Optimized iLID membrane anchors for local optogenetic protein recruitment

Abstract

Optogenetic 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.

Optogenetic relaxation of actomyosin contractility uncovers mechanistic roles of cortical tension during cytokinesis

“…1c). The Stargazin-mEGFP-iLID is suited for the subcellular protein recruitment, because the large N-terminal transmembrane anchor limits the diffusion of SspB proteins 20 . Alternatively, we developed a cryptochrome 2 (CRY2)-based OptoMYPT system, in which CRY2-mCherry-PP1BD was recruited to the plasma membrane with blue light through binding to the plasma membrane localizer CIBN-EGFP-KRasCT 21 .…”

Designing Single-Component Optogenetic Membrane Recruitment Systems: The Rho-Family GTPase Signaling Toolbox

“…While membrane ruffling is known to be dependent on Tiam1-and Rac1-dependent signaling, 19−21 consistent sheet-like projections were expected as the predominant cytoskeletal response, as was the case with opto-Rac1 under all conditions. Others have reported that dynamic membrane recruitment of Tiam1 with the optogenetic heterodimerizer iLID preferentially induces membrane ruffles 4 and filopodia, 22 without resolution of the inconsistency. It is known, though, that low cytoskeletal tension and focal adhesion strength in serum-starved cells result in easier cytoskeletal remodeling 23,24 that is permissive of lamellipodial persistence, and that under conditions of poor lamellipodial persistence, ruffling will instead occur.…”

“…The spatiotemporal resolution of these BcLOV4based tools is anticipated to be generally favorable to existing optogenetic Rho-family tools, owing to rapid membrane interaction kinetics and submobile lateral diffusion of BcLOV4 that together determine characteristic membrane diffusional lengths. 4,26,27 Additionally, the excess binding site availability along the sink-like endogenous inner leaflet may minimize cytosolic-diffusional resolution loss known to be critical in optogenetic heterodimerization when binding sitelimited 4 (i.e., common stoichiometric expression conditions). However, as other approaches have been utilized to great effect to study Rho-family GTPase signaling, the benefit of the toolbox described herein lies in its completeness and experimental simplicity.…”

Opto-RhoGEFs: an optimized optogenetic toolbox to reversibly control Rho GTPase activity on a global to subcellular scale, enabling precise control over vascular endothelial barrier strength

“…1D). As suggested in a previous study, a N-terminal membrane tag is more beneficial than a C-terminal tag (Natwick and Collins, 2021). The conventional C-terminal CaaX tag was compared to the N-terminal Lck tag.…”

“…However, this elegant one component system is temperature dependent and therefore does not allow sustained activation at 37°C and does not allow the choice of other targets, other than the plasma membrane for follow up studies (Benman et al, 2022). We continued to work with the iLID tool and could improve its recruitment efficiency by using the Lck membrane tag instead of the CaaX box as a previous study suggested (Natwick and Collins, 2021). Furthermore, we introduced the HaloTag to the system and labeled the mobile prey component with it.…”