High-content Functional Screen to Identify Proteins that Correct F508del-CFTR Function

Cystic Fibrosis is caused by mutations in CFTR, with a deletion of a phenylalanine at position 508 (F508del-CFTR) representing the most common mutation. The F508del-CFTR protein exhibits a trafficking defect and is retained in the endoplasmic reticulum. Here we describe the development of a high-content screen based on a functional assay to identify proteins that correct the F508del-CFTR defect. Using a HEK293 MSR GripTite cell line that stably expresses F508del-CFTR, we individually co-expressed ∼450 unique proteins fused to the Cl−-sensitive YFP(H148Q/I152L) mutant. We then tested correction of F508del-CFTR function by the CI−/l− exchange method following stimulation with forskolin/IBMX/genistein, using quantitative recordings in multiple individual cells with a high-content (high-throughput) Cellomics KSR imaging system. Using this approach, we identified several known and novel proteins that corrected F508del-CFTR function, including STAT1, Endothelin 1, HspA4, SAPK substrate protein 1, AP2M1, LGALS3/galectin-3, Trk-fused gene, Caveolin 2, PAP/REG3α, and others. The ability of these correctors to rescue F508del-CFTR trafficking was then validated by demonstrating their enhancement of maturation (appearance of band C) and by cell surface expression of F508del-CFTR bearing HA tag at the ectodomain using confocal microscopy and flow cytometry. These data demonstrate the utility of high-content analyses for identifying proteins that correct mutant CFTR and discover new proteins that stimulate this correction. This assay can also be utilized for RNAi screens to identify inhibitory proteins that block correction of F508del-CFTR, small molecule, and peptide screens. Cystic Fibrosis is caused by mutations in CFTR, with a deletion of a phenylalanine at position 508 (F508del-CFTR) representing the most common mutation. The F508del-CFTR protein exhibits a trafficking defect and is retained in the endoplasmic reticulum. Here we describe the development of a high-content screen based on a functional assay to identify proteins that correct the F508del-CFTR defect. Using a HEK293 MSR GripTite cell line that stably expresses F508del-CFTR, we individually co-expressed ∼450 unique proteins fused to the Cl−-sensitive YFP(H148Q/I152L) mutant. We then tested correction of F508del-CFTR function by the CI−/l− exchange method following stimulation with forskolin/IBMX/genistein, using quantitative recordings in multiple individual cells with a high-content (high-throughput) Cellomics KSR imaging system. Using this approach, we identified several known and novel proteins that corrected F508del-CFTR function, including STAT1, Endothelin 1, HspA4, SAPK substrate protein 1, AP2M1, LGALS3/galectin-3, Trk-fused gene, Caveolin 2, PAP/REG3α, and others. The ability of these correctors to rescue F508del-CFTR trafficking was then validated by demonstrating their enhancement of maturation (appearance of band C) and by cell surface expression of F508del-CFTR bearing HA tag at the ectodomain using confocal microscopy and flow cytometry. These data demonstrate the utility of high-content analyses for identifying proteins that correct mutant CFTR and discover new proteins that stimulate this correction. This assay can also be utilized for RNAi screens to identify inhibitory proteins that block correction of F508del-CFTR, small molecule, and peptide screens. Cystic fibrosis (CF) 1The abbreviations used are: CF, cystic fibrosis; 293MSR-GT, HEK293 macrophage scavenger receptor GripTite cell; FIG, forskolin/IBMX/genistein; CFTR, cystic fibrosis transmembrane conductance regulator; RNAi, RNA interference; esiRNA, endoribonuclease-prepared siRNA; ConA, concanavalin A; PBS, phosphate-buffered saline; FBS, fetal bovine serum; BHK, Baby hamster kidney; ET-1, endothelin 1; HA, hemagglutinin; WT, wild type; PAP, pancreatitis-associated protein 1; GFP, green 1The abbreviations used are: CF, cystic fibrosis; 293MSR-GT, HEK293 macrophage scavenger receptor GripTite cell; FIG, forskolin/IBMX/genistein; CFTR, cystic fibrosis transmembrane conductance regulator; RNAi, RNA interference; esiRNA, endoribonuclease-prepared siRNA; ConA, concanavalin A; PBS, phosphate-buffered saline; FBS, fetal bovine serum; BHK, Baby hamster kidney; ET-1, endothelin 1; HA, hemagglutinin; WT, wild type; PAP, pancreatitis-associated protein 1; GFP, green is the most common in the is caused by mutations in the CFTR gene, a in Cystic CFTR function and for