We thank Dr. Mark E. Schurdak and University of Pittsburgh Drug Discovery Institute for providing the high-content imager and initial trainings. Dr. Krzysztof Palczewski (Case Western Reserve University) generously shared the 1D4 and B630 anti-rhodopsin antibodies. The plasmid containing the cDNA of mouse rhodopsin-Venus construct was shared by Dr. Nevin Lambert (Augusta University). This work was supported by the National Institute of Health grant EY024992 to YC and P30EY008098 from University of Pittsburgh Vision Research Core grant.

NOTE: The rhodopsin transport assay. 
1. Preparation and Culture of Cells
<ol>
	<li>Revive cryo-preserved U2OS stable cells expressing the wild type (WT) or mutant mouse rhodopsin-Venus fusion proteins. Thaw the cells at 37 &#176;C until only small ice crystals are left in the vial. 
	NOTE: The U2OS cells are used in this protocol because there is no photoreceptor cell line available for in vitro studies and the pre-ciliary biosynthesis of rhodopsin is regulate...

<ol>
	<li>Gregersen, N., Bross, P., Vang, S., Christensen, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+misfolding+and+human+disease.">Protein misfolding and human disease.</a> Annual Review of Genomics and Human Genetics. 7, 103-124 (2006).</li><li>Daiger, S. P., Bowne, S. J., Sullivan, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspective+on+genes+and+mutations+causing+retinitis+pigmentosa.">Perspective on genes and mutations causing retinitis pigmentosa.</a> Archives of Ophthalmology. 125 (2), 151-158 (2007).</li><li>Daiger, S. P., Sullivan, L. S., Bowne, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+and+mutations+causing+retinitis+pigmentosa.">Genes and mutations causing retinitis pigmentosa.</a> Clinical Genetics. 84 (2), 132-141 (2013).</li><li>Stenson, P. D., 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=The+Human+Gene+Mutation+Database:+towards+a+comprehensive+repository+of+inherited+mutation+data+for+medical+research,+genetic+diagnosis+and+next-generation+sequencing+studies.">The Human Gene Mutation Database: towards a comprehensive repository of inherited mutation data for medical research, genetic diagnosis and next-generation sequencing studies.</a> Human Genetics. 136 (6), 665-677 (2017).</li><li>Sung, C. H., Schneider, B. G., Agarwal, N., Papermaster, D. S., Nathans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+heterogeneity+of+mutant+rhodopsins+responsible+for+autosomal+dominant+retinitis+pigmentosa.">Functional heterogeneity of mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa.</a> Proceedings of the National Academy of Sciences of the United States of America. 88 (19), 8840-8844 (1991).</li><li>Athanasiou, D., 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=The+molecular+and+cellular+basis+of+rhodopsin+retinitis+pigmentosa+reveals+potential+strategies+for+therapy.">The molecular and cellular basis of rhodopsin retinitis pigmentosa reveals potential strategies for therapy.</a> Progress in Retinal and Eye Research. 62, 1-23 (2018).</li><li>Chiang, W. 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=Robust+Endoplasmic+Reticulum-Associated+Degradation+of+Rhodopsin+Precedes+Retinal+Degeneration.">Robust Endoplasmic Reticulum-Associated Degradation of Rhodopsin Precedes Retinal Degeneration.</a> Molecular Neurobiology. 52 (1), 679-695 (2015).</li><li>Sakami, 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=Probing+mechanisms+of+photoreceptor+degeneration+in+a+new+mouse+model+of+the+common+form+of+autosomal+dominant+retinitis+pigmentosa+due+to+P23H+opsin+mutations.">Probing mechanisms of photoreceptor degeneration in a new mouse model of the common form of autosomal dominant retinitis pigmentosa due to P23H opsin mutations.</a> Journal of Biological Chemistry. 286 (12), 10551-10567 (2011).</li><li>Dryja, T. P., 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=Mutations+within+the+rhodopsin+gene+in+patients+with+autosomal+dominant+retinitis+pigmentosa.">Mutations within the rhodopsin gene in patients with autosomal dominant retinitis pigmentosa.</a> New England Journal of Medicine. 323 (19), 1302-1307 (1990).</li><li>Sohocki, M. M., 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=Prevalence+of+mutations+causing+retinitis+pigmentosa+and+other+inherited+retinopathies.">Prevalence of mutations causing retinitis pigmentosa and other inherited retinopathies.</a> Human Mutation. 17 (1), 42-51 (2001).</li><li>Chen, Y., 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=A+novel+small+molecule+chaperone+of+rod+opsin+and+its+potential+therapy+for+retinal+degeneration.">A novel small molecule chaperone of rod opsin and its potential therapy for retinal degeneration.