Methods in Cell Biology
Bacterial cytokinesis is commonly initiated by the Z-ring, a dynamic cytoskeletal structure that assembles at the site of division. Its primary component is FtsZ, a tubulin-like GTPase, that like its eukaryotic relative forms protein filaments in the presence of GTP. Since the discovery of the Z-ring 25 years ago, various models for the role of FtsZ have been suggested. However, important information about the architecture and dynamics of FtsZ filaments during cytokinesis is still missing. One reason for this lack of knowledge has been the small size of bacteria, which has made it difficult to resolve the orientation and dynamics of individual FtsZ filaments in the Z-ring. While superresolution microscopy experiments have helped to gain more information about the organization of the Z-ring in the dividing cell, they were not yet able to elucidate a mechanism of how FtsZ filaments reorganize during assembly and disassembly of the Z-ring. In this chapter, we explain how to use an in vitro reconstitution approach to investigate the self-organization of FtsZ filaments recruited to a biomimetic lipid bilayer by its membrane anchor FtsA. We show how to perform single-molecule experiments to study the behavior of individual FtsZ monomers during the constant reorganization of the FtsZ-FtsA filament network. We describe how to analyze the dynamics of single molecules and explain why this information can help to shed light onto possible mechanism of Z-ring constriction. We believe that similar experimental approaches will be useful to study the mechanism of membrane-based polymerization of other cytoskeletal systems, not only from prokaryotic but also eukaryotic origin.
Natalia Baranova is supported by an EMBO Long-Term Fellowship (EMBO ALTF 1163-2015) and Martin Loose by an ERC Starting Grant (ERCStG-2015-SelfOrganiCell).
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Baranova NS, Loose M. Single-molecule measurements to study polymerization dynamics of FtsZ-FtsA copolymers. In: Echard A, ed. Cytokinesis. Vol 137. Academic Press; 2017:355-370. doi:10.1016/bs.mcb.2016.03.036
Baranova, N. S., & Loose, M. (2017). Single-molecule measurements to study polymerization dynamics of FtsZ-FtsA copolymers. In A. Echard (Ed.), Cytokinesis (Vol. 137, pp. 355–370). Academic Press. https://doi.org/10.1016/bs.mcb.2016.03.036
Baranova, Natalia S., and Martin Loose. “Single-Molecule Measurements to Study Polymerization Dynamics of FtsZ-FtsA Copolymers.” In Cytokinesis, edited by Arnaud Echard, 137:355–70. Academic Press, 2017. https://doi.org/10.1016/bs.mcb.2016.03.036.
N. S. Baranova and M. Loose, “Single-molecule measurements to study polymerization dynamics of FtsZ-FtsA copolymers,” in Cytokinesis, vol. 137, A. Echard, Ed. Academic Press, 2017, pp. 355–370.
Baranova NS, Loose M. 2017.Single-molecule measurements to study polymerization dynamics of FtsZ-FtsA copolymers. In: Cytokinesis. Methods in Cell Biology, vol. 137, 355–370.
Baranova, Natalia S., and Martin Loose. “Single-Molecule Measurements to Study Polymerization Dynamics of FtsZ-FtsA Copolymers.” Cytokinesis, edited by Arnaud Echard, vol. 137, Academic Press, 2017, pp. 355–70, doi:10.1016/bs.mcb.2016.03.036.