Observing Tyrosine Kinase Function by Polarization Resolved Fluorescence Microscopy

Abstract

Receptor tyrosine kinases are membrane proteins crucial for cell signaling processes. Using techniques of molecular biology, we are trying to develop fluorescently labeled receptor kinases molecules that would allow observations of receptor kinase activation by two-photon polarization microscopy (2PPM). 2PPM is an advanced optical microscopy technique developed by our laboratory that allows observing changes in orientation of fluorescent labels attached to membrane proteins, such as due to conformational changes in the membrane proteins. 2PPM works by analyzing fluorescence images acquired with two perpendicular linear polarizations of the excitation light. Differences between the two images can be used to gain insights into the orientation of the observed fluorescent label, and hence into the conformation of the labeled protein. We have now applied the technique to several tyrosine kinase constructs prepared by other laboratories, as well as to constructs we prepared ourselves. We are now in the process of modifying existing constructs in order to be able to visualize activation of several tyrosine kinase receptors important in disease (such as the insulin receptor in diabetes) and in drug discovery. The poster summarizes our progress and current results.