</a> Nature Communications. 9 (1), (2018).</li><li>Chen, Y., Tang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+assays+to+identify+small+molecules+preventing+photoreceptor+degeneration+caused+by+the+rhodopsin+P23H+mutation.">High-throughput screening assays to identify small molecules preventing photoreceptor degeneration caused by the rhodopsin P23H mutation.</a> Methods in Molecular Biology. 1271, 369-390 (2015).</li><li>Chen, Y., 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=A+High-Throughput+Drug+Screening+Strategy+for+Detecting+Rhodopsin+P23H+Mutant+Rescue+and+Degradation.">A High-Throughput Drug Screening Strategy for Detecting Rhodopsin P23H Mutant Rescue and Degradation.</a> Investigative Ophthalmology &amp; Visual Science. 56 (4), 2553-2567 (2015).</li><li>Noorwez, S. M., 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=Pharmacological+chaperone-mediated+in+vivo+folding+and+stabilization+of+the+P23H-opsin+mutant+associated+with+autosomal+dominant+retinitis+pigmentosa.">Pharmacological chaperone-mediated in vivo folding and stabilization of the P23H-opsin mutant associated with autosomal dominant retinitis pigmentosa.</a> Journal of Biological Chemistry. 278 (16), 14442-14450 (2003).</li><li>Saliba, R. S., Munro, P. M., Luthert, P. J., Cheetham, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cellular+fate+of+mutant+rhodopsin:+quality+control,+degradation+and+aggresome+formation.">The cellular fate of mutant rhodopsin: quality control, degradation and aggresome formation.</a> Journal of Cell Science. 115, 2907-2918 (2002).</li><li>Kaushal, S., Khorana, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+in+rhodopsin.+7.+Point+mutations+associated+with+autosomal+dominant+retinitis+pigmentosa.">Structure and function in rhodopsin. 7. Point mutations associated with autosomal dominant retinitis pigmentosa.</a> Biochemistry. 33 (20), 6121-6128 (1994).</li><li>Chen, Y., Brooks, M. J., Gieser, L., Swaroop, A., Palczewski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+profiling+of+NIH3T3+cell+lines+expressing+opsin+and+the+P23H+opsin+mutant+identifies+candidate+drugs+for+the+treatment+of+retinitis+pigmentosa.">Transcriptome profiling of NIH3T3 cell lines expressing opsin and the P23H opsin mutant identifies candidate drugs for the treatment of retinitis pigmentosa.</a> Pharmacological Research. 115, 1-13 (2016).</li><li>Adamus, G., 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=Anti-rhodopsin+monoclonal+antibodies+of+defined+specificity:+characterization+and+application.">Anti-rhodopsin monoclonal antibodies of defined specificity: characterization and application.</a> Vision Research. 31 (1), 17-31 (1991).</li><li>Goodson, H. V., Dzurisin, J. S., Wadsworth, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+stable+cell+lines+expressing+GFP-tubulin+and+photoactivatable-GFP-tubulin+and+characterization+of+clones.">Generation of stable cell lines expressing GFP-tubulin and photoactivatable-GFP-tubulin and characterization of clones.</a> Cold Spring Harbor Protocols. 2010 (9), (2010).</li><li>Zhang, J. H., Chung, T. D., Oldenburg, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Statistical+Parameter+for+Use+in+Evaluation+and+Validation+of+High+Throughput+Screening+Assays.">A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.</a> Journal of Biomolecular Screening. 4 (2), 67-73 (1999).</li><li>Bray, M. A., Carpenter, A., Sittampalam, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+Assay+Development+Guidelines+for+Image-Based+High+Content+Screening+and+Analysis.">Advanced Assay Development Guidelines for Image-Based High Content Screening and Analysis.</a> Assay Guidance Manual. , (2004).</li><li>Sung, C. H., Davenport, C. M., Nathans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhodopsin+mutations+responsible+for+autosomal+dominant+retinitis+pigmentosa.+Clustering+of+functional+classes+along+the+polypeptide+chain.">Rhodopsin mutations responsible for autosomal dominant retinitis pigmentosa. Clustering of functional classes along the polypeptide chain.</a> Journal of Biological Chemistry. 268 (35), 26645-26649 (1993).</li><li>Krebs, M. P., 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=Molecular+mechanisms+of+rhodopsin+retinitis+pigmentosa+and+the+efficacy+of+pharmacological+rescue.">Molecular mechanisms of rhodopsin retinitis pigmentosa and the efficacy of pharmacological rescue.</a> Journal of Molecular Biology. 395 (5), 1063-1078 (2010).</li></ol>

