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

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.

Recent evolution of a TET-controlled and DPPA3/STELLA-driven pathway of passive DNA demethylation in mammals

“…To quantify this observation, we calculated the coefficient of variation (CV) of nuclear UHRF1-GFP among wt, Dppa3KO, and T12CM ESCs. The CV of a fluorescent signal correlates with its distribution, with low CV values reflecting more homogenous distributions and high CV values corresponding to more heterogeneous distributions 86,87 . Indeed, the pronounced focal accumulation of UHRF1-GFP observed in Dppa3KO and T12CM ESCs corresponded with a highly significant increase in the CV values of nuclear UHRF1-GFP compared with wt ESCs ( Supplementary Fig.…”

Mps1-mediated release of Mad1 from nuclear pores ensures the fidelity of chromosome segregation

“…PCRs were performed with Phusion polymerase, followed by digestion with DpnI restriction enzyme (New England Biolabs). The constructs H2B-mCherry, H2B-GFP, mCherry-α-tubulin, Mad1-EGFP, pHW-CIB-MP-HRW-CRY2-V H H, and pHGW-aPKC have been previously described (Conde et al, 2013;Moura et al, 2017;Osswald et al, 2019). Plasmids were transfected into S2 cells using Effectene Transfection Reagent (Qiagen), according to the manufacturer's instructions.…”

Microtubule disassembly by caspases is the rate-limiting step of cell extrusion

“…Optogenetics can be used to trigger fast clustering and sequestration of proteins of interest. We used the LARIAT system (Light-Activated Reversible Inhibition by Assembled Trap) to trigger clustering of a GFP-tagged α-tubulin upon blue light exposure [43][44][45] . Since the GFP-tagged α-tubulin knock-in is not viable 46 , we instead used the overexpression of GFP-α-tubulin and LARIAT in clones to trigger a partial (endogenous Tubulin is still present) but significant depletion of α-Tubulin.…”