Replenishment of target GVs with dNTPs from conveyer GV

To replenish the depleted substrates of daughter GVs, we constructed a ‘vesicular delivery system’ in which a ‘target’ GV containing all of the reagents needed for DNA replication, except for dNTPs, adhered and fused with a ‘conveyer’ GV filled with dNTPs when triggered by a pH change of the vesicular dispersion (Fig. 1c; Supplementary Fig. 1a; Supplementary Methods). Thus, the daughter model protocell could potentially acquire a sustainable recursive ability to proliferate when external stimuli, such as sequential thermal cycles, and the addition of V* were applied to it.

We simulated the target GVs as newborn daughter GVs, which depleted the substrates (dNTPs). To compare with the membrane composition of the newborn GVs after consumption of membrane precursor, the membrane composition of the target GV was adjusted to POPC:POPG:membrane molecule V:catalyst C:cholesterol=30:7.5:55:5:2.5 mol% (the molecular structures are shown in Fig. 1a). Hence, the surface charge of the target GV was positive. On the other hand, the surface charge of the conveyer GVs (POPC:POPG:catalyst C:cholesterol=25:60:10:5 mol%) that contained dNTPs was negative because anionic POPG was abundant in the membrane. When these two kinds of GVs were mixed and the pH of the vesicular dispersion was lowered, the GVs adhered (Supplementary Fig. 2) and fused during incubation with gentle stirring at pH=3 overnight. Although vesicular fusion between GVs with opposite surface charges has been documented15,19, this vesicular fusion was triggered by pH lowering because the charge of the POPC of the target GV became positive under the acidic condition (pH=3) produced by the partial protonation of the zwitterionic POPC (pK a =ca. 1)20, whereas that of the conveyer GV remained negative even though almost half of the POPG molecules were protonated (pK a =ca. 3)20. In the current case, the charge of the target GV was positive due to the incorporation of cationic lipid V (Supplementary Fig. 1a), the adhesion might occur even before the acidification of the vesicular dispersion, but the fusion occurred only after the acidification, suggesting that the protonation of POPC is crucial for the membrane reorganization19.

After neutralization of the dispersion containing the fused GVs, we subjected the vesicular dispersion to thermal cycles. DNA amplification was clearly detected by assessing the fluorescence emission from a SYBR Green I–double-stranded DNA (dsDNA) complex in the fused GV (Supplementary Fig. 1), suggesting that no deactivation of polymerase occurred due to the proton impermeability of vesicular membranes of the target GV (Supplementary Fig. 3). This observation provided strong evidence for the ingestion of dNTPs by the target GV from the conveyer GV. When the lipid precursor V* was added (Fig. 1b), the fused GVs containing the amplified DNA exhibited a budding-like deformation and subsequently divided (Supplementary Fig. 4). These observations indicate that our GV-based model protocell acquired recursive ability in its self-proliferative cycle. Statistical analysis of flow cytometry data showed that these sequential dynamics, such as adhesion and fusion between the target and the conveyer GVs and division after amplification of DNA using ingested dNTPs, occurred as ubiquitous events (Supplementary Fig. 5; Supplementary Methods).

Recursive GV-proliferation over three generations

The recursive ability of our model protocell was confirmed by the production of a granddaughter GV in the presence of the vesicular transport system (Fig. 2). The vesicular membrane of the original GV was the same as that used in the previous experiment7 and was stained with a rhodamine-tagged lipid (Rhod-DOPE, 0.1 mol%) to distinguish the offspring of the original GV from accidentally formed GVs, whereas the conveyer GVs with the negative surface charge contained dNTPs and SYBR Green I (Fig. 2a). These two types of GVs with opposite charges adhered and fused upon co-incubation, as occurred in the previous experiment (Methods).

Figure 2: Repeated self-proliferation cycle to produce GV-based model protocell of the 3rd generation. (a) Self-proliferation of GV-based model protocell from 1st generation to 3rd generation. DNA amplification in mother GV was followed by the first division to give rise to daughter GVs. Ingestion of dNTP in conveyer GV by daughter GVs and DNA amplification in daughter GV led the second division to give granddaughter GVs (bottom). (b) Differential interface contrast microscope image of DNA-amplified daughter GV (left). Fluorescence microscope images of the red fluorescence emitted from the vesicular membrane (center) and the green fluorescence from inside the daughter GV (right). Scale bar, 10 μm. (c) Division of the daughter GV to afford granddaughter GVs by the addition of precursor V* of the membrane lipid. Scale bar, 20 μm. Full size image

After neutralization of the GV dispersion, the GVs were subjected to thermal cycles according to the protocol (Methods). Fluorescence microscopy images of the PCR-subjected GVs showed intense green fluorescence from the dsDNA and SYBR Green I complex inside the GV and red fluorescence from the membranes stained with Rhod-DOPE (Fig. 2b). These data unequivocally demonstrate the amplification of DNA in the fused GV. We already confirmed that most of the mother GVs containing amplified DNA divided into daughter GVs when V* was added, as previously reported7 (Methods). Hence, GVs that emitted green fluorescence from the dsDNA and SYBR Green I complex and red fluorescence from the membrane stained with Rhod-DOPE could be accurately designated as daughter GVs bearing amplified DNA (Fig. 2c).

