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<div class="texttitle">四、 We are seeking for more applications with organelles platform
  
 
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                                     <p>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.</p>
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                                     <p>TWe have tested several functions of synthetic organelles platform. But more functions have not been tested owing to the time limit. Here we'll show some expectations of potential applications:</p>
 
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                                     <h3>FKBP and FRB</h3>
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                                     <h3>Real-time sensor of small molecules in cells</h3>
 
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                                     <p>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 the FKBP-rapamycin binding domain (FRB). Thus, we chose them as a pair of interaction modules. And in order to make synthetic organelles visualization, we chose mCherry (red fluorescent protein) and yEGFP (yeast-enhanced green fluorescent protein) as reporters. Then, we fused mCherry to the C-terminus of FKBP and to the N-terminus of HOTag3, similarly, we fused yEGFP to the C-terminus of FRB and to the N-terminus of HOTag6. We transformed them into yeast and proved that they can stably express. And then, only if we add rapamycin to the yeast can we see red droplets colocalize with green droplets in cells through fluorescence microscope. Synthetic organelles come true! </p>
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                                     <p>As we demonstrate before, SPOT can act as a sensor as they response to the environment rapidly and sensitively, so we wonder that they can be used to sense small molecules semi quantitatively in real-time scale in living cells. Our plan includes an NAD+ sensor in the future, which plays an important role in the study of cell's growth and metabolism. By using interaction modules that could be induced by NAD+, our synthetic organelles can work well.<br/>
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SPOT also have the potential to detect the posttranslational modification of protein. There are some modification such as ubiquitination and SUMOylation. Measure the ubiquitination and SUMOylation of a protein can be a hard work including protein extract, western blot etc. Using the protein target to the substrate and ubiquitin as interaction modules, we might have the chance the catching the dynamic change of ubiquitination in the cell.
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                                     <h3>SUMO and SIM</h3>
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                                     <h3>Reaction hub for special need</h3>
 
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                                     <p>Post-translational modifications by the small ubiquitin-like modifier (SUMO) are crucial events in cellular response to radiation and a wide range of DNA-damaging agents. Previous studies have shown that SUMO mediates protein-protein interactions by binding to a SUMO-interacting motif (SIM) on receptor proteins. And recent studies have shown that a protein with ten repeats of human SUMO3 (polySUMO) and a protein with ten repeats of SIM (polySIM) can drive interacting multivalent scaffolds in vitro. Therefore, we chose SUMO3 and SIM as a pair of interaction modules and they can spontaneously drive the formation of synthetic organelles. For plasmid construction, we did the same as FKBP and FRB. What’s more, different from inducer-mediated interactions like FKBP and FRB, we used Tet07 promoter, an inducible promoter, to initiate the expression of SIM. Then, if we add dox (the inducer of Tet07) into yeast, we will see an magical scene: yeast with two colors replaces yeast with only one color before, and red droplets colocalize with green droplets in cells through fluorescence microscope. Synthetic organelles come true! </p>
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                                     <p>Now we can indicate that enzymatic reactions can happen normally in synthetic organelles. So what's the fittest situation of such a reaction hub? Beyond just accelerating the reaction rates, there are much more functions it can perform. By accelerating part of the reaction pathway, we can change the final product of the engineered cells. Besides, some intermediates in metabolism pathways are toxic to cells, which limits the application in engineered cells. If the enzymes of the reaction are recruited into the organelles, the toxicity problem may be solved just as what happens in lysosome. </p>
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                                     <h3>Phytohormone</h3>
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                                     <h3> Signal amplifier</h3>
 
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                                     <p>Gibberellin (GA) is a well-known phytohormone, whose perception is mediated by GID1 (GA-INSENSITIVE-DWARF1). And a key event in GA signaling is the degradation of DELLA proteins, which are negative regulators of GA response that interact with GID1 in a GA-dependent manner. Since this interaction seems to be a simple biochemical reaction that does not require additional factors, we try to do further research to make full use of it. Fortunately, it has been reported that GAI (GA-INSENSITIVE) is one of DELLA family proteins in Arabidopsis and the affinity of the GID-GA interaction can increase about 100-fold by GAI, suggesting that the GID1-GA complex is stabilized by DELLA proteins. So we chose GID1 and GAI as a pair of interaction modules. And we assembled them on to yeast plasmid as the same as the construction of FKBP and FRB and transformed them into yeast. And then, only if we add GA to the yeast can we see red droplets colocalize with green droplets in cells through fluorescence microscope. Synthetic organelles come true! </p>
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                                     <p>Phase separation process shows sensitive dynamics so that we think our synthetic organelles can be introduced into artificial signal pathway as a signal amplifier. </p>
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                                    <p>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.
 
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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. 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. 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.
 
