A Rhodopsin Transport Assay by High-Content Imaging Analysis

Summary

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.

Abstract

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.

Introduction

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 Mutati....

Protocol

NOTE: The rhodopsin transport assay.

1. Preparation and Culture of Cells

1. Revive cryo-preserved U2OS stable cells expressing the wild type (WT) or mutant mouse rhodopsin-Venus fusion proteins. Thaw the cells at 37 °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 regulated by similar molecular mechanisms in mammalian cells. Additionally, the U2OS cells attach tightly to the bottom of the plate and have large cell bodie....

Results

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.......

Discussion

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 rhodopsin^5,14,15,16. However, the quantification of images taken by the traditional imaging methods is limi.......

Materials

List of materials used in this article

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