce pore formation in bilayers at low (PEAA) ($<$ 20 $\mu$g/ml) in a pH-dependent manner. Discrete events resembling those characteristic of biological channels are observed. PEAA channels are found to be cation-selective, as demonstrated by comparing the relative Na$\sp+$ and Cl$\sp-$ permeabilities of the channels, but the Na$\sp+$/Cl$\sp-$ permeability ratio spans a wide range, indicating the existence of channels of different ion selectivities. Point amplitude histogram analysis of one type of channel event reveals at least four separate single-channel conductance states. The interpretation of ligand-receptor equilibrium binding data collected from a population of cells is often ambiguous. A method using quantitative fluorescence microscopy to measure EGF/receptor binding kinetics at the single cell level is developed. The binding of f-EGF to single cells is recorded over time and fitted to models with photobleaching correction and either one monovalent binding site or two independent monovalent binding sites. The results disprove the one-site model; therefore, the reported EGF receptor affinity heterogeneity exists at the single cell level.

The dissertation is focused on the role of chemotaxis receptor played in the transmembrane signaling. The first project accomplished was to study the interaction between serine receptor and methyltransferase. As an effort to probe the regulation mechanism of methyltransferase CheR, Isothermal Titration Calorimetry (ITC) was applied to characterize any possible regulation associated allosteric effect between methyltransferase CheR's multiple interactions. It was found that methyltransferase CheR binds to its receptor docking site and substrate SAH independently, further experiments are proposed to explore the interaction between the receptor methylation region and methyltransferase, CheR. It is speculated that conformational change in the receptor upon ligand binding controls the accessibility of the receptor methylation region to methyltransferase. The second project was carried out as a continued effort to determine the receptor ligand binding activity in the ternary complex with the adaptor protein CheW and the histidine kinase CheA, and it was expected that some cooperativity between receptor dimers would be observed. Surprisingly, in the absence of the cytoplasmic signaling proteins, CheW and CheA, serine receptors exhibited negative cooperativity in ligand binding between dimers, and the negative cooperavtivity disappeared in detergent solution. For the first time, direct evidence was found as a strong support the transmembrane signaling mechanism that involves receptor clusters. Chemoreceptors form patches at the poles of bacteria in the presence and absence of signaling proteins, and the active interaction between receptor dimers could play a significant role in information integration when passing through the bacterial membrane. Experiments are also designed for further clarify the function of receptor cluster in the future.