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Biomolecules 2019, 9(2), 61; https://doi.org/10.3390/biom9020061

Light-Induced Protein Clustering for Optogenetic Interference and Protein Interaction Analysis in Drosophila S2 Cells

1
Epithelial Polarity and Cell Division, i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal
2
IBMC, Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal
*
Author to whom correspondence should be addressed.
Received: 11 December 2018 / Revised: 30 January 2019 / Accepted: 30 January 2019 / Published: 12 February 2019
(This article belongs to the Special Issue Cytoskeleton and Regulation of Mitosis)
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Abstract

Drosophila Schneider 2 (S2) cells are a simple and powerful system commonly used in cell biology because they are well suited for high resolution microscopy and RNAi-mediated depletion. However, understanding dynamic processes, such as cell division, also requires methodology to interfere with protein function with high spatiotemporal control. In this research study, we report the adaptation of an optogenetic tool to Drosophila S2 cells. Light-activated reversible inhibition by assembled trap (LARIAT) relies on the rapid light-dependent heterodimerization between cryptochrome 2 (CRY2) and cryptochrome-interacting bHLH 1 (CIB1) to form large protein clusters. An anti-green fluorescent protein (GFP) nanobody fused with CRY2 allows this method to quickly trap any GFP-tagged protein in these light-induced protein clusters. We evaluated clustering kinetics in response to light for different LARIAT modules, and showed the ability of GFP-LARIAT to inactivate the mitotic protein Mps1 and to disrupt the membrane localization of the polarity regulator Lethal Giant Larvae (Lgl). Moreover, we validated light-induced co-clustering assays to assess protein-protein interactions in S2 cells. In conclusion, GFP-based LARIAT is a versatile tool to answer different biological questions, since it enables probing of dynamic processes and protein-protein interactions with high spatiotemporal resolution in Drosophila S2 cells. View Full-Text
Keywords: Drosophila; Schneider 2 cells; optogenetics; LARIAT; mitosis; cell polarity; Mps1; aPKC Drosophila; Schneider 2 cells; optogenetics; LARIAT; mitosis; cell polarity; Mps1; aPKC
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Osswald, M.; Santos, A.F.; Morais-de-Sá, E. Light-Induced Protein Clustering for Optogenetic Interference and Protein Interaction Analysis in Drosophila S2 Cells. Biomolecules 2019, 9, 61.

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