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An investigation at the level of the individual A431 cell: The ATP-induced calcium response and the kinetics of EGF and EGF-R binding
Abstract
A fluorescence microscope and a digital imaging system including a charge coupled device and a computer were used to address two lines of inquiry each using individual A431 cells as the subject. The ATP-induced calcium response was approached using ratio imaging techniques and fura-2, a fluorescent calcium ion indicator. We demonstrated that ATP induced a calcium response in a dose and calcium-dependent manner. The calcium stores in A431 cells were selectively emptied and refilled under experimental control. A431 cells were shown to be desensitized upon repeated stimulation with ATP. Calcium pool emptying and desensitization were found to be concurrent processes. The second pursuit was to develop and employ an approach for measuring the kinetics of ligand and receptor association and dissociation on individual cells using fluorescence microscopy and digital imaging techniques. The strategy was used to study EGF binding to its receptors on individual cells. Binding curves were fit to single and two site exponential functions which assumed first order kinetics binding to independent binding sites. The values for k$\sb{\rm on}$ and k$\sb{\rm off}$ were similar to those found in the literature. However the assumptions that the k$\sb{\rm off}$ rate is independent of (EGF) and that there are two independent populations of EGF-R in respect to affinity for EGF were challenged. EGF-receptor density was found to vary from cell to cell, while the on- and off-rates did to a lesser extent. This approach increased the temporal resolution of binding phenomenon and can be used to analyze a variety of ligand and receptor dynamics.
Subject Area
Cellular biology|Biophysics|Molecular biology
Recommended Citation
Sciaky, Noah, "An investigation at the level of the individual A431 cell: The ATP-induced calcium response and the kinetics of EGF and EGF-R binding" (1993). Doctoral Dissertations Available from Proquest. AAI9329666.
https://scholarworks.umass.edu/dissertations/AAI9329666