Quantitative Analysis of Phosphoinositide 3‐Kinase (PI3K) Signaling Using Live‐Cell Total Internal Reflection Fluorescence (TIRF) Microscopy

Heath E. Johnson1, Jason M. Haugh1

1 Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 14.14
DOI:  10.1002/0471143030.cb1414s61
Online Posting Date:  December, 2013
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Abstract

This unit focuses on the use of total internal reflection fluorescence (TIRF) microscopy and image analysis methods to study the dynamics of signal transduction mediated by class I phosphoinositide 3‐kinases (PI3Ks) in mammalian cells. The first four protocols cover live‐cell imaging experiments, image acquisition parameters, and basic image processing and segmentation. These methods are generally applicable to live‐cell TIRF experiments. The remaining protocols outline more advanced image analysis methods, which were developed in our laboratory for the purpose of characterizing the spatiotemporal dynamics of PI3K signaling. These methods may be extended to analyze other cellular processes monitored using fluorescent biosensors. Curr. Protoc. Cell Biol. 61:14.14.1‐14.14.24. © 2013 by John Wiley & Sons, Inc.

Keywords: signal transduction; image analysis; image processing; cell migration; cell biophysics

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Preparation of Cells and Glass Surfaces for Imaging
  • Basic Protocol 2: Live‐Cell Imaging Using TIRF Microscopy
  • Basic Protocol 3: Image Processing
  • Basic Protocol 4: Segmentation of PI3K Signaling Hot Spots
  • Basic Protocol 5: Calculating the Macroscopic PI3K Signaling Vector
  • Basic Protocol 6: PI3K Hot Spot Tracking
  • Basic Protocol 7: Constructing a Spatiotemporal Map of PI3K Signaling
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Cells and Glass Surfaces for Imaging

  Materials
  • Cell line stably expressing a biosensor or cell culture to be transiently transfected, e.g., NIH 3T3 cells (American Type Culture Collection), to express a biosensor
  • DNA plasmid encoding the biosensor of interest (e.g., Addgene, cat. no. 21218)
  • Appropriate cell culture medium with fetal bovine serum (FBS) and antibiotics (e.g., see unit 1.2)
  • Opti‐MEM serum‐free medium, Plus Reagent, and Lipofectamine (Life Technologies) or other appropriate transfection agents (units 20.3 20.7, 24.4)
  • Fibronectin (BD Biosciences), poly‐D‐lysine (Sigma‐Aldrich), or other adhesion‐promoting molecules, as required
  • Deionized water, sterile
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Trypsin/EDTA (Life Technologies), if required
  • Imaging buffer (see recipe)
  • 35‐mm tissue culture–treated dishes (e.g., BD Biosciences)
  • 20/35‐mm glass‐bottom dishes (MatTek)
  • 37°C incubator or water bath
  • 1.5‐ml microcentrifuge tubes
  • Hemacytometer or Coulter counter

Basic Protocol 2: Live‐Cell Imaging Using TIRF Microscopy

  Materials
  • Dye solution (e.g., Alexa‐conjugated dextrans, Life Technologies)
  • Lens paper
  • Methanol
  • Immersion oil (n = 1.52)
  • Transfected cells plated on coated cover slips or glass‐bottom dishes ( protocol 1)
  • TIRF microscope setup (see Strategic Planning), including
    • Appropriate lasers and filters
    • Heating system for stage or enclosure
  • Cooled charge‐coupled device (CCD) or complementary metal oxide semiconductor (CMOS) digital camera with appropriate acquisition software (e.g., Hamamatsu)
  • Humidification system (optional)
  • Image acquisition software (e.g., MetaMorph, Molecular Devices)

Basic Protocol 3: Image Processing

  MaterialsHardware
  • Computer workstation (system requirements for MATLAB 2009b available at http://www.mathworks.com/support/sysreq/release2009bi>; additional RAM may be required for processing many or large images)
Software
  • ImageJ (http://rsb.info.nih.gov/ij/download.html)
  • MATLAB (MathWorks)
Files
  • Image stacks or sequences ( protocol 2)

Basic Protocol 4: Segmentation of PI3K Signaling Hot Spots

  MaterialsHardware
  • Computer workstation (system requirements for MATLAB 2009b available at http://www.mathworks.com/support/sysreq/release2009b; additional RAM may be required for processing many or large images)
Software
  • MATLAB (MathWorks)
Files
  • Images in MATLAB ( protocol 3)

Basic Protocol 5: Calculating the Macroscopic PI3K Signaling Vector

  Materials listHardware
  • Computer workstation (system requirements for MATLAB 2009b available at http://www.mathworks.com/support/sysreq/release2009b; additional RAM may be required for processing many or large images)
Software
  • MATLAB (MathWorks)
Files
  • Hot spots identified by segmentation ( protocol 4)

Basic Protocol 6: PI3K Hot Spot Tracking

  MaterialsHardware
  • Computer workstation (system requirements for MATLAB 2009b available at http://www.mathworks.com/support/sysreq/release2009b; additional RAM may berequired for processing many or large images)
Software
  • MATLAB (MathWorks)
Files
  • Hot spots identified by segmentation ( protocol 4)

Basic Protocol 7: Constructing a Spatiotemporal Map of PI3K Signaling

  MaterialsHardware
  • Computer workstation (system requirements for MATLAB 2009b available at http://www.mathworks.com/support/sysreq/release2009b; additional RAM may be required for processing many or large images)
Software
  • MATLAB (MathWorks)
Files
  • Segmented PI3K hot spots ( protocol 4)
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Figures

Videos

Literature Cited

Literature Cited
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