Tubulin Dimers Oligomerize before Their Incorporation into Microtubules

In the presence of GTP, purified dimers of α- and β-tubulin will interact longitudinally and laterally to self-assemble into microtubules (MTs). This property provides a powerful in vitro experimental system to describe MT dynamic behavior at the micrometer scale and to study effects and functioning of a large variety of microtubule associated proteins (MAPs). Despite the plethora of such data produced, the molecular mechanisms of MT assembly remain disputed. Electron microscopy (EM) studies suggested that tubulin dimers interact longitudinally to form short oligomers which form a tube by lateral interaction and which contribute to MT elongation. This idea is however challenged: Based on estimated association constants it was proposed that single dimers represent the major fraction of free tubulin. This view was recently supported by measurements suggesting that MTs elongate by addition of single tubulin dimers. To solve this discrepancy, we performed a direct measurement of the longitudinal interaction energy for tubulin dimers. We quantified the size distribution of tubulin oligomers using EM and fluorescence correlation spectroscopy (FCS). From the distribution we derived the longitudinal interaction energy in the presence of GDP and the non-hydrolysable GTP analog GMPCPP. Our data suggest that MT elongation and nucleation involves interactions of short tubulin oligomers rather than dimers. Our approach provides a solid experimental framework to better understand the role of MAPs in MT nucleation and growth

Enteric Species F Human Adenoviruses use Laminin-Binding Integrins as Co-Receptors for Infection of Ht-29 Cells

The enteric species F human adenovirus types 40 and 41 (HAdV-40 and -41) are the third most common cause of infantile gastroenteritis in the world. Knowledge about HAdV-40 and -41 cellular infection is assumed to be fundamentally different from that of other HAdVs since HAdV-40 and -41 penton bases lack the aV-integrin-interacting RGD motif. This motif is used by other HAdVs mainly for internalization and endosomal escape. We hypothesised that the penton bases of HAdV-40 and -41 interact with integrins independently of the RGD motif. HAdV-41 transduction of a library of rodent cells expressing specific human integrin subunits pointed to the use of laminin-binding alpha 2-, alpha 3- and alpha 6- containing integrins as well as other integrins as candidate co-receptors. Specific laminins prevented internalisation and infection, and recombinant, soluble HAdV-41 penton base proteins prevented infection of human intestinal HT-29 cells. Surface plasmon resonance analysis demonstrated that HAdV-40 and -41 penton base proteins bind to alpha 6-containing integrins with an affinity similar to that of previously characterised penton base:integrin interactions. With these results, we propose that laminin-binding integrins are co-receptors for HAdV-40 and -41

