Summary

PHluorin изображений с метками рецепторов вставки в плазматическую мембрану в первичной культуре нейронов мыши

Published: November 20, 2012
doi:

Summary

По теги внеклеточных доменов мембранных рецепторов с superecliptic pHluorin, и эти изображения слияния рецепторов в культуре нейронов мыши, мы можем напрямую визуализировать отдельные везикулярного события вставки рецепторы клеточной мембраны. Эта техника будет играть важную роль в выяснении молекулярных механизмов, регулирующих рецепторов вставки в плазматическую мембрану.

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 resolution1-6 . 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. Подготовка культуры мышь Glia для нейронов принадлежности культуры T75 колбы покрыты раствора коллагена (1:3 разбавление Purecol в DDH 2 O). Колбы устанавливаются вертикально и оставили на ночь сушиться в капюшоне культуры ткани. Препарирование буфера (ACSF: 119 мм NaCl, 5 мМ KCl, 1 мМ MgCl …

Discussion

По неизвестным причинам, мыши нейроны всегда сложнее, чем крысы культуре нейронов. По нашему опыту, смешанной культуре нейронов и глии хорошо работает для первичной культуре нейронов мыши. Однако такая смешанная культура не подходят для экспериментов TIRF изображений, так как в этом тип?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Эта работа поддерживается запуск средств из лаборатории Джексона.

Materials

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

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Cite This Article
Li, Y., Roy, B. D., Wang, W., Zhang, L., Sampson, S. B., Lin, D. Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons. J. Vis. Exp. (69), e4450, doi:10.3791/4450 (2012).

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