Photosynthetic artificial organelles sustain and control ATP-dependent reactions in a protocellular system

Abstract

Inside cells, complex metabolic reactions are distributed across the modular compartments of organelles1,2. Reactions in organelles have been recapitulated in vitro by reconstituting functional protein machineries into membrane systems3,4,5. However, maintaining and controlling these reactions is challenging. Here we designed, built, and tested a switchable, light-harvesting organelle that provides both a sustainable energy source and a means of directing intravesicular reactions. An ATP (ATP) synthase and two photoconverters (plant-derived photosystem II and bacteria-derived proteorhodopsin) enable ATP synthesis. Independent optical activation of the two photoconverters allows dynamic control of ATP synthesis: red light facilitates and green light impedes ATP synthesis. We encapsulated the photosynthetic organelles in a giant vesicle to form a protocellular system and demonstrated optical control of two ATP-dependent reactions, carbon fixation and actin polymerization, with the latter altering outer vesicle morphology. Switchable photosynthetic organelles may enable the development of biomimetic vesicle systems with regulatory networks that exhibit homeostasis and complex cellular behaviors.

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Figure 1: Design and applications of the photosynthetic artificial organelle.
Figure 2: Light-driven ATP synthesis in the artificial organelle.
Figure 3: Optical control mechanism of the artificial organelle.
Figure 4: Control of actin polymerization and membrane shape of the photosynthetic protocellular system by light.

References

  1. 1

    Purnick, P.E.M. & Weiss, R. The second wave of synthetic biology: from modules to systems. Nat. Rev. Mol. Cell Biol. 10, 410–422 (2009).

    CAS  Article  Google Scholar 

  2. 2

    Adamala, K.P., Martin-Alarcon, D.A., Guthrie-Honea, K.R. & Boyden, E.S. Engineering genetic circuit interactions within and between synthetic minimal cells. Nat. Chem. 9, 431–439 (2017).

    CAS  Article  Google Scholar 

  3. 3

    Liu, A.P. & Fletcher, D.A. Biology under construction: in vitro reconstitution of cellular function. Nat. Rev. Mol. Cell Biol. 10, 644–650 (2009).

    CAS  Article  Google Scholar 

  4. 4

    Rivas, G., Vogel, S.K. & Schwille, P. Reconstitution of cytoskeletal protein assemblies for large-scale membrane transformation. Curr. Opin. Chem. Biol. 22, 18–26 (2014).

    CAS  Article  Google Scholar 

  5. 5

    Wang, L., Roth, J.S., Han, X. & Evans, S.D. Photosynthetic proteins in supported lipid bilayers: towards a biokleptic approach for energy capture. Small 11, 3306–3318 (2015).

    CAS  Article  Google Scholar 

  6. 6

    Gardner, P.M., Winzer, K. & Davis, B.G. Sugar synthesis in a protocellular model leads to a cell signalling response in bacteria. Nat. Chem. 1, 377–383 (2009).

    CAS  Article  Google Scholar 

  7. 7

    Elani, Y., Law, R.V. & Ces, O. Vesicle-based artificial cells as chemical microreactors with spatially segregated reaction pathways. Nat. Commun. 5, 5305 (2014).

    CAS  Article  Google Scholar 

  8. 8

    Rossman, J.S., Jing, X., Leser, G.P. & Lamb, R.A. Influenza virus M2 protein mediates ESCRT-independent membrane scission. Cell 142, 902–913 (2010).

    CAS  Article  Google Scholar 

  9. 9

    Lingwood, D. & Simons, K. Lipid rafts as a membrane-organizing principle. Science 327, 46–50 (2010).

    CAS  Article  Google Scholar 

  10. 10

    Keber, F.C. et al. Topology and dynamics of active nematic vesicles. Science 345, 1135–1139 (2014).

    CAS  Article  Google Scholar 

  11. 11

    Carvalho, K. et al. Cell-sized liposomes reveal how actomyosin cortical tension drives shape change. Proc. Natl. Acad. Sci. USA 110, 16456–16461 (2013).

    CAS  Article  Google Scholar 

  12. 12

    Liu, A.P. et al. Membrane-induced bundling of actin filaments. Nat. Phys. 4, 789–793 (2008).

    CAS  Article  Google Scholar 

  13. 13

    Choi, H.-J. & Montemagno, C.D. Artificial organelle: ATP synthesis from cellular mimetic polymersomes. Nano Lett. 5, 2538–2542 (2005).

