Denis Chrétien

EB1 regulates microtubule dynamics and tubulin sheet closure in vitro

End binding 1 (EB1) is a plus-end-tracking protein (+TIP) that localizes to microtubule plus ends where it modulates their dynamics and interactions with intracellular organelles. Although the regulating activity of EB1 on microtubule dynamics has been studied in cells and purified systems, the molecular mechanisms involved in its specific activity are still unclear. Here, we describe how EB1 regulates the dynamics and structure of microtubules assembled from pure tubulin. We found that EB1 stimulates spontaneous nucleation and growth of microtubules, and promotes both catastrophes (transitions from growth to shrinkage) and rescues (reverse events). Electron cryomicroscopy showed that EB1 induces the initial formation of tubulin sheets, which rapidly close into the common 13-protofilament-microtubule architecture. Our results suggest that EB1 favours the lateral association of free tubulin at microtubule-sheet edges, thereby stimulating nucleation, sheet growth and closure. The reduction of sheet length at microtubule growing-ends together with the elimination of stressed microtubule lattices may account for catastrophes. Conversely, occasional binding of EB1 to the microtubule lattice may induce rescues.

New data on the microtubule surface lattice

Summary— The in vitro polymerisation of tubulin is a remarkable example of protein self‐assembly in thet several closely related microtubule structures coexist on the polymerisation plateau. Unfixed and unstained in vitro assembled microtubules were observed in vitreous ice by cryo‐electron microscopy. New results are reported that considerably extend previous observations [47]. In ice, microtubule images have a distinctive contrast related to the number and skew of the photofilaments. The microtubules observed have from twelve to seventeen protofilaments. Comparison with thin sections of pelleted material allows a direct identification of images from microtubules with thirteen, fourteen and fifteen protofilaments. A surface lattice accommodation mechanism, previously proposed to explain how variable numbers of protofilaments can be incorporated into the basic thirteen protofilament structure, is described in detail. Our new experimental results are shown to be in overall agreement with the theoretical predictions. Only thirteen protofilament microtubules have unskewed protofilaments, this was confirmed by observations on axoneme fragments. The results imply that the microtubule surface lattice is based on a mixed packing which combines features of the standard A and B lattices.

Characterization of microtubule protofilament numbers: How does the surface lattice accommodate?☆

Frozen-hydrated specimens of microtubules assembled in vitro were observed by cryoelectron microscopy. Specimens were of both pure tubulin, and of microtubule protein isolated by three cycles of assembly and disassembly. It is shown that the characteristic image contrast of individual microtubules allows the microtubule protofilament number to be determined unambiguously. Microtubules with 13, 14 and 15 protofilaments are observed to coexist in specimens prepared under various assembly conditions. Confirmation of these results is obtained by observations of thin sections of pelleted samples fixed and stained using the glutaraldehyde/tannic acid technique. Images of individual microtubules show both characteristic contrast profiles across their width and typical variations of these profiles along their length. The profiles across the images indicate the protofilament number of the microtubule. The lengthwise variations indicate how the protofilaments are aligned with respect to the microtubule axis giving what has previously been called a supertwist. In 13 protofilament microtubules the protofilaments are paraxial. In 14 and 15 protofilament microtubules, the protofilaments are skewed with respect to the microtubule axis. The skew is greater for the 15 protofilament case than for 14 protofilaments. The skew allows the extra protofilaments to be accommodated by the surface lattice. These results should also be relevant to situations in vivo.

Procentriole assembly revealed by cryo-electron tomography

Centrosomes are cellular organelles that have a major role in the spatial organisation of the microtubule network. The centrosome is comprised of two centrioles that duplicate only once during the cell cycle, generating a procentriole from each mature centriole. Despite the essential roles of centrosomes, the detailed structural mechanisms involved in centriole duplication remain largely unknown. Here, we describe human procentriole assembly using cryo‐electron tomography. In centrosomes, isolated from human lymphoblasts, we observed that each one of the nine microtubule triplets grows independently around a periodic central structure. The proximal end of the A‐microtubule is capped by a conical structure and the B‐ and C‐microtubules elongate bidirectionally from its wall. These observations suggest that the gamma tubulin ring complex (γ‐TuRC) has a fundamental role in procentriole formation by nucleating the A‐microtubule that acts as a template for B‐microtubule elongation that, in turn, supports C‐microtubule growth. This study provides new insights into the initial structural events involved in procentriole assembly and establishes the basis for determining the molecular mechanisms of centriole duplication on the nanometric scale.

CLIP-170/Tubulin-Curved Oligomers Coassemble at Microtubule Ends and Promote Rescues

BACKGROUND CLIP-170 is a microtubule binding protein specifically located at microtubule plus ends, where it modulates their dynamic properties and their interactions with intracellular organelles. The mechanism by which CLIP-170 is targeted to microtubule ends remains unclear today, as well as its precise effect on microtubule dynamics. RESULTS We used the N-terminal part of CLIP-170 (named H2), which contains the microtubule binding domains, to investigate how it modulates in vitro microtubule dynamics and structure. We found that H2 primarily promoted rescues (transitions from shrinkage to growth) of microtubules nucleated from pure tubulin and isolated centrosomes, and stimulated microtubule nucleation. Electron cryomicroscopy revealed that H2 induced the formation of tubulin rings in solution and curved oligomers at the extremities of microtubules in assembly conditions. CONCLUSIONS These results suggest that CLIP-170 targets specifically at microtubule plus ends by copolymerizing with tubulin and modulates microtubule nucleation, polymerization, and rescues by the same basic mechanism with tubulin oligomers as intermediates.

