1.A spontaneous organelle can be formed with HOTags and SUMO-SIM interaction module

Through the time-lapse fluorescence video results, we noticed there was a great time delay of gene expression, usually about several hours. To make our SPOT system response to environment rapidly, we designed an inducible SPOT which relies on FKBP-rapamycin-Frb system, short as RapaSPOT. FKBP and Frb can combine together in the presence of rapamycin. (Figure.4A) We constructed strains with FKBP-mCherry-HOTag3 and Frb-yEGFP-HOTag6 and used 10 μM rapamycin to induce the RapaSPOT formation. Minutes after adding rapamycin, granules appeared and became larger gradually. (Figure.4B,4C) The merge images of granules confirmed that the granules consisted of both components as we expect.

2.The toxicity of inducer will not affect SPOT much

Rapamycin induced phase separation can make the synthetic organelles controllable at protein level. However, rapamycin is a typical antifungal which is toxic to yeast, since its complex with FKBP will activate the TOR pathway and the cell cycle will be arrested in G1. So what will happen when rapamycin is 'blocked' in our phase separation system? We assume that this effect is strong enough to prevent yeast cell death so that rapamycin induction can be applied in bioengineering.

We measured the cell growth curves with the two strains: yeasts with both FKBP-mCherry-HOTag3 and Frb-yEGFP-HOTag6 and wild type yeasts with different concentrations of rapamycin as control group. (Figure.4D) The results show that wild-type yeast cannot grow and reproduce normally in the presence of rapamycin, while the yeast with both FKBP-mCherry-HOtag3 and Frb-yEGFP-HOtag6, which can form SPOT, could still grow, especially in a relatively low concentration of rapamycin. We then observed the cells that had been treated with rapamycin for 12 hours under the microscope and confirmed that the yeast cells are in good condition and the synthetic organelles didn’t disappear. Therefore, we believe that the design of RapaSPOT is feasible, and sequestration is a potential function for SPOT.

Figure4. Chemical-induced SPOT can be formed by using rapamycin to induce FKBP-Frb interactions (A) The structure of FKBP and Frb. Rapamycin can induce the interaction between them. (B) Design of RapaSPOT. FKBP is fused with mCherry and HOTag3 while Frb is fused with yEGFP and HOTag6. After adding rapamycin, they are expected to self-organize to form large assemblies, which will be an organelle in cells. (C) granules in chemical-induced SPOT after adding rapamycin. Ura3-FKBP-HOTag3(mCherry) and PDH3-Frb-HOTag6 with yEGFP are transferred and expressed in S. cerevisiae. Fluorescence images in both GFP and mCherry channels of cells are taken after the addition of 10 μM rapamycin. Granules occurs in less than 20 minutes and the two fluorescence channels merge well. (D) Growth curves of wild-type yeasts (black curve) and yeast strains with Ura3-FKBP-HOTag3 and PDH3-Frb-HOTag6 (red curve). They are measured after adding different concentration of rapamycin for 24 hours. Wild-type yeasts cannot grow and reproduce normally while engineered yeasts with FKBP-HOTag3 and Frb-HOTag6 can survive when exposed to rapamycin.\ Movie2 The formation process of chemical-induced SPOT after adding rapamycin. Granules occurs in less than 20 minutes and the two fluorescence channels merge well.

3.Different concentration of components and rapamycin vary the kinetics of phase separation

As our Modeling work predicts, the kinetics of a system depends on the concentration of the components and the interaction strength. (Figure.5A) By verifying these theoretical predictions by experiment, we have made our SPOT a more tunable and modularized system for applications.

The kinetics of SPOT system depend on the concentrations of components. The concentration can be regulated by controlling the gene expression level of components, so we design a 3*3 promoters combinations in our RapaSPOT to research some principles for engineering. Ura3, Tef2, and PDH3 were chosen to provide concentration gradient and their strength was measured by flow cytometry. (Figure.5B) The interaction between FKBP and Frb was induced by 10μM rapamycin and observations of aggregates were conducted under fluorescence microscope since all the components are fused with yEGFP. We found that the aggregates appeared only when FKBP-HOTags were expressed at a low level of Frb-HOTags were at a high level, which fit our simulation well.

