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Systematic genetic interaction screens uncover cell polarity regulators and functional redundancy

Abstract

Although single-gene loss-of-function analyses can identify components of particular processes, important molecules are missed owing to the robustness of biological systems. Here we show that large-scale RNAi screening for suppression interactions with functionally related mutants greatly expands the repertoire of genes known to act in a shared process and reveals a new layer of functional relationships. We performed RNAi screens for 17 Caenorhabditis elegans cell polarity mutants, generating the most comprehensive polarity network in a metazoan, connecting 184 genes. Of these, 72% were not previously linked to cell polarity and 80% have human homologues. We experimentally confirmed functional roles predicted by the network and characterized through biophysical analyses eight myosin regulators. In addition, we discovered functional redundancy between two unknown polarity genes. Similar systematic genetic interaction screens for other biological processes will help uncover the inventory of relevant genes and their patterns of interactions.

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Figure 1: Suppressor genetic screen strategy.
Figure 2: Cell polarity genetic network.
Figure 3: Suppressor screen identifies a new layer of functional relationships.
Figure 4: PKA signalling antagonizes PKC-3 function.
Figure 5: Characterization of new PAR protein regulators acting upstream and downstream of PARs.
Figure 6: Biophysical characterization of new actomyosin regulators.
Figure 7: NOP-1 (F25B5.2) regulates actomyosin localization and dynamics, affects cell polarity establishment and acts redundantly with PLST-1 during polarity maintenance.

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Acknowledgements

This work was supported by a Wellcome Trust Senior Research Fellowship (054523, to J.A.), postdoctoral fellowships from the Human Frontier Science Program (to B.T.F. and J.R.), a Herchel Smith Post-doctoral fellowship (to J.R.) and an EMBO fellowship (to B.T.F.). We thank P. Mains (Department of Biochemistry and Molecular Biology, University of Calgary, Canada) for providing let-502(ts) and mel-11(ts) mutants, and K. Kemphues (Department of Molecular Biology and Genetics, Cornell University, USA) for NMY-2-expressing bacteria. We thank F. Antigny, D. Lefer, V. Karabacak, S. Kroschwald, A. Maffioletti and A. Sayadian for their contribution in visual scoring and S. Fürthauer for help with hydrodynamic length measurements. We thank the media team of the Gurdon Institute for preparing screen reagents. We also thank C. Dix and R. Durbin for comments on the manuscript. Some nematode strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR). The P4A1 monoclonal antibody developed by J. Priess was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242.

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Contributions

B.T.F. and J.R. designed and carried out the screen, analysed the network, characterized polarity candidates and drafted the manuscript. C.L. participated in the primary screen experiments. S.N. and S.G. designed and performed the biophysical analysis of myosin regulators. E.Z. generated the GFP::NOP-1 transgenic strain. T.I. and M.S. isolated the nmy-2(ts) and pkc-3(ts) mutants. J.A. participated in the design and coordination of the study and edited the manuscript.

Corresponding author

Correspondence to Julie Ahringer.

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Time-lapse movie of NMY-2::GFP expressed in a wild-type control embryo.

Cortical projection of NMY-2::GFP in a wild-type control embryo. Images were acquired every 5 s. (MOV 1596 kb)

Time-lapse movie of NMY-2::GFP expressed in a cdc-42(RNAi) embryo.

Cortical projection of NMY-2::GFP in a cdc-42(RNAi) embryo. Images were acquired every 5 s. (MOV 1863 kb)

Time-lapse movie of NMY-2::GFP expressed in a csnk-1(RNAi) embryo.

Cortical projection of NMY-2::GFP in a csnk-1(RNAi) embryo. Images were acquired every 5 s. (MOV 1665 kb)

Time-lapse movie of NMY-2::GFP expressed in a mlc-5(RNAi) embryo.

Cortical projection of NMY-2::GFP in a mlc-5(RNAi) embryo. Images were acquired every 5 s. (MOV 2316 kb)

Time-lapse movie of NMY-2::GFP expressed in a unc-45(RNAi) embryo.

Cortical projection of NMY-2::GFP in a unc-45(RNAi) embryo. Images were acquired every 5 s. (MOV 1433 kb)

Time-lapse movie of NMY-2::GFP expressed in a Y54H5A.2(RNAi) embryo.

Cortical projection of NMY-2::GFP in a Y54H5A.2(RNAi) embryo. Images were acquired every 5 s. (MOV 3730 kb)

Time-lapse movie of NMY-2::GFP expressed in a plst-1(RNAi) embryo.

Cortical projection of NMY-2::GFP in a plst-1(RNAi) embryo. Images were acquired every 5 s. (MOV 1718 kb)

Time-lapse movie of NMY-2::GFP expressed in a unc-59(RNAi) embryo.

Cortical projection of NMY-2::GFP in a unc-59(RNAi) embryo. Images were acquired every 5 s. (MOV 1427 kb)

Time-lapse movie of NMY-2::GFP expressed in a erm-1(RNAi) embryo.

Cortical projection of NMY-2::GFP in a erm-1(RNAi) embryo. Images were acquired every 5 s. (MOV 1252 kb)

Time-lapse movie of NMY-2::GFP expressed in a cnt-2(RNAi) embryo.

Cortical projection of NMY-2::GFP in a cnt-2(RNAi) embryo. Images were acquired every 5 s. (MOV 1864 kb)

Time-lapse movie of NMY-2::GFP expressed in a gsp-1(RNAi) embryo.

Cortical projection of NMY-2::GFP in a gsp-1(RNAi) embryo. Images were acquired every 5 s. (MOV 1642 kb)

Time-lapse movie of NMY-2::GFP expressed in a nop-1(RNAi) embryo.

Cortical projection of NMY-2::GFP in a nop-1(RNAi) embryo. Images were acquired every 5 s. (MOV 1495 kb)

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Fievet, B., Rodriguez, J., Naganathan, S. et al. Systematic genetic interaction screens uncover cell polarity regulators and functional redundancy. Nat Cell Biol 15, 103–112 (2013). https://doi.org/10.1038/ncb2639

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