Dimerization of iLID optogenetic proteins observed using 3D single-molecule tracking in live E. coli

3D single-molecule tracking microscopy has enabled measurements of protein diffusion in living cells, offering information about protein dynamics and cellular environments. For example, different diffusive states can be resolved and assigned to protein complexes of different size and composition. However, substantial statistical power and biological validation, often through genetic deletion of binding partners, are required to support diffusive state assignments. When investigating cellular processes, real-time perturbations to protein spatial distributions is preferable to permanent genetic deletion of an essential protein. For example, optogenetic dimerization systems can be used to manipulate protein spatial distributions that could offer a means to deplete specific diffusive states observed in single-molecule tracking experiments. Here, we evaluate the performance of the iLID optogenetic system in living E. coli cells using diffraction-limited microscopy and 3D single-molecule tracking. We observed a robust optogenetic response in protein spatial distributions after 488 nm laser activation. Surprisingly, 3D single-molecule tracking results indicate activation of the optogenetic response when illuminating with high-intensity light with wavelengths at which there is minimal photon absorbance by the LOV2 domain. The preactivation can be minimized through the use of iLID system mutants, and titration of protein expression levels.SIGNIFICANCE We describe the combination of 3D single-molecule tracking microscopy and optogenetic manipulation of protein spatial distributions in living cells. Such a combination is impactful, because optogenetic systems enable sample perturbation in real time using light, which provides more flexibility than gene deletion (i.e., knockout mutants) and gene editing approaches that result in permanent changes to the specimen. We specifically investigate the performance of the iLID optogenetic system in knocksideways experiments (as opposed to knockout approaches), in which cytosolic prey proteins (SspB) are sequestered to the membrane by interacting with membrane-anchored bait proteins (iLID). We quantified the magnitude of the optogenetic effect using both diffraction-limited imaging and 3D single-molecule tracking microscopy. We found, surprisingly, that the iLID optogenetic response is activated substantially by high-intensity light at wavelengths for which there is minimal photon absorption by the iLID protein. Quantification of this alternative activation mechanism is a necessary component before optogenetic tools, such as iLIDs, are employed in single-molecule knocksideways experiments that are designed to provide new biological insights.