This work was supported in part by the National Institute on Drug Abuse T32 DA 016176, National Institute on Drug Abuse DA 038817, and National Institute on Drug Abuse DA 040047.

1. Cell Culture and Transfection
<ol>
	<li>Maintain mouse neuroblastoma 2a (N2a) cells in growth media.
	<ol>
		<li>Make 500 ml N2a growth media from 200 ml Dulbecco&#39;s Eagle medium (DMEM) with high glucose, 250 ml reduced serum media, 50 ml fetal bovine serum (FBS), and 5 ml penicillin/streptomycin (100x).</li>
		<li>Maintain cells in a T75 flask at 37 &#176;C in a 5% CO2 incubator.</li>
		<li>Split cells 1:15 when necessary, or at approximately 80-90% confluency. This is typically 2-3 times a week.</li>
	</ol>
	</li>
	<li>Coat 35 mm glass bottom dish with poly-D-lysine.
	<ol>
		<li>In a laminar flow biosafety cabinet, add 200 &#181;l of 0.1 &#181;g/ml poly-D-lysine onto glass bottom region of sterile 35 mm dish.</li>
		<li>Place dish in a 37 &#176;C incubator for 1 hr.</li>
		<li>Carefully rinse dish with ddH2O 3-4 times.</li>
		<li>Allow dishes to completely dry for more than 1 hr.</li>
		<li>Sterilize dishes using UV light in the biosafety cabinet if necessary.</li>
	</ol>
	</li>
	<li>Plate N2a cells for TIRFM imaging.
	<ol>
		<li>Remove growth media from adherent cells.</li>
		<li>Detach cells from the flask by incubating with 1 ml 1x trypsin (+EDTA) for 5 min at 37 &#176;C in a 5% CO2 incubator.</li>
		<li>Inactivate trypsin by adding 9 ml growth media. Mix media, trypsin, and detached cells using a pipette.</li>
		<li>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.</li>
		<li>Add 2 ml growth media to each glass-bottom dish containing 90,000 cells.</li>
		<li>Incubate the dish at 37 &#176;C in a 5% CO2 incubator for 16-24 hr.</li>
	</ol>
	</li>
	<li>N2a transfection
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Add 500 ng of each desired plasmid construct to 250 &#181;l reduced serum media (tube 1).</li>
		<li>Add 2 &#181;l of the transfection reagent to a separate tube containing 250 &#181;l 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.</li>
		<li>Combine tubes 1 and 2 to make a solution containing 500 &#181;l of transfection reagent and plasmid mix. Incubate at room temperature for 25 min.</li>
		<li>Add the 500 &#181;l transfection mix to pre-plated cells for a total volume of 2 ml per dish.</li>
		<li>Incubate cells at 37 &#176;C in a 5% CO2 incubator for 24 hr.</li>
		<li>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.</li>
		<li>Incubate cells at 37 &#176;C in a 5% CO2 incubator for 24 hr.</li>
		<li>Image cells 48 hr after transfection.</li>
	</ol>
	</li>
</ol>
2. Live Cell Imaging by Total Internal Reflection Fluorescence Microscopy (TIRFM)
<ol>
	<li>Imaging set up
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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).</li>
	</ol>
	</li>
	<li>Prepare the imaging solution
	<ol>
		<li>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.</li>
		<li>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).</li>
		<li>Add 2 ml of ECS (pH 7.4) to the transfected cells before imaging.</li>
	</ol>
	</li>
	<li>Live cell imaging in TIRF
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>In the imaging program, set the exposure time to 200 msec and optimize the fluorescence intensity by setting the EM gain.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Imaging SEP to determine subcellular localization
	<ol>
		<li>Locate healthy, transfected, single cells in the imaging plane.</li>
		<li>Acquire a focused image of cell at pH 7.4. SEP labeled receptors on the plasma membrane and endoplasmic reticulum should be visible.</li>
