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 t...

<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. E., 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=Cortical+&#945;7+nicotinic+acetylcholine+receptor+and+&#946;-amyloid+levels+in+early+alzheimer+disease.">Cortical &#945;7 nicotinic acetylcholine receptor and &#946;-amyloid levels in early alzheimer disease.</a> Arch Neurol. 66 (5), 646-651 (2009).</li><li>Richards, C. 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. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nicotine+metabolite,+cotinine,+alters+the+assembly+and+trafficking+of+a+subset+of+nicotinic+acetylcholine+receptors.">The nicotine metabolite, cotinine, alters the assembly and trafficking of a subset of nicotinic acetylcholine receptors.</a> J Biol Chem. 290 (40), 24403-24412 (2015).</li><li>Henderson, B. J., 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+exploits+a+COPI-mediated+process+for+chaperone-mediated+up-regulation+of+its+receptors.">Nicotine exploits a COPI-mediated process for chaperone-mediated up-regulation of its receptors.</a> J Gen Physiol. 143 (1), 51-66 (2014).</li><li>Henderson, B. J., 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=Menthol+Alone+Upregulates+Midbrain+nAChRs,+Alters+nAChR+Subtype+Stoichiometry,+Alters+Dopamine+Neuron+Firing+Frequency,+and+Prevents+Nicotine+Reward.">Menthol Alone Upregulates Midbrain nAChRs, Alters nAChR Subtype Stoichiometry, Alters Dopamine Neuron Firing Frequency, and Prevents Nicotine Reward.</a> J Neurosci. 36 (10), 2957-2974 (2016).</li><li>Ameen, N., Silvis, M., Bradbury, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytic+trafficking+of+CFTR+in+health+and+disease.">Endocytic trafficking of CFTR in health and disease.</a> J. Cyst. Fibros. 6 (1), 1-14 (2007).</li><li>Pauly, J. R., Marks, M. J., Robinson, S. F., van de Kamp, J. L., Collins, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+nicotine+and+mecamylamine+treatment+increase+brain+nicotinic+receptor+binding+without+changing+alpha+4+or+beta+2+mRNA+levels.">Chronic nicotine and mecamylamine treatment increase brain nicotinic receptor binding without changing alpha 4 or beta 2 mRNA levels.</a> J Pharmacol Exp Ther. 278 (1), 361-369 (1996).</li><li>Govind, A. P., Walsh, H., Green, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotine-induced+upregulation+of+native+neuronal+nicotinic+receptors+is+caused+by+multiple+mechanisms.">Nicotine-induced upregulation of native neuronal nicotinic receptors is caused by multiple mechanisms.</a> J Neurosci. 32 (6), 2227-2238 (2012).</li><li>Mazzo, F., 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-modulated+subunit+stoichiometry+affects+stability+and+trafficking+of+alpha3beta4+nicotinic+receptor.">Nicotine-modulated subunit stoichiometry affects stability and trafficking of alpha3beta4 nicotinic receptor.</a> J Neurosci. 33 (30), 12316-12328 (2013).</li><li>Moroni, M., Zwart, R., Sher, E., Cassels, B. K., Bermudez, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#945;4&#946;2+nicotinic+receptors+with+high+and+low+acetylcholine+sensitivity:+pharmacology,+stoichiometry,+and+sensitivity+to+long-term+exposure+to+nicotine.">&#945;4&#946;2 nicotinic receptors with high and low acetylcholine sensitivity: pharmacology, stoichiometry, and sensitivity to long-term exposure to nicotine.</a> Mol Pharmacol. 70 (2), 755-768 (2006).</li><li>Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+green+fluorescent+protein.">The green fluorescent protein.</a> Annu Rev Biochem. 67, 509-544 (1998).</li><li>Miesenbock, G., De Angelis, D. A., Rothman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+secretion+and+synaptic+transmission+with+pH-sensitive+green+fluorescent+proteins.">Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.</a> Nature. 394 (6689), 192-195 (1998).</li><li>Fox-Loe, A. M., Dwoskin, L. P., Richards, C. I., Ming, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuromethods: Nicotinic Acetylcholine Receptor Technologies. 117, (2016).</li><li>Khiroug, S. S., 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=Dynamic+visualization+of+membrane-inserted+fraction+of+pHluorin-tagged+channels+using+repetitive+acidification+technique.">Dynamic visualization of membrane-inserted fraction of pHluorin-tagged channels using repetitive acidification technique.</a> BMC Neurosci. 10 (141), (2009).</li><li>Araki, Y., Lin, D. T., Huganir, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+membrane+insertion+of+the+AMPA+receptor+GluA2+subunit+is+regulated+by+NSF+binding+and+Q/R+editing+of+the+ion+pore.">Plasma membrane insertion of the AMPA receptor GluA2 subunit is regulated by NSF binding and Q/R editing of the ion pore.</a> Proc Natl Acad Sci U S A. 107 (24), 11080-11085 (2010).</li><li>Yudowski, G. 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=Real-time+imaging+of+discrete+exocytic+events+mediating+surface+delivery+of+AMPA+receptors.">Real-time imaging of discrete exocytic events mediating surface delivery of AMPA receptors.</a> J Neurosci. 27 (41), 11112-11121 (2007).</li><li>Mattheyses, A. L., Simon, S. M., Rappoport, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+with+total+internal+reflection+fluorescence+microscopy+for+the+cell+biologist.">Imaging with total internal reflection fluorescence microscopy for the cell biologist.</a> J Cell Sci. 123, 3621-3628 (2010).</li><li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+Internal+Reflection+Fluorescence+Microscopy.">Total Internal Reflection Fluorescence Microscopy.</a> Methods Cell Biol. 89, 169-221 (2008).</li><li>Paroutis, P., Touret, N., Grinstein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pH+of+the+secretory+pathway:+measurement,+determinants,+and+regulation.">The pH of the secretory pathway: measurement, determinants, and regulation.</a> Physiol (Bethesda). 19, 207-215 (2004).</li></ol>

