title: High affinity receptor labeling based on basic leucine zipper domain peptides conjugated with pH-sensitive fluorescent dye: Visualization of AMPA-type glutamate receptor endocytosis in living neurons.
abstract: Techniques to visualize receptor trafficking in living neurons are important, but currently available methods are limited in their labeling efficiency, specificity and reliability. Here we report a method for receptor labeling with a basic leucine zipper domain peptide (ZIP) and a binding cassette specific to ZIP. Receptors are tagged with a ZIP-binding cassette at their extracellular domain. Tagged receptors expressed in cultured cells were labeled with exogenously applied fluorescently labeled ZIP with low background and high affinity. To test if ZIP labeling is useful in monitoring endocytosis and intracellular trafficking, we next conjugated ZIP with a pH-sensitive dye RhP-M (ZIP-RhP-M). ZIP binding to its binding cassette was pH-resistant and RhP-M fluorescence dramatically increased in acidic environment. Thus AMPA-type glutamate receptors (AMPARs) labeled by ZIP-RhP-M can report receptor endocytosis and subsequent intracellular trafficking. Application of ZIP-RhP-M to cultured hippocampal neurons expressing AMPARs tagged with a ZIP-binding cassette resulted in appearance of fluorescent puncta in PSD-95-positive large spines, suggesting local endocytosis and acidification of AMPARs in individual mature spines. This spine pool of AMPARs in acidic environment was distinct from the early endosomes labeled by transferrin uptake. These results suggest that receptor labeling by ZIP-RhP-M is a useful technique for monitoring endocytosis and intracellular trafficking. This article is part of the Special Issue entitled 'Synaptopathy--from Biology to Therapy'.
authors.affiliation: Department of Cellular Neurobiology, Graduate School of Medicine, The University of Tokyo, Japan; CREST, JST, Japan.
authors.forename: Ayako
authors.initials: A
authors.lastname: Hayashi
authors.affiliation: Department of Neurobiology, Graduate School of Medicine, The University of Tokyo, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Asanuma
authors.affiliation: Laboratory of Chemical Biology and Molecular Imaging, Graduate School of Medicine, The University of Tokyo, Japan.
authors.forename: Mako
authors.initials: M
authors.lastname: Kamiya
authors.affiliation: Laboratory of Chemical Biology and Molecular Imaging, Graduate School of Medicine, The University of Tokyo, Japan.
authors.forename: Yasuteru
authors.initials: Y
authors.lastname: Urano
authors.affiliation: Department of Cellular Neurobiology, Graduate School of Medicine, The University of Tokyo, Japan; CREST, JST, Japan. Electronic address: okabe@m.u-tokyo.ac.jp.
authors.forename: Shigeo
authors.initials: S
authors.lastname: Okabe
mesh_terms: D000345:Affinity Labels / Q000493:pharmacokinetics; D000818:Animals; D002478:Cells, Cultured; D049229:Dendritic Spines / Q000502:physiology*; D004705:Endocytosis; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D006624:Hippocampus / Q000166:cytology / Q000502:physiology; D016350:Leucine Zippers; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011522:Protons; D018091:Receptors, AMPA / Q000502:physiology*; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D000345:Affinity Labels; D005456:Fluorescent Dyes; D011522:Protons; D018091:Receptors, AMPA
keywords: AMPA-type glutamate receptor; Cell surface receptor; Endocytosis; Imaging; Neuron