Imaging the Insertion of Superecliptic pHluorin‐Labeled Dopamine D2 Receptor Using Total Internal Reflection Fluorescence Microscopy

Kathryn M. Daly1, Yun Li1, Da‐Ting Lin2

1 Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, Maryland, 2 The Jackson Laboratory, Bar Harbor, Maine
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 5.31
DOI:  10.1002/0471142301.ns0531s70
Online Posting Date:  January, 2015
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A better understanding of mechanisms governing receptor insertion to the plasma membrane (PM) requires an experimental approach with excellent spatial and temporal resolutions. Here we present a strategy that enables dynamic visualization of insertion events for dopamine D2 receptors into the PM. This approach includes tagging a pH‐sensitive GFP, superecliptic pHluorin, to the extracellular domain of the receptor. By imaging pHluorin‐tagged receptors under total internal reflection fluorescence microscopy (TIRFM), we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This novel imaging approach can be applied to both secreted proteins and many membrane proteins with an extracellular domain labeled with superecliptic pHluorin, and will ultimately allow for detailed dissections of the key mechanisms governing secretion of soluble proteins or the insertion of different membrane proteins to the PM. © 2015 by John Wiley & Sons, Inc.

Keywords: TIRF microscopy; superecliptic pHluorin; protein trafficking; insertion; GPCR

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Table of Contents

  • Introduction
  • Basic Protocol 1: Interfacial Neuron‐Glia Culture
  • Basic Protocol 2: Neuronal Transfection with Lipofectamine 2000 Using Plasmid cDNA
  • Basic Protocol 3: Total Internal Reflection Fluorescence Imaging
  • Basic Protocol 4: Manual Data Analysis for Tirfm Visualization of Insertion Events
  • Alternate Protocol 1: Data Analysis for TIRFM Visualization of Insertion Events with Wombat Software
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Interfacial Neuron‐Glia Culture

  • Purified bovine collagen solution, Type I, 3 mg/ml (Advanced Biomatrix, cat. no. 5005‐B)
  • Dissection buffer (see recipe)
  • Glia culture medium (see recipe)
  • Neonatal mice
  • Papain solution (see recipe)
  • Phosphate‐buffered saline (PBS; Life Technologies, cat. no. AM9625), 10×
  • 0.05% trypsin‐EDTA (1×) (Life Technologies, cat. no. 25300‐054)
  • 70% nitric acid
  • Sterile deionized water
  • Poly‐L‐lysine solution (see recipe)
  • Neuronal plating media (see recipe)
  • E15‐18 pregnant mice
  • CO 2
  • 75‐cm2 culture flasks
  • 37°C water bath
  • Dissection microscope
  • 15‐ml conical tubes
  • Fire‐polished glass Pasteur pipets
  • 70‐μm cell strainer
  • 37°C cell culture incubator
  • Culture inserts (Millipore, cat. no. PICM03050)
  • 6‐well plates
  • 25‐mm round coverslips (Warner Instruments)
  • Thomas Cover Glass Staining Outfits (Thomas Scientific)
  • Large glass dish
  • Isotemp oven
  • Hemacytometer

Basic Protocol 2: Neuronal Transfection with Lipofectamine 2000 Using Plasmid cDNA

  • Lipofectamine 2000 (Life Technologies, cat. no. 11668‐019)
  • Neurobasal medium (Life Technologies, cat. no. 21103‐049)
  • Plasmid cDNA superecliptic pHluorin Drd2 (see Li et al., 2012)
  • Neuronal culture medium + B27 (see recipe)
  • Neuronal‐glia culture (see protocol 1)
  • 1.5‐ml microcentrifuge tubes
  • 50‐ml conical tubes
  • 37°C incubator

