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
Graphical abstract
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.].
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