Elsevier

Neuropharmacology

Volume 100, January 2016, Pages 66-75
Neuropharmacology

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

https://doi.org/10.1016/j.neuropharm.2015.07.026Get rights and content

Highlights

  • A basic leucine zipper domain peptide (ZIP) can be used for surface receptor labeling.

  • ZIP tagged with pH-sensitive fluorescent dye (RhP-M) detects receptor endocytosis.

  • Both ionotropic and metabotropic glutamate receptors can be labeled by ZIP-RhP-M.

  • ZIP-RhP-M detects internalized AMPA receptors within dendrites in living neurons.

  • Majority of AMPA receptor-containing endosomes are localized at dendritic spines.

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’.

Introduction

The number, distribution and modulation of cell surface receptors are main determinants of cellular responses to extracellular signals (Craig et al., 1993, Das and Banker, 2006, Kopec et al., 2007). Receptors bind their ligands on the cell surface at neutral pH of the extracellular environment. The fates of receptors after ligand binding are diverse but some undergo endocytosis (Bonifacino and Traub, 2003). Immediately after endocytosis, the pH in the endocytotic vesicles drops rapidly by V-type H+-ATP-dependent proton pumps (Murata et al., 2002). Receptors are dissociated from their ligands in acidic environment and sorted either to the degradation pathway or recycled back to the plasma membrane. Intracellular trafficking and recycling of receptors are complex processes and full understanding of their regulation requires development of novel techniques of tracing receptors in living cells.

AMPA type glutamate receptors (AMPARs) play a major role in the expression of synaptic plasticity (Kessels and Malinow, 2009, Malinow and Malenka, 2002). Postsynaptic AMPAR response can be modulated either by phosphorylation or recruitment of new AMPARs from intracellular pools (Liao et al., 1999). Accumulating evidences indicate that rapid insertion and removal of synaptic AMPARs take place in response to plasticity-inducing stimuli (Carroll et al., 1999, Luscher et al., 2007). Recycling of AMPARs is also important in brain diseases, including neurodegenerative disorders such as Alzheimer's disease (Wu et al., 2011) and developmental disorders such as Angelman syndrome (Greer et al., 2010) and fragile X-syndrome (Nakamoto et al., 2007).

Determination of exact location of AMPAR recycling is critical in clarifying the mechanisms of synaptic plasticity. However, there are few available techniques for live detection of AMPAR recycling (Chen et al., 2005). Lateral diffusion of AMPARs on the cell membrane can be monitored by the technique of single particle tracking. These analyses reported that lateral diffusion and capture at the postsynaptic density (PSD) are important in AMPAR replenishment (Triller and Choquet, 2008). Supply of diffusing extrasynaptic AMPARs is regulated by the balance between exocytosis and endocytosis within or outside of spines. Exocytosis of AMPARs can be monitored by labeling AMPARs with Super-Ecliptic pHluorin (SEP). Their endocytosis can be monitored by pulse-chase labeling of surface AMPARs and subsequent visualization of internalized receptors. These two techniques are useful in describing spatiotemporal pattern of AMPAR exocytosis and endocytosis, but potential problems associated with these techniques were also reported (Lin et al., 2000, Rathje et al., 2013).

Here we report a novel method for the labeling of the receptors by using a basic leucine zipper domain peptide (ZIP) and a binding cassette specific to ZIP (Moll et al., 2001, Maruo et al., 2008). We conjugated a fluorescent pH indicator RhP-M (Asanuma et al., 2014) to ZIP (ZIP-RhP-M) and applied it exogenously to the cells expressing AMPARs tagged with ZIP binding cassette. We found that the AMPARs labeled with ZIP-RhP-M were fluorescent in the acidic environment of internalized endosomes and can be monitored in living neurons. Fluorescence signals from AMPARs labeled with ZIP-RhP-M were enriched in large dendritic spines positive with PSD-95. Our results indicate that the ZIP system is a useful tool for real-time tracing of receptor recycling in living neurons.

