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.