The Role and Regulation of FtsZ Filaments in Bacterial Cytokinesis

Abstract

Like all cells, bacteria must divide in order to proliferate. This process must be carefully controlled in space and time to ensure that these cells maintain the appropriate size, shape, and DNA content, and respond appropriately to environmental stressors. Additionally, cytokinesis in bacteria is a complex process, requiring cells to simultaneously remodel their membrane and synthesize new cell wall at the division site. Cytokinesis in most bacteria is carried out by a group of proteins collectively known as the divisome. The best-studied member of the divisome is the tubulin homolog FtsZ, which forms the bacterial cytokinetic ring, called the Z ring. FtsZ plays a central role in bacterial cell division: it has been proposed to function as a scaffold for the other division proteins, a regulator of cell division, and a force generator that pulls the membrane inward during cytokinesis. Early in the cell cycle, FtsZ binding proteins are recruited to the Z ring and are thought to regulate its assembly and dynamics. A second group of proteins arrives later, including cell wall synthesis enzymes. As cells divide, these enzymes synthesize a new cell wall, called a septum, between the two daughter cells. While many divisome proteins have been identified, the mechanisms by which they collectively execute cell division have remained unclear. In this thesis, I investigate the properties of FtsZ filaments, how these properties are established, and their role in cytokinesis. I begin by characterizing the motions of FtsZ filaments in Bacillus subtilis cells. We find that these filaments move around the cell by treadmilling, and that this motion also occurs within the Z ring. We demonstrate that this treadmilling motion drives processive single-molecule movement of the essential septal synthesis enzyme Pbp2B. We then show that this motion plays a key role in cytokinesis: the speed at which FtsZ filaments treadmill controls Pbp2B’s movement speed, the rate and distribution of peptidoglycan synthesis at the septum, and the overall speed at which cytokinesis proceeds. Next, I implement a single molecule lifetime assay to characterize the properties of FtsZ filaments in live cells. Past in vivo studies of FtsZ dynamics have been limited by spatial resolution, but the lifetime assay avoids these issues and allows us to analyze FtsZ’s treadmilling behavior more precisely. We demonstrate that this assay works robustly in live cells, and confirm that it reports on filament treadmilling. Additionally, combining this assay with measurements of FtsZ treadmilling velocity allows us to estimate other parameters for FtsZ filaments in live cells and compare them to values obtained in vitro. These measurements suggest that FtsZ filaments in cells treadmill at their native steady state, and that cellular factors do not affect FtsZ’s fundamental treadmilling behavior. Finally, I investigate the role of the FtsZ binding proteins EzrA, SepF, and ZapA. These proteins have been previously described as regulators of both FtsZ’s dynamics and filament bundling. We find that in vivo, these proteins have no effect on FtsZ’s treadmilling behavior. Instead, they bundle FtsZ filaments together to form the Z ring. This Z ring condensation is required for cytokinesis, and enhances the recruitment of cell wall synthesis enzymes to the division site. Together, these data provide new insights into FtsZ’s filament properties in vivo, and the fundamental role that these properties play in dividing the bacterial cell.