		<li>Quickly block the laser beam from reaching the sample to prevent photobleaching.</li>
		<li>Memorize the stage positions corresponding to each cell using microscope imaging software capable of recording multiple xy locations.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Carefully add 2 ml of pH 5.4 ECS to the dish and wait 10 min. During this time, save previously acquired images.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Imaging single vesicle insertion events in live cells
	<ol>
		<li>Replace growth media of transfected cells with 2 ml pH 7.4 ECS, or with Leibovitz&#39;s L-15 media. The pH of Leibovitz&#39;s L-15 media is CO2 independent, allowing imaging to take place over a long period of time if necessary.</li>
		<li>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.</li>
		<li>If equipped, set the autofocus so the focus does not drift over the period of imaging.</li>
		<li>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.</li>
		<li>Locate another single cell and repeat the above step. Reset autofocus if necessary.</li>
	</ol>
	</li>
</ol>
3. Image Analysis and Data Processing
<ol>
	<li>Analyzing SEP fluorescence to determine subcellular localization
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Measure the cell area, mean intensity, and integrated density using a plugin in ImageJ or using the built in &#34;Measure&#34; function under the Analyze tab.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Analyzing single vesicle insertion events
	<ol>
		<li>Open the series of 1,000 tiff images using image analysis software. Subtract the background from all frames using the rolling ball setting.</li>
		<li>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 (&#62;600 msec).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Lester, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotine+is+a+selective+pharmacological+chaperone+of+acetylcholine+receptor+number+and+stoichiometry.+Implications+for+drug+discovery.">Nicotine is a selective pharmacological chaperone of acetylcholine receptor number and stoichiometry. Implications for drug discovery.</a> AAPS J. 11 (1), 167-177 (2009).</li><li>Henderson, B. J., Lester, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inside-out+neuropharmacology+of+nicotinic+drugs.">Inside-out neuropharmacology of nicotinic drugs.</a> Neuropharmacology. 96 (Pt B), 178-193 (2015).</li><li>Banerjee, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+expression+of+alpha7+nicotinic+acetylcholine+receptor+protein+in+the+temporal+cortex+in+Alzheimer's+and+Parkinson's+disease--a+stereological+approach.">Cellular expression of alpha7 nicotinic acetylcholine receptor protein in the temporal cortex in Alzheimer's and Parkinson's disease--a stereological approach.</a> Neurobiol Dis. 7 (6 Pt B), 666-672 (2000).</li><li>Ikonomovic, M. D., Wecker, L., Abrahamson, E. 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I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trafficking+of+&#945;4*+Nicotinic+Receptors+Revealed+by+Superecliptic+Phluorin.">Trafficking of &#945;4* Nicotinic Receptors Revealed by Superecliptic Phluorin.</a> J Biol Chem. 286 (36), 31241-31249 (2011).</li><li>Kuryatov, A., Luo, J., Cooper, J., Lindstrom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotine+acts+as+a+pharmacological+chaperone+to+up-regulate+human+a4b2+acetylcholine+receptors.">Nicotine acts as a pharmacological chaperone to up-regulate human a4b2 acetylcholine receptors.</a> Mol Pharmacol. 68 (6), 1839-1851 (2005).</li><li>Fox, A. M., Moonschi, F. H., Richards, C. 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The authors have no conflicts of interest to declare.