    CAS  Article  Google Scholar 

  14. 14

    Feng, X., Jia, Y., Cai, P., Fei, J. & Li, J. Coassembly of photosystem II and ATPase as artificial chloroplast for light-driven ATP Synthesis. ACS Nano 10, 556–561 (2016).

    CAS  Article  Google Scholar 

  15. 15

    Kurihara, K. et al. A recursive vesicle-based model protocell with a primitive model cell cycle. Nat. Commun. 6, 8352 (2015).

    CAS  Article  Google Scholar 

  16. 16

    Tsuji, G., Fujii, S., Sunami, T. & Yomo, T. Sustainable proliferation of liposomes compatible with inner RNA replication. Proc. Natl. Acad. Sci. USA 113, 590–595 (2016).

    CAS  Article  Google Scholar 

  17. 17

    Chan, V., Novakowski, S.K., Law, S., Klein-Bosgoed, C. & Kastrup, C.J. Controlled transcription of exogenous mRNA in platelets using protocells. Angew. Chem. Int. Ed. 54, 13590–13593 (2015).

    CAS  Article  Google Scholar 

  18. 18

    Noireaux, V. & Libchaber, A. A vesicle bioreactor as a step toward an artificial cell assembly. Proc. Natl. Acad. Sci. USA 101, 17669–17674 (2004).

    CAS  Article  Google Scholar 

  19. 19

    Kapoor, V. & Wendell, D. Engineering bacterial efflux pumps for solar-powered bioremediation of surface waters. Nano Lett. 13, 2189–2193 (2013).

    CAS  Article  Google Scholar 

  20. 20

    Hohmann-Marriott, M.F. & Blankenship, R.E. Evolution of photosynthesis. Annu. Rev. Plant Biol. 62, 515–548 (2011).

    CAS  Article  Google Scholar 

  21. 21

    Umena, Y., Kawakami, K., Shen, J.R. & Kamiya, N. Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 Å. Nature 473, 55–60 (2011).

    CAS  Article  Google Scholar 

  22. 22

    Béjà, O. et al. Bacterial rhodopsin: evidence for a new type of phototrophy in the sea. Science 289, 1902–1906 (2000).

    Article  Google Scholar 

  23. 23

    Bamann, C., Bamberg, E., Wachtveitl, J. & Glaubitz, C. Proteorhodopsin. Biochim. Biophys. Acta 1837, 614–625 (2014).

    CAS  Article  Google Scholar 

  24. 24

    Tunuguntla, R. et al. Lipid bilayer composition can influence the orientation of proteorhodopsin in artificial membranes. Biophys. J. 105, 1388–1396 (2013).

    CAS  Article  Google Scholar 

  25. 25

    Bogdanov, M., Xie, J., Heacock, P. & Dowhan, W. To flip or not to flip: lipid-protein charge interactions are a determinant of final membrane protein topology. J. Cell Biol. 182, 925–935 (2008).

    CAS  Article  Google Scholar 

  26. 26

    Tomashek, J.J., Glagoleva, O.B. & Brusilow, W.S.A. The Escherichia coli F1F0 ATP synthase displays biphasic synthesis kinetics. J. Biol. Chem. 279, 4465–4470 (2004).

    CAS  Article  Google Scholar 

  27. 27

    Pollard, T.D. & Borisy, G.G. Cellular motility driven by assembly and disassembly of actin filaments. Cell 112, 453–465 (2003).

    CAS  Article  Google Scholar 

  28. 28

    Fletcher, D.A. & Mullins, R.D. Cell mechanics and the cytoskeleton. Nature 463, 485–492 (2010).

    CAS  Article  Google Scholar 

  29. 29

    Rafelski, S.M. & Marshall, W.F. Building the cell: design principles of cellular architecture. Nat. Rev. Mol. Cell Biol. 9, 593–602 (2008).

    CAS  Article  Google Scholar 

  30. 30

    Walde, P. Building artificial cells and protocell models: experimental approaches with lipid vesicles. BioEssays 32, 296–303 (2010).

    CAS  Article  Google Scholar 

  31. 31

    Caffarri, S., Kouril, R., Kereïche, S., Boekema, E.J. & Croce, R. Functional architecture of higher plant photosystem II supercomplexes. EMBO J. 28, 3052–3063 (2009).

