Team:Peking/Functions D
Data Page-Functions
一、 Spatial segmentation and organization
The most basic function of a synthetic organelle is to form a segmentation of the space, changing the position of the substance, thereby causing a corresponding effect.
(一) Substance condensation
The components used to construct the synthetic organelles can condense via phase separation, resulting in different concentrations of these components in the synthetic organelles and cytosol. When 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 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 given a sufficient concentration of doxorubicin, the SIM component expresses. Then expression is such that the SUMO components aggregate due to the formation of SPOT.( fig 1)
Such condensation allows these 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 poisoning 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 as an inducer to conduct the function of induce the formation of synthetic organelles, and because of the presence of synthetic organelles, the yeast also gain the ability to resist rapamycin and growth normally ( fig 2)
Fig1 fig2
(二) Organization hub
The substance that does not participate in the construction of synthetic organelles can aggregate at the synthetic organelles via the diverse interaction.
With protein-protein interactions, the synthetic organelles can be treat as a platform that organize the other substance. For example, the yeast with the synthetic organelles is a modular system to regulate the spatial distribution of substance. Anti-GFP nanobody is a protein that can bind GFP. We can fuse the protein to be controlled with anti-GFP nanobody to aggregate it at the synthetic organelles.(fig3)
We verified the function by fusing CFP with nanobody, and we found the co-localization of the blue and green fluorescence.
This system is modular and flexible. We can fuse almost any protein with nanobody and then it can aggregate in the synthetic organelles. What’s more, this strategy avoids fusing protein in the large system, which might result in the loss of functions because of structure change. This system also have the potential to aggregate the endogenous protein and even micromolecules by fusing the ligand of the substance with nanobody as medium.
Fig 3
二、 Sensor
Rapamycin can induce the formation of the synthetic organelles. Think differently, rapamycin can be detected by the synthetic organelles. If the substance to be detected can bind with two proteins (homologous or heterologous) simultaneous, these proteins can be use as the interaction module to construct a synthetic organelle. The existence of the detected molecule can be shown by the formation of the synthetic organelles. As shown before, synthetic organelles show up when given rapamycin, which means the presence of synthetic organelles corresponding to the presence of rapamycin.
Thus we first verified this function with rapamycin first. We culture the yeast in different concentration of rapamycin to make sure the rapamycin have enough time to diffuse into the yeast and the formation of SPOT is in the equilibrium state. Then we observe the yeast under microscope and have a statistics on the proportion of the yeast with SPOT formation. As the result, the formation of the SPOT stated the concentration of rapamycin well.
Since rapamycin can be detected by SPOT, we go further and consider whether SPOT can also detect other molecule. Here we verified this potential by detecting ABA.
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 assumption, if we use PYL1/ABI1 as interaction modules, we will see the SPOT forming in the cell after given ABA. However, we found the green fluorescence, which shows the location of PYL1, condensed into a granule like the SPOT before given ABA.
We then manage to work out the reason. Previous structural and biochemical observations have provided insight into PYL-mediated ABA signaling which can be explained by a 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 CL2loop. 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 forms.
This information explained this strange phenomenon, also gave a prediction. In the absence of ABA, SPOT forming only with PYL1 for the homodimers of PYL1, We will observe the green fluorescence in SPOT, but no red fluorescence colocalization. And after we added ABA into yeast, ABI1 entered the organelles through the interaction of ABI1 and PYL1, and we saw red droplets colocalize with green droplets in cells under fluorescence microscope. We then verified this ABA sensor through the forming of red fluorescence, The pattern met the prediction perfectly and the system also detected ABA with a high sensitivity.
This experiment also inspire us of a new regulate method: controling the location of one of the component instead of controlling the formation of SPOT. As what the experiment demonstration, new components can enter the original organelles and the time of occurrence can be regulated as it is inducer-mediated regulation. So it gave our designs and functions more possibilities.
Figure 6 Interaction of PYL and PP2C
As shown by the results, SPOT can be used to detect molecules in vivo, in contrast to the tradition sensor, which rely on the expression of the reporter gene and have a delay of hours, this system have a much lessen delay time of only minutes when given reagent in yeast medium. The delay can be further shorten if the detected molecule are right in the cells. Thus, the synthetic organelles can be a quick response sensor in vivo.
三、 Metabolism regulation
Our synthetic organelles base on phase separation. As a liquid-liquid phase separation in cell, the synthetic organelles and the cytosol will form a boundary. This boundary has some special properties like absorbance effect. The synthetic organelles also condense many substances, which result in different reaction environment and different concentration between the synthetic organelles and the cytosol. All effects above can affect the reaction rate and the direction of reaction in the synthetic organelles.
Since the effect on reactions of synthetic organelles can be diverse for all those factors referred above, we examine the produce of β-carotene as our first step. The produce of β-carotene is a widely used metabolism pathway in biology. (fig 4) It is robust and visible for β-carotene is orange. We fused the three enzymes that for produce β-carotene into the synthetic organelles. In the presence of rapamycin, they can condense into organelles. We set the free enzymes and free enzymes with synthetic organelles as the controls. As the result.
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