several of in CFTR identified to Cystic the most common in of is a deletion of phenylalanine at position 508 (F508del-CFTR) of the cystic fibrosis The F508del-CFTR mutant is a trafficking mutant that is retained in the endoplasmic of in cells of The common of cystic fibrosis transmembrane conductance is by and in a a for maturation of the to correction of this trafficking defect can be by the cells with of mutant cystic fibrosis transmembrane conductance is the of the most common cystic fibrosis These be used to the several screens to identify small that can correct the trafficking and functional of the F508del-CFTR and CFTR identified by of based on and CFTR identified by of cystic fibrosis transmembrane conductance conductance identified by small correctors of correctors of identified by of protein trafficking identified by a novel of identified a novel of the F508del-CFTR trafficking and of and CFTR by a novel of trafficking and in cystic fibrosis by small of these in identifying small that correct the trafficking defect of F508del-CFTR can be a identify the protein that is by the small we on the development of a functional assay that for the of proteins that correct F508del-CFTR function in multiple individual cells using Cellomics We a HEK293 MSR GripTite cell line that stably expresses F508del-CFTR and individually several proteins fused to the Cl−-sensitive YFP(H148Q/I152L) small correctors of with and to for CFTR in a Here we describe the development of the assay and the of proteins that co-expressed with F508del-CFTR rescue function by and phosphate-buffered with and fetal bovine and was and was and concanavalin was was The was the Cystic Fibrosis by and was and and was The was and the was The and the and and new was by the with in the the then by this new assay of and proteins using for and was RNA by with RNA in MSR GripTite cells stably with wild F508del-CFTR in using the cells and and of wild CFTR mutant F508del-CFTR was validated by using cells in with FBS, and at in Baby hamster cells stably wild mutant protein with the tag at the ectodomain a in with and at The cells in the for a of the cells in the The the cells on with of using the method and at was to and the was at for the cells F508del-CFTR and and the with for and using cell cells in to the The cells then using with of of the cells in on in to the of cells using to of proteins and protein was in in in a with the was with of in the of the at of of was of and in was using the Cellomics at The assay was also on cells at with for using the The was to Cellomics based on for on the of the was for a was used for data of to that rescue of at in and protein and rescue the was in of of the F508del-CFTR for and to the cells in and in on to and with with with in and with was with cells with PBS, with phosphate-buffered and then with and with and bovine for The was in the and at for with and with for The and on cells then by confocal cells and in with the cell cells with a for at the cells with the and with at for then and in with The was using The data cells and with this we describe the development of a functional screen to identify proteins that with F508del-CFTR correct This assay can also be used to identify proteins that CFTR function rescue of F508del-CFTR at for RNAi small peptide screens. the ability of the Cellomics to data cells that the protein cells the of this of assay is for screens RNAi that to the of on proteins on small is that can identify novel in CFTR of the proteins that we tested to CFTR maturation to the of CFTR in cystic The in CFTR CFTR and and of these also identified F508del-CFTR rescue in proteins by several for their of their that in their their is correctors we identified several HspA4, and proteins and trafficking proteins AP2M1, proteins and STAT1, ET-1, proteins in the and proteins with function and in of these proteins correction the a known of F508del-CFTR correctors of identified by of the F508del-CFTR protein we identified is and of is a in the of of in the of known of with also that in cell to a in CFTR expression CFTR expression in of cystic fibrosis transmembrane conductance by in cells and in expression of is to a in and for this of and of of and protein in cystic fibrosis correction of and expression in cystic fibrosis The of protein in F508del-CFTR and a new to of the of the in and the by the cells to correct the to STAT1, the proteins identified correctors of the F508del-CFTR defect was to CFTR in and of in and that also trafficking of F508del-CFTR to the is in is