    CAS  Article  Google Scholar 

  32. 32

    Wang, Z.G., Xu, T.H., Liu, C. & Yang, C.H. Fast isolation of highly active photosystem II core complexes from spinach. J. Integr. Plant Biol. 52, 793–800 (2010).

    CAS  Article  Google Scholar 

  33. 33

    Kim, S.Y., Waschuk, S.A., Brown, L.S. & Jung, K.H. Screening and characterization of proteorhodopsin color-tuning mutations in Escherichia coli with endogenous retinal synthesis. Bba-Bioenergetics 1777, 504–513 (2008).

    CAS  Article  Google Scholar 

  34. 34

    Dioumaev, A.K. et al. Proton transfers in the photochemical reaction cycle of proteorhodopsin. Biochemistry 41, 5348–5358 (2002).

    CAS  Article  Google Scholar 

  35. 35

    Hicks, D.B. & Krulwich, T.A. Purification and reconstitution of the F1F0-ATP synthase from alkaliphilic Bacillus firmus OF4. Evidence that the enzyme translocates H+ but not Na+. J. Biol. Chem. 265, 20547–20554 (1990).

    CAS  Google Scholar 

  36. 36

    Rigaud, J.L. & Lévy, D. Reconstitution of membrane proteins into liposomes. Methods Enzymol. 372, 65–86 (2003).

    CAS  Article  Google Scholar 

  37. 37

    Porra, R.J. The chequered history of the development and use of simultaneous equations for the accurate determination of chlorophylls a and b. Photosynth. Res. 73, 149–156 (2002).

    CAS  Article  Google Scholar 

  38. 38

    Béjà, O., Spudich, E.N., Spudich, J.L., Leclerc, M. & DeLong, E.F. Proteorhodopsin phototrophy in the ocean. Nature 411, 786–789 (2001).

    Article  Google Scholar 

  39. 39

    Walde, P., Cosentino, K., Engel, H. & Stano, P. Giant vesicles: preparations and applications. ChemBioChem 11, 848–865 (2010).

    CAS  Article  Google Scholar 

  40. 40

    Angelova, M.I. & Dimitrov, D.S. Liposome electroformation. Faraday Discuss. Chem. Soc. 81, 303–311 (1986).

    CAS  Article  Google Scholar 

  41. 41

    Girard, P. et al. A new method for the reconstitution of membrane proteins into giant unilamellar vesicles. Biophys. J. 87, 419–429 (2004).

    CAS  Article  Google Scholar 

  42. 42

    Schmid, E.M., Richmond, D.L. & Fletcher, D.A. Reconstitution of proteins on electroformed giant unilamellar vesicles. Methods Cell Biol. 128, 319–338 (2015).

    CAS  Article  Google Scholar 

  43. 43

    Kaiser, H.J. et al. Order of lipid phases in model and plasma membranes. Proc. Natl. Acad. Sci. USA 106, 16645–16650 (2009).

    CAS  Article  Google Scholar 

  44. 44

    Seksek, O., Henry-Toulmé, N., Sureau, F. & Bolard, J. SNARF-1 as an intracellular pH indicator in laser microspectrofluorometry: a critical assessment. Anal. Biochem. 193, 49–54 (1991).

    CAS  Article  Google Scholar 

  45. 45

    Heider, E.C., Myers, G.A. & Harris, J.M. Spectroscopic microscopy analysis of the interior pH of individual phospholipid vesicles. Anal. Chem. 83, 8230–8238 (2011).

    CAS  Article  Google Scholar 

  46. 46

    Kreir, M., Farre, C., Beckler, M., George, M. & Fertig, N. Rapid screening of membrane protein activity: electrophysiological analysis of OmpF reconstituted in proteoliposomes. Lab Chip 8, 587–595 (2008).

    CAS  Article  Google Scholar 

  47. 47

    Gutsmann, T., Heimburg, T., Keyser, U., Mahendran, K.R. & Winterhalter, M. Protein reconstitution into freestanding planar lipid membranes for electrophysiological characterization. Nat. Protoc. 10, 188–198 (2015).

    Article  Google Scholar 

  48. 48

    Pitard, B., Richard, P., Duñach, M., Girault, G. & Rigaud, J.L. ATP synthesis by the F0F1 ATP synthase from thermophilic Bacillus PS3 reconstituted into liposomes with bacteriorhodopsin. 1. Factors defining the optimal reconstitution of ATP synthases with bacteriorhodopsin. Eur. J. Biochem. 235, 769–778 (1996).

