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Summary

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

Abstract

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Introduction

Changes in receptor expression, distribution, and assembly have been connected to a wide variety of diseases, including Alzheimer's disease, Parkinson's disease, cystic fibrosis, and drug addiction^1,2,3,4,5. For example, nicotine and other nicotinic ligands influence the trafficking of nicotinic acetylcholine receptors (nAChRs) leading to changes in trafficking, expression, and upregulation^{1,2,5,6,7,8,9,10}. Nicotine increases the total number of assembled nAChRs within a cell, increases trafficking towards the plasma membrane, and alters the assembly of subunits to favor a high sensitivity version of some subtypes. Resolving distinct changes in trafficking, assembly, and expression of receptors in a disease model provides crucial mechanistic details that are essential to define drug targets. An ideal approach would rapidly differentiate between intracellular receptors and those localized on the plasma membrane. This is particularly challenging in cases where a majority of a particular protein resides intracellularly, such as with nAChRs. Since the majority of nAChRs are localized to the endoplasmic reticulum, traditional measurements lack the spatio-temporal resolution necessary to pinpoint localization and trafficking changes along the secretory pathway. Receptor trafficking and expression studies of nAChRs have primarily been conducted using radioligand binding¹¹, biotinylation assays¹², western blotting¹³, or immunoprecipitation techniques¹². These depend on the binding specificity of a reporter molecule or fixation of cells and lack the ability to simultaneously distinguish between plasma membrane resident and intracellular receptors. Therefore, studies of ion channel assembly and vesicle dynamics have largely relied on low-throughput electrophysiological techniques¹⁴.

Superior spatial and temporal resolution is possible with advances in fluorescence microscopy. Genetically encoded reporter molecules, such as green fluorescent protein (GFP) and its variants, eliminate nonspecific binding issues and increase sensitivity¹⁵. A pH sensitive variant of GFP, known as superecliptic pHluorin (SEP), can be used to exploit inherent pH differences between compartments within a cell to determine localization^{5,7,8,9,16,17,18}. SEP fluoresces when the pH is higher than 6, but remains in an off state at lower pH. Therefore, receptors tagged with SEP on their luminal side are detected when present in the endoplasmic reticulum (ER) or upon insertion into the plasma membrane (PM), but not when confined to a trafficking vesicle. Manipulation of the extracellular pH in contact with receptors on the plasma membrane consequently alters the fluorescence and therefore detection of these receptors. If the same cell is sequentially imaged at both a neutral extracellular pH and then a pH lower than 6, the difference between the images is attributed to receptors located on the plasma membrane. This allows a simultaneous measurement of intracellular (peripheral ER) and plasma membrane resident receptors^5,7,8,9. Single vesicle insertion events can also be resolved when the extracellular pH is neutral. Once a low pH trafficking vesicle fuses with the plasma membrane, the luminal side of the vesicle is exposed to the neutral extracellular solution, causing a transition detected as a burst of fluorescence^7,18,19,20. SEP enables the measurement of receptors localized to the plasma membrane and peripheral endoplasmic reticulum, and provides a means to measure trafficking of receptors between these subcellular regions^5,7,18.

To achieve higher resolution at the plasma membrane, a receptor with SEP genetically encoded is imaged by total internal reflection florescence microscopy (TIRFM). This method is particularly useful if the majority of receptors are localized to intracellular regions, since TIRFM increases the visibility of the plasma membrane. TIRFM also enables the resolution of trafficking dynamics of single vesicles carrying SEP-labeled receptors upon insertion into the PM. Total internal reflection occurs at the interface of materials with different refractive indices, such as between a cell and a glass cover-slip^21,22. SEP fluoresces when irradiated with 488 nm excitation, which is oriented to achieve total internal reflection at the interface of the glass and cell solution. This produces an evanescent wave that penetrates approximately 150 nm into the sample, only exciting fluorophores within this volume. Only SEP containing receptors in a neutral pH environment within this range of excitation are detected, corresponding to those residing on the plasma membrane or peripheral endoplasmic reticulum. Since detection is limited to excitation by the evanescent wave, background fluorescence from the intracellular region is reduced and the signal to noise ratio is increased^21,22. In addition, since radiation does not penetrate the bulk of the cell, photodamage is minimized which allows live cell imaging over the course of time. As a result, TIRFM coupled with genetically encoded SEP provides the high resolution and sensitivity required to measure subcellular localization and trafficking dynamics of membrane receptors along the secretory pathway.

