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Measuring Membrane Lipid Turnover with the pH-sensitive Fluorescent Lipid Analog ND6
In This Article
Summary
This protocol presents a fluorescence imaging method that uses a class of pH-sensitive lipid fluorophores to monitor lipid membrane trafficking during cell exocytosis and the endocytosis cycle.
Abstract
Exo-/endocytosis is a common process mediating the exchange of biomolecules between cells and their environment and among different cells. Specialized cells use this process to execute vital body functions such as insulin secretion from β cells and neurotransmitter release from chemical synapses. Owing to its physiological significance, exo-/endocytosis has been one of the most studied topics in cell biology. Many tools have been developed to study this process at the gene and protein level, because of which much is known about the protein machinery participating in this process. However, very few methods have been developed to measure membrane lipid turnover, which is the physical basis of exo-/endocytosis.
This paper introduces a class of new fluorescent lipid analogs exhibiting pH-dependent fluorescence and demonstrates their use to trace lipid recycling between the plasma membrane and the secretory vesicles. Aided by simple pH manipulations, those analogs also allow the quantification of lipid distribution across the surface and the intracellular membrane compartments, as well as the measurement of lipid turnover rate during exo-/endocytosis. These novel lipid reporters will be of great interest to various biological research fields such as cell biology and neuroscience.
Introduction
The lipid bilayer is one of the most common biomolecule assemblies and is indispensable for all cells. Outside cells, it forms the plasma membrane interfacing cells and their environment; inside cells, it compartmentalizes various organelles specialized for designated functionalities. Rather dynamic than still, lipid membranes constantly experience fusion and fission, which mediates biomaterial transport, organelle reform, morphology change, and cellular communication. Undoubtedly, the lipid membrane is the physical foundation for almost all cellular processes, and its dysfunction plays a crucial role in various disorders ranging from cancer1 t....
Protocol
The following protocol includes (1) a simplified procedure for establishing mouse hippocampal and cortical cultures based on a well-established protocol22, (2) a brief introduction to an epifluorescence microscope setup for live neurons, (3) a detailed description of loading and imaging ND6 in mouse neurons, (4) a discussion about the quantification of membrane trafficking by ND6 signal. All procedures follow the biosafety and IACUC guidelines at the Florida Atlantic University. The synthesis of N.......
Representative Results
SVs are specialized for neurotransmitter release via evoked exo-/endocytosis27. SVs have highly acidic lumen (i.e., pH 5.5), which is ideal for ND6. We used high K+ stimulation to evoke SV exo-/endocytosis in order to allow ND6 to access SV. Expectedly, bright green fluorescent puncta along neuronal processes showed up after loading (Figure 9A). The line profile shown in Figure 4B demonstrated a strong overlap between ND6 (green curve) and .......
Discussion
Lipid-based dyes, such as 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI) and 3,3′-Dioctadecyloxacarbocyanine perchlorate (DiO), have long been used to illustrate cell morphology and track cellular processes such as the axon projections of neurons. Styryl dyes, such as FM1-43, have been invented and used successfully for the study of exocytosis34. Due to their low membrane affinity, they selectively label endocytosed vesicles where they are trapped while dyes r.......
Acknowledgements
This work was supported by Florida Atlantic University Office of Undergraduate Research and Inquiry grant (M.J.S.), Florida Department of Health Ed and Ethel Moore Pilot Grant 20A17 (Q.Z.), Alzheimer's Association grant AARG-NTF-19-618710 (Q.Z.), and NIA R21 grant AG061656-01A1 (Q.Z.).
....Materials
| Name | Company | Catalog Number | Comments |
| Digidata 1440A Data Acquistion System | Molecular Devices | Digidata 1440A | For synchronized stimulation and solution exchange |
| Dual Channel Temperature Controller | Warner Instruments | TC-344B | For live-cell imaging |
| Fetal Bovine Serum | OMEGA Scientific | FB-01 | For making H+20 solution used in dissection and tissue culture |
| Hamamatsu Flash4.0 sCOMS camera | Hamamatsu Inc. | C13440-20CU | high-sensitivity camera |
| Hank's Balanced Salt Solution | Sigma | H6648 | For making H+20 solution used in dissection and tissue culture |
| Heated Platform | Warner Instruments | PH-1 | For live-cell imaging |
| Matrigel | BD Biosciences | 354234 | For tissue culture |
| Micro-G Vibration Isolation Table | TMC | 63-564 | For live-cell imaging |
| Micro-manager | https://micro-manager.org/ | NA | For image acquisition control |
| Multi-Line In-Line Solution Heater | Warner Instruments | SHM-6 | For live-cell imaging |
| Neurobasal Plus Medium | THermoFisher Scientific | A3582901 | For tissue culture |
| Nikon Ti-E Inverted Microscope | Nikon | Ti-E/B | For live-cell imaging |
| ORCA-Flash4.0 Digital CMOS camera | Hamamatsu | C1340-20CU | For live-cell imaging |
| Perfusion Chamber | Warner Instruments | RC-26G | For live-cell imaging |
| Six-Channel Valve Control Perfusion System | Warner Instruments | VC-6 | For solution exchange |
| Square Pulse Stimulator | Grass Instrument | SD9 | For electric field stimulation |
References
- Polo, S., Pece, S., Di Fiore, P. P. Endocytosis and cancer. Current Opinion in Cell Biology. 16 (2), 156-161 (2004).
- Eckert, G. P., Wood, W. G., Muller, W. E. Lipid membranes and beta-amyloid: a harmful connection. Current Protein and Pept ....
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