in cystic fibrosis and a to in and of the This at a is also a ET-1, the identified protein correctors known to CFTR maturation pancreatitis-associated protein and is a protein with the expression of is in the of in that and in of protein in to for cystic of in for cystic using pancreatitis-associated protein and The of was is known to be in to in the that is with to cystic fibrosis transmembrane conductance for of for to cystic for cystic fibrosis of to is that screen also identified proteins that maturation of F508del-CFTR at The proteins that we identified The function of is with the and is for of proteins to their the for and of the by novel protein the to is that of to of F508del-CFTR, is to in rescue of this mutant of the proteins identified in the screen known to with CFTR function, the of the novel and the by F508del-CFTR trafficking and function known and small that correct the maturation defect of F508del-CFTR be a identify the protein that is by the small screen can identify novel in CFTR F508del-CFTR and to known to of the can then be tested for correction of F508del-CFTR of HspA4, and proteins correct F508del-CFTR cell surface expression and function to the ability of that the including stimulation of to rescue that can correct F508del-CFTR function, in with a by and of endoplasmic fibrosis transmembrane and and with a that rescue of mutant CFTR with the utility of for of the cystic fibrosis is used for the of multiple be to for is to that F508del-CFTR function in the of that function a and of of a of F508del-CFTR cell surface expression and function, be the of known to STAT1, on rescue of can be in several of in the of rescue of F508del-CFTR of and the of in the correction of and expression in cystic fibrosis the that the is in the rescue of be the of on rescue of and that the in cells by correction of and expression in cystic fibrosis correctors we identified corrected F508del-CFTR to their of at that correction of of CFTR be to function of for of function in cystic the of rescue of F508del-CFTR is in with cystic fibrosis and and function in with cystic rescue of F508del-CFTR function the development of a high-content assay to identify correctors of F508del-CFTR and the of several known and novel proteins that co-expressed with F508del-CFTR to rescue of trafficking and functional the by these novel rescue of F508del-CFTR, the of these proteins on cells Cystic fibrosis (CF) 1The abbreviations used are: CF, cystic fibrosis; 293MSR-GT, HEK293 macrophage scavenger receptor GripTite cell; FIG, forskolin/IBMX/genistein; CFTR, cystic fibrosis transmembrane conductance regulator; RNAi, RNA interference; esiRNA, endoribonuclease-prepared siRNA; ConA, concanavalin A; PBS, phosphate-buffered saline; FBS, fetal bovine serum; BHK, Baby hamster kidney; ET-1, endothelin 1; HA, hemagglutinin; WT, wild type; PAP, pancreatitis-associated protein 1; GFP, green 1The abbreviations used are: CF, cystic fibrosis; 293MSR-GT, HEK293 macrophage scavenger receptor GripTite cell; FIG, forskolin/IBMX/genistein; CFTR, cystic fibrosis transmembrane conductance regulator; RNAi, RNA interference; esiRNA, endoribonuclease-prepared siRNA; ConA, concanavalin A; PBS, phosphate-buffered saline; FBS, fetal bovine serum; BHK, Baby hamster kidney; ET-1, endothelin 1; HA, hemagglutinin; WT, wild type; PAP, pancreatitis-associated protein 1; GFP, green is the most common in the is caused by mutations in the CFTR gene, a in Cystic CFTR function and for several of in CFTR identified to Cystic the most common in of is a deletion of phenylalanine at position 508 (F508del-CFTR) of the cystic fibrosis The F508del-CFTR mutant is a trafficking mutant that is retained in the endoplasmic of in cells of The common of cystic fibrosis transmembrane conductance is by and in a a for maturation of the to correction of this trafficking defect can be by the cells with of mutant cystic fibrosis transmembrane conductance is the of the most common cystic fibrosis These be used to the several screens to identify small that can correct the trafficking and functional of the F508del-CFTR and CFTR identified by of based on and CFTR identified by of cystic fibrosis transmembrane conductance conductance identified by small correctors of correctors of identified by of protein trafficking identified by a novel of identified a novel of the F508del-CFTR trafficking and of and CFTR by a novel of trafficking and in cystic fibrosis by small of these in identifying small that correct the trafficking defect of F508del-CFTR can be a