Development of a pipeline for automated, high-throughput analysis of paraspeckle proteins reveals specific roles for importin a proteins

作者：Major Andrew T; Miyamoto Yoichi; Lo Camden Y; Jans David A; Loveland Kate L

来源：Scientific Reports, 2017, 7(1): 43323.

DOI：10.1038/srep43323

We developed a large-scale, unbiased analysis method to measure how functional variations in importin (IMP) alpha 2, IMP alpha 4 and IMP alpha 6 each influence PSPC1 and SFPQ nuclear accumulation and their localization to paraspeckles. This addresses the hypothesis that individual IMP protein activities determine cargo nuclear access to influence cell fate outcomes. We previously demonstrated that modulating IMP alpha 2 levels alters paraspeckle protein 1 (PSPC1) nuclear accumulation and affects its localization into a subnuclear domain that affects RNA metabolism and cell survival, the paraspeckle. An automated, high throughput, image analysis pipeline with customisable outputs was created using Imaris software coupled with Python and R scripts; this allowed non-subjective identification of nuclear foci, nuclei and cells. HeLa cells transfected to express exogenous full-length and transport-deficient IMPs were examined using SFPQ and PSPC1 as paraspeckle markers. Thousands of cells and > 100,000 nuclear foci were analysed in samples with modulated IMPa functionality. This analysis scale enabled discrimination of significant differences between samples where paraspeckles inherently display broad biological variability. The relative abundance of paraspeckle cargo protein(s) and individual IMPs each influenced nuclear foci numbers and size. This method provides a generalizable high throughput analysis platform for investigating how regulated nuclear protein transport controls cellular activities.

• 出版日期2017-2-27
• 单位河北医科大学