Actin plays crucial parts in cell motility through a dynamic process driven by polymerization and depolymerization, that is, the globular (G) to fibrous (F) actin transition. Although our knowledge about the actin-based cellular functions and the molecules that regulate the G- to F-actin transition is growing, the structural aspects of the transition remain enigmatic. We created a model of F-actin using X-ray fibre diffraction intensities obtained from well oriented sols of rabbit skeletal muscle F-actin to 3.3 A in the radial direction and 5.6 A along the equator. Here we show that the G- to F-actin conformational transition is a simple relative rotation of the two major domains by about 20 degrees. As a result of the domain rotation, the actin molecule in the filament is flat. The flat form is essential for the formation of stable, helical F-actin. Our F-actin structure model provides the basis for understanding actin polymerization as well as its molecular interactions with actin-binding proteins.

ParM is a prokaryotic actin homologue, which ensures even plasmid segregation before bacterial cell division. In vivo, ParM forms a labile filament bundle that is reminiscent of the more complex spindle formed by microtubules partitioning chromosomes in eukaryotic cells. However, little is known about the underlying structural mechanism of DNA segregation by ParM filaments and the accompanying dynamic instability. Our biochemical, TIRF microscopy and high-pressure SAX observations indicate that polymerization and disintegration of ParM filaments is driven by GTP rather than ATP and that ParM acts as a GTP-driven molecular switch similar to a G protein. Image analysis of electron micrographs reveals that the ParM filament is a left-handed helix, opposed to the righthanded actin polymer. Nevertheless, the intersubunit contacts are similar to those of actin. Our atomic model of the ParM-GMPPNP filament, which also fits well to X-ray fibre diffraction patterns from oriented gels, can explain why after nucleotide release, large conformational changes of the protomer lead to a breakage of intra-and interstrand interactions, and thus to the observed disintegration of the ParM filament after DNA segregation.

In vivo cell division protein FtsZ from E. coli forms rings and spirals which have only been observed by low resolution light microscopy. We show that these suprastructures are likely formed by molecular crowding which is a predominant factor in prokaryotic cells and enhances the weak lateral bonds between proto-filaments. Although FtsZ assembles into single proto-filaments in dilute aqueous buffer, with crowding agents above a critical concentration, it forms polymorphic supramolecular structures including rings and toroids (with multiple protofilaments) about 200 nm in diameter, similar in appearance to DNA toroids, and helices with pitches of several hundred nm as well as long, linear bundles. Helices resemble those observed in vivo, whereas the rings and toroids may represent a novel energy minimized state of FtsZ, at a later stage of Z-ring constriction. We shed light on the molecular arrangement of FtsZ filaments within these suprastructures using high resolution electron microscopy.

In vivo fluorescence microscopy studies of bacterial cells have shown that the bacterial shape-determining protein and actin homolog, MreB, forms cable-like structures that spiral around the periphery of the cell. The molecular structure of these cables has yet to be established. Here we show by electron microscopy that Thermatoga maritime MreB forms complex, several m long multilayered sheets consisting of diagonally interwoven filaments in the presence of either ATP or GTP. This architecture, in agreement with recent rheological measurements on MreB cables, may have superior mechanical properties and could be an important feature for maintaining bacterial cell shape. MreB polymers within the sheets appear to be single-stranded helical filaments rather than the linear protofilaments found in the MreB crystal structure. Sheet assembly occurs over a wide range of pH, ionic strength, and temperature. Polymerization kinetics are consistent with a cooperative assembly mechanism requiring only two steps: monomer activation followed by elongation. Steady-state TIRF microscopy studies of MreB suggest filament treadmilling while high pressure small angle x-ray scattering measurements indicate that the stability of MreB polymers is similar to that of F-actin filaments. In the presence of ADP or GDP, long, thin cables formed in which MreB was arranged in parallel as linear protofilaments. This suggests that the bacterial cell may exploit various nucleotides to generate different filament structures within cables for specific MreB-based functions.Despite usually being constrained by a cell wall, bacterial shapes are highly diverse, reflecting the large phylogenetic range. For example Escherichia coli, Bacillus subtilis, and Thermatoga maritime are straight rods, Vibrio cholera is a curved rod, Borrelia burgdorferi forms flat waves, whereas Spiroplasma species are helical. One of the main cytoskeletal proteins involved in determining the shapes of bacteria is thought to be MreB an actin homolog whose atomic structure is very similar to G-actin, despite the low sequence homology (1). It can assemble into polymers both in vitro (1) and in vivo (2). Several studies suggest that MreB plays roles in chromosome segregation (3), polar localization of proteins (4), and maintenance of cell shape and resistance to external mechanical stresses (2). Peptidoglycan cell wall synthesis has been linked to the role of the MreB homolog MbI in B. subtilis (5); however, mechanisms by which MreB may provide mechanical support either directly to the cell or indirectly by affecting peptidoglycan wall integrity remain unclear.In vivo studies of MreB have mainly been limited to visualization under the fluorescence microscope (2, 5). At the low resolution of this technique (ϳ0.2 m), MreB was seen to form cable-like structures, which spiral around the periphery of the cell in B. subtilis, presumably just underneath the cytoplasmic membrane. By electron microscopy (EM), 2 MreB has been observed in vitro to form straight or curved sheets and bundles (1...

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The actin filament is quite dynamic in the cell. To determine the relationship between the structure and the dynamic properties of the actin filament, experiments using actin mutants are indispensable. We focused on Gln 137 to understand the relationships between two activities: the conformational changes relevant to the G-to F-actin transition and the activation of actin ATPase upon actin polymerization. To elucidate the function of Gln 137 in these activities, we characterized Gln 137 mutants of human cardiac muscle ␣-actin. Although all of the single mutants, Q137E, Q137K, Q137P, and Q137A, as well as the wild type were expressed by a baculovirus-based system, only Q137A and the wild type were purified to high homogeneity. The CD spectrum of Q137A was similar to that of the wild type, and Q137A showed the typical morphology of negatively stained Q137A F-actin images. However, Q137A had an extremely low critical concentration for polymerization. Furthermore, we found that Q137A polymerized 4-fold faster, cleaved the ␥-phosphate group of bound ATP 4-fold slower, and depolymerized 5-fold slower, as compared with the wild-type rates. These results suggest that Gln 137 plays dual roles in actin polymerization, in both the conformational transition of the actin molecule and the mechanism of ATP hydrolysis.

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