identify the protein that is by the small we on the development of a functional assay that for the of proteins that correct F508del-CFTR function in multiple individual cells using Cellomics We a HEK293 MSR GripTite cell line that stably expresses F508del-CFTR and individually several proteins fused to the Cl−-sensitive YFP(H148Q/I152L) small correctors of with and to for CFTR in a Here we describe the development of the assay and the of proteins that co-expressed with F508del-CFTR rescue function by and phosphate-buffered with and fetal bovine and was and was and concanavalin was was The was the Cystic Fibrosis by and was and and was The was and the was The and the and and new was by the with in the the then by this new assay of and proteins using for and was RNA by with RNA in MSR GripTite cells stably with wild F508del-CFTR in using the cells and and of wild CFTR mutant F508del-CFTR was validated by using cells in with FBS, and at in Baby hamster cells stably wild mutant protein with the tag at the ectodomain a in with and at The cells in the for a of the cells in the The the cells on with of using the method and at was to and the was at for the cells F508del-CFTR and and the with for and using cell cells in to the The cells then using with of of the cells in on in to the of cells using to of proteins and protein was in in in a with the was with of in the of the at of of was of and in was using the Cellomics at The assay was also on cells at with for using the The was to Cellomics based on for on the of the was for a was used for data of to that rescue of at in and protein and rescue the was in of of the F508del-CFTR for and to the cells in and in on to and with with with in and with was with cells with PBS, with phosphate-buffered and then with and with and bovine for The was in the and at for with and with for The and on cells then by confocal cells and in with the cell cells with a for at the cells with the and with at for then and in with The was using The data cells and with and phosphate-buffered with and fetal bovine and was and was and concanavalin was was The was the Cystic Fibrosis by and was and and was The was and the was The and the and phosphate-buffered with and fetal bovine and was and was and concanavalin was was The was the Cystic Fibrosis by and was and and was The was and the was The and the and and new was by the with in the the then by this new assay of and proteins using for and was RNA by with RNA in new was by the with in the the then by this new assay of and proteins using for and was RNA by with RNA in MSR GripTite cells stably with wild F508del-CFTR in using the cells and and of wild CFTR mutant F508del-CFTR was validated by using cells in with FBS, and at in Baby hamster cells stably wild mutant protein with the tag at the ectodomain a in with and at The cells in the for a of HEK293 MSR GripTite cells stably with wild F508del-CFTR in using the cells and and of wild CFTR mutant F508del-CFTR was validated by using cells in with FBS, and at in Baby hamster cells stably wild mutant protein with the tag at the ectodomain a in with and at The cells in the for a of the cells in the The the cells on with of using the method and at was to and the was at for the cells F508del-CFTR and and the with for and using cell cells in to the The cells then using with of of the cells in on in to the of cells using to the cells in the The the cells on with of using the method and at was to and the was at for the cells F508del-CFTR and and the with for and using cell cells in to the The cells then using with of of the cells in on in to the of cells using to Cellomics of proteins and protein was in in in a with the was with of in the of the at of of was of and in was using the Cellomics at The assay was also on cells at with for using the The was to of proteins and protein was in in in a with the was with of in the of the at of of was of and in was using the Cellomics at The assay was also on cells at with for using the The was to Cellomics based on for on the of the was for a was used for data of to that rescue of at in and protein and rescue the was in The Cellomics based on for on the of the was for a was used for data of to that rescue of at in and protein and rescue the was in of of the F508del-CFTR for and to the cells in and in on to and with with with in and with was with cells with PBS, with phosphate-buffered and then with and with and bovine for The was in the and at for with and with for The and on cells then by confocal cells and in with the cell