Naht Bindung, ein Neuartiger Mechanismus zur Stabilisierung von Mikrotubuli

Microtubules are a fascinating component of the cellular scaffold protein network, the cytoskeleton. These hollow tubular structures are assembled of laterally associated proto-filaments containing ab-tubulin heterodimers in a head to tail arrangement. Accordingly microtubules have a defined polarity, which sets the base for the polarity of the cell. The microtubule lattice can be arranged in two conformations: In the more abundant B-lattice conformation, where the protofilaments interact laterally through a- to a- and b- to b-tubulin contacts and in the less stable A-lattice conformation, where a-tubulin interacts laterally with b-tubulin. In cells the microtubules generally contain 13 protofilaments of which usually one pair interacts in the A-lattice conformation, forming the so-called lattice seam. Microtubule dynamics and interactions are strongly regulated by micro-tubule associate proteins (MAPs). Structural investigations on MAPs and microtubule associated motor proteins in complex with microtubules have become possible in combination with modern electron microscopy (EM) and image processing. We have used biochemistry and different advanced EM techniques to study the interaction between microtubules and the MAP Mal3p in vitro. Mal3p is the sole member of the end-binding protein 1 (EB1) protein family in the fission yeast Schizosaccharomyces pombe. Previous in vivo studies have shown that Mal3p promotes microtubule growth. Our studies with high-resolution unidirectional shadowing EM revealed that Mal3p interacts with the microtubule lattice in a novel way, using binding sites on the microtubule that are different from those reported for other MAPs or motor proteins. Full-length Mal3p preferentially binds between two protofilaments on the microtubule lattice, leaving the rest of the lattice free. A case where Mal3p was found in two adjacent protofilament, revealed an A-lattice conformation on the microtubules, surprisingly indicating specific binding of Mal3p to the microtubule seam. With a lattice enhancer, in form of a b-tubulin binding kinesin motor domain, it was demonstrated that Mal3p stabilizes the seam which is thought to be the weakest part of a microtubule. Further, the presence of Mal3p during microtubule polymerization enhances the closure of protofilament sheets into a tubular organization. Cryo-EM and 3-D helical reconstruction on a monomeric microtubule binding domain of Mal3p, confirm the localization in between the protofilament and result in an accurate localization on the microtubule lattice. The results also indicate Mal3p’s capacity to influence the microtubule lattice conformation. Together, studies approached in vitro demonstrate that an EB1-family homolog not only interacts with the microtubule plus end, but also with the microtubule lattice. The structure of Mal3p interacting with microtubules reveals a new mechanism for microtubule stabilization and further insight on how plus end binding proteins are able promote microtubule growth. These findings further suggest that microtubules exhibit two distinct reaction platforms on their surface that can independently interact with selected MAPs or motors.Mikrotubuli sind eine faszinierende Komponente des Zytoskeletts einer Zelle. Ihre Struktur entspricht der eines Hohlzylinders. Sie sind aus seitlich assoziierten Proto-filamenten zusammengesetzt, die aus a- und b-Tubulin Untereinheiten bestehen. Diese Heterodimere sind gerichtet, bedingt durch ihre Kopf-Schwanz Anordnung. Folglich besitzen Mikrotubuli eine definierte Polarität, welche die Basis für die Polarität der Zelle bildet. Die Anordnung der Untereinheiten zu einem so genannten Mikrotubulus Gitter kann in zwei Konformationen vorkommen: In der häufigeren B-Gitter Formation, in welcher die Protofilamente seitlich durch a- zu a- und b- zu b-Tubulin interagieren und in der weniger stabilen A-Gitter Konformation, in der a-Tubulin lateral mit b-Tubulin wechselwirkt. In der Zelle vorkommende Mikrotubuli haben grundsätzlich 13 Proto-filamente. Mindestens ein Paar dieser Protofilamente interagiert in der A-Gitter Kon-formation und bildet die so genannte Gitter-Naht (lattice seam). Mikrotubuli Dynamik und Interaktionen sind streng durch Mikrotubuli assoziierte Proteine (MAPs) reguliert. Die Kombination aus moderner Elektronenmikroskopie (EM) und Bild-verarbeitung macht strukturelle Untersuchungen an MAPs und Motorproteinen im Zusammenhang mit Mikrutubuli möglich. Wir haben biochemische und hoch entwickelte EM Techniken benutzt, um die Interaktion zwischen Mikrotubuli und dem Mikrotubuli assoziierten Protein Mal3 in vitro zu untersuchen. Mal3p ist ein Homolog des konservierten Ende-Bindungs Protein 1 (EB1) in der Spalthefe Schizosaccharomyces pombe. Es wurde bereits gezeigt, dass EB1 die Struktur von Mikrotubuli stabilisiert. Mit Hilfe einer speziellen, hochauflösenden EM Schattierungstechnik haben wir demonstriert, dass Mal3p auf neuartige Weise mit dem Mikrotubulus Gitter interagiert. Dabei besetzt Mal3p Bindungsstellen am Mikrotubulus, die sich von denen der anderen MAPs oder Motorproteinen unterscheiden. Mal3p bevorzugt die Bindung zwischen zwei Proto-filamenten, lässt jedoch das übrigen Gitter unbesetzt. In seltenen Fällen wurde Mal3p in zwei nebeneinander angrenzenden Protofilamenten gefunden. An diesen Stellen zeigt sich überraschenderweise eine A-Gitter-Konformation am Mikrotubulus, was auf eine spezifische Naht-Bindung hinweist. Mit Hilfe einer Gitterverstärkung in Form einer Kinesin-Motor-Domäne, die an jede b-Untereinheit bindet, konnte gezeigt werden, dass Mal3p die Naht, den schwächsten Teil eines Mikrotubulus, stabilisiert. Des Weiteren unterstützt die Anwesenheit von Mal3p während der Mikrotubulus Polymerisation die Formierung zur Bildung des Hohlzylinders. Die Untersuchung der monomeren Mikrotubuli-Bindungs-Domäne von Mal3p unter Anwendung von Kryo-EM und anschließender 3-D helikalen Rekonstruktion, führte zur genauen Lokalisierung des Proteins auf dem Mikrotubulus Gerüst. Hierbei bestätigte sich auch die Lokalisation zwischen den Protofilamenten. Des Weiteren konnte gezeigt werden, dass Mal3p die Fähigkeit besitzt, die Konformation des Mikrotubulus Gitters zu beeinflussen. Zusammenfassend lässt sich sagen, dass das EB1-Homolog nicht nur an das Mikrotubulus Plus Ende, sondern auch an der Naht entlang des ganzen Mikrotubulus bindet. Die Art wie Mal3p mit den Mikrotubuli interagiert, zeigt einen neuen Mecha-nismus der Mikrotubuli Stabilisierung und eröffnet weitere Sichtweisen, wie Plus End Bindungsproteine die Dynamik von Mikrotubuli beeinflussen. Die Ergebnisse belegen, dass Mikrotubuli zwei definierte Reaktionsplattformen auf ihrer Oberfläche besitzen, die unabhängig mit verschiedenen MAPs und Motorproteinen interagiere