Visualization of cell microtubules in their native state

Background information. Over the past decades, cryo‐electron microscopy of vitrified specimens has yielded a detailed understanding of the tubulin and microtubule structures of samples reassembled in vitro from purified components. However, our knowledge of microtubule structure in vivo remains limited by the chemical treatments commonly used to observe cellular architecture using electron microscopy.

Modeling elastic properties of microtubule tips and walls

Abstract Electron micrographs of tips of growing and shrinking microtubules are analyzed and interpreted. The many shapes observed are all consistent with a simple mechanical model, a flexible tube with competing intrinsic curvatures. Observations are also consistent with growing and shrinking microtubules having the same intrinsic curvature for protofilaments, the one observed in oligomers peeling off shrinking microtubules. If this is so, the lateral bonds between protofilaments are responsible for the difference between shapes of tips on growing and shrinking microtubules.

Structural microtubule cap: stability, catastrophe, rescue, and third state

Microtubules polymerize from GTP-liganded tubulin dimers, but are essentially made of GDP-liganded tubulin. We investigate the tug-of-war resulting from the fact that GDP-liganded tubulin favors a curved configuration, but is forced to remain in a straight one when part of a microtubule. We point out that near the end of a microtubule, the proximity of the end shifts the balance in this tug-of-war, with some protofilament bending as result. This somewhat relaxes the microtubule lattice near its end, resulting in a structural cap. This structural cap thus is a simple mechanical consequence of two well-established facts: protofilaments made of GDP-liganded tubulin have intrinsic curvature, and microtubules are elastic, made from material that can yield to forces, in casu its own intrinsic forces. We explore possible properties of this structural cap, and demonstrate 1) how it allows both polymerization from GTP-liganded tubulin and rapid depolymerization in its absence; 2) how rescue can occur; 3) how a third, meta-stable intermediate state is possible and can explain some experimental results; and 4) how the tapered tips observed at polymerizing microtubule ends are stabilized during growth, though unable to accommodate a lateral cap. This scenario thus supports the widely accepted GTP-cap model by suggesting a stabilizing mechanism that explains the many aspects of dynamic instability.

Determination of microtubule polarity by cryo-electron microscopy

BACKGROUND Microtubules are tubular polymers of tubulin dimers, which are arranged head-to-tail in protofilaments that run lengthwise along the microtubules, giving them an overall structural polarity. Many of the functions of microtubules depend on this polarity, including directed intracellular transport and chromosome segregation during mitosis. The determination of microtubule polarity for lengthwise views of microtubules observed by electron microscopy has not previously been possible. Here, we present methods for directly determining the polarity of individual microtubules imaged by cryo-electron microscopy. RESULTS When observed in vitreous ice by cryo-electron microscopy, microtubules with skewed protofilaments show arrowhead moiré patterns. We have used centrosome nucleated microtubules to relate the directionality of the moiré patterns to microtubule polarity. We show that the arrowheads point towards the plus end of microtubules with protofilaments having a right-handed skew, and towards the minus end of microtubules with protofilaments having a left-handed skew. We describe two methods for determining the handedness of the protofilament skew. The first method uses two or more tilted views. The second method involves analysis of the diffraction patterns of the microtubule images. CONCLUSIONS It is now possible to determine directly the polarity of in vitro assembled microtubules from cryo-electron micrographs. This will be helpful in a number of types of studies, including studies of the three-dimensional structure of microtubules interacting with motor proteins, as knowledge of the polarity of the microtubule is essential to understand motor directionality.

The 90-kDa Heat Shock Protein Hsp90 Protects Tubulin against Thermal Denaturation

Hsp90 and tubulin are among the most abundant proteins in the cytosol of eukaryotic cells. Although Hsp90 plays key roles in maintaining its client proteins in their active state, tubulin is essential for fundamental processes such as cell morphogenesis and division. Several studies have suggested a possible connection between Hsp90 and the microtubule cytoskeleton. Because tubulin is a labile protein in its soluble form, we investigated whether Hsp90 protects it against thermal denaturation. Both proteins were purified from porcine brain, and their interaction was characterized in vitro by using spectrophotometry, sedimentation assays, video-enhanced differential interference contrast light microscopy, and native polyacrylamide gel electrophoresis. Our results show that Hsp90 protects tubulin against thermal denaturation and keeps it in a state compatible with microtubule polymerization. We demonstrate that Hsp90 cannot resolve tubulin aggregates but that it likely binds early unfolding intermediates, preventing their aggregation. Protection was maximal at a stoichiometry of two molecules of Hsp90 for one of tubulin. This protection does not require ATP binding and hydrolysis by Hsp90, but it is counteracted by geldanamycin, a specific inhibitor of Hsp90.