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.
Highlights
Understanding the mechanisms that control the dynamic behavior of most cellular processes requires an experimental setup that enables detailed visualization with high-resolution microscopy and that is amenable to genetic manipulation
To build a toolbox for interference with protein function through optogenetic clustering in Drosophila Schneider 2 (S2) cells, we adapted Light-activated reversible inhibition by assembled trap (LARIAT) from a set of constructs previously developed for human cells [11]
We found that efficient removal of Lethal Giant Larvae (Lgl) from the cortex requires high levels ofofRFP-cryptochrome 2 (CRY2)
Summary
Understanding the mechanisms that control the dynamic behavior of most cellular processes requires an experimental setup that enables detailed visualization with high-resolution microscopy and that is amenable to genetic manipulation. S2 cells provide a reduced system for the molecular dissection at the cell autonomous level of processes that require reorganization of the cytoskeleton and the plasma membrane in a particular axis, such as cell motility, cell polarity, and oriented cell division [6,7,8] Investigation of these very dynamic cellular processes requires progression from established genetic approaches to methodologies that perturb protein function with high spatial and temporal control. Binding to CIBN and the formation of clusters to trap GFP-tagged proteins. Lgl and to determine interactions within the aPKC complex living cells This new tool expands the power of Drosophila S2 cells as a model for spatiotemporal investigation of mechanisms controlling cell division and cell polarity, which are two interconnected. Demands the ability to interfere with protein function and to evaluate protein interactions with high temporal control
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