Visualization of Cellular Phosphoinositide Pools with GFP‐Fused Protein‐Domains

Tamas Balla1, Péter Várnai2

1 National Institutes of Health, Bethesda, Maryland, 2 Semmelweis University of Medical School, Budapest, Hungary
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 24.4
DOI:  10.1002/0471143030.cb2404s42
Online Posting Date:  March, 2009
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This unit describes the method of following phosphoinositide dynamics in live cells. Inositol phospholipids have emerged as universal signaling molecules present in virtually every membrane of eukaryotic cells. Phosphoinositides are present in only tiny amounts as compared to structural lipids, but they are metabolically very active as they are produced and degraded by the numerous inositide kinase and phosphatase enzymes. Phosphoinositides control the membrane recruitment and activity of many membrane protein signaling complexes in specific membrane compartments, and they have been implicated in the regulation of a variety of signaling and trafficking pathways. It has been a challenge to develop methods that allow detection of phosphoinositides at the single‐cell level. The only available technique in live cell applications is based on the use of the same protein domains selected by evolution to recognize cellular phosphoinositides. Some of these isolated protein modules, when fused to fluorescent proteins, can follow dynamic changes in phosphoinositides. While this technique can provide information on phosphoinositide dynamics in live cells with subcellular localization, and it has rapidly gained popularity, it also has several limitations that must be taken into account when interpreting the data. This unit summarizes the design and practical use of these constructs and also reviews important considerations for interpretation of the data obtained by this technique. Curr. Protoc. Cell Biol. 42:24.4.1‐24.4.27. © 2009 by John Wiley & Sons, Inc.

Keywords: phosphoinositide; FRET; live‐cell imaging; green fluorescent proteins; pleckstrin homology domain; fluorescence microscopy

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Expression of Fluorescent Proteins in Mammalian Cells
  • Basic Protocol 2: Verifying Structural Integrity of the Fusion Construct
  • Basic Protocol 3: Observe GFP Signal by Microscopy
  • Basic Protocol 4: Live‐Cell Imaging
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Expression of Fluorescent Proteins in Mammalian Cells

  • 98% ethanol
  • 0.1% (w/v) poly‐L‐lysine in water (Sigma cat. no. P‐8920)
  • Cultured cells of interest
  • Culture medium with serum and antibiotics (depending on the cells)
  • Plasmid DNA (usually a midiprep)
  • Transfection reagents (depending on the cells in use; units 20.3, 20.4, 20.5, 20.6, & 20.7)
  • Phosphate buffered saline (PBS)
  • 2% (w/v) paraformaldehyde (see recipe)
  • Blocking solution (10% FBS in PBS made fresh)
  • Primary antibody against fluorescent protein (available from several commercial sources)
  • 0.2% saponin
  • Fluorophore‐conjugated secondary antibody
  • Aqua‐Poly/Mount (Polysciences)
  • Clear nail polish
  • 25‐mm coverslips (PGC Scientific cat. no. 60‐4884‐25 or Warner Instruments cat. no. 64‐0705) for TIRF applications
  • 6‐well culture dishes
  • Glass microscope slides
  • Fluorescent microscope

Basic Protocol 2: Verifying Structural Integrity of the Fusion Construct

  • Cultured cells of interest
  • Culture medium with serum and antibiotics (depending on the cells)
  • Plasmid DNA (usually a midiprep)
  • Transfection reagents (depending on cells used)
  • Phosphate buffered saline (PBS)
  • Laemmli buffer
  • 10‐cm SDS‐PAGE acrylamide gel
  • Gel running buffer
  • 12‐well culture plates
  • 1.5‐ml microcentrifuge tubes
  • Sonicator
  • SDS gel apparatus
  • Phosphorimager (or reagents and apparatus for immunoblotting)

Basic Protocol 3: Observe GFP Signal by Microscopy

  • Coverslips holding transfected cells
  • Medium appropriate for cells (e.g., modified Krebs‐Ringer solution; see recipe)
  • Immersion oil
  • Chambers to hold coverslips (e.g., metal Atto chambers, Invitrogen)
  • Kimwipes
  • Wide‐field fluorescence microscope equipped with sensitive camera and appropriate software for data acquisition or confocal microscope
  • Lens cleaning paper
  • Objective heater and heated stage (Bioptechs, or a complete temperature control enclosure
  • Computer controlled valve‐system and perifusion (optional)
  • Forceps
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Literature Cited

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