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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. 
 
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                                    <p>The above describes a lot of components used as interaction modules, but they all have a common disadvantage——irreversible! Now, this problem can be solved be the Arabidopsis red light-inducible phytochrome (PHYB-PIF) system, which comprises the phytochrome B (PHYB) protein and the basic-helix-loop-helix (bHLH) transcription factor phytochrome interaction factor (PIF; PIF3 or PIF6). These two domains are induced to bind under far infrared and the binding is reversed within seconds of exposure to infrared light but is otherwise stable for hours in the dark. What’s more, the phytochrome system has a 10-100  larger dynamic range than the cryptochrome and LOV-based systems, and the affinity of its light-gated interaction is tighter than others. Therefore, we chose PHYB and PIF6 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. After that, synthetic organelles can form and disappear under the regulation of far infrared.    </p>
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<div class="texttitle">Two function sites
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                                    <p>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.</p>
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                                    <h3>Kidnapping——direct integration into the skeleton</h3>
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                                    <p>Just as we characterize synthetic organelles with fluorescent proteins, we can fuse function modules to the C-terminus of interaction modules and to the N-terminus of HOTags. Then, the function modules can be “kidnapped” into the synthetic organelles to fulfill intended functions.</p>
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                                    <h3> Welcome——with nanobody</h3>
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                                    <p>We introduce a magic protein, anti-GFP nanobody, which is very small (only 13-kDa, 1.5nm 2.5nm) and high-affinity (0.59nM) camelid antibody to GFP. So we can use its characteristic to improve our designs. We can fuse GFP to the C-terminus of interaction modules and to the N-terminus of HOTags, and fuse function modules to the C-terminus of anti-GFP nanobodies. Then, with the help of interaction between anti-GFP nanobodies and GFP, synthetic organelles will “welcome” function modules, expected functions can be realized. You may ask: How does anti-GFP nanobody improve the design? Firstly, it will not make the protein extremely large and will reduce the effect on the structure of function modules, which can ensure the quality of functions. Secondly, it can bring components not belonging to the original structure to synthetic organelles, which can enlarge the enrichment range of synthetic organelles. Thirdly, it is easy to regulate the expression of target proteins. So you can see, nanobodies may do better and give you a surprise!</p>
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                                    <p>We artificially designed phase separation in cells and synthesized membraneless organelles. And the main work to synthesize an organelle is to fulfill phase separation in a cell, so we stress the importance of interactions and multivalency. For these two aspects, we gave our ideas and the feasibility was analyzed. At last, we proposed two ideas to implement functions. We believe that in the near future, “millions of dollars” will no longer be a dream!</p>
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1. Yin P, Fan H, Hao Q, et al. Structural insights into the mechanism of abscisic acid signaling by PYL proteins[J]. Nature Structural & Molecular Biology, 2009, 16(12):1230-1236.
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2. Park S Y, Fung P, Nishimura N, et al. Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins[J]. Science, 2009, 324(5930):1068-1071.<br/>
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3. 冯婵莹, 王永飞. 植物脱落酸PYR/PYL/RCAR受体[J]. 生命的化学, 2015(6):721-726.<br/>
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4. Nambara E, Marion-Poll A. Abscisic acid biosynthesis and catabolism.[J]. Annual Review of Plant Biology, 2005, 56(56):165-185.<br/>
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5. Hirano K, Ueguchi-Tanaka M, Matsuoka M. GID1-mediated gibberellin signaling in plants[J]. Trends in Plant Science, 2008, 13(4):192-199.<br/>
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6. Nelis S, Conti L, Zhang C, et al. A functional Small Ubiquitin-like Modifier (SUMO) interacting motif (SIM) in the gibberellin hormone receptor GID1 is conserved in cereal crops and disrupting this motif does not abolish hormone dependency of the DELLA-GID1 interaction[J]. Plant Signaling & Behavior, 2015, 10(2):e987528.<br/>
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7. Ueguchi-Tanaka M, Nakajima M, Motoyuki A, et al. Gibberellin receptor and its role in gibberellin signaling in plants.[J]. Annual Review of Plant Biology, 2007, 58(1):183-198.<br/>
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8. Ries J, Kaplan C, Platonova E, et al. A simple, versatile method for GFP-based super-resolution microscopy via nanobodies.[J]. Nature Methods, 2012, 9(6):582-584.<br/>
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9. Caussinus E, Kanca O, Affolter M. Fluorescent fusion protein knockout mediated by anti-GFP nanobody[J]. Nature Structural & Molecular Biology, 2011, 19(1):117-121.<br/>
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10. Zhang K, Cui B. Optogenetic control of intracellular signaling pathways[J]. Trends in Biotechnology, 2015, 33(2):92-100.<br/>
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11. Buckley C, Moore R, Reade A, et al. Reversible Optogenetic Control of Subcellular Protein Localization in a Live Vertebrate Embryo[J]. Developmental Cell, 2016, 36(1):117.<br/>
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12. Banaszynski L A, And C W L, Wandless T J. Characterization of the FKBP·Rapamycin·FRB Ternary Complex  [J. Am. Chem. Soc. 2005, 127, 4715−4721].[J]. Journal of the American Chemical Society, 2006, 128(49).<br/>
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13. Woolfson D N, Bartlett G J, Burton A J, et al. De novo protein design: how do we expand into the universe of possible protein structures?[J]. Current Opinion in Structural Biology, 2015, 33:16-26.<br/>
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14. Banani S F, Rice A M, Peeples W B, et al. Compositional Control of Phase-Separated Cellular Bodies[J]. Cell, 2016, 166(3):651-663.<br/>
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15. Zhang Q, Huang H, Lu Q, et al. Visualizing Dynamics of Cell Signaling InVivo, with a Phase Separation-Based Kinase Reporter[J]. Molecular Cell, 2018, 69(2):347.<br/>
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Revision as of 18:00, 14 October 2018

