title: Development of a pipeline for automated, high-throughput analysis of paraspeckle proteins reveals specific roles for importin α proteins.
abstract: We developed a large-scale, unbiased analysis method to measure how functional variations in importin (IMP) α2, IMPα4 and IMPα6 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 IMPα2 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 IMPα 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.
authors.affiliation: Department of Anatomy and Developmental Biology, Monash University, Melbourne, Australia.|The ARC Centre of Excellence in Biotechnology and Development, Australia.
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Major
authors.affiliation: National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan.|Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Miyamoto
authors.affiliation: Monash Micro Imaging Facility, Monash University, Melbourne, Australia.|Centre for Reproductive Health, Hudson Institute of Medical Research, Melbourne, Australia.
authors.forename: Camden Y
authors.initials: CY
authors.lastname: Lo
authors.affiliation: The ARC Centre of Excellence in Biotechnology and Development, Australia.|Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia.
authors.forename: David A
authors.initials: DA
authors.lastname: Jans
authors.affiliation: Department of Anatomy and Developmental Biology, Monash University, Melbourne, Australia.|The ARC Centre of Excellence in Biotechnology and Development, Australia.|Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Australia.|Centre for Reproductive Health, Hudson Institute of Medical Research, Melbourne, Australia.|Department of Molecular and Translational Sciences, School of Clinical Sciences, Monash University, Melbourne, Australia.
authors.forename: Kate L
authors.initials: KL
authors.lastname: Loveland
mesh_terms: D021581:Active Transport, Cell Nucleus; D057205:Automation, Laboratory; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D002470:Cell Survival; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D057166:High-Throughput Screening Assays / Q000295:instrumentation*; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D000072670:PTB-Associated Splicing Factor / Q000235:genetics* / Q000378:metabolism; D025341:Principal Component Analysis; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D028901:alpha Karyopherins / Q000235:genetics* / Q000378:metabolism
chemical_list: C490630:KPNA2 protein, human; D009687:Nuclear Proteins; C450059:PSPC1 protein, human; D000072670:PTB-Associated Splicing Factor; D020033:Protein Isoforms; D016601:RNA-Binding Proteins; D028901:alpha Karyopherins; D049452:Green Fluorescent Proteins