Monitoring nucleolar-nucleoplasmic protein shuttling in living cells by high-content microscopy and automated image analysis

Author:

Engbrecht Marina1,Grundei David1ORCID,Dilger Asisa M2,Wiedemann Hannah1,Aust Ann-Kristin1,Baumgärtner Sarah1,Helfrich Stefan3,Kergl-Räpple Felix3,Bürkle Alexander1,Mangerich Aswin12ORCID

Affiliation:

1. Molecular Toxicology, Department of Biology, University of Konstanz , 78457 Konstanz , Germany

2. Nutritional Toxicology, Institute of Nutritional Science, University of Potsdam , 14469 Potsdam , Germany

3. KNIME GmbH , Reichenaustr. 11, 78467 Konstanz , Germany

Abstract

Abstract The nucleolus has core functions in ribosome biosynthesis, but also acts as a regulatory hub in a plethora of non-canonical processes, including cellular stress. Upon DNA damage, several DNA repair factors shuttle between the nucleolus and the nucleoplasm. Yet, the molecular mechanisms underlying such spatio-temporal protein dynamics remain to be deciphered. Here, we present a novel imaging platform to investigate nucleolar-nucleoplasmic protein shuttling in living cells. For image acquisition, we used a commercially available automated fluorescence microscope and for image analysis, we developed a KNIME workflow with implementation of machine learning-based tools. We validated the method with different nucleolar proteins, i.e., PARP1, TARG1 and APE1, by monitoring their shuttling dynamics upon oxidative stress. As a paradigm, we analyzed PARP1 shuttling upon H2O2 treatment in combination with a range of pharmacological inhibitors in a novel reporter cell line. These experiments revealed that inhibition of SIRT7 results in a loss of nucleolar PARP1 localization. Finally, we unraveled specific differences in PARP1 shuttling dynamics after co-treatment with H2O2 and different clinical PARP inhibitors. Collectively, this work delineates a highly sensitive and versatile bioimaging platform to investigate swift nucleolar-nucleoplasmic protein shuttling in living cells, which can be employed for pharmacological screening and in-depth mechanistic analyses.

Funder

University of Konstanz

University of Potsdam

PANDOTA-Promotionsförderung

AFF UKN

Publisher

Oxford University Press (OUP)

Reference81 articles.

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