EXCEED THE SPACE PROVIDED. The long term objectives of this research remain unchanged. The goals are to determine the molecular mechanisms involved in bacterial cell division and the underlying spatial and temporal regulatory mechanisms. Our efforts have focused on the FtsZ protein which assembles into a cytoskeletal ring that recruits other proteins to the division site. FtsZ is the ancestral homologue of eukaryotic tubulins and undergoes dynamic assembly during the cell cycle. It is the target of several endogenous inhibitors that participate in the regulation of cell division as part of the normal cell cycle or in response to DNA damage. Our recent work has shown that SulA and MinC are inhibitors of FtsZ assembly, however, they function quite differently. SulA is synthesized following DNA damage and sequesters FtsZ preventing it from assembling. In contrast, MinC is part of a sophisticated spatial regulatory system that oscillates between the poles of the cell to prevent FtsZ from assembling near the cell poles. In vitro, MinC can prevent FtsZ assembly and acts by destabilizing FtsZ filaments. In vivo MinC requires MinD to be a functional inhibitor. MinD activates MinC by recruiting it to the membrane and conferring upon it a high affinity for a septal component. Our studies have demonstrated that MinD binds to the membrane through a C-terminal amphipathic helix. We have also shown that MinE can displace MinC from a MinCD vesicle complex and stimulate the MinD ATPase releasing MinD from the vesicle. We have also shown that MinD can polymerize on vesicles causing tubulation. Our studies have also defined the sites on MinC involved in its binding to MinD and in its binding to the septum. In the present proposal we will use genetic and biochemical approaches to further define the interactions among the Min proteins and between the Min proteins and FtsZ that are necessary to regulate cell division. We will also use the knowledge gained from our study of the E. coil Min system to explore the Min system from B. subtilis and a Par protein.
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