Characterisation of Schizosaccharomyces pombe alpha-actinin

The actin cytoskeleton plays a fundamental role in eukaryotic cells. Its reorganization is regulated by a plethora of actin-modulating proteins, such as a-actinin. In higher organisms, alpha-actinin is characterized by the presence of three distinct structural domains: an N-terminal actin-binding domain and a C-terminal region with EF-hand motif separated by a central rod domain with four spectrin repeats. Sequence analysis has revealed that the central rod domain of alpha-actinin from the fission yeast Schizosaccharomyces pombe consists of only two spectrin repeats. To obtain a firmer understanding of the structure and function of this unconventional alpha-actinin, we have cloned and characterized each structural domain. Our results show that this alpha-actinin isoform is capable of forming dimers and that the rod domain is required for this. However, its actin-binding and cross-linking activity appears less efficient compared to conventional alpha-actinins. The solved crystal structure of the actin-binding domain indicates that the closed state is stabilised by hydrogen bonds and a salt bridge not present in other a-actinins, which may reduce the affinity for actin

Characterisation of Schizosaccharomyces pombe alpha-actinin

The actin cytoskeleton plays a fundamental role in eukaryotic cells. Its reorganization is regulated by a plethora of actin-modulating proteins, such as a-actinin. In higher organisms, alpha-actinin is characterized by the presence of three distinct structural domains: an N-terminal actin-binding domain and a C-terminal region with EF-hand motif separated by a central rod domain with four spectrin repeats. Sequence analysis has revealed that the central rod domain of alpha-actinin from the fission yeast Schizosaccharomyces pombe consists of only two spectrin repeats. To obtain a firmer understanding of the structure and function of this unconventional alpha-actinin, we have cloned and characterized each structural domain. Our results show that this alpha-actinin isoform is capable of forming dimers and that the rod domain is required for this. However, its actin-binding and cross-linking activity appears less efficient compared to conventional alpha-actinins. The solved crystal structure of the actin-binding domain indicates that the closed state is stabilised by hydrogen bonds and a salt bridge not present in other a-actinins, which may reduce the affinity for actin

Exploring the bacterial nano-universe

Since the days of the first acknowledged microscopist, Antonie van Leeuwenhoek, the 'animalcules', that is, bacteria and other microbes have been subject to increasingly detailed visualization. With the currently most sophisticated molecular imaging method; cryo electron tomography (Cryo-ET), we are reaching the milestone of being able to image an entire organism in a single dataset at nanometer resolution. Cryo-ET will enable the next revolution in our understanding of bacterial cells, their ultra-structure and intricate molecular nanomachines. Here, we highlight recent research discoveries based on constantly progressing technology developments. We discuss advantages and challenges of using Cryo-ET to visualize spatial structure of microorganisms and macromolecular complexes in their native environment

