A recursive vesicle-based model protocell with a primitive model cell cycle

Abstract

Self-organized lipid structures (protocells) have been proposed as an intermediate between nonliving material and cellular life. Synthetic production of model protocells can demonstrate the potential processes by which living cells first arose. While we have previously described a giant vesicle (GV)-based model protocell in which amplification of DNA was linked to self-reproduction, the ability of a protocell to recursively self-proliferate for multiple generations has not been demonstrated. Here we show that newborn daughter GVs can be restored to the status of their parental GVs by pH-induced vesicular fusion of daughter GVs with conveyer GVs filled with depleted substrates. We describe a primitive model cell cycle comprising four discrete phases (ingestion, replication, maturity and division), each of which is selectively activated by a specific external stimulus. The production of recursive self-proliferating model protocells represents a step towards eventual production of model protocells that are able to mimic evolution.

Figure 1. Concept of a self-proliferative GV-based model protocell.

(a) Membrane lipids consisting of vesicular membrane of self-reproductive GV. Cationic membrane lipid V, amphiphilic catalyst C and phospholipids (POPC and POPG) (right). The membrane lipid V and electrolyte molecule E are generated through the hydrolysis of the membrane lipid precursor V*. (b) The production of cationic membrane lipid V from its precursor V*. The cationic membrane V is produced together with the electrolyte E at an active site comprised of amplified DNA and amphiphilic catalyst C in the giant vesicular membrane. The proposed structure of the active site, comprised of amplified DNA, cationic membrane lipid V and amphiphilic catalyst C, for production of membrane lipids is shown in the bottom. (c) pH lowering induced adhesion and fusion between the target GV and the conveyer GV. The surface charge of the target GV changes to cationic due to the protonation of the POPC as well as the increase of the cationic membrane lipid V from its precursor, and the target GV adheres to the conveyer GV with a negative surface charge at pH=3. These two types of GVs fuse, and the transport of dNTP from the conveyer GV to the target GV proceeds.

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.

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.