High-Content RNAi Phenotypic Screening Unveils the Involvement of Human Ubiquitin-Related Enzymes in Late Cytokinesis
Mikaël Boullé,
Laurianne Davignon,
Keïs Nabhane Saïd Halidi,
Salomé Guez,
Emilie Giraud,
Marcel Hollenstein,
Fabrice Agou
Affiliations
Mikaël Boullé
Center for Technological Resources and Research (C2RT), Department of Structural Biology and Chemistry, Chemogenomic and Biological Screening Core Facility, Institut Pasteur, Université Paris Cité, CNRS UMR 3523, F-75015 Paris, France
Laurianne Davignon
Center for Technological Resources and Research (C2RT), Department of Structural Biology and Chemistry, Chemogenomic and Biological Screening Core Facility, Institut Pasteur, Université Paris Cité, CNRS UMR 3523, F-75015 Paris, France
Keïs Nabhane Saïd Halidi
Center for Technological Resources and Research (C2RT), Department of Structural Biology and Chemistry, Chemogenomic and Biological Screening Core Facility, Institut Pasteur, Université Paris Cité, CNRS UMR 3523, F-75015 Paris, France
Salomé Guez
Center for Technological Resources and Research (C2RT), Department of Structural Biology and Chemistry, Chemogenomic and Biological Screening Core Facility, Institut Pasteur, Université Paris Cité, CNRS UMR 3523, F-75015 Paris, France
Emilie Giraud
Center for Technological Resources and Research (C2RT), Department of Structural Biology and Chemistry, Chemogenomic and Biological Screening Core Facility, Institut Pasteur, Université Paris Cité, CNRS UMR 3523, F-75015 Paris, France
Marcel Hollenstein
Laboratory for Bioorganic Chemistry of Nucleic Acids, Department of Structural Biology and Chemistry, Institut Pasteur, Université Paris Cité, CNRS UMR 3523, F-75015 Paris, France
Fabrice Agou
Center for Technological Resources and Research (C2RT), Department of Structural Biology and Chemistry, Chemogenomic and Biological Screening Core Facility, Institut Pasteur, Université Paris Cité, CNRS UMR 3523, F-75015 Paris, France
CEP55 is a central regulator of late cytokinesis and is overexpressed in numerous cancers. Its post-translationally controlled recruitment to the midbody is crucial to the structural coordination of the abscission sequence. Our recent evidence that CEP55 contains two ubiquitin-binding domains was the first structural and functional link between ubiquitin signaling and ESCRT-mediated severing of the intercellular bridge. So far, high-content screens focusing on cytokinesis have used multinucleation as the endpoint readout. Here, we report an automated image-based detection method of intercellular bridges, which we applied to further our understanding of late cytokinetic signaling by performing an RNAi screen of ubiquitin ligases and deubiquitinases. A secondary validation confirmed four candidate genes, i.e., LNX2, NEURL, UCHL1 and RNF157, whose downregulation variably affects interconnected phenotypes related to CEP55 and its UBDs, as follows: decreased recruitment of CEP55 to the midbody, increased number of midbody remnants per cell, and increased frequency of intercellular bridges or multinucleation events. This brings into question the Notch-dependent or independent contributions of LNX2 and NEURL proteins to late cytokinesis. Similarly, the role of UCHL1 in autophagy could link its function with the fate of midbody remnants. Beyond the biological interest, this high-content screening approach could also be used to isolate anticancer drugs that act by impairing cytokinesis and CEP55 functions.
