A STUDY OF NGF TRAFFICKING USING A COMBINATION OF MICROSCOPY TECHNIQUES

MARÍA M. ECHARTE; SILVIO J. LUDUEÑA; LÍA I. PIETRASANTA

Rio de Janeiro

Workshop; Workshop; 2nd Internacional workshop on spectroscopy for biology.; 2003; 2004

Our general aim is to study the structure and dynamics of biological macromolecules by single molecule spectroscopy and imaging. The approach is to combine atomic force microscopy, confocal and wide-field fluorescence microscopy. In particular we focus on the molecular tracking of nerve growth factor (NGF) in living PC12 cells. To achieve our proposals we use quantum dots (QDs) as topographic and fluorescent probes to visualize the binding, internalization and processing of NGF. Quantum dots are nanometer-scale crystals of semiconductor material. Their high photostability and the fact that they are conjugated to streptavidin make them ideal probes for cellular imaging over long time periods. Lidke et al. (2004) showed that QDs, bearing natural ligands as effector molecules, provide the means for prolonged real-time visualizations of different steps in signaling mechanism in living cells. So far we have observed that NGF-biotin-QDs ligands bind specifically to differenciated PC12 cells and that they are slowly internalized. Using AFM we have also imaged the topography of these cells under different conditions, and plan to explore the potential of this technique to monitor the dynamics of QDs in this system. The types of data gathered from atomic force, confocal and wide-field microscopies are complementary allowing us to build up a more complete picture of NGF dynamics.