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.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-2770 |
| Date | 01 January 1996 |
| Creators | Chung, Johnson Cheng-Chun |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
Page generated in 0.0017 seconds