ABSTRACT
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.
Subject(s)
Archaea/cytology , Bacteria/cytology , Cytoskeleton/metabolism , Archaeal Proteins/metabolism , Bacterial Proteins/metabolismABSTRACT
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.
Subject(s)
Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , Cell Shape , Haloferax volcanii/cytology , Haloferax volcanii/metabolism , Tubulin/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Cell Division , Cell Membrane/metabolism , Crystallography, X-Ray , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/metabolism , Models, Molecular , Molecular Sequence Data , Movement , Tubulin/chemistryABSTRACT
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.
Subject(s)
Adenosine Triphosphatases/chemistry , Bacterial Proteins/chemistry , Cell Cycle Proteins/chemistry , Cytoskeletal Proteins/chemistry , Cytoskeleton/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/chemistry , Membrane Proteins/chemistry , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Division , Cell Membrane/chemistry , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Crystallography, X-Ray , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Cytoskeleton/metabolism , Cytoskeleton/ultrastructure , Escherichia coli/metabolism , Escherichia coli/ultrastructure , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Gene Expression , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Liposomes/chemistry , Liposomes/ultrastructure , Membrane Proteins/genetics , Membrane Proteins/metabolism , Microscopy, Electron , Models, Molecular , Mutation , Polymerization , Protein Binding , Protein Transport , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
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.
Subject(s)
Pseudomonas Phages/chemistry , Viral Proteins/chemistry , Viral Proteins/metabolism , Amino Acid Sequence , Cryoelectron Microscopy , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Protein Multimerization , Sequence AlignmentABSTRACT
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.
Subject(s)
Actins/metabolism , Biological Evolution , Cytoskeleton/metabolism , Microtubules/metabolism , Tubulin/metabolism , Amino Acid Sequence , Animals , Humans , Models, Molecular , Molecular Sequence Data , Sequence Homology, Amino AcidABSTRACT
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.
Subject(s)
Microtubules/drug effects , Microtubules/ultrastructure , Tubulin Modulators/pharmacology , Tubulin/chemistry , Tubulin/metabolism , Binding Sites , Humans , Microtubules/chemistry , Microtubules/metabolism , Tubulin/ultrastructure , Tubulin Modulators/chemistry , Tubulin Modulators/metabolismABSTRACT
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.
Subject(s)
Bacillus thuringiensis/chemistry , Bacterial Proteins/chemistry , Cytoskeleton/chemistry , Sequence Homology, Amino Acid , Tubulin/chemistry , Bacterial Proteins/ultrastructure , Catalytic Domain , Crystallography, X-Ray , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/ultrastructure , Cytoskeleton/ultrastructure , Escherichia coli/metabolism , Magnesium/metabolism , Models, Molecular , Phosphates/metabolism , Protein Stability , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Subunits/chemistry , Recombinant Fusion Proteins/chemistry , Reproducibility of Results , Tubulin/ultrastructureABSTRACT
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.
ABSTRACT
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.
Subject(s)
Microscopy, Electron/methods , DNA/ultrastructure , DNA-Binding Proteins/ultrastructure , Rec A Recombinases/ultrastructureABSTRACT
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.
Subject(s)
Bacterial Proteins/chemistry , Dynamins/chemistry , Nostoc/chemistry , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cryoelectron Microscopy , Dynamins/genetics , Dynamins/metabolism , GTP Phosphohydrolases/metabolism , Humans , Models, Molecular , Nostoc/metabolism , Protein Interaction Domains and Motifs , RatsABSTRACT
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.
Subject(s)
Biological Evolution , Cytoskeleton/genetics , Eukaryotic Cells/ultrastructure , Prokaryotic Cells/ultrastructure , Animals , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/ultrastructure , Cytoskeleton/chemistry , Models, Molecular , Tubulin/chemistry , Tubulin/genetics , Tubulin/ultrastructureSubject(s)
Dyneins/chemistry , Dyneins/metabolism , Microtubules/metabolism , Adenosine Triphosphate/metabolism , Crystallization , Crystallography, X-Ray , Microscopy, Electron , Microtubules/ultrastructure , Models, Molecular , Protein Folding , Protein Structure, Tertiary , Protein Subunits/chemistry , Protein Subunits/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolismABSTRACT
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 4.9 nm. Here we show that Mal3, an EB1 family tip-tracking protein, drives tubulin to assemble in vitro into exclusively 13-protofilament microtubules with a high proportion of A-lattice protofilament contacts. We present a three-dimensional cryo-EM reconstruction of a purely A-lattice microtubule decorated with Mal3, in which Mal3 occupies the groove between protofilaments and associates closely with one tubulin monomer. We propose that Mal3 promotes assembly by binding to freshly formed tubulin polymer and particularly favors any with A-lattice arrangement. These results reopen the question of microtubule structure in cells.
Subject(s)
Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Amino Acid Sequence , Cryoelectron Microscopy , Gene Deletion , Microtubule-Associated Proteins/chemistry , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/ultrastructure , Microtubules/chemistry , Microtubules/ultrastructure , Models, Molecular , Protein Binding , Schizosaccharomyces/chemistry , Schizosaccharomyces/genetics , Schizosaccharomyces/ultrastructure , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/ultrastructureABSTRACT
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. Tektins assemble into continuous rods 2 nm in diameter that are probably equivalent to subfilaments of the 10 nm diameter IFs. Tektin and IF rod sequences both have a repeating pattern of charged amino acids superimposed on the seven-amino-acid hydrophobic pattern of coiled-coil proteins. The length of the repeat segment matches that of tubulin subunits, suggesting that tektins and tubulins may have coevolved, and that lamins and IFs may have emerged later as modified forms of tektin. Unlike IFs, tektin sequences include one copy of a conserved peptide of nine amino acids that may bind tubulin. The 2 nm filaments associate closely with tubulin in doublet and triplet microtubules of axonemes and centrioles, respectively, and help to stabilize these structures. Their supply restricts the assembled lengths of cilia and flagella. In doublet microtubules, the 2 nm filaments may also help to organize the longitudinal spacing of accessory structures, such as groups of inner dynein arms and radial spokes.
Subject(s)
Microtubule Proteins/chemistry , Microtubule Proteins/physiology , Animals , Evolution, Molecular , Humans , Microtubule Proteins/genetics , Microtubules/chemistry , Microtubules/ultrastructure , PhylogenyABSTRACT
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.
Subject(s)
Microscopy, Electron/methods , Microtubules/chemistry , Microtubules/ultrastructure , Animals , Brain/ultrastructure , Models, Molecular , Particle Size , Protein Structure, Tertiary , Swine , Tubulin/chemistry , Tubulin/isolation & purificationABSTRACT
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. A 3D structure of the microtubule-bound Kar3 motor domain in a nucleotide-free state has revealed dramatic changes not yet reported for any crystal structure, including melting of the switch II helix, that may be part of the mechanism by which information is transmitted. A nucleotide-dependent movement of helix alpha6, first seen in crystal structures of Kif1a, appears to bring it into contact with tubulin and may provide another communication link. A microtubule-induced movement of loop L7 and a related distortion of the central beta-sheet, detected only in the empty state, may also send a signal to the region of the motor core that interacts with the neck. Earlier images of a kinesin-1 dimer in the empty state, showing a close interaction between the two motor heads, can now be interpreted in terms of a communication route from the active site of the directly bound head via its central beta-sheet to the tethered head.
Subject(s)
Kinesins/chemistry , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Dimerization , Humans , Kinesins/ultrastructure , Microtubules/metabolism , Microtubules/ultrastructure , Protein Binding , Protein Structure, Tertiary , Tubulin/metabolismABSTRACT
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.