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Liposome division by a simple bacterial division machinery.

ABSTRACT: We previously reconstituted Z rings in tubular multilamellar liposomes with FtsZ-YFP-mts, where mts is a membrane-targeting amphiphilic helix. These reconstituted Z rings generated a constriction force but did not divide the thick-walled liposomes. Here we developed a unique system to observe Z rings in unilamellar liposomes. FtsZ-YFP-mts incorporated inside large, unilamellar liposomes formed patches that produced concave distortions when viewed at the equator of the liposome. When viewed en face at the top of the liposome, many of the patches were seen to be small Z rings, which still maintained the concave depressions. We also succeeded in reconstituting the more natural, two-protein system, with FtsA and FtsZ-YFP (having the FtsA-binding peptide instead of the mts). Unilamellar liposomes incorporating FtsA and FtsZ-YFP showed a variety of distributions, including foci and linear arrays. A small fraction of liposomes had obvious Z rings. These Z rings could constrict the liposomes and in some cases appeared to complete the division, leaving a clear septum between the two daughter liposomes. Because complete liposome divisions were not seen with FtsZ-mts, FtsA may be critical for the final membrane scission event. We demonstrate that reconstituted cell division machinery apparently divides the liposome in vitro.

SUBMITTER: Osawa M

PROVIDER: S-EPMC3703997 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Liposome division by a simple bacterial division machinery.

Osawa Masaki M Erickson Harold P HP

Proceedings of the National Academy of Sciences of the United States of America 20130617 27

We previously reconstituted Z rings in tubular multilamellar liposomes with FtsZ-YFP-mts, where mts is a membrane-targeting amphiphilic helix. These reconstituted Z rings generated a constriction force but did not divide the thick-walled liposomes. Here we developed a unique system to observe Z rings in unilamellar liposomes. FtsZ-YFP-mts incorporated inside large, unilamellar liposomes formed patches that produced concave distortions when viewed at the equator of the liposome. When viewed en fa ...[more]

PMID: 23776220

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Project description:Cell division in most prokaryotes is mediated by the well-studied fts genes, with FtsZ as the principal player. In many archaeal species, however, division is orchestrated differently. The Crenarchaeota phylum of archaea features the action of the three proteins, CdvABC. This Cdv system is a unique and less-well-studied division mechanism that merits closer inspection. In vivo, the three Cdv proteins form a composite band that contracts concomitantly with the septum formation. Of the three Cdv proteins, CdvA is the first to be recruited to the division site, while CdvB and CdvC are thought to participate in the active part of the Cdv division machinery. Interestingly, CdvB shares homology with a family of proteins from the eukaryotic ESCRT-III complex, and CdvC is a homolog of the eukaryotic Vps4 complex. These two eukaryotic complexes are key factors in the endosomal sorting complex required for transport (ESCRT) pathway, which is responsible for various budding processes in eukaryotic cells and which participates in the final stages of division in Metazoa. There, ESCRT-III forms a contractile machinery that actively cuts the membrane, whereas Vps4, which is an ATPase, is necessary for the turnover of the ESCRT membrane-abscission polymers. In contrast to CdvB and CdvC, CdvA is unique to the archaeal Crenarchaeota and Thaumarchaeota phyla. The Crenarchaeota division mechanism has often been suggested to represent a simplified version of the ESCRT division machinery thus providing a model system to study the evolution and mechanism of cell division in higher organisms. However, there are still many open questions regarding this parallelism and the division mechanism of Crenarchaeota. Here, we review the existing data on the role of the Cdv proteins in the division process of Crenarchaeota as well as concisely review the ESCRT system in eukaryotes. We survey the similarities and differences between the division and abscission mechanisms in the two cases. We suggest that the Cdv system functions differently in archaea than ESCRT does in eukaryotes, and that, unlike the eukaryotic case, the Cdv system's main function may be related to surplus membrane invagination and cell-wall synthesis.

Project description:UnlabelledThe synthesis of peptidoglycan (PG) in bacteria is a crucial process controlling cell shape and vitality. In contrast to bacteria such as Escherichia coli that grow by dispersed lateral insertion of PG, little is known of the processes that direct polar PG synthesis in other bacteria such as the Rhizobiales. To better understand polar growth in the Rhizobiales Agrobacterium tumefaciens, we first surveyed its genome to identify homologs of (~70) well-known PG synthesis components. Since most of the canonical cell elongation components are absent from A. tumefaciens, we made fluorescent protein fusions to other putative PG synthesis components to assay their subcellular localization patterns. The cell division scaffolds FtsZ and FtsA, PBP1a, and a Rhizobiales- and Rhodobacterales-specific l,d-transpeptidase (LDT) all associate with the elongating cell pole. All four proteins also localize to the septum during cell division. Examination of the dimensions of growing cells revealed that new cell compartments gradually increase in width as they grow in length. This increase in cell width is coincident with an expanded region of LDT-mediated PG synthesis activity, as measured directly through incorporation of exogenous d-amino acids. Thus, unipolar growth in the Rhizobiales is surprisingly dynamic and represents a significant departure from the canonical growth mechanism of E. coli and other well-studied bacilli.ImportanceMany rod-shaped bacteria, including pathogens such as Brucella and Mycobacteriu, grow by adding new material to their cell poles, and yet the proteins and mechanisms contributing to this process are not yet well defined. The polarly growing plant pathogen Agrobacterium tumefaciens was used as a model bacterium to explore these polar growth mechanisms. The results obtained indicate that polar growth in this organism is facilitated by repurposed cell division components and an otherwise obscure class of alternative peptidoglycan transpeptidases (l,d-transpeptidases). This growth results in dynamically changing cell widths as the poles expand to maturity and contrasts with the tightly regulated cell widths characteristic of canonical rod-shaped growth. Furthermore, the abundance and/or activity of l,d-transpeptidases appears to associate with polar growth strategies, suggesting that these enzymes may serve as attractive targets for specifically inhibiting growth of Rhizobiales, Actinomycetales, and other polarly growing bacterial pathogens.

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Liposome division by a simple bacterial division machinery.

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Liposome division by a simple bacterial division machinery.

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