Basic Protocol 3: Total Internal Reflection Fluorescence Imaging

  • Artificial cerebrospinal fluid (aCSF) (see recipe)
  • Lens cleaning solution
  • Transfected neurons (see protocol 2)
  • Immersion oil (n = 1.52)
  • 37°C water bath
  • TIRF microscope setup including:
    • Inverted microscope (e.g., Zeiss AxioObserver D1)
    • Light source
      • 488‐nm excitation laser (e.g., Newport Excelsior 200 mW)
      • Epi‐fluorescent light source (e.g., Zeiss metal halide lamp HXP 120V)
    • Laser beam shutter and controller (e.g., Vincent Associates Uniblitz LS6 shutter, VCM–D1 controller)
    • Filter cubes in microscope (e.g., Chroma Technology Corporation)
    • EGFP: excitation filter (ET470/40×) dichroic mirror (Z488RDC), emission filter (ET525/50 m)
    • A‐Plan 100× Objective lens (e.g., Zeiss, NA 1.46)
    • sCMOS camera (e.g., Hamamatsu Photonics ORCA Flash 4.0 v2)
    • Image acquisition software (μManager)
    • Heating insert for live imaging (specific model depends on microscope platform used)
    • Live Cell Imaging Chamber (specific model depends on microscope platform used)
  • Lens paper
NOTE: The imaging system described here and in Figure is the setup used by the authors’ laboratory. In principle, any commercial TIRF imaging platform with sufficient laser excitation and either a sCMOS or Electron Multiplying Charge Coupled Device (EMCCD) camera should provide comparable imaging performance.

Basic Protocol 4: Manual Data Analysis for Tirfm Visualization of Insertion Events

  • Recombinant GFP (Clontech)
  • Artificial Cerebrospinal Fluid (aCSF) (see recipe)
  • TIRF microscope setup (see protocol 3)
  • Computer
  • Image processing software (ImageJ)
  • Microsoft Excel

Alternate Protocol 1: Data Analysis for TIRFM Visualization of Insertion Events with Wombat Software

  Additional Materials (also see protocol 4)
  • Wombat insertion analysis software
  • Microsoft Excel or similar spreadsheet software (optional)
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Literature Cited