Significance of the four phases in the self-proliferative GVs

This self-proliferative cycle can be divided into four discrete phases: ingestion, replication, maturity and division (Fig. 3), which could be considered as a primitive model cell cycle. These phases are described and discussed in detail below.

Figure 3: Primitive model cell cycle of self-proliferative model protocell with four discrete phases. (a) In the ingestion phase, the GV of the next generation ingests substrates through vesicular fusion with conveyer GV containing dNTP, triggered by a pH jump. (b) In the replication phase, the replication of DNA in the next-generation GV proceeds using ingested dNTP. (c) In the maturity phase, the catalytic ability of the vesicular membrane matures in a sense that a complex between amplified DNA, amphiphilic catalyst C and cationic lipids V intrudes into the vesicular membrane, forming an active site for converting membrane precursor V* to lipid membrane V. (d) In the division phase, the self-proliferative GV grows and exhibits a budding deformation and an equivolume division when the precursor V* of the membrane lipid is added to the exterior of GVs. Full size image

In the ingestion phase, the protocell ingests the depleted substrates (dNTPs) via the vesicular delivery system, in which conveyer GVs transport the required substrates to the original (target) GV. This delivery system is dependent on selective adhesion and fusion between GVs with different surface charges (Fig. 3a). We confirmed that this vesicular delivery system could also replenish depleted DNA polymerase (Supplementary Fig. 6; Supplementary Methods), and other reagents that would need to be ingested after several rounds of division. Of note, the membrane composition of GVs varies during the cycle due to the incorporation of cationic membrane lipids derived from their precursors. However, it is possible to restore the membrane composition of the daughter GV, as in the case for the dNTP substrates, by fusing the daughter GV with conveyer GV containing the complimentary lipids. Therefore, it would be a trivial matter for vesicles to oscillate their constitutions through cycles.

In the replication phase, DNA is replicated only in the GVs that have ingested dNTPs through the vesicular fusion that occurred during the ingestion phase (Fig. 3b). DNA replication is driven by periodic changes of temperature (–(94–68 °C) 20 –)7,21 (Supplementary Fig. 7; Supplementary Methods). Efficient DNA replication depends on the status of the inner cavity of the GVs. Specifically, if the inner cavity of the GV is large and sufficient reagents are encapsulated, the efficiency of DNA amplification is optimal, and DNA is amplified by even a smaller number of thermal cycles7,21. The amplified DNA in the GV shifts the equilibrium between the adhered DNA on the inner membrane surface and the dissolved DNA in the inner water phase to the adhered side, which prepares the active sites (see next paragraph) for membrane lipid formation within the vesicular membrane. The replication phase ceases when the dNTP is depleted.

The maturity phase is characterized by maturation of the catalytic activity of the vesicular membrane and the DNA–catalyst complex22,23. This phase is not triggered by an external stimulus as in the ingestion or replication phases; it is activated by an internal stimulus corresponding to an increase in the concentration of the amplified DNA in the inner water pool of the GV. The division frequency of GVs that contain amplified DNA is much greater than that of GVs, which do not contain amplified DNA7. This finding strongly suggests that amplified DNA accelerates GV growth and division by the DNA–catalyst complex, which consists of amplified DNA, cationic lipid V and amphiphilic catalyst C. The DNA–catalyst complex intrudes into the vesicular membrane, creating an ‘active site’ for the production of membrane lipid V from its precursor V* (Fig. 3c). During the maturity phase, sufficient membrane lipid V is locally provided around active sites. Immediately after the local concentration of V becomes high, the next phase (the division phase) starts. Of note, only GVs in which the active site is constructed can undergo growth and division.

The division phase is the phase of proliferation of the GV containing the amplified DNA after the addition of membrane precursor. The DNA–catalyst complex not only serves as the pseudo-enzyme to produce membrane lipid V from its precursor V*, but also acts as a scaffold for GV division, because a series of divisions occurs around the same position in the vesicular membrane. The divided daughter GVs contain partitioned DNA (Fig. 3d). Only one type of DNA is present in our GV-based model protocell, but the role of DNA in division may be crucial, because the DNA–catalyst complex serves as a pseudo-enzyme that directly participates in division dynamics.