In just the last few years, fluorescent light microscopy has shown that in bacteria, many proteins are dynamically regulated in both time and space, certain chromosomal loci are strictly positioned, and numerous cytoskeletal filaments exist. Thus instead of being simple """"""""bags"""""""" of enzymes, even prokaryotic cells have substantial internal organization that enables their life cycle. The molecular basis of this organization is still unclear, but two key functions of the bacterial cytoskeleton have been hypothesized: determination of cell shape and segregation of chromosomes during cell division. In support of these hypotheses, several shape-altering mutations have been found in proteins which form cytoskeletal filaments just inside the cytoplasmic membrane, including MreB, Mbl, CreS, and FtsZ. In addition, it is now clear that at least some bacterial plasmids are segregated by prokaryotic actin homologs that form filamentous mitotic machineries, and specific molecular models have been proposed. A growing body of evidence suggests that prokaryotic chromosomes are also actively segregated and positioned by protein filaments. Concurrent to these discoveries, electron cryotomography has emerged as a powerful new tool to visualize the three- dimensional structure of intact, small cells to """"""""molecular resolution"""""""" (~4-8 nm) in a life-like state. Capitalizing on the recent installation of a one-of-a-kind, state-of-the-art electron cryomicroscope at the California Institute of Technology, we have, for the first time just last year, visualized bacterial cytoskeletal filaments directly within intact cells. Here we propose to extend these results and test the hypotheses above by determining the structure of the bacterial cytoskeleton in several model species throughout their life cycles by electron cryotomography. We believe the most exciting result will be direct visualization of mitotic machineries involved in bacterial chromosome segregation. Because this work will exploit prototype new instrumentation, a significant component of the effort will be technology development. This will include optimizing and refining strategies for collecting dual-axis tilt-series of frozen-hydrated cells as well as development of software to optimally merge the images into a three-dimensional reconstruction. ? ?
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