2.2 Light-induced recruitment of POI into LLPS-based compartments
To recruit POI into compartments rapidly and dynamically, the light-responsive module was designed to sense the dynamic light signal. Together with the phase module, the system was termed the Photo-Activated Switch in E. coli (PhASE) (Figure 3A). The phase module acted as a scaffold to form isolated compartments from the cytosol of E. coli , while the light-responsive module remained evenly distributed in the cell until triggered by induction signals. After the light induction, the light-responsive module would be recruited into the compartments formed by the phase module (Figure 1C). Light-responsive protein pair CIB1 and CRY2 were chosen to respond to the blue light signal, between which the binding affinity would increase with the light intensity[38]. CIB1 was included as a part of the phase module, localizing permanently in compartments isolated from the cytosol, while CRY2 was included as a part of the light-responsive module, distributing evenly inside the cell in darkness. POI was represented by mCherry (Figure 3A). During the light induction process, the light-responsive module was captured and fixed when diffused into the LLPS-based compartment. Since protein diffusion is the major time-consuming step in this process, the regulation can be completed in the time scale of seconds theoretically, estimated by the protein diffusion rate in E. coli [39, 40]. Indeed, it was found that right after 8 seconds of 488 nm laser intense stimulation, the reorganization of mCherry into the compartments was completed. The concentration of mCherry inside could reach more than 15 folds compared to the cytosol (Figure 3B) and maintained almost unchanged over 30 minutes (data not shown). The LLPS-based compartments labeled with GFP were captured after the stimulation process, showing perfect overlap with mCherry signal (Figure 3B). When stimulated with the laser of lower intensity, the recruited amount of mCherry reduced, with the enrichment reaching only about 2 folds (Figure 3C). Furthermore, this state was highly reversible, with the fastest recovery time less than 10 minutes, and a fully reversed state was attained within less than 15 minutes (Figure 3C, Video S3). We next tried to stimulate the system several times after reversion and found that the induction effect was consistent and robust at least in the first three rounds (Figure 3C, Video S3). The overall reduction of the mCherry signal could be the effect of laser bleaching, yet the fold enrichment of POI in the compartment remained roughly the same under the same intensity of light during different induction rounds. Remarkably, like other condensates formed by proteins[41], their aging and solidification were observed. Their fluidity decreased along time and was almost undetectable after 10-hour IPTG induction at 16oC. However, under such states, POIs could still be recruited in seconds, suggesting a different mechanism for protein entrance other than the dynamic component exchange between interior and exterior layer (Figure 3, Supplementary Figure 4). In summary, PhASE#1 was verified reorganizing POI within seconds in a reversible manner.
PhASE#2 system was constructed using similar light-responsive components as PhASE#1 but substituting FUSLCD with tandem SIM and SUMO repeats (Figure 3D), known for phase separating via multi-valent interactions[42]. The fold enrichment of POI inside the compartment after light induction could be over 10-folds (Figure 3E). Additionally, the interaction between phase module and light-responsive module in this system can be conveniently redesigned by adding different number (valency) of SIM or SUMO to light-responsive module genetically[42], so that the fold enrichment of POI can still be tuned even light source is fixed.