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 vesicles5,7,8,9,18,19,20. Several techniques including surface biotin...

Changes in receptor expression, distribution, and assembly have been connected to a wide variety of diseases, including Alzheimer&#39;s disease, Parkinson&#39;s disease, cystic fibrosis, and drug addiction1,2,3,4,5. For example, nicotine and other nicotinic ligands influence the trafficking of nicotinic acetylcholine receptors (nAChRs) leading to changes in trafficking, expression, and upregulation1,2,5,6,7,8,9,10. 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 localization5,7,8,9,16,17,18. 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 receptors5,7,8,9. 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 fluorescence7,18,19,20. 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 regions5,7,18.
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-slip21,22. 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 increased21,22. 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.

<table border="0" cellpadding="0" cellspacing="0" width="861">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Reagent/Material</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Culture Flasks with Filter Cap, Sterile, Greiner Bio One, 75 cm^2</td>
			<td>VWR</td>
			<td>82050-856</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:413px;">35 mm glass bottom petri dishes, sterile</td>
			<td style="width:163px;">Cell E&#38;G</td>
			<td style="width:119px;">GBD00002-200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Poly-D-lysine</td>
			<td style="width:163px;">vwr</td>
			<td align="right">215017510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dulbecco Modified Eagle Medium&#8206; (DMEM), High Glucose</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td>11-965-084&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Opti-MEM I Reduced Serum Medium</td>
			<td style="width:163px;">Gibco / Fisher Scientific</td>
			<td>31-985-088</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Fetal Bovine Serum, Certified, US Origin, Standard (Sterile-Filtered)&#160;</td>
			<td style="width:163px;">Gibco / Fisher Scientific</td>
			<td>16-000-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TrypLE Express Enzyme (1X), no phenol red</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">12604-021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin Solution</td>
			<td>VWR</td>
			<td>45000-652</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:413px;">Leibovitz&#39;s L-15 Medium, no phenol red</td>
			<td style="width:163px;">Gibco / Fisher Scientific</td>
			<td style="width:119px;">21083027</td>
			<td>Optional</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Lipofectamine</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">11668030</td>
			<td>Gently mix; Do not vortex</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td>BP358-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium chloride</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td>P217-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium chloride</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td>BP214-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium chloride</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td>BP310-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-Glucose</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td>D16-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Objective immersion oil&#160;</td>
			<td style="width:163px;">Olympus</td>
			<td style="width:119px;">Type F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Name</td>
			<td style="width:163px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Equipment</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Microscope</td>
			<td style="width:163px;">Olympus</td>
			<td style="width:119px;">IX81</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Camera</td>
			<td style="width:163px;">Andor</td>
			<td>iXon Ultra 897</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">60x, 1.49 NA oil immersion objective</td>
			<td style="width:163px;">Olympus</td>
			<td>APON 60XOTIRF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Motorized stage</td>
			<td style="width:163px;">Prior</td>
			<td>IXPROXY</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Motorized actuator (stepper motor)</td>
			<td style="width:163px;">Thorlabs</td>
			<td>ZST213</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">MetaMorph (or other imaging program)</td>
			<td style="width:163px;">Metamorph</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">488 nm laser</td>
			<td style="width:163px;">Market Tech</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Single mode fiber</td>
			<td style="width:163px;">Thorlabs</td>
			<td style="width:119px;">SM450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Mirrors</td>
			<td style="width:163px;">Thorlabs</td>
			<td style="width:119px;">BB1-E01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Dichroic 488 nm LP</td>
			<td style="width:163px;">Semrock</td>
			<td>Di02-R488-25x36</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Bandpass filter, 488 nm</td>
			<td style="width:163px;">Semrock</td>
			<td>LL01-488-12.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Bandpass filter, 525/50</td>
			<td style="width:163px;">Semrock</td>
			<td>FF03-525/50-25</td>
			<td></td>
		</tr>
	</tbody>
</table>

utilizing phluorin-tagged receptors to monitor subcellular localization and trafficking

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.

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
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Watch this Scientific Journal Video about Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking at JoVE.com

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

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.

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

utilizing-phluorin-tagged-receptors-to-monitor-subcellular

Research

JoVE Journal

Biology

8.7K Views.  University of Kentucky. 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.

Video: Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a fluorescently tagged-protein of interest is transiently expressed in plant tissue following biolistic delivery of its encoding DNA construct. The intra- and intercellular distribution of the tagged protein is then analyzed by confocal microscopy. We describe this technology in detail, providing step-by-step protocols to assay and evaluate the extent of symplastic protein transport in three plant species, Arabidopsis thaliana, Nicotiana benthamiana and N. tabacum (tobacco).

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Macromolecular trafficking between plant cells can be assessed by transiently expressing a fluorescently-tagged protein of interest and analyzing its intra- and intercellular distribution by confocal microscopy.

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a ...