  Alberts, B., Bray, D., Johnson, A., Lewis, J., Raff, M., Roberts, K., and Walter, P. 1998. Cell communication. In Essential Cell Biology: An Introduction to the Molecular Biology of the Cell (M. Robertson, ed.) p. 481‐512. Garland Publishing, New York.
  Axelrod, D. 2001a. Selective imaging of surface fluorescence with very high aperture microscope objectives. J. Biomed. Opt. 6:6‐13.
  Axelrod, D. 2001b. Total internal reflection fluorescence microscopy in cell biology. Traffic 2:764‐774.
  Axelrod, D. 2013. Evanescent excitation and emission in fluorescence microscopy. Biophys. J. 104:1401‐1409.
  Binda, A.V., Kabbani, N., Lin, R., and Levenson, R. 2002. D2 and D3 dopamine receptor cell surface localization mediated by interaction with protein 4.1N. Mol. Pharmacol. 62:507‐513.
  Blum, K., Sheridan, P.J., Wood, R.C., Braverman, E.R., Chen, T.J., Cull, J.G., and Comings, D.E. 1996. The D2 dopamine receptor gene as a determinant of reward deficiency syndrome. J. R. Soc. Med. 89:396‐400.
  Brady, R.J., Damer, C.K., Heuser, J.E., and O'Halloran, T.J. 2010. Regulation of Hip1r by epsin controls the temporal and spatial coupling of actin filaments to clathrin‐coated pits. J. Cell Sci. 123:3652‐3661.
  Carlsson, A. 1988. The current status of the dopamine hypothesis of schizophrenia. Neuropsychopharmacology 1:179‐186.
  Drake, M.T., Shenoy, S.K., and Lefkowitz, R.J. 2006. Trafficking of G protein‐coupled receptors. Circ. Res. 99:570‐582.
  Evans, N. 2004. Methods of measuring internalization of G protein‐coupled receptors. Curr. Protoc. Pharmacol. 24:12.6.1‐12.6.22.
  Filmore, D. 2004. It's a GPCR world. Modern Drug Discov. 7:24‐28.
  Free, R.B., Hazelwood, L.A., Cabrera, D.M., Spalding, H.N., Namkung, Y., Rankin, M.L., and Sibley, D.R. 2007. D1 and D2 dopamine receptor expression is regulated by direct interaction with the chaperone protein calnexin. J. Biol. Chem. 282:21285‐21300.
  Funatsu, T., Harada, Y., Tokunaga, M., Saito, K., and Yanagida, T. 1995. Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution. Nature 374:555‐559.
  Genedani, S., Carone, C., Guidolin, D., Filaferro, M., Marcellino, D., Fuxe, K., and Agnati, L.F. 2010. Differential sensitivity of A2A and especially D2 receptor trafficking to cocaine compared with lipid rafts in cotransfected CHO cell lines. Novel actions of cocaine independent of the DA transporter. J. Mol. Neurosci. 41:347‐357.
  Jaiswal, J.K. and Simon, S.M. 2003. Total internal reflection fluorescence microscopy for high‐resolution imaging of cell‐surface events. Curr. Protoc. Cell Biol. 20:4.12.1‐4.12.15.
  Johnson, D.S., Jaiswal, J.K., and Simon, S. 2012. Total internal reflection fluorescence (TIRF) microscopy illuminator for improved imaging of cell surface events. Curr. Protoc. Cytom. 61:12.29.1‐12.29.19.
  Karam, C.S., Ballon, J.S., Bivens, N.M., Freyberg, Z., Girgis, R.R., Lizardi‐Ortiz, J.E., Markx, S., Lieberman, J.A., and Javitch, J.A. 2010. Signaling pathways in schizophrenia: Emerging targets and therapeutic strategies. Trends Pharmacol. Sci. 31:381‐390.
  Kim, C.H. and Lisman, J.E. 2001. A labile component of AMPA receptor‐mediated synaptic transmission is dependent on microtubule motors, actin, and N‐ethylmaleimide‐sensitive factor. J. Neurosci. 21:4188‐4194.
  Kotowski, S.J., Hopf, F.W., Seif, T., Bonci, A., and von Zastrow, M. 2011. Endocytosis promotes rapid dopaminergic signaling. Neuron 71:278‐290.
  Lappano, R. and Maggiolini, M. 2011. G protein‐coupled receptors: Novel targets for drug discovery in cancer. Nat. Rev. Drug Discov. 10:47‐60.
  Lawford, B.R., Young, R., Noble, E.P., Kann, B., and Ritchie, T. 2006. The D2 dopamine receptor (DRD2) gene is associated with co‐morbid depression, anxiety and social dysfunction in untreated veterans with post‐traumatic stress disorder. Eur. Psychiatry 21:180‐185.
  Li, Y., Roy, B.D., Wang, W., Zhang, L., Sampson, S.B., Yang, Y., and Lin, D.T. 2012. Identification of two functionally distinct endosomal recycling pathways for dopamine D2 receptor. J. Neurosci. 32:7178‐7190.
  Lin, J.T., Chang, W.C., Chen, H.M., Lai, H.L., Chen, C.Y., Tao, M.H., and Chern, Y. 2013. Regulation of feedback between protein kinase A and the proteasome system worsens Huntington's disease. Mol. Cell Biol. 33:1073‐1084.
  Lin, R., Canfield, V., and Levenson, R. 2002. Dominant negative mutants of filamin A block cell surface expression of the D2 dopamine receptor. Pharmacology 66:173‐181.
  Luoma, J.I., Kelley, B.G., and Mermelstein, P.G. 2011. Progesterone inhibition of voltage‐gated calcium channels is a potential neuroprotective mechanism against excitotoxicity. Steroids 76:845‐855.
  Macey, T.A., Gurevich, V.V., and Neve, K.A. 2004. Preferential Interaction between the dopamine D2 receptor and Arrestin2 in neostriatal neurons. Mol. Pharmacol. 66:1635‐1642.
  Miesenböck, G., De Angelis, D.A., and Rothman, J.E. 1998. Visualizing secretion and synaptic transmission with pH‐sensitive green fluorescent proteins. Nature 394:192‐195.
  Millet, L.J. and Gillette, M.U. 2012. Over a century of neuron culture: From the hanging drop to microfluidic devices. Yale J. Biol. Med. 85:501‐521.
  Park, H.J., Kim, S.T., Yoon, D.H., Jin, S.H., Lee, S.J., Lee, H.J., and Lim, S. 2005. The association between the DRD2 TaqI A polymorphism and smoking cessation in response to acupuncture in Koreans. J. Altern. Complement. Med. 11:401‐405.
  Piston, D.W. 1998. Choosing objective lenses: The importance of numerical aperture and magnification in digital optical microscopy. Biol. Bull. 195:1‐4.
  Sankaranarayanan, S., De Angelis, D., Rothman, J.E., and Ryan, T.A. 2000. The use of pHluorins for optical measurements of presynaptic activity. Biophys. J. 79:2199‐2208.
  Tirotta, E., Fontaine, V., Picetti, R., Lombardi, M., Samad, T.A., Oulad‐Abdelghani, M., Edwards, R., and Borrelli, E. 2008. Signaling by dopamine regulates D2 receptors trafficking at the membrane. Cell Cycle 7:2241‐2248.
Internet Resources
  μManager: open source microscopy software.
  ImageJ: Image processing and analysis in Java.
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