De novo synthesized Min proteins drive oscillatory liposome deformation and regulate FtsA-FtsZ cytoskeletal patterns

Abstract

The Min biochemical network regulates bacterial cell division and is a prototypical example of self-organizing molecular systems. Cell-free assays relying on purified proteins have shown that MinE and MinD self-organize into surface waves and oscillatory patterns. In the context of developing a synthetic cell from elementary biological modules, harnessing Min oscillations might allow us to implement higher-order cellular functions. To convey hereditary information, the Min system must be encoded in a DNA molecule that can be copied, transcribed, and translated. Here, the MinD and MinE proteins are synthesized de novo from their genes inside liposomes. Dynamic protein patterns and accompanying liposome shape deformation are observed. When integrated with the cytoskeletal proteins FtsA and FtsZ, the synthetic Min system is able to dynamically regulate FtsZ patterns. By enabling genetic control over Min protein self-organization and membrane remodeling, our methodology offers unique opportunities towards directed evolution of bacterial division processes in vitro.

Cell-free biogenesis of bacterial division proto-rings that can constrict liposomes

“…Liposomes were formed by natural swelling, with a composition of zwitterionic PC and PE phospholipids, anionic PG and cardiolipin, and a small fraction of TexasRed-conjugated lipid for membrane imaging 35 . Such a lipid mixture and liposome preparation method have proved compatible with the cell-free synthesis of membrane-associated enzymes 10 , DNA replication proteins 11 and division proteins 12 . Liposome size distribution ranges from~1 µm up to over 15 µm in diameter, which provides a more relevant bacterial cell-size compartment than >20 µm liposomes produced with other methods 23,33 .…”

“…Notable achievements include the reconstitution of the minimal translation machinery from Escherichia coli 1 , the yeast DNA replication apparatus 2 , filopodial structures 3 , cytoskeleton self-organization and centrosome positioning 4 , egg cytokinesis signaling 5 , DNA segregation with Par 6 , and clathrincoated buds 7 . Encouraged by the many cellular pieces that have already been reconstituted in vitro, synthetic biologists have now engaged in the construction of an entire cell [8][9][10][11][12] .…”

Genetically controlled membrane synthesis in liposomes

“…Activity of single or multiple enzymes in the pathway, or substrate selectivity, could be improved by generating a library of mutagenized genes and selecting for PS-enriched liposomes by fluorescence-activated cell sorting 54 . This strategy may become decisive when combining membrane growth with other functional modules, such as DNA replication 55 and liposome division 56,57 .…”

Bulk-surface coupling reconciles Min-protein pattern formationin vitroandin vivo

“…Local oscillations in the in vitro reconstituted Min system were theoretically predicted (39) and experimentally observed in vesicles (25,43) where they manifest as homogeneous "pulsing" of the Min-protein density on the vesicle surface. In contrast, such pulsing was never observed in cells, indicating that there are no local oscillations in vivo.…”

“…The Min system was discovered in E. coli (14,15), and subsequently purified and reconstituted in vitro on supported lipid bilayers that mimic the cell membrane (16). This reconstitution provides a minimal system that enables precise control of reaction parameters and geometrical constraints (16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27). This enabled the study of the patternformation process and its molecular mechanism in a well-controlled manner, and showed the ability of the Min system to form a rich plethora of dynamic patterns, predominantly travelling waves and spirals, but also "mushrooms", "snakes", "amoebas", "bursts" (16,17,28) as well as quasi-static labyrinths, spots, and mesh-like patterns (26,27).…”