Perspective

It will show our future plan

More interaction modules can be used in our organelles

In our design, the interaction module is replaceable. We have used SUMO-SIM modules and FKBP-Frb modules to build spontaneous and induced synthetic organelles. To enrich our platform, more interaction modules are being considered. such as the GA-induced GID1 (GA-insensitive dwarf1) and GAI (GA-insensitive) systems In addition to the use of small chemical molecules to induce organelle formation, photoinduction is also a promising strategy. Its advantage is that it has a faster response to the environment and can make more precise regulation. At the same time, the optical guidance system usually ha We will select phyB / PIF6 system and use red light to induce dimerization to test the feasibility of the light-induced organelle. It may be a useful tool in synthetic biology.

Synthetic organelles can perform complex behaviors in the future

We have observed the formation process of synthetic organelles. However, in our previous work, the inducer rapamycin is difficult to be removed from the system, so we have not seen the disassociation process of organelles. We think that the formation and disassociation of synthetic organelles can be both controlled by using light-induced interaction module or chemical-induced module with weaker strength. We believe that our synthetic organelles can perform more functions with this ability.
Another assumption is that two orthogonal synthetic organelles can co-exist in cells. Two organelles can be induced and perform functions independently.

How to recruited function modules to synthetic organelles?

When we try to use synthetic organelles to accelerated reaction, we found the enzyme fused to the system cannot perform well as free enzyme. So it inspires us to develop a new method to load function modules to the whole systems, where the organelle acts as an organization hub.
In the design, the function modules will not be fused into the components, and be recruited into the organelles by protein-protein interactions. We have tested the feasibility of the design by using anti-GFP nanobody. 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.
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. These effects will be tested in the future, especially in the metabolism regulation protein.
This system also has the potential to aggregate the endogenous protein and even macromolecules by fusing the ligand of the substance with nanobody as medium.
Another assumption is that two orthogonal synthetic organelles can co-exist in cells. Two organelles can be induced and perform functions independently.

四、 We are seeking for more applications with organelles platform

TWe have tested several functions of synthetic organelles platform. But more functions have not been tested owing to the time limit. Here we'll show some expectations of potential applications:

1.

Real-time sensor of small molecules in cells

As we demonstrate before, SPOT can act as a sensor as they response to the environment rapidly and sensitively, so we wonder that they can be used to sense small molecules semi quantitatively in real-time scale in living cells. Our plan includes an NAD+ sensor in the future, which plays an important role in the study of cell's growth and metabolism. By using interaction modules that could be induced by NAD+, our synthetic organelles can work well.
SPOT also have the potential to detect the posttranslational modification of protein. There are some modification such as ubiquitination and SUMOylation. Measure the ubiquitination and SUMOylation of a protein can be a hard work including protein extract, western blot etc. Using the protein target to the substrate and ubiquitin as interaction modules, we might have the chance the catching the dynamic change of ubiquitination in the cell.

2.

Reaction hub for special need

Now we can indicate that enzymatic reactions can happen normally in synthetic organelles. So what's the fittest situation of such a reaction hub? Beyond just accelerating the reaction rates, there are much more functions it can perform. By accelerating part of the reaction pathway, we can change the final product of the engineered cells. Besides, some intermediates in metabolism pathways are toxic to cells, which limits the application in engineered cells. If the enzymes of the reaction are recruited into the organelles, the toxicity problem may be solved just as what happens in lysosome.

3.

Signal amplifier

Phase separation process shows sensitive dynamics so that we think our synthetic organelles can be introduced into artificial signal pathway as a signal amplifier.