Protocol

1. Cell Culture and Transfection Maintain mouse neuroblastoma 2a (N2a) cells in growth media. Make 500 ml N2a growth media from 200 ml Dulbecco's Eagle medium (DMEM) with high glucose, 250 ml reduced serum media, 50 ml fetal bovine serum (FBS), and 5 ml penicillin/streptomycin (100x). Maintain cells in a T75 flask at 37 °C in a 5% CO2 incubator. Split cells 1:15 when necessary, or at approximately 80-90% confluency. This is typically 2-3 times a week. Coat 35 mm glass bottom dish with poly-D-lysine. In a laminar flow biosafety cabinet, add 200 µl of 0.1 µg/ml poly-D-lysine onto glass bottom region of sterile 35 mm dish. Place dish in a 37 °C incubator for 1 hr. Carefully rinse dish with ddH2O 3-4 times. Allow dishes to completely dry for more than 1 hr. Sterilize dishes using UV light in the biosafety cabinet if necessary. Plate N2a cells for TIRFM imaging. Remove growth media from adherent cells. Detach cells from the flask by incubating with 1 ml 1x trypsin (+EDTA) for 5 min at 37 °C in a 5% CO2 incubator. Inactivate trypsin by adding 9 ml growth media. Mix media, trypsin, and detached cells using a pipette. Visually count cells using a hemocytometer and calculate the correct volume to add 90,000 cells to each poly-D-lysine coated glass bottom dish. This density is required for efficient transfection and single cell TIRF imaging. Add 2 ml growth media to each glass-bottom dish containing 90,000 cells. Incubate the dish at 37 °C in a 5% CO2 incubator for 16-24 hr. N2a transfection Obtain a plasmid construct containing an SEP fluorophore incorporated into the extracellular region of the protein of interest. Standard cloning techniques such as PCR amplification5 can be used to generate the construct. NOTE: SEP should be incorporated into the extracellular region of the membrane protein so that it resides within the lumen of the endoplasmic reticulum or trafficking vesicles and is exposed to extracellular solution when on the plasma membrane. SEP is similar in size to GFP and similar cloning strategies can be used. Replace the growth media on plated cells with 1.5 ml reduced serum media (e.g., Opti-MEM), approximately 30 min before the addition of the transfection reagent. NOTE: To study drug induced changes in receptors, an appropriate concentration of drug can be added at the time of the transfection. Add 500 ng of each desired plasmid construct to 250 µl reduced serum media (tube 1). Add 2 µl of the transfection reagent to a separate tube containing 250 µl of reduced serum media. Incubate tube 2 at room temperature for 5 min. The ratio of transfection reagent to plasmid DNA should be optimized to express each protein of interest. Combine tubes 1 and 2 to make a solution containing 500 µl of transfection reagent and plasmid mix. Incubate at room temperature for 25 min. Add the 500 µl transfection mix to pre-plated cells for a total volume of 2 ml per dish. Incubate cells at 37 °C in a 5% CO2 incubator for 24 hr. After 24 hr, remove the transfection mix and rinse the cells with growth media and then add 2 ml of growth media to each dish. NOTE: If a drug was added at the time of transfection, it can be replenished again at this step. Incubate cells at 37 °C in a 5% CO2 incubator for 24 hr. Image cells 48 hr after transfection. 2. Live Cell Imaging by Total Internal Reflection Fluorescence Microscopy (TIRFM) Imaging set up Perform imaging with an inverted fluorescence microscope set-up with a scanning stage as shown in Figure 1. This requires alignment of a 488 nm DPSS laser source, a stepper motor to adjust 488 nm beam position, and a high numerical aperture (1.49 NA) 60X or 100X oil immersion objective. Pass the laser beam through the appropriate excitation filter (bandpass 488/10 nm). Align the polarized laser beam through a single mode fiber connected to a launcher mounted to a stepper motor. In epifluorescence mode, the excitation beam is centered in the middle of the back aperture of the objective. To attain TIRF, use the stepper motor to translate the focused laser beam across the back aperture of the objective lens until the critical angle is reached and beam is no longer transmitted through the sample and is instead totally reflected off the glass-cell interface. Capture images using an EMCCD (512 x 512 pixels) controlled with imaging software (e.g. Metamorph) with the appropriate emission filter mounted in the emission pathway (525/50 nm). Prepare the imaging solution Prepare 200 ml extracellular solution (ECS) by mixing 150 mM NaCl, 4 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM HEPES and 10 mM glucose in ddH2O. Stock solutions of NaCl, KCl, MgCl2, and CaCl2 can be prepared in advance and combined with fresh HEPES and glucose on the day of imaging. Adjust the pH of a solution containing 100 ml ECS to pH 7.4. Adjust the remaining 100 ml of ECS to a pH of 5.4. Rinse the transfected cells with 2 ml of ECS (pH 7.4). Add 2 ml of ECS (pH 7.4) to the transfected cells before imaging. Live cell imaging in TIRF Turn on the entire