cells with a for at the cells with the and with at for then and in with The was using The data cells and with The for and to the cells in and in on to and with with with in and with was with cells with PBS, with phosphate-buffered and then with and with and bovine for The was in the and at for with and with for The and on cells then by confocal cells and in with the cell cells with a for at the cells with the and with at for then and in with The was using The data cells and with the cells in and in on to and with with with in and with was with the cells in and in on to and with with with in and with was with cells with PBS, with phosphate-buffered and then with and with and bovine for The was in the and at for with and with for The and on cells then by confocal cells with PBS, with phosphate-buffered and then with and with and bovine for The was in the and at for with and with for The and on cells then by confocal cells and in with the cell cells with a for at the cells with the and with at for then and in with The was using The data cells and with cells and in with the cell cells with a for at the cells with the and with at for then and in with The was using The data cells and with this we describe the development of a functional screen to identify proteins that with F508del-CFTR correct This assay can also be used to identify proteins that CFTR function rescue of F508del-CFTR at for RNAi small peptide screens. the ability of the Cellomics to data cells that the protein cells the of this of assay is for screens RNAi that to the of on proteins on small is that can identify novel in CFTR of the proteins that we tested to CFTR maturation to the of CFTR in cystic The in CFTR CFTR and and of these also identified F508del-CFTR rescue in proteins by several for their of their that in their their is correctors we identified several HspA4, and proteins and trafficking proteins AP2M1, proteins and STAT1, ET-1, proteins in the and proteins with function and in of these proteins correction the a known of F508del-CFTR correctors of identified by of the F508del-CFTR protein we identified is and of is a in the of of in the of known of with also that in cell to a in CFTR expression CFTR expression in of cystic fibrosis transmembrane conductance by in cells and in expression of is to a in and for this of and of of and protein in cystic fibrosis correction of and expression in cystic fibrosis The of protein in F508del-CFTR and a new to of the of the in and the by the cells to correct the to STAT1, the proteins identified correctors of the F508del-CFTR defect was to CFTR in and of in and that also trafficking of F508del-CFTR to the is in is in cystic fibrosis and a to in and of the This at a is also a ET-1, the identified protein correctors known to CFTR maturation pancreatitis-associated protein and is a protein with the expression of is in the of in that and in of protein in to for cystic of in for cystic using pancreatitis-associated protein and The of was is known to be in to in the that is with to cystic fibrosis transmembrane conductance for of for to cystic for cystic fibrosis of to is that screen also identified proteins that maturation of F508del-CFTR at The proteins that we identified The function of is with the and is for of proteins to their the for and of the by novel protein the to is that of to of F508del-CFTR, is to in rescue of this mutant of the proteins identified in the screen known to with CFTR function, the of the novel and the by F508del-CFTR trafficking and function known and small that correct the maturation defect of F508del-CFTR be a identify the protein that is by the small screen can identify novel in CFTR F508del-CFTR and to known to of the can then be tested for correction of F508del-CFTR of HspA4, and proteins correct F508del-CFTR cell surface expression and function to the ability of that the including stimulation of to rescue that can correct F508del-CFTR function, in with a by and of endoplasmic fibrosis transmembrane and and with a that rescue of mutant CFTR with the utility of for of the cystic fibrosis is used for the of multiple be to for is to that F508del-CFTR function in the of that function a and of of a of F508del-CFTR cell surface expression and function, be the of known to STAT1, on rescue of can be in several of in the of rescue of F508del-CFTR of and the of in the correction of and expression in cystic fibrosis the that the is in the rescue of be the of on rescue of and that the in cells by correction of and expression in cystic fibrosis