Overview of the Diverse Roles of Bacterial and Archaeal Cytoskeletons.

As discovered over the past 25 years, the cytoskeletons of bacteria and archaea are complex systems of proteins whose central components are dynamic cytomotive filaments. They perform roles in cell division, DNA partitioning, cell shape determination and the organisation of intracellular components. The protofilament structures and polymerisation activities of various actin-like, tubulin-like and ESCRT-like proteins of prokaryotes closely resemble their eukaryotic counterparts but show greater diversity.

CetZ tubulin-like proteins control archaeal cell shape.

Tubulin is a major component of the eukaryotic cytoskeleton, controlling cell shape, structure and dynamics, whereas its bacterial homologue FtsZ establishes the cytokinetic ring that constricts during cell division. How such different roles of tubulin and FtsZ evolved is unknown. Studying Archaea may provide clues as these organisms share characteristics with Eukarya and Bacteria.

MinCD cell division proteins form alternating copolymeric cytomotive filaments.

During bacterial cell division, filaments of the tubulin-like protein FtsZ assemble at midcell to form the cytokinetic Z-ring. Its positioning is regulated by the oscillation of MinCDE proteins. MinC is activated by MinD through an unknown mechanism and prevents Z-ring assembly anywhere but midcell.

Structure of the tubulin/FtsZ-like protein TubZ from Pseudomonas bacteriophage ΦKZ.

Pseudomonas ΦKZ-like bacteriophages encode a group of related tubulin/FtsZ-like proteins believed to be essential for the correct centring of replicated bacteriophage virions within the bacterial host. In this study, we present crystal structures of the tubulin/FtsZ-like protein TubZ from Pseudomonas bacteriophage ΦKZ in both the monomeric and protofilament states, revealing that ΦKZ TubZ undergoes structural changes required to polymerise, forming a canonical tubulin/FtsZ-like protofilament. Combining our structures with previous work, we propose a polymerisation-depolymerisation cycle for the Pseudomonas bacteriophage subgroup of tubulin/FtsZ-like proteins.

New insights into the mechanisms of cytomotive actin and tubulin filaments.

Dynamic, self-organizing filaments are responsible for long-range order in the cytoplasm of almost all cells. Actin-like and tubulin-like filaments evolved independently in prokaryotes but have converged in terms of many important properties. They grow, shrink, and move directionally within cells, using energy and information provided by nucleotide hydrolysis.

What tubulin drugs tell us about microtubule structure and dynamics.

A wide range of small molecules, including alkaloids, macrolides and peptides, bind to tubulin and disturb microtubule assembly dynamics. Some agents inhibit assembly, others inhibit disassembly. The binding sites of drugs that stabilize microtubules are discussed in relation to the properties of microtubule associated proteins.

Filament structure of bacterial tubulin homologue TubZ.

Low copy number plasmids often depend on accurate partitioning systems for their continued survival. Generally, such systems consist of a centromere-like region of DNA, a DNA-binding adaptor, and a polymerizing cytomotive filament. Together these components drive newly replicated plasmids to opposite ends of the dividing cell.

Articulated tubes.

High quality images of microtubules with different numbers of protofilaments, and hence substantially different curvatures, have been reconstructed from electron microscopy (EM) data (Sui and Downing, 2010). The data show how three versatile loops that mediate lateral interactions allow microtubules to be strong without being brittle.

Structure of a bacterial dynamin-like protein lipid tube provides a mechanism for assembly and membrane curving.

Proteins of the dynamin superfamily mediate membrane fission, fusion, and restructuring events by polymerizing upon lipid bilayers and forcing regions of high curvature. In this work, we show the electron cryomicroscopy reconstruction of a bacterial dynamin-like protein (BDLP) helical filament decorating a lipid tube at approximately 11 A resolution. We fitted the BDLP crystal structure and produced a molecular model for the entire filament.

Electron microscopy of helical filaments: rediscovering buried treasures in negative stain.

Although negative stain electron microscopy is a wonderfully simple way of directly visualizing protein complexes and other biological macromolecules, the images are not really comparable to those of objects seen in everyday life. The failure to appreciate this has recently led to an incorrect interpretation of RecA-family filament structures.

Mal3, the Schizosaccharomyces pombe homolog of EB1, changes the microtubule lattice.

In vitro studies of pure tubulin have suggested that tubulin heterodimers in cells assemble into B-lattice microtubules, where the 8-nm dimers in adjacent protofilaments are staggered by 0.9 nm. This arrangement requires the tube to close by forming a seam with an A-lattice, in which the protofilaments are staggered by 4.