system, including 488 nm laser, camera, translation stage, and imaging program. Focus the epifluorescence beam and adjust the power to approximately 1 mW at the 60X objective. Add oil to the objective and place a dish of transfected cells on the translation stage. Secure the dish in place using stage mounts to ensure it does not move with respect to the stage so the cells can be imaged multiple times. In epifluorescence mode, focus the microscope and locate the fluorescent, transfected cells. Cells will remain focused across several focus planes. Locate single, isolated cells to proceed with TIRF. In the imaging program, set the exposure time to 200 msec and optimize the fluorescence intensity by setting the EM gain. Transition the laser beam into TIRF by translating the beam across the objective in a stepwise manner using the stepper motor. As the critical angle approaches, the beam will visibly translate across the edge of the dish until it converges at the point of total internal reflection on the sample plane. Verify that cells are in TIRF mode by adjusting the focus knob. In TIRF, only one plane of cells can be focused (i.e. approximately 150 nm from glass interface), producing a very defined image with high resolution of the plasma membrane. Imaging SEP to determine subcellular localization Locate healthy, transfected, single cells in the imaging plane. Acquire a focused image of cell at pH 7.4. SEP labeled receptors on the plasma membrane and endoplasmic reticulum should be visible. Quickly block the laser beam from reaching the sample to prevent photobleaching. Memorize the stage positions corresponding to each cell using microscope imaging software capable of recording multiple xy locations. Repeat steps 2.4.1-2.4.4 for 20-30 cells per dish while using the software to record the position of each cell. After all the cell images at pH 7.4 are collected, manually remove the pH 7.4 ECS solution using a pipette. Do not touch the dish as this could move the memorized stage positions. Carefully add 2 ml of pH 5.4 ECS to the dish and wait 10 min. During this time, save previously acquired images. Under the identical set of conditions used to collect images at pH 7.4, move the stage to each saved position and acquire an image of the same cell at pH 5.4. Cells should look less defined since all detected fluorescence is originating from endoplasmic reticulum confined SEP labeled receptors. Save the pH 5.4 cell images. Imaging single vesicle insertion events in live cells Replace growth media of transfected cells with 2 ml pH 7.4 ECS, or with Leibovitz's L-15 media. The pH of Leibovitz's L-15 media is CO2 independent, allowing imaging to take place over a long period of time if necessary. Place the dish of transfected cells on the stage of the microscope. Secure and focus a single cell in TIRF following Step 2.3 above. If equipped, set the autofocus so the focus does not drift over the period of imaging. Record a series of 1,000 frames, continuously capturing images at a frame rate of 200 msec. Bursts of fluorescence will be visible during this time, corresponding to low pH trafficking vesicles fusing with the plasma membrane, exposing the SEP to the extracellular pH 7.4. Locate another single cell and repeat the above step. Reset autofocus if necessary. 3. Image Analysis and Data Processing Analyzing SEP fluorescence to determine subcellular localization Open the cell images using an image analysis software such as Metamorph or ImageJ (http://imagej.nih.gov/ij/). Subtract the background from both pH 5.4 and pH 7.4 images using the rolling ball setting. Use an intensity based threshold to quantify fluorescence from a single cell. Manually select a region of interest around the cell in the pH 7.4 image. Measure the cell area, mean intensity, and integrated density using a plugin in ImageJ or using the built in "Measure" function under the Analyze tab. Repeat steps 3.1.1-3.1.3 for the same cell at pH 5.4, carefully adjusting the intensity based threshold and region of interest in the same manner. After the integrated density is obtained for cell images at both pH 7.4 and 5.4, calculate the plasma membrane integrated density by subtracting the pH 5.4 value from the pH 7.4 value. The difference corresponds to the relative number receptors on the plasma membrane within the TIRF excitation region. As a measure of trafficking, calculate the relative percentage of SEP labeled receptors located on the plasma membrane compared to the remaining receptors visible in the TIRF excitation volume by dividing the plasma membrane integrated density by the total integrated density at pH 7.4, multiplied by 100. Analyzing single vesicle insertion events Open the series of 1,000 tiff images using image analysis software. Subtract the background from all frames using the rolling ball setting. Adjust the color balance of the recording to maximize intense regions corresponding to vesicle insertion events. Manually count the bursts of fluorescence lasting longer than 3 frames (>600 msec).

