1. Light-Induced Protein Clustering for Optogenetic Interference and Protein Interaction Analysis in Drosophila S2 Cells
- Author
-
Eurico Morais-de-Sá, A. Filipa Santos, Mariana Osswald, and Instituto de Investigação e Inovação em Saúde
- Subjects
Light ,Cell division ,lcsh:QR1-502 ,Regulator ,Mitosis ,Optogenetics ,Biochemistry ,lcsh:Microbiology ,Cell Line ,Green fluorescent protein ,aPKC ,03 medical and health sciences ,0302 clinical medicine ,Cell polarity ,Animals ,Drosophila Proteins / chemistry ,Schneider 2 cells ,optogenetics ,Mps1 ,Molecular Biology ,030304 developmental biology ,APKC ,mitosis ,0303 health sciences ,Polarity (international relations) ,Chemistry ,LARIAT ,Drosophila Proteins / metabolism ,Cell biology ,cell polarity ,Drosophila ,030217 neurology & neurosurgery ,Protein Binding - 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. This research was funded by the National Funds through Fundação para a Ciência e a Tecnologia (FCT) under the project (PTDC/BEX-BCM/0432/2014). E.M. holds an FCT Investigator position. M.O. is supported by a fellowship from the GABBA PhD program from the University of Porto, PD/BD/105746/2014.
- Published
- 2019
- Full Text
- View/download PDF