Molecular architecture of the bacterial actin cytoskeleton. (360G-Wellcome-095514_Z_11_Z)

1) What is the molecular arrangement of the MreB filaments of the bacterial cytoskeleton in the cell? Bacteria possess both actin- and tubulin-like filamentous proteins that function in cell shape determination, cell division and DNA dynamics and that constitute the bacterial cytoskeleton (Figure A) (1). It is currently not known what filament structures are formed at the molecular level in cells, besides the longitudinal protofilament contacts that are conserved from prokaryotic to eukaryotic actin and tubulin (Figure F) (2, 3). Here, we aim to elucidate the exact in vivo molecular structures of the actin-like MreB filaments and their locations in E. coli bacterial cells. We will use a combination of X-ray crystallography and electron cryomicroscopy of molecules and filaments, and, importantly, electron cryotomography (4) of whole cells to achieve this goal. We will develop new in vivo labelling technology and new holders of bacterial cells. Furthermore, in vitro biochemistry and in vivo mutants will link our data together on the functional level. We aim at a molecular description of which filaments are formed where in the cell by what mechanism. 2) How do the MreB filaments of the bacterial cytoskeleton drive their biological function of cell shape maintenance? MreB drives cell shape determination in nearly all non-spherical bacteria (5) and we will investigate how the filaments of MreB accomplish this task. This will involve a thorough investigation of the interaction of MreB with RodZ, MreC, MreD and RodA and the downstream proteins in the periplasm, most notably penicillin-binding proteins (PBPs) that make the cell wall (6, 7). This will involve integration of the cellular tomography data from aim 1) with biochemical and X-ray crystallographic data of the molecular complexes, providing a cell-wide picture of the process. This work will be complemented with assays to investigate interactions and mutants in vivo, in order to test any hypothesis derived from i n vitro data. The final goal is a precise, molecular description of the process that transmits the genetically encoded information of the shape of the cell from the inside to the outside, where the cell wall is made. 3) Combining results from both aims 1) and 2), reconstitution of the filament system in liposomes will be attempted, eventually including downstream proteins so that cell wall synthesis can be initiated on the outside.