Abstract
A major challenge towards the realization of an autonomous synthetic cell resides in the encoding of a division machinery in a genetic programme. In the bacterial cell cycle, the assembly of cytoskeletal proteins into a ring defines the division site. At the onset of the formation of the Escherichia coli divisome, a proto-ring consisting of FtsZ and its membrane-recruiting proteins takes place. Here, we show that FtsA-FtsZ ring-like structures driven by cell-free gene expression can be reconstituted on planar membranes and inside liposome compartments. Such cytoskeletal structures are found to constrict the liposome, generating elongated membrane necks and budding vesicles. Additional expression of the FtsZ cross-linker protein ZapA yields more rigid FtsZ bundles that attach to the membrane but fail to produce budding spots or necks in liposomes. These results demonstrate that gene-directed protein synthesis and assembly of membrane-constricting FtsZ-rings can be combined in a liposome-based artificial cell.
Highlights
A major challenge towards the realization of an autonomous synthetic cell resides in the encoding of a division machinery in a genetic programme
We find that in our low-volume supported lipid bilayer (SLB) assays with ZipA and ≤3 μM FtsZ, the generic crowding agent Ficoll[70] is necessary to elicit bundle formation
To obtain quantitative insights about the concentration of cellfree synthesized FtsA, pre-ran PURE system samples were analyzed by liquid chromatography-coupled mass spectrometry (LC-MS) (Supplementary Fig. 2, Supplementary Table 1)
Summary
A major challenge towards the realization of an autonomous synthetic cell resides in the encoding of a division machinery in a genetic programme. We show that FtsA-FtsZ ring-like structures driven by cell-free gene expression can be reconstituted on planar membranes and inside liposome compartments. Such cytoskeletal structures are found to constrict the liposome, generating elongated membrane necks and budding vesicles. Additional expression of the FtsZ cross-linker protein ZapA yields more rigid FtsZ bundles that attach to the membrane but fail to produce budding spots or necks in liposomes These results demonstrate that gene-directed protein synthesis and assembly of membrane-constricting FtsZ-rings can be combined in a liposome-based artificial cell. At an earlier stage of cytokinesis, a proto-ring composed of the FtsZ, FtsA, and ZipA proteins, assembles on the inner leaflet of the cytoplasmic membrane at the future division site[14,15,16]. Whether FtsZ filaments alone exert a contractile force and contribute to the final stage of division remains a subject of debate[31,32] and evidence for complete liposome division is still lacking
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
