Live-cell imaging of single receptor composition using zero-mode waveguide nanostructures.

We exploit the optical and spatial features of subwavelength nanostructures to examine individual receptors on the plasma membrane of living cells. Receptors were sequestered in portions of the membrane projected into zero-mode waveguides. Using single-step photobleaching of green fluorescent protein incorporated into individual subunits, the resulting spatial isolation was used to measure subunit stoichiometry in α4β4 and α4β2 nicotinic acetylcholine and P2X2 ATP receptors. We also show that nicotine and cytisine have differential effects on α4β2 stoichiometry.

[1]  Hervé Rigneault,et al.  Bright unidirectional fluorescence emission of molecules in a nanoaperture with plasmonic corrugations. , 2011, Nano letters.

[2]  E. Perry,et al.  Differential nicotinic acetylcholine receptor subunit expression in the human hippocampus , 2003, Journal of Chemical Neuroanatomy.

[3]  Erik A. Rodriguez,et al.  Single-molecule imaging of a fluorescent unnatural amino acid incorporated into nicotinic receptors. , 2009, Biophysical journal.

[4]  H. Lester,et al.  Characterizing functional α6β2 nicotinic acetylcholine receptors in vitro: mutant β2 subunits improve membrane expression, and fluorescent proteins reveal responsive cells. , 2011, Biochemical pharmacology.

[5]  J. Changeux,et al.  Nicotine Upregulates Its Own Receptors through Enhanced Intracellular Maturation , 2005, Neuron.

[6]  S. Turner,et al.  Zero-Mode Waveguides for Single-Molecule Analysis at High Concentrations , 2003, Science.

[7]  J. Lindstrom,et al.  Ca2+ Permeability of the (α4)3(β2)2 Stoichiometry Greatly Exceeds That of (α4)2(β2)3 Human Acetylcholine Receptors , 2006, Molecular Pharmacology.

[8]  H. Lester,et al.  Neural Systems Governed by Nicotinic Acetylcholine Receptors: Emerging Hypotheses , 2011, Neuron.

[9]  H. Lester,et al.  Trafficking of alpha4* nicotinic receptors revealed by superecliptic phluorin: effects of a beta4 amyotrophic lateral sclerosis-associated mutation and chronic exposure to nicotine. , 2011, The Journal of biological chemistry.

[10]  H. Lester,et al.  Nicotine up-regulates α4β2 nicotinic receptors and ER exit sites via stoichiometry-dependent chaperoning , 2011, The Journal of general physiology.

[11]  A. C. Collins,et al.  Effects of chronic nicotine infusion on tolerance development and nicotinic receptors. , 1983, The Journal of pharmacology and experimental therapeutics.

[12]  E. Isacoff,et al.  Subunit counting in membrane-bound proteins , 2007, Nature Methods.

[13]  Toshio Yanagida,et al.  Single-molecule imaging of EGFR signalling on the surface of living cells , 2000, Nature Cell Biology.

[14]  A. Kuryatov,et al.  Nicotine Acts as a Pharmacological Chaperone to Up-Regulate Human α4β2 Acetylcholine Receptors , 2005, Molecular Pharmacology.

[15]  T. Ebbesen,et al.  Plasmonic antennas for directional sorting of fluorescence emission. , 2011, Nano letters.

[16]  D. Bertrand,et al.  Chronic Exposure to Nicotine Upregulates the Human α4β2 Nicotinic Acetylcholine Receptor Function , 2001, The Journal of Neuroscience.

[17]  P. Selvin,et al.  Counting bungarotoxin binding sites of nicotinic acetylcholine receptors in mammalian cells with high signal/noise ratios. , 2010, Biophysical journal.

[18]  J. Lippincott-Schwartz,et al.  Imaging Intracellular Fluorescent Proteins at Nanometer Resolution , 2006, Science.

[19]  H. Craighead,et al.  An array of planar apertures for near-field fluorescence correlation spectroscopy. , 2011, Biophysical journal.

[20]  Barbara Baird,et al.  High spatial resolution observation of single-molecule dynamics in living cell membranes. , 2005, Biophysical journal.

[21]  B. Hille,et al.  Functional stoichiometry of the unitary calcium-release-activated calcium channel , 2008, Proceedings of the National Academy of Sciences.

[22]  H. Vogel,et al.  Correlated Optical and Electrical Single‐Molecule Measurements Reveal Conformational Diffusion from Ligand Binding to Channel Gating in the Nicotinic Acetylcholine Receptor , 2011, Chembiochem : a European journal of chemical biology.

[23]  R. D. Schwartz,et al.  In Vivo Regulation of [3H]Acetylcholine Recognition Sites in Brain by Nicotinic Cholinergic Drugs , 1985, Journal of neurochemistry.

[24]  H. Lester,et al.  Pharmacological Chaperoning of Nicotinic Acetylcholine Receptors Reduces the Endoplasmic Reticulum Stress Response , 2012, Molecular Pharmacology.

[25]  F. Pinaud,et al.  High affinity scFv-hapten pair as a tool for quantum dot labeling and tracking of single proteins in live cells. , 2008, Nano letters.

[26]  J. Frydman,et al.  Action of the chaperonin GroEL/ES on a non-native substrate observed with single-molecule FRET. , 2010, Journal of molecular biology.

[27]  X. Zhuang,et al.  Superresolution Imaging of Chemical Synapses in the Brain , 2010, Neuron.

[28]  E. Gouaux,et al.  Crystal structure of the ATP-gated P2X4 ion channel in the closed state , 2009, Nature.

[29]  J. Lindstrom,et al.  Alternate Stoichiometries of α4β2 Nicotinic Acetylcholine Receptors , 2003 .

[30]  H. Craighead,et al.  Zero-mode waveguides: sub-wavelength nanostructures for single molecule studies at high concentrations. , 2008, Methods.

[31]  D. K. Berg,et al.  Lateral Mobility of Nicotinic Acetylcholine Receptors on Neurons Is Determined by Receptor Composition, Local Domain, and Cell Type , 2010, The Journal of Neuroscience.

[32]  Hervé Rigneault,et al.  Diffusion analysis within single nanometric apertures reveals the ultrafine cell membrane organization. , 2007, Biophysical journal.

[33]  J. Taraska,et al.  Fluorescence Applications in Molecular Neurobiology , 2010, Neuron.

[34]  E. Sher,et al.  α4β2 Nicotinic Receptors with High and Low Acetylcholine Sensitivity: Pharmacology, Stoichiometry, and Sensitivity to Long-Term Exposure to Nicotine , 2006, Molecular Pharmacology.