Section snippets

Plasmid construction

A cDNA construct comprising mGluR1a containing an extracellular tag insertion site was generated by inserting a NheI-XhoI site at N terminal domain of mGluR1. Oligonucleotide pairs encoding a high-affinity peptide 2 (HAP2)-tag with a glycine tetramer linker were annealed and then ligated into the NheI-XhoI site. The DNA fragments of ZIP and ZIP-binding peptide (ZBP) were generated by PCR using artificially synthesized ZIP and ZBP DNA (GeneScript) as templates. The respective sequences of ZIP,

Labeling of cell surface receptors by the ZIP system

Both ZIP and ZBP are short peptides that form a leucine zipper with six pairs of oppositely charged amino acids (arginine and glutamic acid) (Fig. 1A). ZIP and ZBP form tight heterodimers, while self-dimerization of ZIP and ZBP was minimal. In this study, we mainly used ZIP as a motif for synthesizing fluorescently labeled probes and ZBP as a binding cassette tagged at the extracellular domains of glutamate receptors. To test efficiency and specificity of interaction between ZIP and ZBP-tagged

Discussion

Here we report a novel labeling method that enables visualizing membrane proteins on cell surface and subsequent endosomal trafficking. The ZIP-based labeling offers multiple advantages over available membrane protein labeling systems (Moll et al., 2001, Maruo et al., 2008). The ZIP-tag is much smaller than other molecular tags, including antibodies, fluorescent proteins and αBTx. Heteromeric binding of ZIP peptides is based on 14 α-helical motifs that are wrapped around each other to form a

Author contribution

AH carried out the imaging. DA, MK and YU synthesized RhP-M. AH, DA, MK, YU and SO designed the project. AH and SO wrote the paper. ALL author contributed the writing of the paper.

Acknowledgments

We thank to Dr. Iwasaki and Dr. Tanaka for advice on the project and Ms. Muranaga, Ms. Ohkubo and Ms. Urushido for making dissociated culture of neurons. This work was supported by Grants-in-Aid for Scientific Research (25117006, and 26250014 [to S.O.]) and by Core Research for Evolutional Science and Technology from the Japanese Science and Technology Agency [to S.O.].

References (38)

  • S. Usui et al.

    Synaptic targeting of PSD-Zip45 (Homer 1c) and its involvement in the synaptic accumulation of F-actin

    J. Biol. Chem.

    (2003)
  • J. Wu et al.

    Arc/Arg3.1 regulates an endosomal pathway essential for activity-dependent b-amyloid generation

    Cell

    (2011)
  • D. Asanuma et al.

    Acidic-pH-activatable fluorescence probes for visualizing exocytosis dynamics

    Angew. Chem. Int. Ed.

    (2014)
  • J.S. Bonifacino et al.

    Signals for sorting of transmembrane proteins to endosomes and lysosomes

    Annu. Rev. Biochem.

    (2003)
  • R.C. Carroll et al.

    Dynamin-dependent endocytosis of ionotropic glutamate receptors

    Proc. Natl. Acad. Sci. U. S. A.

    (1999)
  • I. Chen et al.

    Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase

    Nat. Methods

    (2005)
  • J.R. Cooney et al.

    Endosomal compartments serve multiple hippocampal dendritic spines from a widespread rather than a local store of recycling membrane

    J. Neurosci.

    (2002)
  • S.S. Das et al.

    The role of protein interaction motifs in regulating the polarity and clustering of the metabotropic glutamate receptor mGluR1a

    J. Neurosci.

    (2006)
  • M. Heine et al.

    Surface mobility of postsynaptic AMPARs tunes synaptic transmission

    Science

    (2008)
  • Cited by (6)

    • Importance of the orthogonal structure between porphyrin and aniline moieties on the pH-activatable porphyrin derivative for photodynamic therapy

      2018, Journal of Photochemistry and Photobiology A: Chemistry
      Citation Excerpt :

      These inactive and active states are called “ON” and “OFF” states, respectively. This type of activation functions have been well developed in the field of fluorescence probes [21–28]. In many cases, a fluorophore is combined with a quencher unit, and fluorescence is in the OFF state due to quenching.

    • Targetable fluorescent sensors for advanced cell function analysis

      2017, Journal of Photochemistry and Photobiology C: Photochemistry Reviews
      Citation Excerpt :

      This is an advantage for both probe synthesis and for use in live cell experiments, in which membrane permeability is very important. One rare example of the development of a sensor target based on non-covalent labeling is based on use of the leucine zipper [77]. In this example, a basic leucine zipper peptide (ZIP) was chemically conjugated with a fluorescent pH sensor, and a ZIP-binding peptide (ZBP) was genetically fused with an extracellular domain from the glutamate receptor.

    • Fluorescence lifetime imaging of cancer in vivo

      2016, Methods in Molecular Biology
    View full text