Name: measuring membrane lipid turnover with the ph-sensitive fluorescent lipid analog nd6
Uploaded: 2021-07-29T14:00:00
Description: Watch this Scientific Journal Video about Measuring Membrane Lipid Turnover with the pH-sensitive Fluorescent Lipid Analog ND6 at JoVE.com

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&#39;s Association grant AARG-NTF-19-618710 (Q.Z.), and NIA R21 grant AG061656-01A1 (Q.Z.).

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...

<ol>
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Lipid-based dyes, such as 1,1&#8242;-dioctadecyl-3,3,3&#8242;,3&#8242;-tetramethylindocarbocyanine (DiI) and 3,3&#8242;-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...

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&#39;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 exocytosis3,4,5. 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-/endocytosis7,8,9. 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 sensors16,17,18.
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 state19,20. 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&#39;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&#39;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&#39;s solutions (Figure 4D). This protocol facilitates the quantitative measurement of the assembled exocytosis and endocytosis rates under different circumstances (Figure 4C).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Digidata 1440A Data Acquistion System</td>
			<td>Molecular Devices</td>
			<td>Digidata 1440A</td>
			<td>For synchronized stimulation and solution exchange</td>
		</tr>
		<tr>
			<td>Dual Channel Temperature Controller</td>
			<td>Warner Instruments</td>
			<td>TC-344B</td>
			<td>For live-cell imaging</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>OMEGA Scientific</td>
			<td>FB-01</td>
			<td>For making H+20 solution used in dissection and tissue culture</td>
		</tr>
		<tr>
			<td>Hamamatsu Flash4.0 sCOMS camera</td>
			<td>Hamamatsu Inc.</td>
			<td>C13440-20CU</td>
			<td>high-sensitivity camera</td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution</td>
			<td>Sigma</td>
			<td>H6648</td>
			<td>For making H+20 solution used in dissection and tissue culture</td>
		</tr>
		<tr>
			<td>Heated Platform</td>
			<td>Warner Instruments</td>
			<td>PH-1</td>
			<td>For live-cell imaging</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BD Biosciences</td>
			<td>354234</td>
			<td>For tissue culture</td>
		</tr>
		<tr>
			<td>Micro-G Vibration Isolation Table</td>
			<td>TMC</td>
			<td>63-564</td>
			<td>For live-cell imaging</td>
		</tr>
		<tr>
			<td>Micro-manager</td>
			<td>https://micro-manager.org/</td>
			<td>NA</td>
			<td>For image acquisition control</td>
		</tr>
		<tr>
			<td>Multi-Line In-Line Solution Heater</td>
			<td>Warner Instruments</td>
			<td>SHM-6</td>
			<td>For live-cell imaging</td>
		</tr>
		<tr>
			<td>Neurobasal Plus Medium</td>
			<td>THermoFisher Scientific</td>
			<td>A3582901</td>
			<td>For tissue culture</td>
		</tr>
		<tr>
			<td>Nikon Ti-E Inverted Microscope</td>
			<td>Nikon</td>
			<td>Ti-E/B</td>
			<td>For live-cell imaging</td>
		</tr>
		<tr>
			<td>ORCA-Flash4.0 Digital CMOS camera</td>
			<td>Hamamatsu</td>
			<td>C1340-20CU</td>
			<td>For live-cell imaging</td>
		</tr>
		<tr>
			<td>Perfusion Chamber</td>
			<td>Warner Instruments</td>
			<td>RC-26G</td>
			<td>For live-cell imaging</td>
		</tr>
		<tr>
			<td>Six-Channel Valve Control Perfusion System</td>
			<td>Warner Instruments</td>
			<td>VC-6</td>
			<td>For solution exchange</td>
		</tr>
		<tr>
			<td>Square Pulse Stimulator</td>
			<td>Grass Instrument</td>
			<td>SD9</td>
			<td>For electric field stimulation</td>
		</tr>
	</tbody>
</table>

measuring membrane lipid turnover with the ph-sensitive fluorescent lipid analog nd6

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 &#946; 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.

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 ...

Watch this Scientific Journal Video about Measuring Membrane Lipid Turnover with the pH-sensitive Fluorescent Lipid Analog ND6 at JoVE.com

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

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.

Exo-/endocytosis is a common process mediating the exchange of biomolecules between cells and their environment and among different cells. Specialized ...

Research

JoVE Journal

Neuroscience

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real ...

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate ...

Online Transcranial Magnetic Stimulation Protocol for Measuring Cortical Physiology Associated with Response Inhibition

We describe the development of a reproducible, child-friendly motor response inhibition task suitable for online Transcranial Magnetic Stimulation (TMS) ...

A Micro-agar Salt Bridge Electrode for Analyzing the Proton Turnover Rate of Recombinant Membrane Proteins

To date, more than 50% of all pharmacological drugs target the transport kinetics of membrane proteins. The electrophysiological characterization of ...

Lipid Bilayer Experiments with Contact Bubble Bilayers for Patch-Clampers

Lipid bilayers provide a unique experimental platform for functional studies of ion channels, allowing the examination of channel-membrane interactions ...

Measuring and Manipulating Functionally Specific Neural Pathways in the Human Motor System with Transcranial Magnetic Stimulation

Understanding interactions between brain areas is important for the study of goal-directed behavior. Functional neuroimaging of brain connectivity has ...