FtsZ, a homolog of tubulin, is the major cytoskeletal protein in bacterial cell division. It assembles into protofilaments (pfs) that are~30 subunits (120 nm) long. In vivo these pfs are further assembled into a Z ring, which encircles the cell at mid-point, and eventually constricts to divide the cell. We have recently shown that FtsZ is extremely dynamic-turnover with a half time of 8 seconds both in vivo and in vitro. We propose here 4 new projects to explore assembly dynamics and the mechanism of division. (1) We will use TIRF microscopy to image single FtsZ pfs and follow growth and shrinking. We will first study pfs of M. tuberculosis FtsZ, which form pfs that are~5 um long, easily visible in the light microscope. Once we have developed the technology we will apply it to the much more challenging FtsZ of E. coli, whose pfs are shorter than the resolution of the light microscope. We expect an assembly mechanism based on dynamic instability, and the results with FtsZ should shed light on the mechanism of the GTP cap in microtubules. (2) We will attempt to reconstitute the FtsZ division machine in mitochondria. Mitochondria should be an ideal vesicle for this because they are the right size and shape, and they originally used FtsZ for division. We will co-express FtsZ and FtsA in mitochondria of CHO cells and yeast, and expect that these should be sufficient to assemble a Z ring. We will test the hypothesis that FtsZ is sufficient to develop the constriction force. (3) We will apply the fluorescence techniques that we have recently developed for E. coli FtsZ to M. tuberculosis, whose FtsZ pfs are structurally very different. We will obtain a complete characterization of initial assembly kinetics and turnover in vitro and in vivo, to compare with those of E. coli. (4) We have initiated studies of the newly discovered bacterial tubulins, BtubA and BtubB, and characterized their assembly into pf pairs. We will develop solution fluorescence assays similar to those used for FtsZ, to determine the assembly dynamics of BtubA/B protofilaments. The pf pairs assembled from BtubA/B will be ideal subjects for single molecule TIRF microscopy. Again we believe the mechanism will be related to microtubule dynamic instability. (5) We will pursue our quest to understand how the single-stranded FtsZ pfs can show cooperative assembly. We will use high concentrations of the independently folding N-and C-terminal domains, to determine how they poison pf assembly and dynamics. We will also attempt to re-create the dimer nucleus from these domains.