Structure and function of the prokaryotic cytoskeleton by electron cryotomography

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DESCRIPTION (provided by applicant): Bacteria are nearly ubiquitous, play vital roles in industry and the environment, and are important actors in both health and disease for humans and other organisms. Given their importance, it is surprising how much we still don't understand about basic bacterial cell biology. We still don't know, for instance, how bacteria generate and maintain their characteristic shapes, establish polarity, organize their genomes, segregate their chromosomes, divide, and in some cases move. In eukaryotes, all these tasks are performed by cytoskeletal filaments, but because previous imaging technologies failed to reveal analogous structures in bacteria, it was long thought that bacteria don't possess cytoskeletons. More recently, fluorescence microscopy has shown that bacteria have substantial internal order. In the first cycle of this grant, we used another young technology, electron cryotomography (ECT), to produce three- dimensional images of intact bacterial cells in a near-native state to "molecular" (~4-6 nm) resolution. Using ECT we directly visualized hundreds of cytoskeletal filaments in ~20 different bacterial species, proving that the bacterial cytoskeleton is in fact both general and complex. Here we propose to develop and apply new correlated light and electron microscopy techniques to identify these filaments generally. This should allow us to resolve key discrepancies between the existing light and electron microscopical results and generate much-needed insight into the structures and functions of the bacterial cytoskeleton. We also propose work to improve EM image quality generally and expand the number of filaments being studied.