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. The BDLP GTPase domain dimerizes and forms the tube surface, the GTPase effector domain (GED) mediates self-assembly, and the paddle region contacts the lipids and promotes curvature. Association of BDLP with GMPPNP and lipid induces radical, large-scale conformational changes affecting polymerization. Nucleotide hydrolysis seems therefore to be coupled to polymer disassembly and dissociation from lipid, rather than membrane restructuring. Observed structural similarities with rat dynamin 1 suggest that our results have broad implication for other dynamin family members.

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. Here we report the structure and function of proteins from a distinct family related to tubulin and FtsZ, named CetZ, which co-exists with FtsZ in many archaea. CetZ X-ray crystal structures showed the FtsZ/tubulin superfamily fold, and one crystal form contained sheets of protofilaments, suggesting a structural role. However, inactivation of CetZ proteins in Haloferax volcanii did not affect cell division. Instead, CetZ1 was required for differentiation of the irregular plate-shaped cells into a rod-shaped cell type that was essential for normal swimming motility. CetZ1 formed dynamic cytoskeletal structures in vivo, relating to its capacity to remodel the cell envelope and direct rod formation. CetZ2 was also implicated in H. volcanii cell shape control. Our findings expand the known roles of the FtsZ/tubulin superfamily to include archaeal cell shape dynamics, suggesting that a cytoskeletal role might predate eukaryotic cell evolution, and they support the premise that a major function of the microbial rod shape is to facilitate swimming.

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. Their activities are modulated by a wide range of accessory proteins but these do not include homologues of the motor proteins that supplement filament dynamics to aid eukaryotic cell motility. Numerous other filamentous proteins, some related to eukaryotic IF-proteins/lamins and dynamins etc, seem to perform structural roles similar to those in eukaryotes.

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. The activities of assembly inhibitors are discussed in relation to different nucleotide states of tubulin family protein structures.

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. The Bacillus thuringiensis plasmid pBToxis relies on a filament of the tubulin/FtsZ-like protein TubZ for its segregation. By combining crystallography and electron microscopy, we have determined the structure of this filament. We explain how GTP hydrolysis weakens the subunit-subunit contact and also shed light on the partitioning of the plasmid-adaptor complex. The double helical superstructure of TubZ filaments is unusual for tubulin-like proteins. Filaments of ParM, the actin-like partitioning protein, are also double helical. We suggest that convergent evolution shapes these different types of cytomotive filaments toward a general mechanism for plasmid separation.

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. The dynein-activator complex dynactin has an essential role in axonal transport and mutations in its gene are associated with lower motor neuron disease. We show here for the first time that the N-terminal projection domain of tau binds to the C-terminus of the p150 subunit of the dynactin complex. Tau and dynactin show extensive colocalization, and the attachment of the dynactin complex to microtubules is enhanced by tau. Mutations of a conserved arginine residue in the N-terminus of tau, found in patients with FTDP-17, affect its binding to dynactin, which is abnormally distributed in the retinal ganglion cell axons of transgenic mice expressing human tau with a mutation in the microtubule-binding domain. These findings, which suggest a direct involvement of tau in axonal transport, have implications for understanding the pathogenesis of tauopathies.

Structural/functional homology between the bacterial and eukaryotic cytoskeletons.

Structural proteins are now known to be as necessary for controlling cell division and cell shape in prokaryotes as they are in eukaryotes. Bacterial ParM and MreB not only have atomic structures that resemble eukaryotic actin and form similar filaments, but they are also equivalent in function: the assembly of ParM drives intracellular motility and MreB maintains the shape of the cell. FtsZ resembles tubulin in structure and in its dynamic assembly, and is similarly controlled by accessory proteins. Bacterial MinD and eukaryotic dynamin appear to have similar functions in membrane control. In dividing eukaryotic organelles of bacterial origin, bacterial and eukaryotic proteins work together.

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.