    CAS  Article  Google Scholar 

  49. 49

    Hazard, A. & Montemagno, C. Improved purification for thermophilic F1F0 ATP synthase using n-dodecyl beta-D-maltoside. Arch. Biochem. Biophys. 407, 117–124 (2002).

    CAS  Article  Google Scholar 

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Acknowledgements

This work was supported by the Mid-Career Researcher Programs (2016R1A2B3015239 and 2011-0017539), Foreign Research Institute Recruitment Program (2013K1A4A3055268), 2015R1D1A1A01058917, and DRC-14-03-KRICT through the National Research Foundation, funded by the Ministry of Science and ICT, Korea. K.Y.L. and T.K.A. acknowledge the support provided by the Woo Jang Chun Special Project of the Rural Development Administration, Korea (PJ009106022013).

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K.Y.L., S.-J.P., K.-H.J., T.K.A., K.K.P., and K.S. developed the concept and supervised experiments. K.Y.L., K.A.L., and S.-H.K. carried out purification of photoconverters and ATP synthase. K.Y.L. and H.K. calibrated intravesicular pH. K.Y.L. and S.-J.P. performed photocurrent measurements. K.Y.L., S.-J.P., and K.S. designed the photosynthetic protocellular system with artificial organelles and performed reconstitution experiments. Y.M., K.Y.L., S.-J.P., and L.M. worked out the theoretical description. All authors discussed the results and contributed to the writing of the final manuscript.

Corresponding authors

Correspondence to Kwang-Hwan Jung or Tae Kyu Ahn or Kevin Kit Parker or Kwanwoo Shin.

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The authors declare no competing financial interests.

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Supplementary Text and Figures

Supplementary Figures 1–24 (PDF 5602 kb)

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Supplementary Notes

Supplementary Notes 1–2 (PDF 329 kb)

Regulation of intravesicular pH of a PSII-PR co-reconstituted proteoliposome by selectively activating PSII with red light and PR with green light.

Time-lapse video of a PSII-PR co-reconstituted proteoliposome stimulated by alternating white (400–700 nm), red (660 ± 52 nm), and green (540 ± 27 nm) light. The intravesicular pH rapidly decreased from an initial pH of 8.2 with the activation of both PSII and PR with white light (0–28 min) and decreased further with the activation of PSII with red light (28–36 and 44–50 min) but increased with the activation of PR with green light (36–44 and 50–60 min). The intravesicular pH of the proteoliposome was measured using a ratiometric pH SNARF-1 indicator. Scale bar = 10 μm. (MOV 1231 kb)

Three-dimensional reconstruction of microscopic images of actin filaments in a protocellular system.

The actins (white lines) encapsulated inside a protocellular system were polymerized when they were coupled via ATP synthesis of the artificial organelles (green dots) inside the membrane system (membrane: red outer boundary). (MOV 11015 kb)

Actin polymerization in the protocellular system during white light illumination.

Time-lapse video of a protocellular system (red) containing photosynthetic organelles (green) and G-actin showing polymerization of actin filaments (white). Optical stimulation initiated G-actin nucleation (~15 min) and F-actin elongation (~60 min), leading to formation of a single F-actin sphere (~135 min). The F-actin sphere grew until the membrane ruptured (275 min). Scale bar = 10 μm. (MOV 4702 kb)

Regulation of actin polymerization in a protocellular system via the organelle-based optical regulatory mechanism.

ATP-dependent actin (white lines) polymerization in a protocellular system (membrane: red outer boundary) was coupled with the ATP regulatory mechanism of the artificial organelles (green dots). The projection area of a F-actin sphere increased when ATP synthesis was facilitated via PSII activation with red light (120–180 and 240–300 min), but the F-actin area remained the same or decreased when ATP synthesis was impeded via PR activation with green light (180–240 min). Scale bar = 10 μm. (MOV 2026 kb)

Regulation of actin polymerization in a protocellular system via the light-on and light-off protocols.

ATP-dependent actin (white lines) polymerization in a protocellular system (membrane: red outer boundary) was coupled with the ATP regulatory mechanism of the artificial organelles (green dots). The projection area of a F-actin sphere increased in the light-on condition (white light, 120–180 min), and the F-actin area continuously increased even after the start of the light-off condition (180–200 min). The quenching rate of actin polymerization in the light-off case was slower than that of the green-light case. Scale bar = 10 μm. (MOV 1814 kb)

Failure of actin polymerization in a protocellular system in the absence of light stimulation (negative control).

