Measuring Membrane Lipid Turnover with the pH-sensitive Fluorescent Lipid Analog ND6

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 ND6 has been described previously²⁰.

1. Preparation of mouse hippocampal and cortical cultures

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.......

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