Representative Results

SEP incorporation into a receptor allows direct detection of that receptor in a live cell. Coupled with TIRFM, this allows the evaluation of the relative expression levels on the plasma membrane and the distribution of receptors within the subcellular locations in the region of TIRF excitation. Single vesicle trafficking events can also be resolved. Relative Expression Levels of SEP-labeled Receptors on Plasma Membrane <p class…

Discussion

The pH sensitivity of SEP enables receptors residing on the plasma membrane to be distinguished from intracellular receptors in the endoplasmic reticulum, and it can be used to resolve insertion events of receptor-carrying vesicles^{5,7,8,9,18,19,20}. Several techniques including surface biotin…

Materials

Reagent/Material
Cell Culture Flasks with Filter Cap, Sterile, Greiner Bio One, 75 cm^2	VWR	82050-856
35 mm glass bottom petri dishes, sterile	Cell E&G	GBD00002-200
Poly-D-lysine	vwr	215017510
Dulbecco Modified Eagle Medium‎ (DMEM), High Glucose	Fisher Scientific	11-965-084
Opti-MEM I Reduced Serum Medium	Gibco / Fisher Scientific	31-985-088
Fetal Bovine Serum, Certified, US Origin, Standard (Sterile-Filtered)	Gibco / Fisher Scientific	16-000-044
TrypLE Express Enzyme (1X), no phenol red	Fisher Scientific	12604-021
Penicillin-Streptomycin Solution	VWR	45000-652
Leibovitz's L-15 Medium, no phenol red	Gibco / Fisher Scientific	21083027	Optional
Lipofectamine	Fisher Scientific	11668030	Gently mix; Do not vortex
Sodium chloride	Fisher Scientific	BP358-1
Potassium chloride	Fisher Scientific	P217-10
Magnesium chloride	Fisher Scientific	BP214-500
Calcium chloride	Fisher Scientific	C79-500
HEPES	Fisher Scientific	BP310-500
D-Glucose	Fisher Scientific	D16-1
Objective immersion oil	Olympus	Type F
Name	Company	Catalog Number	Comments
Equipment
Microscope	Olympus	IX81
Camera	Andor	iXon Ultra 897
60x, 1.49 NA oil immersion objective	Olympus	APON 60XOTIRF
Motorized stage	Prior	IXPROXY
Motorized actuator (stepper motor)	Thorlabs	ZST213
MetaMorph (or other imaging program)	Metamorph
488 nm laser	Market Tech
Single mode fiber	Thorlabs	SM450
Mirrors	Thorlabs	BB1-E01
Dichroic 488 nm LP	Semrock	Di02-R488-25×36
Bandpass filter, 488 nm	Semrock	LL01-488-12.5
Bandpass filter, 525/50	Semrock	FF03-525/50-25