Petra A. Levin - US grants

[unreadable] DESCRIPTION (provided by applicant): How cells determine when and where to divide remains one of the great mysteries of modern biology. Spatially, division is tightly regulated to ensure the accurate positioning of septa. Temporally, division is coordinated with DNA replication and chromosome segregation. From bacteria to yeast to humans, cell division is initiated by the formation of a ring of a cytoskeletal protein at the nascent division site. This ring establishes the location of the division septum and serves as a framework for assembly of the division apparatus. In bacteria this ring is composed of the essential tubulin-like GTPase FtsZ. This proposal focuses on the regulatory networks that govern FtsZ ring formation in the soil bacterium B. subtilis. The factors that establish the division site and couple FtsZ ring formation to the cell cycle remain unknown. Comprehending the spatial and temporal regulation of bacterial division thus requires the identification of the cellular and molecular mechanisms that stimulate FtsZ ring formation at midcell in response to cell cycle cues and inhibit FtsZ ring formation at all other sites. EzrA, a factor that helps restrict FtsZ ring formation to midcell, was identified through classical genetic screens. Preliminary biochemical data suggest that EzrA interacts directly with FtsZ to inhibit ring formation by destabilization of FtsZ polymers. This proposal has three major goals. One, to characterize the molecular mechanism by which EzrA prevents ectopic FtsZ ring formation. Two, to clone and to characterize the gene identified by wee2, a mutation that uncouples cell division from growth, leading to the formation of small cells, many less than 25% the size of wild type B. subtilis. Three, to extend genetic screens to identify additional factors that (i) promote FtsZ ring formation at midcell, (ii) couple FtsZ ring formation to the cell cycle, and (iii) inhibit FtsZ ring formation at inappropriate sites. As essential components of the bacterial cell division machinery, FtsZ and the factors governing its activity hold promise as potential targets for the development of new antibiotics. Furthermore, this work should illuminate not only bacterial cell division, but also aspects of cytokinesis fundamental to all organisms. Understanding the molecular mechanisms that normally control cell division will help identify why they fail during oncogenesis, leading to the aberrant divisions and rapid proliferation characteristic of cancer cells.

Research in the Levin laboratory seeks to understand how bacterial cells integrate external and internal signals to determine when and where to divide. The focus of this work is a protein called FtsZ that is found in most bacteria. When a cell is ready to divide, FtsZ forms a ring that establishes the location of the nascent septum. During division the FtsZ ring constricts, like a drawstring, ultimately splitting the cell in two. This project seeks to characterize the signals that tell FtsZ to form a ring initiating the process of cell division, and the guideposts that direct FtsZ to midcell ensuring daughter cells are of the appropriate size and shape. The impact of this work is two-fold. Mechanistically, the process of cell division is conserved across evolutionary boundaries. Thus, dissecting the regulatory networks that control division in a simple bacterium, should reveal general principles that coordinate cell division with growth and differentiation in all organisms. Moreover, because cell division is an essential process, FtsZ and the factors governing its activity hold promise as potential targets for antibiotic development. The educational component of this project is directed towards developing a sequence analysis module for students taking Microbiology at Washington University. In addition to reinforcing concepts covered in lecture, this module will provide the means to introduce a significant number of undergraduates (~65 to 70 per semester) to key aspects of basic research, an essential component of a well-rounded education in biology.

DESCRIPTION (provided by applicant): How cells determine when and where to divide is one of the great questions in modern biology. Spatially, division is tightly regulated to ensure th accurate positioning of septa. Temporally, division is coordinated with cell growth, DNA replication, and chromosome segregation to ensure that daughter cells are the appropriate size and have complete genomes. In organisms from humans to bacteria, cells initiate division by the formation of a cytoskeletal protein ring at the nascent division site. In bacteria this ring is composed of the essential tubulin-like GTPase FtsZ. Bacteria achieve precise control over division primarily through the concerted actions of factors that modulate FtsZ assembly dynamics. Comprehending the spatial and temporal regulation of bacterial division, thus, requires the identification and characterization of factors that modulate FtsZ assembly. While we have begun to understand the factors responsible for preventing FtsZ assembly at aberrant subcellular locations and for maintaining integrity of the FtsZ ring, much less is known about the mechanisms responsible for coordinating FtsZ ring formation with cell growth and the cell cycle. This proposal has three primary objectives: first, to dissect the nutrient-dependent mechanisms governing the activity of UgtP and OpgH, division inhibitors that contribute to growth rate-dependent increases in cell size in B. subtilis and E. coli respectively; second, to identify and characterize additional components of the regulatory circuit responsible for E. coli cell size homeostasis; and, third, to assess the contribution of FtsZ's unstructured C-terminal domain -- a primary site of interaction between FtsZ and its modulatory proteins -- to the assembly and integrity of the cytokinetic ring via an integrated approach employing genetics, biochemistry, and superresolution microscopy. This project should also help shed light upon questions of broader scientific importance. FtsZ and the factors governing its activity are essential components of the bacterial cell division machinery and are therefore attractive targets for the development of new antibiotics. Furthermore, comparative analysis of the factors responsible for the spatial and temporal control of cell division in E. coli and B. subtilis, two highly divergent model organisms, promises to reveal underlying aspects of cell cycle regulation fundamental to all domains of life. Finally, understanding the molecular mechanisms that normally control cell division should help identify why these mechanisms fail during oncogenesis, and lead to the aberrant divisions and rapid cell proliferation characteristic of cancer.

PROJECT SUMMARY The Project Summary is contained within the Research Strategy per PA-21-071 guidelines.