Bacterial cell division requires formation of the cytokinetic cell division septum at the mid-cell position, a process that is determined by three Min proteins; MinC, MinD and MinE. Regulation of cell division by Min proteins occurs via a multi-step process involving interactions between various Min proteins, as well as the membrane. In this cycle, ATP-bound MinD binds to the membrane surface where it can recruit MinC to inhibit formation of the cell division septum. MinE binding to this complex displaces MinC and stimulates ATP hydrolysis, leading to the dissociation of MinD from the membrane. These interactions give rise to a dynamic pattern of Min protein localization that appears to involve a polymeric state that is designed to create a zone that is permissive to cell division at the mid-point of the cell. The interaction between MinD and the membrane is a critical aspect of this cycle, yet the role of the lipid bilayer in MinD activation, localization and polymerization is not well understood. To probe the role of membrane charge and fluidity on MinD activation and polymerization, we developed a kinetic assay of MinE-stimulated MinD ATPase activity. We found that membrane charge is essential for MinD activation and that differences in membrane fluidity give rise to changes in its activity. Moreover, a burst phase was also observed during the first few minutes of reaction, but only on the most fluid anionic lipid tested. To help determine if the observed membrane-dependent changes in MinD activity are linked to any changes in MinD polymer structure, we have begun to develop a method to identify surface exposed regions of MinD through a combination of covalent labeling and mass spectrometry. Optimization of various steps for the assay has been done, and the assay can be applied to the future characterization of MinD polymer structure. Results from this assay, in combination with those from the kinetic measurements described here, will help to improve understanding about how membrane properties modulate MinD ATPase activity, and how this can influence the Min protein oscillation that is required to ensure normal bacterial cell division.

Bacterial cell division requires formation of the cytokinetic cell division septum at the mid-cell position, a process that is determined by three Min proteins; MinC, MinD and MinE. Regulation of cell division by Min proteins occurs via a multi-step process involving interactions between various Min proteins, as well as the membrane. In this cycle, ATP-bound MinD binds to the membrane surface where it can recruit MinC to inhibit formation of the cell division septum. MinE binding to this complex displaces MinC and stimulates ATP hydrolysis, leading to the dissociation of MinD from the membrane. These interactions give rise to a dynamic pattern of Min protein localization that appears to involve a polymeric state that is designed to create a zone that is permissive to cell division at the mid-point of the cell. The interaction between MinD and the membrane is a critical aspect of this cycle, yet the role of the lipid bilayer in MinD activation, localization and polymerization is not well understood. To probe the role of membrane charge and fluidity on MinD activation and polymerization, we developed a kinetic assay of MinE-stimulated MinD ATPase activity. We found that membrane charge is essential for MinD activation and that differences in membrane fluidity give rise to changes in its activity. Moreover, a burst phase was also observed during the first few minutes of reaction, but only on the most fluid anionic lipid tested. To help determine if the observed membrane-dependent changes in MinD activity are linked to any changes in MinD polymer structure, we have begun to develop a method to identify surface exposed regions of MinD through a combination of covalent labeling and mass spectrometry. Optimization of various steps for the assay has been done, and the assay can be applied to the future characterization of MinD polymer structure. Results from this assay, in combination with those from the kinetic measurements described here, will help to improve understanding about how membrane properties modulate MinD ATPase activity, and how this can influence the Min protein oscillation that is required to ensure normal bacterial cell division.

Bacterial cell division requires formation of the cytokinetic cell division septum at the mid-cell position, a process that is determined by three Min proteins; MinC, MinD and MinE. Regulation of cell division by Min proteins occurs via a multi-step process involving interactions between various Min proteins, as well as the membrane. In this cycle, ATP-bound MinD binds to the membrane surface where it can recruit MinC to inhibit formation of the cell division septum. MinE binding to this complex displaces MinC and stimulates ATP hydrolysis, leading to the dissociation of MinD from the membrane. These interactions give rise to a dynamic pattern of Min protein localization that appears to involve a polymeric state that is designed to create a zone that is permissive to cell division at the mid-point of the cell. The interaction between MinD and the membrane is a critical aspect of this cycle, yet the role of the lipid bilayer in MinD activation, localization and polymerization is not well understood. To probe the role of membrane charge and fluidity on MinD activation and polymerization, we developed a kinetic assay of MinE-stimulated MinD ATPase activity. We found that membrane charge is essential for MinD activation and that differences in membrane fluidity give rise to changes in its activity. Moreover, a burst phase was also observed during the first few minutes of reaction, but only on the most fluid anionic lipid tested. To help determine if the observed membrane-dependent changes in MinD activity are linked to any changes in MinD polymer structure, we have begun to develop a method to identify surface exposed regions of MinD through a combination of covalent labeling and mass spectrometry. Optimization of various steps for the assay has been done, and the assay can be applied to the future characterization of MinD polymer structure. Results from this assay, in combination with those from the kinetic measurements described here, will help to improve understanding about how membrane properties modulate MinD ATPase activity, and how this can influence the Min protein oscillation that is required to ensure normal bacterial cell division.