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OCT 26, 2020 11:00 AM EDT

Introduction to FLIM-FRET techniques

Speaker
  • Director Biological Sciences, Sunnybrook Research Institute
    Biography

      Dr. David Andrews is Director of and senior scientist in Biological Sciences at Sunnybrook Research Institute, Professor of Biochemistry and Medical Biophysics at University of Toronto and a Tier 1 Canada Research Chair in Membrane Biogenesis. His research includes, the molecular mechanisms by which Bcl-2 family proteins regulate apoptosis at mitochondria, mechanisms of protein-protein interactions, the assembly of proteins into membranes, high-content screening and development of new microscopes for fluorescence lifetime imaging microscopy.



      Dr. Andrews uses fluorescence spectroscopy, fluorescence lifetime imaging and automated fluorescence microscopy to study protein-protein interactions in live cells and in membranes, protein localization in cells and the effects of drugs on cells. His lab has proposed the now widely accepted model for how Bcl-2 family proteins regulate apoptosis and has discovered and characterized small molecules that inhibit Bcl-2 family protein mediated mitochondrial outer membrane permeabilization that have applications to cancer and regenerative medicine.



      Dr. Andrews is active in the public and private sector. In recent years he was president of the Society for Biomolecular Imaging and Informatics. He is a member of the editorial board of Cell Death and Differentiation. He participated in the start-up of two companies, Fermentas and Isogenica and consults for several other companies and academic centers. His group performs collaborative and contract research for a variety of biotech companies including ABBVIE, Eli Lilly, Johnson and Johnson, Novartis and Celgene. He holds licensed patents in areas such as translational regulation, in vitro evolution, peptide display technologies and optical microscopy.


    Abstract

    Fluorescence Resonance Energy Transfer (FRET) between fluorescence proteins has been implemented for a number of biosensors in which the donor and acceptor are linked in a single sensor.  For example, many sensors have been published to measure caspase activity in live cells using a single molecule consisting of a CFP donor linked via a caspase site to an YFP acceptor. In single molecule sensors FRET can be measured using either stimulated emission or Fluorescence Lifetime Imaging Microscopy (FLIM) because the ratio of donor to acceptor is one. However, to measure protein:protein interactions in live cells is more complicated because the relative concentration of donor and acceptor are unknown. Because fluorescence lifetime is independent of concentration, it is possible to use FLIM FRET to quantify binding between any two proteins in live cells. By successfully automating FLIM FRET assays for high throughput we enabled examining the combinatorial interactions between 4 anti-apoptosis proteins with 6 different pro-apoptotic binding partners and their modulation by 15 drugs at 5 concentrations each.

    In this webinar we will discuss the benefits and limitations of different methods for measuring FRET in cells. I will describe the exceptional utility of this kind of data in early stage drug development. Also discussed will be the steps required to surmount the challenges posed when generating 1800 binding curves in a single experiment with live cells and how to interpret the data to provide the most useful single value to guide medicinal chemistry efforts. Finally, we will explore some of the unexpected insights garnered from our data examining inhibitors on anti-apoptosis proteins in live cells.  

    Learning Objectives:

    1. Understanding what FRET is and how it can be used to measure protein:protein interactions

    2. Understanding how FRET can be measured in live cells using automated microscopy


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