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 to Alzheimer's disease². Although lipid molecules are far less diverse than proteins, membrane research so far has mainly been protein-centric. For example, a lot more is known about protein machinery than about lipids in exocytosis^3,4,5. Moreover, the organization, distribution, dynamics, and homeostasis of lipids across surface and intracellular membranes largely remain unexplored in comparison to membrane proteins⁶.

This is not surprising as modern molecular biology techniques, such as mutagenesis, provide a methodological advantage for studying proteins rather than lipids. For example, transgenic tagging of pH-sensitive green fluorescent protein (a.k.a., pHluorin) to vesicular proteins facilitates the quantitative measurement of the amount and rate of vesicular protein turnover during exo-/endocytosis^7,8,9. However, it is almost impossible to genetically modify membrane lipids in vivo. Moreover, qualitative and even quantitative manipulations of protein amounts and distributions are much more feasible than those of lipids¹⁰. Nevertheless, native and synthetic fluorescent lipids have been isolated and developed to simulate endogenous membrane lipids in vitro and in vivo¹¹. One group of widely used fluorescent lipids are styryl dyes, e.g., FM1-43, which exhibit membrane-enhanced fluorescence and are a powerful tool in studying synaptic vesicle (SV) release in neurons¹². Lately, environment-sensitive lipid dyes have been invented and widely used as a new class of reporters to study various cell membrane properties, including membrane potential¹¹, phase order¹³, and secretion¹⁴.

A new class of lipid mimetics whose fluorescence is both pH-sensitive (e.g., pHluorin) and membrane-sensitive (e.g., FM1-43) was developed to directly measure the lipid distribution in the plasma membrane and endosomes/lysosomes and the lipid traffic during exo-/endocytosis. The well-known solvatochromic fluorophores exhibiting push-pull characteristics due to intramolecular charge transfer were selected for this purpose. Among existing solvatochromic fluorophores, the 1,8-naphthalimide (ND) scaffold is relatively easy to modify, versatile for tagging, and is unique in photo-physics¹⁵ and has therefore been used in DNA intercalators, organic light-emitting diodes, and biomolecule sensors^16,17,18.

Attaching an electron-donating group to the C4 position of the ND scaffold generates a push-pull structure, which leads to an increased dipole moment by redistributing the electron density in the excited state^19,20. Such an intramolecular charge transfer produces large quantum yields and Stokes shifts, resulting in bright and stable fluorescence²¹. This group has recently developed a series of solvatochromic lipid analogs based on the ND scaffold and obtained them with good synthetic yields²⁰.

Spectroscopic characterization shows that among those products, ND6 possesses the best fluorescence properties (Figure 1)²⁰. First, it has well-separated excitation and emission peaks (i.e., ~400 nm and ~520 nm, respectively, in Figure 2A,B) compared to popular fluorophores such as fluorescein isothiocyanate, rhodamine, or GFP, making it spectrally separatable from them and thus useful for multicolor imaging. Second, ND6 fluorescence exhibits a more than eight-fold increase in its fluorescence in the presence of micelles (Figure 2C), suggesting a strong membrane-dependency. Prior live-cell fluorescence imaging studies with different types of cells showed excellent membrane staining by ND6²⁰. Third, when the solution's pH is decreased from 7.5 (commonly found in extracellular or cytosolic environments) to 5.5 (commonly found in endosomes and lysosomes), ND6 shows an approximately two-fold increase in fluorescence (Figure 2D), showing its pH-sensitivity. Moreover, molecular dynamics simulation indicates that ND6 readily integrates into the lipid bilayer with its ND scaffold facing out of the membrane and piperazine residue showing strong interactions with phospholipid head groups (Figure 3). Altogether, these features make ND6 an ideal fluorescent lipid analog to visualize and measure membrane lipid turnover during exo-/endocytosis.

This paper presents a method to study the turnover rate and dynamics of SV lipids using cultured mouse hippocampal neurons. By stimulating neurons with high K⁺ Tyrode's solutions, SVs and the plasma membrane were loaded with ND6 (Figure 4A,B). Subsequently, neurons were re-stimulated with different stimuli followed by NH₄Cl-containing and pH 5.5 Tyrode's solutions (Figure 4D). This protocol facilitates the quantitative measurement of the assembled exocytosis and endocytosis rates under different circumstances (Figure 4C).

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…

Materials

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