Here, we showed a high-content imaging assay used for characterizing hits identified from a HTS. The only automation involved in these protocols is the high-content imager. The immunostaining and fluorescence imaging of rhodopsin have been used commonly to characterize the localization of rhodopsin5,14,15,16. However, the quantification of images taken by the traditional imaging methods is limi...

Protein misfolding is involved in muscular dystrophy, neural degenerations, as well as blinding diseases, including retinitis pigmentosa (RP)1. RP is an inherited and progressive retinal degeneration associated with mutations in over 60 genes affecting the function and homeostasis of rod photoreceptors or the retinal pigmented epitheliums (RPEs)2,3. No effective treatment is currently available for RP. Rhodopsin mutations account for about 25-30% of autosomal dominant (ad) RP cases. Among the more than 150 rhodopsin mutations4 (Human Gene Mutation Database, http:/www.hgmd.cf.ac.uk/), the Class II mutations cause the structural instability of the rhodopsin protein that contributes to the rod photoreceptor death and vision loss5,6,7,8. The P23H is the most frequent rhodopsin mutation in North America, which is also a typical example of the Class II rhodopsin mutations9,10. Due to its inherent structural instability, the misfolded rhodopsin is accumulated in the endoplasmic reticulum (ER) in mammalian cells, whereas the wild type rhodopsin is located on the plasma membrane5. The misfolded rhodopsin P23H mutant exhibits dominant negative cytotoxicity that is not due to haploinsufficiency, but is related to the activation of ER associated protein degradation pathway and the interrupted rod outer segment organization. To alleviate rod photoreceptor cell stress, one strategy is to stabilize the native folding of the mutant rhodopsin using a pharmacological chaperone.
To achieve this goal, we performed a cell-based high-throughput screen (HTSs)11,12,13 using a &#946;-galactosidase fragment complementation assay to quantify the P23H rhodopsin mutant transported on the plasma membrane. The robust and simple protocol of this HTS assay enabled us to explore the activities of about 79,000 small molecules for each screen. However, because this HTS assay reads luminescence signals, false positives including the &#946;-gal inhibitors, colored or cytotoxic compounds are included in the hit list waiting to be identified by a secondary assay.
The traditional immunostaining and fluorescence imaging methods have been used for years to study the rhodopsin transport in mammalian cells5,14,15,16. However, these conventional methods cannot be used to quantify pharmacological effects of more than 10 compounds towards rhodopsin transport because a reliable imaging analysis requires a large number of images taken under a highly consistent condition, which is not amendable by the conventional imaging methods. Here, we developed an immunostaining based high-content imaging protocol as a secondary assay to quantify the cell surface transport of misfolded rhodopsin mutants11,13,17. To label rhodopsin on the plasma membrane, we skipped the step of cell membrane permeabilization and immunostained the rhodopsin mutants by a monoclonal (B6-30) anti-rhodopsin recognizing the N-terminal epitope of rhodopsin at the extracellular side of the cell membrane18. To visualize the mutant rhodopsin in the whole cell, we fused rhodopsin with the Venus fluorescence protein. By the quantification of the fluorescence intensities in different fluorescence channels, we are able to obtain multiple parameters from one single experiment including the total rhodopsin intensity in the whole cell, on the cell surface, and the ratio of rhodopsin fluorescence on the cell surface to that in the whole cell. Applying this method to stable cells expressing a total of six misfolded rhodopsin mutants, we can generate a pharmacological profile of multiple small molecule chaperones towards these mutants. In this protocol, all cells are immunostained in a 384-well plate and imaged using an automated imaging system under a highly consistent imaging condition. An image analysis is performed to each well, containing images of more than 600 cells to reduce variation due to the heterogeneity of the cells with varying cell shape and protein expression level. The workflow of this protocol is summarized in Figure 1. The advantage of this method is that we obtain high-resolution images as well as multi-parameter quantifications from the image-based analysis. In general, this protocol can be modified and applied to quantify the transport of any misfolded membrane protein of interest.