Another variable which could affect the SPOT formation significantly is the strength of interaction between two components. In RapaSPOT, it means the concentration of rapamycin, which will affect the apparent affinity of FKBP and Frb. We conducted a rapamycin gradient induction experiment to investigate the kinetics. Yeast Strains with FKBP-mCherry-HOTag3 and Frb-yEGFP-HOTag6 were induced by rapamycin from 0.01 to 100μM. We found that higher concentration of rapamycin could induce faster SPOT formation which fit our model well. (Figure.5D) At the same time, we had to notice that if the concentration of rapamycin is too high, the SPOT formation process will be restrained.

Figure5. (A) The formation of SPOT can be described as a phase separation process of three components. Two important variable, the concentration of components and the interaction strength are marked in the figure. (B) Flow Cytometry results of three promoters (Ura3, Tef2, and PDH3). The expression level of Ura3 is lowest while PDH3 is the strongest promoter. (C) RapaSPOT of different promoter combinations after 10 μM rapamycin induction. Two axes stand for the expression level of components. After 3 hours, Only SPOT system with high level of Frb can be observed. (D) Proportion of yeasts with granules after rapamycin induction. The rapamycin is from 1μM to 100 μM. Movie3 The formation process of SPOT in different concentration of rapamycin in one experiment. Higher concentration of rapamycin leads to faster formation of phase separation.

1.Spatial segregation and organization

The most basic function of a synthetic organelle is to form a segregated space, changing the position of a substance, and thereby causing a corresponding effect.

The most basic function of a synthetic organelle is to form a segregated space, changing the position of a substance, and thereby causing a corresponding effect.

(1)Aggregation of target substances

The components used to construct the synthetic organelles can condense via phase separation, resulting in different concentrations of their components in the synthetic organelles and the surrounding cytosol. If we control the expression of the SIM component (red fluorescence) in the synthetic organelle system composed of SUMO-SIM, we can regulate the distribution of the SUMO component (green fluorescence) in the cell. We used an inducible promoter to control the expression of the SIM component. When the SIM component was not expressed, the SUMO component was evenly distributed in the cells, and when the cells were treated with a sufficient concentration of doxorubicin, the SIM component was expressed. In that case, the expression is such that the SUMO components aggregate due to the formation of SPOT (Fig. 1).

Such condensation allows the components to perform their functions in a synthetic organelle without affecting the overall state of the cells, which allows some components that are toxic to the cells to function properly in the synthetic organelles without damaging the cells. As an example, high concentrations of rapamycin strongly inhibited the growth of yeast. However, in yeasts with synthetic organelles composed of FKBP/Frb, rapamycin can be used to induce the formation of synthetic organelles, and because of the presence of synthetic organelles, the yeast also gain the ability to resist rapamycin and grow normally in its presence (Fig. 2). Figure removed due to file size Figure removed due to file size Fig. 1 Fig. 2

(2)Organization hub

A substance that does not participate in the construction of synthetic organelles can be aggregated within the synthetic organelles via diverse interactions.

Using protein-protein interactions, the synthetic organelles can be treated as a platform for the organization of other substances, which makes the yeast with the synthetic organelles into a modular system for the regulation of the spatial distribution of substances. The anti-GFP nanobody is a protein that can specifically bind GFP with high affinity. We can fuse the protein to be controlled with the anti-GFP nanobody to aggregate it at the synthetic organelles (Fig. 3).

We verified this function by fusing CFP with the nanobody, and we observed the co-localization of the blue and green fluorescence, as expected.

This system is modular and flexible. We can fuse almost any protein with the nanobody so that it can aggregate within the synthetic organelles. What’s more, this strategy avoids fusing proteins with the large system, which might result in the loss of function because of structural change. This system also has the potential to aggregate endogenous proteins and even small molecules by fusing the ligand of the substance with the nanobody as mediator. Figure removed due to file size Fig. 3

2.Sensor

Rapamycin can be used to induce the formation of the synthetic organelles. Thinking differently, rapamycin can be detected by the synthetic organelles. If the substance to be detected can bind with two proteins (homologous or heterologous) simultaneously, these proteins can be used as the interaction module to construct a synthetic organelle, and the presence of the detected molecule can consequently be shown by the formation of the synthetic organelles. As shown before, synthetic organelles arise when rapamycin is added, which means that the presence of synthetic organelles corresponds to the presence of rapamycin.