Evolution of cytomotive filaments: the cytoskeleton from prokaryotes to eukaryotes.

The basic features of the active filaments that use nucleotide hydrolysis to organise the cytoplasm are remarkably similar in the majority of all cells and are either actin-like or tubulin-like. Nearly all prokaryotic cells contain at least one form of FtsZ, the prokaryotic homologue of tubulin and some bacterial plasmids use tubulin-like TubZ for segregation. The other main family of active filaments, assembled from actin-like proteins, occurs in a wide range of bacterial species as well as in all eukaryotes.

The tektin family of microtubule-stabilizing proteins.

Tektins are insoluble alpha-helical proteins essential for the construction of cilia and flagella and are found throughout the eukaryotes apart from higher plants. Being almost universal but still fairly free to mutate, their coding sequences have proved useful for estimating the evolutionary relationships between closely related species. Their protein molecular structure, typically consisting of four coiled-coil rod segments connected by linkers, resembles that of intermediate filament (IF) proteins and lamins.

Studying the structure of microtubules by electron microscopy.

Although the structures of individual proteins and moderately sized complexes of proteins may be investigated by X-ray crystallography, the interaction between a long polymer, such as a microtubule, and other protein molecules, such as the motor domain of kinesin, need to be studied by electron microscopy. We have used electron cryo-microscopy and image analysis to study the structures of microtubules with and without bound kinesin motor domains and the changes that take place when the motor domains are in different nucleotide states. Among the microtubules that assemble from pure tubulin, we select a minor subpopulation that has perfect helical symmetry, which are the best for three-dimensional reconstruction.

A cool look at the structural changes in kinesin motor domains.

Recently, several 3D images of kinesin-family motor domains interacting with microtubules have been obtained by analysis of electron microscope images of frozen hydrated complexes at much higher resolutions (9-12 A) than in previous reports (15-30 A). The high-resolution maps show a complex interaction interface between kinesin and tubulin, in which kinesin's switch II helix alpha4 is a central feature. Differences due to the presence of ADP, as compared with ATP analogues, support previously determined crystal structures of kinesins alone in suggesting that alpha4 is part of a pathway linking the nucleotide-binding site and the neck that connects to cargo.

High-resolution structural analysis of the kinesin-microtubule complex by electron cryo-microscopy.

To understand the interaction of kinesin and microtubules, it is necessary to study the three-dimensional (3D) structures of the kinesin-microtubule complex at a high enough resolution to identify structural components such as alpha-helices and beta-sheets. Electron cryo-microscopy combined with computer image analysis is the most common method to study such complexes that cannot be crystallized. By selecting microtubules that have a helical symmetry, 3D structures of the complex can be calculated using the helical 3D reconstruction method.

Interaction of tau protein with the dynactin complex.

Tau is an axonal microtubule-associated protein involved in microtubule assembly and stabilization. Mutations in Tau cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), and tau aggregates are present in Alzheimer's disease and other tauopathies. The mechanisms leading from tau dysfunction to neurodegeneration are still debated.

Large conformational changes in a kinesin motor catalyzed by interaction with microtubules.

Kinesin motor proteins release nucleotide upon interaction with microtubules (MTs), then bind and hydrolyze ATP to move along the MT. Although crystal structures of kinesin motors bound to nucleotides have been solved, nucleotide-free structures have not. Here, using cryomicroscopy and three-dimensional (3D) reconstruction, we report the structure of MTs decorated with a Kinesin-14 motor, Kar3, in the nucleotide-free state, as well as with ADP and AMPPNP, with resolution sufficient to show alpha helices.

Microtubules and maps.

Microtubules are very dynamic polymers whose assembly and disassembly is determined by whether their heterodimeric tubulin subunits are in a straight or curved conformation. Curvature is introduced by bending at the interfaces between monomers. Assembly and disassembly are primarily controlled by the hydrolysis of guanosine triphosphate (GTP) in a site that is completed by the association of two heterodimers.

Microtubule structure and its stabilisation.

Microtubules are designed to be dynamically unstable. GTP hydrolysis converts an initially stable polymeric structure into an unstable one in which strain at the interfaces between longitudinal neighbours in the helical lattice of subunits is balanced by lateral interactions. However, stability can be modulated by a variety of factors, including associated proteins and a variety of drug molecules.