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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The authors declare no competing financial interests.

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 ...

This protocol describes the characterization and application of a new pH-sensitive fluorescent lipid probe, specific for cell membranes, which allows researchers to monitor exo and endocytosis in live cells. Most previous reporters on membrane protein based on pH-sensitive green fluorescence protein, which is indirect. As a lipid mimetic, ND6 integrate into the cell membrane, and thus faithfully report lipid membrane trafficking.ND6 probe becomes fluorescent only if embedded in biological membrane environment. The fluorescence intensity of that probe correlates with protonation and deprotonation of the piperazine head group. Thus, it fluoresces much stronger at lower pH.Exocytosis and the endocytosis are ubiquitous processes underlying the secretion and uptake of many signaling molecules. Therefore, ND6 can be used to study some fundamental questions in cell biology and the biomedical sciences. Demonstrating the procedure will be Haley, Oliver, and Jonathan, students from my lab.Begin by preparing an imaging chamber that allows temperature control and solution input output for live cell fluorescence imaging. Set up a programmable device to switch the perfusion solutions and deliver the electric stimulus at defined time points during imaging. Weigh out an appropriate amount of ND6.Dissolve it in dimethyl sulphoxide, and allow it to solubilize at room temperature. Then sonicate the solution briefly. Filter the crude stock solution using a 0.22 micrometer filter to remove large dye aggregates.Using a conventional or micro volume spectrophotometer, determine the dye concentration at 405 nanometers. Before application, dilute the stock solution to a concentration of one milligram per milliliter using the bath solution. Keep the stock solution at room temperature in the dark.Add ND6 stock solution to the culture medium at the final concentration of one microgram per microliter and incubate it to ubiquitously label endocytosed membrane compartments, such as endosomes. To reduce the extent of synaptic vesicles labeling, suppress the spontaneous neuronal activity pharmacologically. After selectively labeling synaptic vesicles as described in the manuscript, mount the cover slip containing cells in the imaging chamber.Connect the temperature sensor, perfusion input, and vacuum tip to the imaging chamber. Adjust the perfusion speed to approximately 0.2 milliliters per second and start the perfusion of prewarmed normal tyrode's solution containing NBQX and DAP5 to remove excessive ND6 in the culture. Adjust the focus and locate the appropriate field of view containing healthy and well spread neurons bearing connected neurites.Avoiding areas containing unresolved Dicolloids. Try imaging ND6 loaded cells with different exposure times to identify the best imaging settings. Set up the stimulation and perfusion protocol, frame interval, and total duration using the information in the text manuscript.Start the image acquisition accompanied by synchronized stimulation and perfusion. Monitor the stimulation and solution exchange during imaging. Stop the perfusion after the imaging ends.Remove the cover slip and clean the imaging chamber for the next trial. Back up and make an electronic copy of all image files. Choose and open the analysis program for data extraction.Open or import an image stack to the analysis program. After generating a binary mask, use the analyze particle function with appropriate area size and circularity to solicit regions of interest, or ROIs, corresponding to cell membranes, endosomes, lysosomes, or synaptic boutons. Select four background ROIs in cell-free regions within the field of view.Then save all selected ROIs. Align all frames within the ND6 time-lapse image stacks. Set the first image as the reference and align the rest to it using rigid registration, which will mitigate artifacts due to XY drifting.Then save the aligned image stacks. Apply the saved ROIs to aligned ND6 image stacks. Measure the average pixel intensities of ND6 fluorescence for every ROI in every frame of the image stack.Then export the results for statistical analysis. Calculate the mean intensity of the background ROIs to obtain the baseline noise, which will be subtracted from all selected ROIs. Average the three highest average intensities for every selected ROI during the application of pH 5.5 tyrode's solution to obtain the maximal fluorescence intensity, defined as 100%for normalization.Average the three lowest average intensities for every selected ROI during the application of 50 millimolar ammonium chloride to set the minimal fluorescence intensity, defined as 0%for normalization. Calculate the relative fluorescence changes for every ROI based on its own 0%and 100%intensities. Derive mean fluorescence changes, change kinetics, and other values or plots from individual ROI data.Bright green fluorescent puncta along the neuronal processes were visible after loading the neuronal cells with ND6. A strong correlation between ND6 and FM464 fluorescence intensities also suggested synaptic vesicle staining by ND6. A decrease was observed in the ND6 fluorescence in response to both electric stimulation and high potassium stimulation, suggesting that ND6 resides in the synaptic vesicle membrane and that the synaptic vesicle lumen is neutralized during the release.Under an exhaustive electric stimulation, the M beta CD treatment significantly reduced synaptic vesicle release and retrieval measured by ND imaging, which suggested that membrane cholesterol facilitates synaptic vesicle exocytosis and endocytosis. Baf A1 prevented ND6 fluorescence recovery after stimulation by acutely blocking the reacidification of retrieved synaptic vesicles. Basic understanding of fluorescence microscopy is a must.Careful planning for physiological conditions to support live cell imaging is very important. It is crucial to precisely determine the concentration of ND stock solution. If possible, remeasure stock solution concentration before every use.Please keep it away from direct sunlight to avoid photo bleaching and probe decomposition.

measuring-membrane-lipid-turnover-with-ph-sensitive-fluorescent-lipid

Research

JoVE Journal

Neuroscience

1.5K Görüntüleme.  The Brain Institute at Florida Atlantic University. Bu protokol, araştırmacıların canlı hücrelerde ekzo ve endositozu izlemesine olanak tanıyan, hücre zarlarına özgü, pH'a duyarlı yeni bir floresan lipid probunun karakterizasyonunu ve uygulamasını açıklar. Membran proteini ile ilgili önceki raporların çoğu, dolaylı olan pH'a duyarlı yeşil floresan proteinine dayanmaktadır. Bir lipid taklitçisi olarak ND6, hücre zarına entegre olur ve böylece lipid zarı trafiğini aslına uygun olarak rapor eder.ND6 probu sad...