<table>
	<tbody>
		<tr>
			<td>U2OS (rhodopsin-Venus) cells</td>
			<td>NA</td>
			<td>NA</td>
			<td>Stable cells generated from U2OS cells</td>
		</tr>
		<tr>
			<td>U2OS (T4R-rhodopsin-Venus) cells</td>
			<td>NA</td>
			<td>NA</td>
			<td>Stable cells generated from U2OS cells</td>
		</tr>
		<tr>
			<td>U2OS (P23H-rhodopsin-Venus) cells</td>
			<td>NA</td>
			<td>NA</td>
			<td>Stable cells generated from U2OS cells</td>
		</tr>
		<tr>
			<td>U2OS (P53R-rhodopsin-Venus) cells</td>
			<td>NA</td>
			<td>NA</td>
			<td>Stable cells generated from U2OS cells</td>
		</tr>
		<tr>
			<td>U2OS (C110Y-rhodopsin-Venus) cells</td>
			<td>NA</td>
			<td>NA</td>
			<td>Stable cells generated from U2OS cells</td>
		</tr>
		<tr>
			<td>U2OS (D190N-rhodopsin-Venus) cells</td>
			<td>NA</td>
			<td>NA</td>
			<td>Stable cells generated from U2OS cells</td>
		</tr>
		<tr>
			<td>U2OS (P267L-rhodopsin-Venus) cells</td>
			<td>NA</td>
			<td>NA</td>
			<td>Stable cells generated from U2OS cells</td>
		</tr>
		<tr>
			<td>DMEM high glucose</td>
			<td>Genesee Scientific</td>
			<td>25-500</td>
			<td>With L-Glutamine, sodium pyruvate</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>16140071</td>
			<td>Heat inactivated</td>
		</tr>
		<tr>
			<td>Plasmocin</td>
			<td>InvivoGen</td>
			<td>ant-mpt</td>
			<td>Mycoplasma elimination reagent</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (100X)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>100X concentrated antibiotic solutions to prevent bacteria contamination of cell cultures</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Genesee Scientific</td>
			<td>25-510</td>
			<td>0.25%, 1mM EDTA in HBSS without calcium and magnesium</td>
		</tr>
		<tr>
			<td>Poly-L-lysine solution</td>
			<td>Sigma-Aldrich</td>
			<td>P4707-50ML</td>
			<td>Mol wt 70,000-150,000, 0.01%, sterile-filtered, BioReagent, suitable for cell culture</td>
		</tr>
		<tr>
			<td>CellCarrier-384 Ultra Microplates</td>
			<td>PerkinElmer</td>
			<td>6057300</td>
			<td>384-well tissue culutre-treated microplates with black well walls and an optically -clear cyclic olefin bottom for imaging cells in high content analysis</td>
		</tr>
		<tr>
			<td>Sterile 96-well plate</td>
			<td>Eppendorf</td>
			<td>30730119</td>
			<td>Tissue culture treated with lid flat bottom, sterile, free of detectable pyrogens, Rnase, DNase and DNA. Non-cytotoxic</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Sailine (PBS)</td>
			<td>Invitrogen</td>
			<td>AM9625</td>
			<td>10 x PBS Buffer, pH 7.4</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D4540</td>
			<td>&#62;99.5%, cell culture tested</td>
		</tr>
		<tr>
			<td>9-cis-retinal</td>
			<td>Sigma-Aldrich</td>
			<td>R5754</td>
			<td></td>
		</tr>
		<tr>
			<td>Compounds tested</td>
			<td>Selleckchem/Life Chemicals/Custom synthesized</td>
			<td>NA</td>
			<td>Compounds were purchased from different vendors or custom synthesized</td>
		</tr>
		<tr>
			<td>B6-30 anti-rhodopsin antibody</td>
			<td>Novus</td>
			<td>NBP2-25160</td>
			<td>Gift from Dr. Krzysztof Palczewski</td>
		</tr>
		<tr>
			<td>Cy3-conjugated goat anti-mouse secondary antibody</td>
			<td>Jackson ImmunoResearch Laboratories, Inc</td>
			<td>115-165-146</td>
			<td></td>
		</tr>
		<tr>
			<td>16% paraformaldehyde</td>
			<td>Thermo Fisher Scientific</td>
			<td>28908</td>
			<td>Methanol-free</td>
		</tr>
		<tr>
			<td>10% Normal Goat Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>50062Z</td>
			<td>Blocking buffer</td>
		</tr>
		<tr>
			<td>Hoechst 33342, Trihydroch</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td>Nuclear staining solution</td>
		</tr>
		<tr>
			<td>High-content imager</td>
			<td>Molecular Devices</td>
			<td>ImageXpress</td>
			<td>ImageXpress&#174; Micro Confocal High-Content Imaging System</td>
		</tr>
		<tr>
			<td>MetaXpress high-content image acquisition and analysis software</td>
			<td>Molecular Devices</td>
			<td>MetaXpress</td>
			<td>High-content image acquisition and analysis software</td>
		</tr>
		<tr>
			<td>Multichannel pipette (0.5-10 &#181;L)</td>
			<td>Rainin</td>
			<td>17013802</td>
			<td>Manual 8-channel pipette, 0.5-10 &#181;L</td>
		</tr>
		<tr>
			<td>Multichannel pipette (0.5-10c</td>
			<td>Rainin</td>
			<td>17013805</td>
			<td>Manual 8-channel pipette, 20-200 &#181;L</td>
		</tr>
		<tr>
			<td>Electronic multichannel pipette (10-200 &#956;L)</td>
			<td>Thermo Scientific</td>
			<td>14-3879-56BT</td>
			<td>Electronic multichanenel pipette for 96- and 384-well microplate pipetting tasks</td>
		</tr>
		<tr>
			<td>50ml Reagent Reservoir</td>
			<td>Genesee Scientific</td>
			<td>28-125</td>
			<td>Reagent reservior for multichannel pippte dispensing</td>
		</tr>
		<tr>
			<td>8-Channel aspirator</td>
			<td>ABC Scientific</td>
			<td>EV503</td>
			<td>8-Channel stainless steel adaptor for aspirating liquids from 96- or 384-well plates</td>
		</tr>
		<tr>
			<td>Excel spreadsheet software</td>
			<td>Microsoft</td>
			<td>Excel2016</td>
			<td>The spreadsheet software for data analysis and heatmap generation</td>
		</tr>
		<tr>
			<td>Origin2018 scientific data analysis and graphing software</td>
			<td>OriginLab</td>
			<td>Origin2018</td>
			<td>The data analysis software for generating the dose response curves</td>
		</tr>
	</tbody>
</table>