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
<p class...

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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 ...

The overall goal of this fluorescence imaging method is to monitor changes in membrane protein trafficking and intracellular distribution. This method provides insight into changes in protein trafficking, such as the effect of genetic mutations or pharmacological agents on the rate of vesicle delivery and protein expression on the cell surface. The main advantage of this technique is that measurements can be performed rapidly, at high resolution, in real time, without destroying the sample.48 hours prior to imaging, mouse neuroblastoma 2A cells are transfected with a plasmid construct containing SEP, a pH sensitive fluorophor incorporated into the extracellular region of the protein of interest. When the transfected cells are ready for imaging, with total internal reflection fluorescence or TIRF microscopy, add two milliliters of pH 7.4 extracellular solution or ECS to the cells. Place a dish of cells on the translation stage and use stage mounts to secure the dish in place.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 milliseconds and optimize the fluorescence intensity by setting the EM gain. Transition the laser beam into TIRF by using the Stepper motor to translate the beam across the objective in a stepwise manner. 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, at which point the beam is no longer visible along the edge of the dish.Verify that cells are in TIRF mode by adjusting the focus knob. In TIRF, only one plane of cells can be focused, producing a highly defined image with high resolution of the plasma membrane. To begin this procedure, locate healthy transfected single cells in the imaging plane.Acquire a focused image of cells 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 photo bleaching.Use microscope imaging software capable of recording multiple XY locations to record the stage positions corresponding to each cell. In this way, capture images of 20 to 30 cells per dish while using the software to record the position of each cell. After all cell images at ph 7.4 are collected, manually remove the pH 7.4 ECS by pipetting without touching the dish.Carefully add two milliliters of pH 5.4 ECS to the dish and wait for 10 minutes. 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.The 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. To image single vesicle insertions, first replace the growth media of transfected cells with two milliliters of pH 7.4 ECS.Next, place the dish of transfected cells on the stage of the microscope and use stage mounts to secure the dish in place. Focus a single cell in TIRF as demonstrated earlier. If equipped, set the auto focus 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 milliseconds. 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. To begin the analysis of SEP fluorescence to determine subcellular localization, open the cell images using an image analysis software such as ImageJ.Subtract the background from both pH 5.4 and pH 7.4 images using the rolling ball setting. Using intensity based threshold to quantify fluorescence from a single cell at pH 7.4, first select image, adjust, and threshold. Then manually select a region of interest around the cell.Using the built in measure function under the analyze tab, measure the cell area, mean intensity, and integrated density. Repeat these measurements for the same cell at pH 5.4. 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 of 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. To analyze 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 three frames or 600 milliseconds.This example of a cell image at pH 7.4 shows receptors located on the plasma membrane and endoplasmic reticulum. At pH 5.4, receptors on the plasma membrane do not fluoresce, so only the endoplasmic reticulum resident receptors are visible. The difference between integrated density of the fluorescence at pH 7.4 and pH 5.4 corresponds to receptors localized to the plasma membrane.Single vesicle insertion events are visualized at pH 7.4 as a burst of fluorescence at the plasma membrane, lasting at least 600 milliseconds. Immediately preceding an insertion, no discernible burst is present. An increase in fluorescence intensity is seen as a transport vesicle carrying SEP arrives.The SEP labeled receptors then diffuse across the plasma membrane until indistinguishable from previously inserted receptors. Once mastered, this technique can be done in 45 minutes per dish, if performed properly. After watching this video, you should have a good understanding of how to monitor changes in protein distribution between the peripheral ER and plasma membrane.

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8.7K vistas.  University of Kentucky. The overall goal of this fluorescence imaging method is to monitor changes in membrane protein trafficking and intracellular distribution. This method provides insight into changes in protein trafficking, such as the effect of genetic mutations or pharmacological agents on the rate of vesicle delivery and protein expression on the cell surface. The main advantage of this technique is that measurements can be performed rapidly, at high resolution, in real time, without destroying the sample.