9513521 Margolin A fundamental problem in biology is how a single cell divides to become two. In bacteria, cytokinesis is a differentiation event: normal longitudinal growth of the cell wall changes to inward growth at the division site, culminating in the separation of daughter cells. This localized wall ingrowth implies that bacterial cells have a cytoplasmic organization and a mechanism for directing proteins involved in cytokinesis to the division site. The FtsZ protein has properties expected of a cytoskeletal element: it is probably present in all prokaryotes; it is related to the eukaryotic cytoskeletal protein tubulin; purified FtsZ forms polymers in the present of GTP; it probably functions as part of a complex with other cell division proteins; and it forms a ring-like polymer at the division site during the initial stages of cytokinesis and it is present at the leading edge of the invaginating wall. The goal of this project is to combine genetic and whole-cell approaches to determine how prokaryotic cell cycle and "cytoskeletal" proteins are targeted to specific cellular sites. Preliminary results indicate that localization of FtsZ in living E. coli can be tracked at high resolution by tagging it with a small fluorescent reporter protein, GFP (green fluorescent protein). The GFP tag will be used to determine localization and structural organization of cell cycle proteins in living bacteria. Specifically, it will be used to define cell cycle protein domains important for cellular targeting, to visualize cytoskeletal structures and dynamics during the cell cycle, to correlate structure and function among FtsZ proteins from diverse species, and to determine whether two other essential cell division proteins, FtsA and FtsQ, localize to the division site. Preliminary results show that the fusion product of FtsZ with GFP at the C-terminus forms the expected ring structures at the division site in living cells, indicating that the GFP tag does not interfere with FtsZ assembly o r localization. Specific FtsZ mutants (deletions, point mutants, and interspecies chimeras) will be constructed with GFP reporter tags and assayed in vivo in order to identify domains important for FtsZ localization and polymerization. This type of analysis will be extended to FtsA and FtsQ, whose function an cellular localization are not yet known. Localization of FtsQ-GFP and FtsA-GFP will be determined in wild type and various cell division mutant cells in an effort to define the sequence of events and participants in the cell division pathway. These studies should make a major contribution to our understanding of cell division mechanisms, protein targeting, and subcellular organization in prokaryotes. The longer-range goal of the research is to understand the mechanism whereby cell cycle proteins become localized to specific regions within the prokaryotic cell. The use of GFP reporter protein tags to track protein spatial dynamics in prokaryotes has the advantage that a large number of genetic constructions can be assayed quickly, easily, and with high sensitivity, without the need for antibodies, fixation of cells, or electron microscopy. The knowledge acquired from these studies should contribute to the basic biology of prokaryotic cell division, as well to further applications of fluorescent proteins in visualizing fundamental processes in living cells. ***