a rhodopsin transport assay by high-content imaging analysis

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding disease that lacks effective treatment. We hypothesize that the cytotoxicity of the misfolded rhodopsin mutant can be alleviated by pharmacologically stabilizing the mutant rhodopsin protein. The P23H mutation, among the other Class II rhodopsin mutations, encodes a structurally unstable rhodopsin mutant protein that is accumulated in the endoplasmic reticulum (ER), whereas the wild type rhodopsin is transported to the plasma membrane in mammalian cells. We previously performed a luminescence-based high-throughput screen (HTS) and identified a group of pharmacological chaperones that rescued the transport of the P23H rhodopsin from ER to the plasma membrane. Here, using an immunostaining method followed by a high-content imaging analysis, we quantified the mutant rhodopsin protein amount in the whole cell and on the plasma membrane. This method is informative and effective to identify true hits from false positives following HTS. Additionally, the high-content image analysis enabled us to quantify multiple parameters from a single experiment to evaluate the pharmacological properties of each compound. Using this assay, we analyzed the effect of 11 different compounds towards six RP associated rhodopsin mutants, obtaining a 2-D pharmacological profile for a quantitative and qualitative understanding about the structural stability of these rhodopsin mutants and efficacy of different compounds towards these mutants.

We characterized the rhodopsin transport with three parameters: the rhodopsin-Venus intensity in the whole cell (Rhodopsin-Venus INT), the immunostaining intensity of rhodopsin on the plasma membrane (Rhodopsin INT on the cell surface), and the ratio of rhodopsin stain on the cell surface to rhodopsin-Venus intensity in the whole cell (MEM-Total Ratio). A representative result of the rhodopsin transport assay is shown in Figure 3 and Figu...

Watch this Scientific Journal Video about A Rhodopsin Transport Assay by High-Content Imaging Analysis at JoVE.com

A Rhodopsin Transport Assay by High-Content Imaging Analysis

Here, we described a high-content imaging method to quantify the transport of rhodopsin mutants associated with retinitis pigmentosa. A multiple-wavelength scoring analysis was used to quantify rhodopsin protein on the cell surface or in the whole cell.

