Gene‐Directed FtsZ Ring Assembly Generates Constricted Liposomes with Stable Membrane Necks

Abstract

vesicles. With a limited number of division factors compared to eukaryotes, the Escherichia coli divisome has been at the center of numerous efforts attempting to split liposomes. [5][6][7][8][9][10][11][12] In vivo, a contractile ring composed of FtsZ, FtsA, and ZipA is formed at midcell. [13,14] FtsZ is a bacterial homologue of tubulin [15][16][17] that polymerizes into self-interacting filaments when bound to GTP. [18][19][20][21] FtsZ lacks a membrane targeting domain, and it is anchored to the cytoplasmic bilayer by FtsA and ZipA.In vitro reconstruction of FtsZ polymers, [15,22] FtsA and ZipA [23] in model membranes has contributed to elucidate the self-organization and dynamics of the E. coli division proteins. In 2008, Osawa and colleagues suggested that Z-rings could generate constriction forces within tubular liposomes in the absence of motor proteins. [5] In this work, FtsZ-mts (a chimeric FtsZ with a membrane targeting sequence fused to the C terminus) caused membrane invagination, though full constriction was not achieved. [5] The authors proposed that the conformational change of FtsZ from straight to curved filaments acts as the driving force. In a follow-up study, the same group claimed that the Z-ring stimulates membrane constriction in giant liposomes to the point of septation. [7] The use of FtsA*, a gain-of-function mutant of FtsA, [24] was required to achieve complete scission, as such division events were not observed with FtsZ-mts. Szwedziak and colleagues showed that purified FtsZ and FtsA from Thermotoga maritima form continuous filament rings encircling the membrane on the inside of small unilamellar vesicles, generating constriction sites but evidences of membrane abscission were not presented. [9] The authors proposed a scenario, where the sliding and shortening of overlapping FtsZ filaments constitute the main force generator. [9] Interestingly, Ramirez Diaz and colleagues reported that FtsZ filaments do not have a single curvature, [25] and that their intrinsic helical structure drives liposome deformation via torsional stress, a mechanism that is reliant on GTP hydrolysis. [12] With the aim to recapitulate a functional Z-ring from gene-encoded proteins in liposomes, our group has demonstrated that ring-like cytoskeletal structures led to membrane constriction into narrow necks connecting budding vesicles, [11] a phenotype that differs from that observed in previous studies by the more pronounced pinching of the liposome membrane. Although no division events could unambiguously be detected, our experimental design was not directed toward addressing Mimicking bacterial cell division in well-defined cell-free systems has the potential to elucidate the minimal set of proteins required for cytoskeletal formation, membrane constriction, and final abscission. Membrane-anchored FtsZ polymers are often regarded as a sufficient system to realize this chain of events. By using purified FtsZ and its membrane-binding protein FtsA or the gain-of-function mutant FtsA* expressed in PURE (Protein syn...

Structural insights into peptidoglycan hydrolysis by the FtsEX system inEscherichia coliduring cell division

“…For example, the overexpression of S. pneumoniae FtsEX in E. coli also indicated an association with E.coli FtsA and could be isolated using styrene-maleic acid isolation and affinity purification as shown in Figure supplement 9C (30,31). If this holds true, it would provide a mechanism for the temporal regulation of PG cleavage at the cell division site, as FtsA and FtsZ are the key components of the constriction ring at the septum site (15,18,27,(32)(33)(34)(35)(36).…”

“…For example, the overexpression of S. pneumoniae FtsEX in E. coli also indicated an association with E.coli FtsA and could be isolated using styrene-maleic acid isolation and affinity purification as shown in Figure supplement 9C (30, 31). If this holds true, it would provide a mechanism for the temporal regulation of PG cleavage at the cell division site, as FtsA and FtsZ are the key components of the constriction ring at the septum site (15, 18, 27, 32–36). However, the identity of this density and the association of FtsEX with other Fts components for divisome formation warrant further investigation to fully elucidate the role of FtsEX in bacterial cell division.…”

Exploring Giant Unilamellar Vesicle Production for Artificial Cells — Current Challenges and Future Directions

“…[173] Swelling on glass beads has proven particularly effective in encapsulating IVTT systems. Recent notable results include the de novo synthesis of MinD and MinE proteins, [132] the formation of FtsA-FtsZ ring-like structures yielding constricting GUVs, [130,131] the assembly of microtubules inside GUVs, [133] and DNA-programmed membrane synthesis [134] from IVTT systems. Despite these promising results with encapsulating IVTT systems, the method has not been used for encapsulation of other biological systems.…”

Leveraging Active Learning to Establish Efficient In Vitro Transcription and Translation from Bacterial Chromosomal DNA

“…Recent advancements in the quest for the minimal synthetic cell have been substantial, especially in critical areas such as physicochemical homeostasis, − DNA replication, − membrane growth, , and membrane scission. ,− The emerging synthetic cell will ultimately harness its own gene expression machinery to regulate these complex features. In vitro transcription-translation (in vitro transcription and translation (IVTT)) is a well-established technology for the synthesis of proteins from plasmids and linear DNA encoding for a small number of genes. − Previous reports on phage genomes and large plasmids have shown that it is possible to express in the order of tens to a few hundred genes using DNA templates longer than 100 kbp. , Whereas these examples are excitingly close to a formerly postulated minimal genome (113 kbp with 151 genes), top-down experimental work on Mycoplasma established that the current minimal synthetic genome requires at least 493 genes. , From a bottom-up perspective, it is conceivable that the construction of synthetic cells requires similarly sized genomes, and these will need to be expressed in vitro.…”