Assembly mechanisms of the bacterial cytoskeletal protein FilP

Despite low-sequence homology, the intermediate filament (IF)–like protein FilP from Streptomyces coelicolor displays structural and biochemical similarities to the metazoan nuclear IF lamin. FilP, like IF proteins, is composed of central coiled-coil domains interrupted by short linkers and flanked by head and tail domains. FilP polymerizes into repetitive filament bundles with paracrystalline properties. However, the cations Na+ and K+ are found to induce the formation of a FilP hexagonal meshwork with the same 60-nm repetitive unit as the filaments. Studies of polymerization kinetics, in combination with EM techniques, enabled visualization of the basic building block — a transiently soluble rod-shaped FilP molecule—and its assembly into protofilaments and filament bundles. Cryoelectron tomography provided a 3D view of the FilP bundle structure and an original assembly model of an IF-like protein of prokaryotic origin, thereby enabling a comparison with the assembly of metazoan IF

Membrane vesicle-mediated release of bacterial RNA

Many Gram-negative bacterial species release outer membrane vesicles (OMVs) that interact with the host by delivering virulence factors. Here, we report for the first time that RNA is among the wide variety of bacterial components that are associated with OMVs. To characterize the RNA profiles of bacterial OMVs, we performed RNA deep sequencing analysis using OMV samples isolated from a wild type Vibrio cholerae O1 El Tor strain. The results showed that RNAs originating from intergenic regions were the most abundant. Our findings reveal a hitherto unrecognised feature of OMVs mimicking eukaryotic exosomes and highlight a need to evaluate the potential role of RNA-containing bacterial membrane vesicles in bacteria-host interactions

Non-sporulating ftsZ mutants in Streptomyces coelicolor reveal amino acid residues critical for FtsZ polymerization dynamics.

During sporulation of Streptomyces coelicolor, the cytokinetic protein FtsZ is assembled into dozens of regularly spaced Z rings, which orchestrate the division of aerial hyphae into spores. We have previously found that a missense allele of ftsZ, ftsZ17(Spo), primarily affects sporulation septation rather than formation of cross-walls in vegetative mycelium. To clarify what aspect of FtsZ function is compromised in such non-sporulating mutants, we here use a genetic strategy to identify new ftsZ(Spo) alleles and describe how some of the mutations affect the biochemical properties of FtsZ. We have established a system for purification of recombinant untagged S. coelicolor FtsZ, and show that it assembles dynamically into single protofilaments, displays a critical concentration indicative of cooperative assembly, and has a rate of GTP hydrolysis that is substantially higher than that of the closely related Mycobacterium tuberculosis FtsZ. Of the nine isolated ftsZ(Spo) mutations, four affect the interface between the two main subdomains of FtsZ that is implicated in the assembly-induced conformational changes thought to mediate the GTP/GDP-driven cooperative assembly of FtsZ. We find that all these four mutations affect the polymerization behavior of FtsZ in vitro. In addition, at least one ftsZ(Spo) mutation at the longitudinal contact surface between subunits in protofilaments strongly affects formation of polymers in vitro. We conclude that the assembly of Z rings during sporulation of S. coelicolor is highly sensitive to disturbances of FtsZ polymerization, and therefore constitutes an excellent system for analysis of the elusive properties of FtsZ that mediate its characteristic polymerization dynamics

Affinity to cellulose is a shared property among coiled-coil domains of intermediate filaments and prokaryotic intermediate filament-like proteins

Coiled-coil domains of intermediate filaments (IF) and prokaryotic IF-like proteins enable oligomerisation and filamentation, and no additional function is ascribed to these coiled-coil domains. However, an IF-like protein from Streptomyces reticuli was reported to display cellulose affinity. We demonstrate that cellulose affinity is an intrinsic property of the IF-like proteins FilP and Scy and the coiled-coil protein DivIVA from the genus Streptomyces. Furthermore, IF-like proteins and DivIVA from other prokaryotic species and metazoan IF display cellulose affinity despite having little sequence homology. Cellulose affinity-based purification is utilised to isolate native FilP protein from the whole cell lysate of S. coelicolor. Moreover, cellulose affinity allowed for the isolation of IF and IF-like protein from the whole cell lysate of C. crescentus and a mouse macrophage cell line. The binding to cellulose is mediated by certain combinations of coiled-coil domains, as demornstrated for FilP and lamin. Fusions of target proteins to cellulose-binding coiled-coil domains allowed for cellulose-based protein purification. The data presented show that cellulose affinity is a novel function of certain coiled-coil domains of IF and IF-like proteins from evolutionary diverse species