इमेजिंग प्राथमिक संवर्धित माउस न्यूरॉन्स में प्लाज्मा झिल्ली रिसेप्टर निवेशन pHluorin टैग
Summary
टैगिंग द्वारा superecliptic pHluorin साथ झिल्ली रिसेप्टर्स की कोशिकी डोमेन, और सभ्य माउस न्यूरॉन्स में इमेजिंग द्वारा इन संलयन रिसेप्टर्स, हम सीधे व्यक्ति vesicular प्रविष्टि घटनाओं प्लाज्मा झिल्ली रिसेप्टर्स की कल्पना कर सकते हैं. इस तकनीक के elucidating आणविक प्लाज्मा झिल्ली रिसेप्टर प्रविष्टि संचालन तंत्र में सहायक होगा.
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
Discussion
अज्ञात कारणों के लिए, माउस न्यूरॉन्स हमेशा चूहा न्यूरॉन्स से अधिक संस्कृति के लिए मुश्किल है. हमारे अनुभव में, न्यूरॉन्स और glia की एक मिश्रित संस्कृति प्राथमिक सभ्य माउस न्यूरॉन्स के लिए अच्छी तरह से का…
Disclosures
The authors have nothing to disclose.
Acknowledgements
यह काम जैक्सन प्रयोगशाला से स्टार्टअप धन के द्वारा समर्थित है.
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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