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. Some bacterial plasmids also use ParM, another actin-like protein. Higher-order filament structures vary from simple to complex depending on the cellular application. Equally, filament-associated proteins vary greatly between species and it is not possible currently to trace their evolution from prokaryotes to eukaryotes. This lack of similarity except in the three-dimensional structures and longitudinal interactions between the filament subunits hints that the most basic cellular function of the filaments is to act as linear motors driven by assembly dynamics and/or bending and hence we term these filament systems 'cytomotive'. The principle of cytomotive filaments seems to have been invented independently for actin- and tubulin-like proteins. Prokaryotes appear to have a third class of cytomotive filaments, typically associated with surfaces such as membranes or DNA: Walker A cytoskeletal ATPases (WACA). A possible evolutionary relationship of WACAs with eukaryotic septins is discussed.

Aaron Klug and the revolution in biomolecular structure determination.

Aaron Klug's group was one of the first to use a combination of X-ray diffraction and electron microscopy to study the structures of macromolecules. He helped to provide the intellectual framework for understanding the self-assembly of regular viruses and developed methods for analyzing their three-dimensional structures from electron microscope images, as well as the structures of helical polymers. He and his coworkers established the basic features of chromatin organization, including the structure of the repeating units (nucleosomes) and how they are stacked together. He studied a variety of molecules that interact with DNA or RNA, including disks of tobacco mosaic virus protein, a tRNA and a ribozyme, and also discovered the zinc-finger motif in nucleic acid-binding proteins. Thus, he has played a major part in developing the ideas and techniques that established structural molecular biology as an exciting new science during the second half of the twentieth century.

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. Details of the interaction are studied by docking the individual crystal structures of the kinesin motor domains and tubulin heterodimer into the 3D maps of the complex. To study the structural changes during ATP hydrolysis, structures of the complexes in the presence and absence of different nucleotides are compared.

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. Gold labeling can be used to mark the positions of certain regions of protein sequence.

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. However, a multitude of associated proteins are able to fine-tune these dynamics so that microtubules are assembled and disassembled where and when they are required by the cell. We review the recent progress that has been made in obtaining a glimpse of the structural interactions involved.

Discodermolide interferes with the binding of tau protein to microtubules.

We investigated whether discodermolide, a novel antimitotic agent, affects the binding to microtubules of tau protein repeat motifs. Like taxol, the new drug reduces the proportion of tau that pellets with microtubules. Despite their differing structures, discodermolide, taxol and tau repeats all bind to a site on beta-tubulin that lies within the microtubule lumen and is crucial in controlling microtubule assembly. Low concentrations of tau still bind strongly to the outer surfaces of preformed microtubules when the acidic C-terminal regions of at least six tubulin dimers are available for interaction with each tau molecule; otherwise binding is very weak.

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. Here, using X-ray crystallography, electron microscopy and in vivo analyses, we show that MinD activates MinC by forming a new class of alternating copolymeric filaments that show similarity to eukaryotic septin filaments. A non-polymerizing mutation in MinD causes aberrant cell division in Escherichia coli. MinCD copolymers bind to membrane, interact with FtsZ and are disassembled by MinE. Imaging a functional msfGFP-MinC fusion protein in MinE-deleted cells reveals filamentous structures. EM imaging of our reconstitution of the MinCD-FtsZ interaction on liposome surfaces reveals a plausible mechanism for regulation of FtsZ ring assembly by MinCD copolymers.

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. Electron cryo-microscopy of ΦKZ TubZ filaments polymerised in vitro implies a long-pitch helical arrangement for the constituent protofilaments. Intriguingly, this feature is shared by the other known subgroup of bacteriophage tubulin/FtsZ-like proteins from Clostridium species, which are thought to be involved in partitioning the genomes of bacteriophages adopting a pseudo-lysogenic life cycle.

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. In the case of microtubules and FtsZ filaments, bending is an essential part of their mechanisms. Both families assemble polar linear protofilaments, with highly conserved interfaces between successive subunits; the bonding at these longitudinal interfaces is nucleotide dependent. Better understanding of the mechanisms by which nucleotide hydrolysis affects the bonding between subunits in filaments, and other structural changes related to the nucleotide hydrolysis cycles, has emerged from recent X-ray crystallographic and electron microscopic structures, showing eukaryotic or prokaryotic protofilaments in various states. Detailed comparisons of the structures of related proteins from eubacteria, archaea, and eukaryotes are helping to illuminate the course of evolution.

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.