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding ...

Rhodopsin misfolding causes progressive retinal degeneration called retinitis pigmentosa. While a traditional imaging method have limited capacity to quantify the rhodopsin transport, this newly-developed image-based assay allows for high throughput screening, eliminating false positives. This technique allows for a quick evaluation of drug pharmacodynamics.In the eye of rescuing the folding of disease-causing rhodopsin, thus accelerating the potential for a treatment for the quietly untreatable and blinding disease, retinitis pigmentosa. The cellular localization of a membrane protein is very important for its function. This method can be easily modified and applied to quantify the transport of any other membrane protein of interest.Make sure you prepare a plate map and gently add and aspirate liquid from each well of the plate, to keep cell number and the immuno state conditions consistent between the cells. This will yield a high Z factor, and reliable results. To begin, seed 5000 cells per well into a black walled, clear bottom, poly lysine treated 384-well-plate.Incubate the plate at 37 degrees Celsius with five percent CO2 for three hours for the cells to attach to the bottom of the plate. Next, use a multichannel pipette to add 10 microliters per well of 5X working solutions, according to a pre-determined plate map like the one shown here. Now, add 10 microliters per well of 2%DMSO to columns 22 and 24.Also, add 10 microliters per well of 25 micromolar 9-cis-retinal to column 23 in a dark room under dim red light. When finished loading all of the various test compounds, cover the plate with aluminum foil, and incubate at 37 degree Celsius for 24 hours. Take out the 384-well-plate in a dark room with dim red light.Here, using 8-channel-aspirator, connected to a vacuum collection bottle, to gentle aspirate the medium from the wells of the plate. Next, use a multichannel pipette to add 20 microliters per well of freshly prepared 4%Paraformaldehyde to the entire 384-well-plate, and fix the cells for 20 minutes at room temperature. After fixation, place the plate into normal light.There, use an 8-channel-aspirator, to aspirate the Paraformaldehyde in each well, and use a multichannel pipette to add 50 microliters per well of PBS. Aspirate and replace the PBS for a total of three wash cycles. Then, add 20 microliters per well of 5%goat serum to each well, and incubate the plate at room temperature for 30 minutes.Following incubation, aspirate the wells, and add 15 microliters of 20 micrograms per milliliter B630 anti-rhodopsin antibody, in 1%goat serum to the wells in rows A to O.Next, add 15 microliters per well of 1%goat serum to row P for the secondary antibody only control group. Incubate the plate at room temperature for 90 minutes, or at four degrees Celsius over night. Then, cover the plate with aluminum foil to avoid photobleaching of the fluorophores.Next, wash the plate three times with 50 microliters per well of PBS. Following the last wash, aspirate the PBS, and add 15 microliters per well of five micrograms per milliliter of Cy3-conjugated goat anti-mouse IgG antibody. Cover the plate with aluminum foil, and incubate the samples at room temperature for one hour.Following the incubation, wash the plate three times. Then, add 50 microliters per well of PBS, containing one microgram per milliliter of Hoechst stain at room temperature for 15 minutes. Finally, seal the well plate with a transparent film, cover it with aluminum foil, and store at four degrees Celsius for up to one week.Remove the foil and place the sample plate into a high content imager, with A1 positioned on the top left corner of the plate. Next, open the image acquisition software to set up parameters for image acquisition. Open the plate acquisition setup window and create new setting or load an existing setup file.Select the 20X objective and set up pixel binning as two, so the calibrated pixel size is 0.80 by 0.80 microns. Then, set the scan lines as 2000, and the image size as 1000 by 1000 pixels per site. Next, select the plate type to be imaged.Use information provided by the manufacturer to fill in the plate dimensions. Now, select the wells to be imaged along with four sites to be imaged per well. Avoid the side of the well, which are touched by the aspiration tips.For imaging, select excitation lasers as 405, 488 and 461 nanometers. Then, select the emission filters for DAPI, FITC and Texas Red channels. Optimize the laser power and gains of each channel, to ensure, the positive control wells are not saturated.Select well to well focus for auto-focus. Then, select the initial well to be acquired, and set the site auto-focus for all sites. Also, select four averages per line for each channel, and optimize the Z-offset value for each channel.Validate, that everything is properly set up, by first testing two to three wells at the corners of the plate, to make sure the images are on focus for