Lo, C.-A. | Search bioRxiv

bioRxiv2024

A Drug Screening Platform for Protein Expression Levels in Neurological Disorders

Neurological and psychiatric diseases and disorders affect more than half of the population. Many of these diseases are caused by the malfunctioning of protein synthesis, where too little or too much production of a protein harms a cell and its functions within the brain. We developed a drug screening platform to identify compounds that target the primary cause of these diseases, namely protein expression amounts. This cellular assay monitors protein expression of a target disease gene along with the protein expression of a control gene using the Protein Quantitation Ratioing (PQR) technique. PQR tracks protein concentration using fluorescence. We used human cells and CRISPR-Cas9 genome editing to insert the Protein Quantitation Reporter into target genes. These cells are used in high-throughput drug screening measuring the fluorescence as the assay. Drug hits can be validated using the same PQR technique or animal models of the disease. HighlightsO_LIThe assay can identify drugs that directly address the molecular cause of a disease. C_LIO_LIThe Protein Quantitation Ratioing (PQR) technique allows for tracking and measuring protein amounts over time in single living cells before, during, and after drug administration. C_LIO_LIGenome editing to insert the PQR into the target gene allows tracking of endogenous protein expression. C_LIO_LIUsing human cell lines allow for faster production of knock-in cells. C_LIO_LIPatient mutations can be replicated using genome editing during the knock-in step. C_LIO_LIUsing induced pluripotent stem cells allow for an unlimited supply of genome edited differentiated cells such as neurons with the PQR knock-in reporter. C_LI

neuroscience↗

bioRxiv2018

Parental Allele-Specific Protein Synthesis Dynamics in Single Cells In Vivo

Allelic expression from each parent-of-origin is important as a backup and to ensure that enough protein products of a gene are produced. Thus far, it is not known how each cell throughout a tissue differs in parental allele expression at the level of protein synthesis. Here, we measure the expression of the Ribosomal protein L13a (Rpl13a) from both parental alleles simultaneously in single cells in the living animal. We use genome-edited Drosophila that have a quantitative reporter of protein synthesis inserted into the endogenous Rpl13a locus. We find that individual cells can have large (>10-fold) differences in protein expression between the two parental alleles. Cells can produce protein from only one allele oftentimes, and time-lapse imaging of protein production from each parental allele in each cell showed that the imbalance in expression from one parental allele over the other can invert over time. One sentence summaryParental allele-specific protein expression varies widely across cells and over time. HighlightsO_LIWe used genome editing to insert a quantifiable protein translation reporter into the endogenous Ribosomal protein L13a gene and thus track the protein expression of both parental alleles simultaneously in every single cell in the awake animal. C_LIO_LICells can have a large difference in protein expression for one parental allele over the other, and this can invert over time, and can occur in clusters of cells within a tissue. C_LIO_LIWe demonstrate the highly variable nature of heterozygous and homozygous definitions across single cells, and over time. C_LIO_LIOur study demonstrates a new paradigm that can be used to examine inherited epigenetic control of expression from a specific parent-of-origin allele across single clonal cells from a common progenitor in vivo. C_LI

cell biology↗