AbstractOptogenetic protein dimerization systems are powerful tools to investigate the biochemical networks that cells use to make decisions and coordinate their activities. These tools, including the improved Light-Inducible Dimer (iLID) system, offer the ability to selectively recruit components to subcellular locations, such as micron-scale regions of the plasma membrane. In this way, the role of individual proteins within signaling networks can be examined with high spatiotemporal resolution. Currently, consistent recruitment is limited by heterogeneous optogenetic component expression, and spatial precision is diminished by protein diffusion, especially over long timescales. Here, we address these challenges within the iLID system with alternative membrane anchoring domains and fusion configurations. Using live cell imaging and mathematical modeling, we demonstrate that the anchoring strategy affects both component expression and diffusion, which in turn impact recruitment strength, kinetics, and spatial dynamics. Compared to the commonly used C-terminal iLID fusion, fusion proteins with large N-terminal anchors show stronger local recruitment, slower diffusion of recruited components, and efficient recruitment over wider gene expression ranges. We also define guidelines for component expression regimes for optimal recruitment for both cell-wide and subcellular recruitment strategies. Our findings highlight key sources of imprecision within light-inducible dimer systems and provide tools that allow greater control of subcellular protein localization across diverse cell biological applications.SignificanceOptogenetic light-inducible dimer systems, such as iLID, offer the ability to examine cellular signaling networks on second timescales and micrometer spatial scales. Confined light stimulation can recruit proteins to subcellular regions of the plasma membrane, and local signaling effects can be observed. Here, we report alternative iLID fusion proteins that display stronger and more spatially confined membrane recruitment. We also define optogenetic component expression regimes for optimal recruitment and show that slow-diffusing iLID proteins allow more robust recruitment in cell populations with heterogenous expression. These tools should improve the spatiotemporal control and reproducibility of optogenetic protein recruitment to the plasma membrane.
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