Time-lapse video of a protocellular system (red) containing photosynthetic organelles (green) and G-actins. Polymerization of actin filaments (white) was not initiated without light illumination, indicating that actin polymerization is completely dependent on ATP synthesized by the organelles. Scale bar = 10 μm (MOV 1078 kb)

Failure of actin polymerization in a protocellular system in the absence of artificial organelles (negative control).

Time-lapse video of a protocellular system (red) containing G-actins. Polymerization of actin filaments (white) was not initiated without the photosynthetic artificial organelles, indicating that actin polymerization is completely dependent on ATP synthesized by the organelles. Scale bar = 10 μm. (MOV 3027 kb)

Failure of actin polymerization inside a protocellular system whose membrane lacked magnesium ionophores.

Time-lapse video of a protocellular system (red) containing photosynthetic organelles (green) and G-actins. Upon illumination, F-actins (white) were polymerized outside the protocellular system. In contrast, actin polymerization inside the protocellular system was not initiated in the absence of magnesium ionophores because magnesium ions were not being transported into the protocellular system. When the membrane ruptured, F-actins inside the cell suddenly polymerized because diffusion of magnesium ions outside the membrane allowed polymerization to occur. Scale bar = 10 μm. (MOV 19852 kb)

Initiation of actin polymerization in a protocellular system by magnesium influx.

Time-lapse video of a protocellular system (red) containing ATP molecules, G-actin, and membrane-bound magnesium ionophores. Actin polymerization occurred when magnesium ions were added outside the protocellular system, showing that magnesium influx through the ionophores initiates actin polymerization. Scale bar = 10 μm. (MOV 1887 kb)

Strong repulsive membrane–actin interaction in a single phase protocellular system made of Lo2 phase membrane mixture.

Time-lapse video of a protocellular system containing photosynthetic organelles (green). The membrane system (red), made of the Lo2 phase mixture (sphingomyelin, polysaturated PEG-2000-PE, and cholesterol), induced a strong repulsive interaction between the membrane and actin filaments (white), resulting in a spherical membrane shape and a gap between the membrane and actin filaments. Scale bar = 10 μm. (MOV 13044 kb)

Weak attractive membrane–actin interaction in a single phase protocellular system cell made of Lo1 phase membrane mixture.

Time-lapse video of a protocellular system containing photosynthetic organelles (green). The membrane system (red), made of the Lo1 phase mixture (sphingomyelin and cholesterol), induced a weak attractive interaction between the outer membrane and actin filaments (white), resulting in attachment of actin filaments to the membrane. Notably, the membrane fragments remained attached to the F-actin sphere even after the membrane ruptured. Scale bar = 10 μm (MOV 3007 kb)

Strong attractive membrane-actin interaction in a single phase protocellular system of Ld phase membrane mixture.

Time-lapse video of a protocellular system containing photosynthetic organelles (green). The membrane system (red), made of the Ld phase mixture (polyunsaturated phospholipids), induced a strong attractive interaction between the membrane and actin filaments (white), resulting in attachment of actin filaments to the membrane and local membrane deformation (wrinkled shape). Scale bar = 10 μm. (MOV 2139 kb)

Shape change in a phase-separated protocellular system made of Ld-Lo2 phase mixtures by membrane–actin interactions.

Time-lapse video of a protocellular system containing photosynthetic organelles (green). The membrane system (red) was made of two different phase membrane mixtures (Ld and Lo2) using the phase-separation method. Polymerized actins (white) were strongly attached to the Ld phase membrane owing to the strong interaction between actin and the Ld phase membrane, but they did not attach to the Lo2 phase membrane owing to the repulsive interaction between actin and the Lo2 phase membrane. The difference between local filament–membrane interactions induced a change in the global protocellular system's curvature, deforming the spherical cell into an asymmetrical dumbbell shape as actin polymerization proceeded. Scale bar = 10 μm. (MOV 3823 kb)

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Lee, K., Park, S., Lee, K. et al. Photosynthetic artificial organelles sustain and control ATP-dependent reactions in a protocellular system. Nat Biotechnol 36, 530–535 (2018). https://doi.org/10.1038/nbt.4140

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