Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

Summary

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

Abstract

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution^1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions ^7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin ¹⁸, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH < 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

Protocol

1. Preparing Mouse Glia Culture for Neuronal Culture Conditioning

1. T75 flasks are coated with collagen solution (1:3 dilution of Purecol in ddH₂O). The flasks are set upright and left to dry overnight in a tissue culture hood.
2. Dissection buffer (aCSF: 119 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 30 mM dextrose, 25 mM HEPES, pH 7.4, without calcium) and glia media (DMEM supplemented with 10% FBS, 10 units/ml penicillin, 10 μg/ml streptomycin, and Glutamax) are prepared and stored at 4 °C until ready for use.
3. On the day of glia culture, the glia media is warmed for at least 30 min in a 37 °C water bath before dissection of mous....

Discussion

For unknown reasons, mouse neurons are always more difficult to culture than rat neurons. In our experience, a mixed culture of neurons and glia works well for primary cultured mouse neurons. However, such a mixed culture is not suitable for TIRF imaging experiments, as in this type of culture neurons and their processes tend to grow on top of glia cells, situating the neuronal somata and dendritic processes beyond the reach of TIRF microscopy. Therefore, a lower-density neuronal culture with few glia on the covers.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
purified bovine collagen solution (Purecol)	Advanced Biomatrix	5005-B
Hank's Balanced Salt Solution (HBSS)	GIBCO, by Life Technologies	14185-045
penicillin-streptomycin (Pen Strep)	GIBCO, by Life Technologies	15140-122
sodium pyruvate	GIBCO, by Life Technologies	11360-070
DMEM High Glucose	GIBCO, by Life Technologies	10313-021
fetal bovine serum (FBS)
GlutaMAX	GIBCO, by Life Technologies	35050-061
papain	Worthington Biochemical Corp.	LS003126
Deoxyribonuclease I from bovine pancreas (DNase I)	Sigma-Aldrich	DN25-10MG
Dulbecco's Phosphate Buffered Saline (DPBS)	GIBCO, by Life Technologies	14190-144
0.05% trypsin	GIBCO, by Life Technologies	25300-054
poly-l-lysine hydrobromide	Sigma-Aldrich	P2636-1G
boric acid	Fisher Scientific	BP 168-500
Neurobasal Medium	GIBCO, by Life Technologies	21103-049
B-27 Serum-Free Supplement	GIBCO, by Life Technologies	17504-044
heat inactive horse serum	GIBCO, by Life Technologies	26050-070
Lipofectamine 2000	Invitrogen	11668 019
HEPES	Fisher Scientific	BP310-500
Culture Insert	EMD Millipore	PICM03050