correctors we identified corrected F508del-CFTR to their of at that correction of of CFTR be to function of for of function in cystic the of rescue of F508del-CFTR is in with cystic fibrosis and and function in with cystic rescue of F508del-CFTR function the development of a high-content assay to identify correctors of F508del-CFTR and the of several known and novel proteins that co-expressed with F508del-CFTR to rescue of trafficking and functional the by these novel rescue of F508del-CFTR, the of these proteins on cells this we describe the development of a functional screen to identify proteins that with F508del-CFTR correct This assay can also be used to identify proteins that CFTR function rescue of F508del-CFTR at for RNAi small peptide screens. the ability of the Cellomics to data cells that the protein cells the of this of assay is for screens RNAi that to the of on proteins on small is that can identify novel in CFTR of the proteins that we tested to CFTR maturation to the of CFTR in cystic The in CFTR CFTR and and of these also identified F508del-CFTR rescue in proteins by several for their of their that in their their is The correctors we identified several HspA4, and proteins and trafficking proteins AP2M1, proteins and STAT1, ET-1, proteins in the and proteins with function and in of these proteins correction the a known of F508del-CFTR correctors of identified by of the F508del-CFTR protein we identified is and of is a in the of of in the of known of with also that in cell to a in CFTR expression CFTR expression in of cystic fibrosis transmembrane conductance by in cells and in expression of is to a in and for this of and of of and protein in cystic fibrosis correction of and expression in cystic fibrosis The of protein in F508del-CFTR and a new to of the of the in and the by the cells to correct the defect. to STAT1, the proteins identified correctors of the F508del-CFTR defect was to CFTR in and of in and that also trafficking of F508del-CFTR to the is in is in cystic fibrosis and a to in and of the This at a is also a ET-1, the identified protein correctors known to CFTR maturation pancreatitis-associated protein and is a protein with the expression of is in the of in that and in of protein in to for cystic of in for cystic using pancreatitis-associated protein and The of was is known to be in to in the that is with to cystic fibrosis transmembrane conductance for of for to cystic for cystic fibrosis of to is that screen also identified proteins that maturation of F508del-CFTR at The proteins that we identified The function of is with the and is for of proteins to their the for and of the by novel protein the to is that of to of F508del-CFTR, is to in rescue of this mutant of the proteins identified in the screen known to with CFTR function, the of the novel and the by F508del-CFTR trafficking and function known and small that correct the maturation defect of F508del-CFTR be a identify the protein that is by the small screen can identify novel in CFTR F508del-CFTR and to known to of the can then be tested for correction of F508del-CFTR of HspA4, and proteins correct F508del-CFTR cell surface expression and function to the ability of that the including stimulation of to rescue that can correct F508del-CFTR function, in with a by and of endoplasmic fibrosis transmembrane and and with a that rescue of mutant CFTR with the utility of for of the cystic fibrosis is used for the of multiple be to for is to that F508del-CFTR function in the of that function a and of of a of F508del-CFTR cell surface expression and function, be the of known to STAT1, on rescue of can be in several of in the of rescue of F508del-CFTR of and the of in the correction of and expression in cystic fibrosis the that the is in the rescue of be the of on rescue of and that the in cells by correction of and expression in cystic fibrosis The correctors we identified corrected F508del-CFTR to their of at that correction of of CFTR be to function of for of function in cystic the of rescue of F508del-CFTR is in with cystic fibrosis and and function in with cystic rescue of F508del-CFTR function the development of a high-content assay to identify correctors of F508del-CFTR and the of several known and novel proteins that co-expressed with F508del-CFTR to rescue of trafficking and functional the by these novel rescue of F508del-CFTR, the of these proteins on cells We and for the on assay development and data for for the and and for the

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