all tested wells. Next, check, that images from all sites per well have cells with more than 40%confluence. Finally, check, that the fluorescence intensities in all of the channels are all about half saturated in the 100%control wells.Then, save the image acquisition method, and run the entire plate. Watch the imager until it finishes capturing images from the first column of the plate to double-check the image quality before leaving the imager. When finished, remove the plate and store it at four degrees Celsius for future use.Pull out image data, using a high content image analysis software. Select one of the 100%control wells in column 23 to set up the parameters. First, select the multi wavelength cell scoring as the analysis method, and start configuring the additional settings.Define the nuclei using images from the DAPI channel. Preview, to make sure the defined nuclei shapes in the selected well fits well with the nuclei images. Next, define the shape of cell in the FITC channel, where Rhodopsin Venus is imaged.To accomplish this, set up minimum and maximum diameters of each cell, minimum fluorescence intensity above background, as well as minimum area of each cell. Now, define the rhodopsin cell surface stain areas in the Texas Red channel, by setting up the minimum and maximum diameters of cell shape, minium fluorescence intensity above background, as well as minimum area of each stained cell. Test the current algorithm in five wells to determine, if the settings are optimized.Then, save the settings and close the configuration window. Run all the wells with the optimized analysis method. When finished, export the object number as the intact cell number.Export the average intensity of the FITC channel as the Rhodopsin Venus intensity, and export the average intensity of the Texas Red Channel as Rhodopsin intensity on the cell surface. Use the spreadsheet software to generate a two-color heat map for each parameter. Arrange the name of cell lines on the x-axis, and the compounds on the y-axis.Here, our images of P23H rhodopsin Venus treated U2OS cells, expressing Venus fluorescence in green, and cell surface immuno-staining in red. Cells were either treated with DMSO or five micromolar 9-cis-retinal. The rhodopsin transport assay, comparing the rhodopsin amount in the whole cell under various conditions, shows a significant recovery in the P23H mutant cell line, when treated with 9-cis-retinal.In addition, the rhodopsin intensity on the cell surface, and the ratio of rhodopsin stain on the cell surface to rhodopsin Venus intensity in the whole cell, are not significantly lower for the P23H mutant, compared to the wildtype rhodopsin treated with DMSO. A heat map is an efficient way to compare the average rhodopsin amount and localization per cell across multiple mutants. Here, wildtype controls and six rhodopsin mutants are listed horizontally, and the effects of compound treatments are compared vertically.In agreement with previous studies, the rhodopsin intensity on the cell surface and the ratio of rhodopsin stain on the cell surface to rhodopsin Venus intensity are lower for the six mutants compared to the wildtype treated with DMSO. Compounds one, two, six and nine significantly increased the ratio of rhodopsin stain on the cell surface to rhodopsin Venus intensity in T4R, P23H, D190N, and P267L rhodopsin mutants, suggesting that these compounds rescue the transport of these rhodopsin mutants to the plasma membrane. Keep in mind, that cells need to be between 50 and 70%confluent before fixation, as this is critical for image analysis.Also, we showed images of a wells across the whole plate. Make sure, that images in focus, and that the fluorescence, that don't exit, have all the threshold value for any channel. Up to a selecting compounds, that rescue misfolded rhodopsin transport, we can validate the process, then pass the efficacy of these compounds in vivo, using a mouse-model expressing the rhodopsin P23H mutant.This method allows us to quantify membrane protein amount, and its localization. Therefore, it is a great tool for micro mistake started on membrane protein per assist save and degradation. Paraformaldehyde, as is in this experiment, has procedures involving the handling of this reagent.It will be done under the hood. Waste including this reagent should be properly disposed of as a hazardous chemical.

a-rhodopsin-transport-assay-by-high-content-imaging-analysis

Research

JoVE Journal

Chemistry

6.4K visualizações.  University of Pittsburgh. O descompactamento da rhodopsina causa degeneração progressiva da retina chamada retinite pigmentosa. Embora um método de imagem tradicional tenha capacidade limitada para quantificar o transporte de rodopsina, este ensaio recém-desenvolvido baseado em imagem permite uma triagem de alto rendimento, eliminando falsos positivos. Esta técnica permite uma avaliação rápida da farmacodinâmica medicamentosa.No olho de resgatar a dobra da rhodopsina causadora de doenças, acelerando a...