Gastrointestinal Motility Monitor (GIMM)

The Gastrointestinal Motility Monitor (GIMM; Catamount Research and Development; St. Albans, VT) is an in vitro system that monitors propulsive motility in isolated segments of guinea pig distal colon. The complete system consists of a computer, video camera, illuminated organ bath, peristaltic and heated water bath circulating pumps, and custom GIMM software to record and analyze data. Compared with traditional methods of monitoring colonic peristalsis, the GIMM system allows for continuous, quantitative evaluation of motility. The guinea pig distal colon is bathed in warmed, oxygenated Krebs solution, and fecal pellets inserted in the oral end are propelled along the segment of colon at a rate of about 2 mm/sec. Movies of the fecal pellet proceeding along the segment are captured, and the GIMM software can be used track the progress of the fecal pellet. Rates of propulsive motility can be obtained for the entire segment or for any particular region of interest. In addition to analysis of bolus-induced motility patterns, spatiotemporal maps can be constructed from captured video segments to assess spontaneous motor activity patterns. Applications of this system include pharmacological evaluation of the effects of receptor agonists and antagonists on propulsive motility, as well as assessment of changes that result from pathophysiological conditions, such as inflammation or stress. The guinea pig distal colon propulsive motility assay, using the GIMM system, is straightforward and simple to learn, and it provides a reliable and reproducible method of assessing propulsive motility.

Gastrointestinal Motility Monitor GIMM

Evaluation of colonic motility in the guinea pig distal colon with the Gastrointestinal Motility Monitor (GIMM) is a straightforward and simple to learn approach to quantitatively evaluate propulsive motility in the gastrointestinal tract.

The Gastrointestinal Motility Monitor (GIMM; Catamount Research and Development; St. Albans, VT) is an in vitro system that monitors propulsive motility ...

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated 1-3. However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. 
Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated 4-7. However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. 
Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot 8-10. 
We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.

We describe a method to localize fluorescently tagged proteins in electron micrographs. Fluorescence is first localized using photo-activated localization microscopy on ultrathin sections. These images are then aligned to electron micrographs of the same section.

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

We describe a modified method for membrane fraction isolation from human cells and sample preparation for the detection of SCAP N-glycosylation and total protein by using western blot. We further introduce a GFP-labeling method to monitor SCAP trafficking using confocal microscopy. This protocol can be used in regular biology laboratories.

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of ...

Live-cell Imaging of Fungal Cells to Investigate Modes of Entry and Subcellular Localization of Antifungal Plant Defensins

Small cysteine-rich defensins are one of the largest groups of host defense peptides present in all plants. Many plant defensins exhibit potent in vitro antifungal activity against a broad-spectrum of fungal pathogens and therefore have the potential to be used as antifungal agents in transgenic crops. In order to harness the full potential of plant defensins for diseases control, it is crucial to elucidate their mechanisms of action (MOA). With the advent of advanced microscopy techniques, live-cell imaging has become a powerful tool for understanding the dynamics of the antifungal MOA of plant defensins. Here, a confocal microscopy based live-cell imaging method is described using two fluorescently labeled plant defensins (MtDef4 and MtDef5) in combination with vital fluorescent dyes. This technique enables real-time visualization and analysis of the dynamic events of MtDef4 and MtDef5 internalization into fungal cells. Importantly, this assay generates a wealth of information including internalization kinetics, mode of entry and subcellular localization of these peptides. Along with other cell biological tools, these methods have provided critical insights into the dynamics and complexity of the MOA of these peptides. These tools can also be used to compare the MOA of these peptides against different fungi.

Plant defensins play an important role in plant defense against pathogens. For effective use of these antifungal peptides as antifungal agents, understanding their modes of action (MOA) is critical. Here, a live-cell imaging method is described to study critical aspects of the MOA of these peptides.

Small cysteine-rich defensins are one of the largest groups of host defense peptides present in all plants. Many plant defensins exhibit potent in vitro ...

Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of conventional light microscopes. As the resolution for a given microscope cannot be easily enhanced, we present protocols to transform the small rod-shaped gram-negative Escherichia coli (E. coli) and gram-positive Bacillus megaterium (B. megaterium) into larger, easily imaged spherical forms called spheroplasts or protoplasts. This transformation allows observers to rapidly and clearly determine whether peptides lodge themselves into the bacterial membrane (i.e., membrane localizing) or cross the membrane to enter the cell (i.e., translocating). With this approach, we also present a systematic method to characterize peptides as membrane localizing or translocating. While this method can be used for a variety of membrane-active peptides and bacterial strains, we demonstrate the utility of this protocol by observing the interaction of Buforin II P11A (BF2 P11A), an antimicrobial peptide (AMP), with E. coli spheroplasts and B. megaterium protoplasts.

Here we present a protocol to produce gram-negative Escherichia coli (E. coli) spheroplasts and gram-positive Bacillus megaterium (B. megaterium) protoplasts to clearly visualize and rapidly characterize peptide-bacteria interactions. This provides a systematic method to define membrane localizing and translocating peptides.

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of ...

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein subcellular localization changes; in particular, a nuclear translocation under a heat stress condition. Proteins react correspondingly to external and internal signals. A common mechanism is to change its subcellular localization. This article describes a protocol to track protein localization that does not require an antibody, radioactive labeling, or a confocal microscope. In this article, GFP is used to tag the target protein EXL-1 in C. elegans, a member of the chloride intracellular channel proteins (CLICs) family, including mammalian CLIC4. An integrated translational exl-1::gfp transgenic line (with a promoter and a full gene sequence) was created by transformation and &#947;-radiation, and stably expresses the gene and gfp. Recent research showed that upon heat stress, not oxidative stress, EXL-1::GFP accumulates in the nucleus. Overlapping the GFP signal with both the nuclei structure and the DAPI signals confirms the EXL-1 subcellular localization changes under stress. This protocol presents two different fixation methods for DAPI staining: ethanol fixation and acetone fixation. The DAPI staining protocol presented in this article is fast and efficient and preserves both the GFP signal and the protein subcellular localization changes. This method only requires a fluorescence microscope with Nomarski, a FITC filter, and a DAPI filter. It is suitable for a small laboratory setting, undergraduate student research, high school student research, and biotechnology classrooms.

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

This protocol demonstrates how to track a protein nuclear translocation under heat stress by using a green fluorescence protein (GFP) fusion protein as a marker and 4',6-diamidino-2-phenylindole (DAPI) staining. The DAPI staining protocol is fast and preserves the GFP and protein subcellular localization signals.

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein ...

Probing Structural and Dynamic Properties of Trafficking Subcellular Nanostructures by Spatiotemporal Fluctuation Spectroscopy

Imaging-derived mean square displacement (iMSD) is used to address the structural and dynamic properties of subcellular nanostructures, such as vesicles involved in the endo/exocytotic trafficking of solutes and biomolecules. iMSD relies on standard time-lapse imaging, is compatible with any optical setup, and does not need to dwell on single objects to extract trajectories. From each iMSD trace, a unique triplet of average structural and dynamic parameters (i.e., size, local diffusivity, anomalous coefficient) is calculated and combined to build the "iMSD signature" of the nanostructure under study. 
The potency of this approach is proved here with the exemplary case of macropinosomes. These vesicles evolve in time, changing their average size, number, and dynamic properties passing from early to late stages of intracellular trafficking. As a control, insulin secretory granules (ISGs) are used as a reference for subcellular structures that live in a stationary state in which the average structural and dynamic properties of the whole population of objects are invariant in time. The iMSD analysis highlights these peculiar features quantitatively and paves the way to similar applications at the sub-cellular level, both in the physiological and pathological states.

Imaging-derived mean square displacement (iMSD) analysis is applied to macropinosomes to highlight their intrinsic time-evolving nature in terms of structural and dynamic properties. Macropinosomes are then compared to insulin secretory granules (ISGs) as a reference for subcellular structures with time-invariant average structural/dynamic properties.

Imaging-derived mean square displacement (iMSD) is used to address the structural and dynamic properties of subcellular nanostructures, such as vesicles ...