In vitro and in vivo Single Molecular Experiments of Biological Systems

University Of California-Berkeley, Berkeley CA

Investigators

Abstract

This project represents the continuation of a program in single molecule studies of polymer and biological physics that began in 1998 and that has resulted in a number of advances in single-molecule approaches to the study of biological systems. The core focus of the continuation consists of three parts. (i) Single molecule fluorescence combined with force measurements will be integrated with structural and biochemical data to develop a mechanistic understanding of translation by the ribosome. (ii) Studies of in vivo cell signaling in dorsal ganglia root (DRG) neurons induced by neural growth factor (NGF) using quantum dot-labeled NGF and the photo-activated green fluorescent protein labeled TrkA receptor will be undertaken. Studies of further downstream signaling activity with high spatial and temporal resolution are part of this effort. (iii) A research program studying the cell adhesion molecule E-cadherin with combined single molecule FRET and atomic force measurements constitutes the third project. A new, broad-area, non-fluorescing afm tip has been developed and will be used to study the formation and structure of the extracellular adhesive complex and the cellular controls of the strength of cadherin adhesion using live cells. The downstream cell signaling functions of cadherin will be investigated with micro-fluidic methods. In terms of broader impact, methods, such as optical tweezers, acousto-optic feedback control of optical tweezers, novel flow cells for polymer dynamics studies, FRET with immobilized molecules in stopped-flow geometries, improved bio-compatible surfaces for single molecule enzymology, and the suppression of dye-photobleaching were developed in the course of this research work and are now widely used by other researchers. Students and postdoctoral fellows trained by this P.I. in biophysics are now professors at major research institutions. The present focus on technique development and the training of students and postdocs is expected to continue this trend. Funding for this award is provided by the Division of Physics in the Directorate for Mathematical and Physical Sciences and by the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences.