Image-Based Analysis of Protein Stability

1024 177 Penn Lab
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Cytometry Part A 2019 November;

DOI: 10.1002/cyto.a.23928


Hickman KA, Hariharan S, de Melo J, Ylanko J, Lustig LC, Penn LZ, Andrews DW.


Short half-life proteins regulate many essential processes, including cell cycle, tran- scription, and apoptosis. However, few well-characterized protein-turnover pathways have been identified because traditional methods to measure protein half-life are time and labor intensive. To overcome this barrier, we developed a protein stability probe and high-content screening pipeline for novel regulators of short half-life proteins using automated image analysis. Our pilot probe consists of the short half-life protein c-MYC (MYC) fused to Venus fluorescent protein (MYC-Venus). This probe enables protein half-life to be scored as a function of fluorescence intensity and distribution. Rapid turnover prevents maximal fluorescence of the probe due to the relatively longer maturation time of the fluorescent protein. Cells expressing the MYC-Venus probe were analyzed using a pipeline in which automated confocal microscopy and image analyses were used to score MYC-Venus stability by two strategies: assaying the per- centage of cells with Venus fluorescence above background, and phenotypic compara- tive analysis. To evaluate this high-content screening pipeline and our probe, a kinase inhibitor library was screened by confocal microscopy to identify known and novel kinases that regulate MYC stability. Compounds identified were shown to increase the half-life of both MYC-Venus and endogenous MYC, validating the probe and pipeline. Fusion of another short half-life protein, myeloid cell leukemia 1 (MCL1), with Venus also demonstrated an increase in percent Venus-positive cells after treatment with inhibitors known to stabilize MCL1. Together, the results validate the use of our auto- mated microscopy and image analysis pipeline of stability probe-expressing cells to rapidly and quantitatively identify regulators of short half-life proteins.