Establishment And Verification Of High Throughput Automated Live Cell Screening For Drugs That Affect Ciliation

Objective: The primary cilium,is a highly conservative,hair-like organelle that protrudes from the surface of the majority of mammalian cells.Biologically,it could be serve as receptor and signaling hub,regulating cell basic physiological and pathological processes,such as cell proliferation,cell polarity,cell migration and cell invasion.The structural integrity and assembly/disassembly capability are essential for primary cilia biological function.The absence or dysfunctions of cilia,or the mutations of ciliaassociated genes could result in severe genetic disorders termed ciliopathies.Recent studies have found that large number of compounds have unexpected activities in regulating ciliation,which may further affect the progression of cilia-related diseases,such as cancer and polycystic kidney disease.However,current cellular models have limitations on evaluating large numbers of drugs that affect ciliation.Therefore,we here construct a cost-effectively microscopy-based high throughput automated live cell screening for identifying molecules that affect ciliation,in order to identify ciliary activities of existing agents,and identify drugs that may have beneficial activity through action on human diseases.Methods: ARL13 B,SMO,MCHR1 were chosen to label primary cilia after literature investigation.Then,stably transfected hTERT-RPE1-ARL13B-EGFP,hTERTRPE1-SMO-EGFP,hTERT-RPE1-MCHR1-TdTomato cell lines were constructed by genetic engineering.Then the structure and biofunctions of cilia on these cell lines were verified by the co-localization between tagged protein and classical cilia marker;and sensitivity of serum induced ciliary disassembly.After verification,we construct a high throughput screening methods for identifying compounds that affect ciliation,through serum induced ciliary disassembly in different density of cells.At last,we verified and optimized this method by testing drugs those have been reported affecting ciliary disassembly.Results: The fluorescent-tagged ARL13 B,SMO and MCHR1 proteins were localized to the primary cilia of hTERT-RPE1 cells in all three stably transfected cell lines.And its expressing almost had no effect on ciliary assembly,disassembly and cilia responses to external stimulation.The high-throughput screening method,which based on ImageXpress imaging system,could effectively identify drugs that regulate ciliary disassembly,at an appropriate cell density.Conclusion: This project initially constructed an automated high-throughput screening method that could effectively and efficiently identify drugs that affect ciliary integrity.With further verification,this system could successfully identify drugs that could significantly affect ciliary disassembly.