Thus, we first verified this function with rapamycin. We cultured the yeast cells with different concentrations of rapamycin to make sure that rapamycin has enough time to diffuse into the calls and the formation of SPOT can reach the equilibrium state. We then observed the yeast cells under a microscope and made statistics on the proportion of the yeast with SPOT formation. The formation of the SPOT reflected the concentration of rapamycin well.

Since rapamycin can be detected by SPOT, we went further and considered whether SPOT can also detect other molecules. Here we verified this potential by detecting 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. Upon binding to ABA, a PYL protein associates with type 2C protein phosphatases (PP2Cs) such as ABI1 and ABI2, inhibiting their activity.

In our design, if we use PYL1/ABI1 as interaction modules, we will see the SPOT forming in the cell after ABA administration. However, we found the green fluorescence, which shows the location of PYL1, condensed into a granule like the SPOT before ABA administration.

We then manage to work out the reason. Previous structural and biochemical observations have provided insights into PYL-mediated ABA signaling, which can be explained by an efficacious 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 the CL2loop. In response to ABA binding, the CL2 loop undergoes a conformational rearrangement to close over the ABA-binding pocket, which enables the interaction between PYLs and PP2Cs.

This information explained this strange phenomenon, and also enabled a prediction. In the absence of ABA, SPOT formed only with PYL1 for the homodimers of PYL1. Thus, we can observe the green fluorescence in SPOT, but no red fluorescence colocalization. After we added ABA to the yeast, ABI1 entered the organelles through the interaction of ABI1 and PYL1, and we saw red aggregates colocalize with green aggregates in cells under the fluorescence microscope. We then verified this ABA sensor through the formation of red fluorescence. The pattern met the prediction perfectly and the system also detected ABA with a high sensitivity. .

This experiment also inspired us to design a new regulation method: controlling the location of one of the component instead of controlling the formation of SPOT. As the experiment demonstrated, new components can enter the original organelles and the time of occurrence can be regulated as it is inducer-mediated. This gave our designs and functions more possibilities. Figure removed due to file size Figure 6 Interaction of PYL and PP2C

As shown by the results, SPOT can be used to detect molecules in vivo. In contrast to traditional sensors, which rely on the expression of the reporter gene and have a delay of hours, this system has a much shorter reaction time of only minutes after the reagent is added to the yeast medium. The delay can be further shortened if the detected molecules are inside the cells. Thus, the synthetic organelles can function as a quick response sensor in vivo.

3.Reaction Hub

Our synthetic organelles are based on phase separation. Due to liquid-liquid phase separation inside cells, the synthetic organelles and the cytosol will form a boundary. This boundary has some special properties, such as absorbance effects. The synthetic organelles also condense many substances, which results in a different reaction environment and different concentration between the synthetic organelles and the cytosol. All these effects can affect the reaction rate and the direction of reaction in the synthetic organelles.

Since the effects of the synthetic organelles on different reactions can be diverse due to all the factors referred to above, we develop a model to find out the key factors that effect this process. The model suggests that a higher solubility of the substrate or intermediate in the droplet phase and a relatively large KM lead to higher reaction rate. ( Please read the model for more details)

According to the conclusion given by the model, we examined the production of β-carotene as a first step. The intermediates of this pathway are hydrophobic and the reaction have a large KM, we suppose SPOT might increase the produce of β-carotene. What’s more, the β-carotene production pathway is widely used in biology (Fig. 4). It is robust and its activity is directly observable since β-carotene is orange. We fused the three enzymes that produce β-carotene into the synthetic organelles. In the presence of rapamycin, they can condense into aggregates.

As the result shows, after adding rapamycin, the yeast accumulate more β-carotene and the color become redder (Fig. 5). To demonstrate this increasing quantitatively, we extract β-carotene from the yeast and measure the concentration with HPLC (High Performance Liquid Chromatography). HPLC result also show an increasing after adding rapamycin.

Here we already demonstrate that SPOT can regulate the reaction rate and further regulate the produce of the pathway. However, there are still too many phenomes that we can not explain in a near future. Through more work on different metabolic pathway, we may one day can using SPOT to control the metabolism in cell.