Then we put forward two questions: Why phase separation in cells can produce membraneless organelles? And how can we design our system to fulfill its intended functions? Like oil in water, the contents of cells can separate into droplets. According to physical principles, the process where material self-assemble into organelles is described as ‘phase separation’, which is the conversion of a single-phase system into a multiphase system. In general, materials flow to regions with low chemical potential instead of low concentration. Finally, the components no longer distribute uniformly but form granules locally which are organelles in the cell. That is to say, the main work to synthesize an organelle is to fulfill phase separation in a cell. Then, how can we do it? Composition can switch rapidly through changes in scaffold concentration or multivalency. And our design was inspired by recent works showing that multivalency drives protein phase separation and formation of synthetic organelles. What’s more, we take our inspiration from existing life systems and previous works. For example, Intrinsic Disordered Regions are the symbol of massive phase separation in the cell. They interact with each other through the van der Waals force, hydrophobic effect and electrostatic attraction. And there are many interactions like this in nature, such as FKBP and FRB, SUMO and SIM, SH3 and PRM, phyB and PIF6. Thus, we can make good use of them to induce our designed organelles and regulate them variously.! In a conclusion，multivalency drives protein’s -self-assemblyies and interaction binds the parts together. It means, interaction can induce phase separation and multivalency can make larger assemblies, which are two essential elementmodules in our design and ensure the formation of synthetic organelles.

To design multivalent modules, it is not ideal to use multiple repeat domains, which not only will make the protein extremely large and bring difficulties to DNA recombination, but also may be problematic for making transgenic animals. Thus, instead of using multiple repeats, we turned to de novo-designed homo-oligomeric coiled coils. And we named these coiled coils as HO-Tag (homo-oligomeric tag). They are short peptides, ~30 amino acids^[1], therefore they are ideal tags to introduce multivalency. There are seven coiled coils previously characterized in protein de novo design studies. They have been proved by previous work of Shu Xiaokun’s lab, and according to their work, HOTag3 and HOTag6 are most robust in driving protein droplet formation over a wide range of protein concentrations, so we choose them.

To design interaction modules, we tried a lot of components and we fused them to the N-terminus of HOTag3 or HOTag6. Some of them are spontaneous and some are inducible. And we can regulate them through various kinds of inducers and different intensities of promoters.

FKBP and Frb

The interaction between FKBP and FRB can be robustly induced by rapamycin. Rapamycin is a 31-membered macrolide antifungal antibiotic. It binds with high affinity (Kd=0.2nM) to the 12-kDa FK506 binding protein (FKBP), as well as to a 100-amino acid domain of the mammalian target of rapamycin (mTOR) known as FKBP-rapamycin binding domain (Frb)^[5]. Thus, we chose them as a pair of interaction modules. And we assembled them on to yeast plasmid as the same as the construction of SUMO and SIM and transformed them into yeast. if we add rapamycin to the yeast, we will see red granules colocalize with green granules in cells under fluorescence microscope. Synthetic organelles come true!

Phytohormone

As mentioned above, interactions can be formed not only by inducers such as rapamycin and gibberellin, but also spontaneously, just as SUMO and SIM. So can we combine these two ways of interactions? To solve this problem, we did further studies about phytohormone and found ABA. Abscisic acid (ABA) is an important phytohormone that regulates plant stress responses. Proteins from the PYR-PYL-PCAR family were identified as ABA receptors^[6]. Upon binding to ABA, a PYL protein associates with type 2C protein phosphatases (PP2Cs) such as ABI1 and ABI2, inhibiting their activity^[7]. Previous structural and biochemical observations have provided insight into PYL-mediated ABA signaling and given rise to a working model. In the absence of ABA signaling, PP2Cs are fully active and PYLs exist as inactive homodimers in cells, unable to bind or inhibit PP2Cs, mainly due to the incompatible conformation of CL2loop^[7]. In response to ABA binding, the CL2 loop undergoes a conformational rearrangement to close onto the ABA-bound pocket, then, the interaction between PYLs and PP2Cs can be formed. Here we chose PYL1 and ABI1 as a pair of interaction modules. Then, we assembled them on to yeast plasmid as the same as the construction of FKBP and FRB and transformed them into yeast. Based on the interaction of PYL1 and ABI1, we can get a wonderful scene: In the absence of ABA, the synthetic organelles composed only of PYL1 appear, because of the homodimers of PYL1. And after we add ABA into yeast, ABI1 can enter the organelles with the interaction of ABI1 and PYL1, and we can see red droplets colocalize with green droplets in cells through fluorescence microscope. In this way, new components can enter the original organelles and the time of occurrence can be regulated as it is inducer-mediated regulation. So it give our designs and functions more possibilities.

Now, we have artificially designed phase separation in cells and synthesized membraneless organelles. But how can we fulfill intended functions with synthetic organelles? Here, we propose two ideas. We reserve two sites to implement functions, which means function modules, such as enzymes in metabolism, proteins in signaling pathway, transcription factors in transcription and so on, have two sites in our designs.