Thomas Surrey

Reconstitution of a microtubule plus-end tracking system in vitro

The microtubule cytoskeleton is essential to cell morphogenesis. Growing microtubule plus ends have emerged as dynamic regulatory sites in which specialized proteins, called plus-end-binding proteins (+TIPs), bind and regulate the proper functioning of microtubules. However, the molecular mechanism of plus-end association by +TIPs and their ability to track the growing end are not well understood. Here we report the in vitro reconstitution of a minimal plus-end tracking system consisting of the three fission yeast proteins Mal3, Tip1 and the kinesin Tea2. Using time-lapse total internal reflection fluorescence microscopy, we show that the EB1 homologue Mal3 has an enhanced affinity for growing microtubule end structures as opposed to the microtubule lattice. This allows it to track growing microtubule ends autonomously by an end recognition mechanism. In addition, Mal3 acts as a factor that mediates loading of the processive motor Tea2 and its cargo, the Clip170 homologue Tip1, onto the microtubule lattice. The interaction of all three proteins is required for the selective tracking of growing microtubule plus ends by both Tea2 and Tip1. Our results dissect the collective interactions of the constituents of this plus-end tracking system and show how these interactions lead to the emergence of its dynamic behaviour. We expect that such in vitro reconstitutions will also be essential for the mechanistic dissection of other plus-end tracking systems.

Thermal fluctuations of grafted microtubules provide evidence of a length-dependent persistence length

We use single-particle tracking to study the elastic properties of single microtubules grafted to a substrate. Thermal fluctuations of the free microtubule’s end are recorded, in order to measure position distribution functions from which we calculate the persistence length of microtubules with contour lengths between 2.6 and 48 μm. We find the persistence length to vary by more than a factor of 20 over the total range of contour lengths. Our results support the hypothesis that shearing between protofilaments contributes significantly to the mechanics of microtubules. PACS numbers: 87.15.Ya, 87.15.La, 87.16.Ka, 36.20.Ey ∗ Corresponding author. FP and GL have equally contributed to this work.

EBs Recognize a Nucleotide-Dependent Structural Cap at Growing Microtubule Ends

Summary Growing microtubule ends serve as transient binding platforms for essential proteins that regulate microtubule dynamics and their interactions with cellular substructures. End-binding proteins (EBs) autonomously recognize an extended region at growing microtubule ends with unknown structural characteristics and then recruit other factors to the dynamic end structure. Using cryo-electron microscopy, subnanometer single-particle reconstruction, and fluorescence imaging, we present a pseudoatomic model of how the calponin homology (CH) domain of the fission yeast EB Mal3 binds to the end regions of growing microtubules. The Mal3 CH domain bridges protofilaments except at the microtubule seam. By binding close to the exchangeable GTP-binding site, the CH domain is ideally positioned to sense the microtubules nucleotide state. The same microtubule-end region is also a stabilizing structural cap protecting the microtubule from depolymerization. This insight supports a common structural link between two important biological phenomena, microtubule dynamic instability and end tracking.

CLIP-170 tracks growing microtubule ends by dynamically recognizing composite EB1/tubulin-binding sites

The microtubule cytoskeleton is crucial for the internal organization of eukaryotic cells. Several microtubule-associated proteins link microtubules to subcellular structures. A subclass of these proteins, the plus end–binding proteins (+TIPs), selectively binds to the growing plus ends of microtubules. Here, we reconstitute a vertebrate plus end tracking system composed of the most prominent +TIPs, end-binding protein 1 (EB1) and CLIP-170, in vitro and dissect their end-tracking mechanism. We find that EB1 autonomously recognizes specific binding sites present at growing microtubule ends. In contrast, CLIP-170 does not end-track by itself but requires EB1. CLIP-170 recognizes and turns over rapidly on composite binding sites constituted by end-accumulated EB1 and tyrosinated α-tubulin. In contrast to its fission yeast orthologue Tip1, dynamic end tracking of CLIP-170 does not require the activity of a molecular motor. Our results demonstrate evolutionary diversity of the plus end recognition mechanism of CLIP-170 family members, whereas the autonomous end-tracking mechanism of EB family members is conserved.

A Minimal Midzone Protein Module Controls Formation and Length of Antiparallel Microtubule Overlaps

During cell division, microtubules are arranged in a large bipolar structure, the mitotic spindle, to segregate the duplicated chromosomes. Antiparallel microtubule overlaps in the spindle center are essential for establishing bipolarity and maintaining spindle stability throughout mitosis. In anaphase, this antiparallel microtubule array is tightly bundled forming the midzone, which serves as a hub for the recruitment of proteins essential for late mitotic events. The molecular mechanism of midzone formation and the control of its size are not understood. Using an in vitro reconstitution approach, we show here that PRC1 autonomously bundles antiparallel microtubules and recruits Xklp1, a kinesin-4, selectively to overlapping antiparallel microtubules. The processive motor Xklp1 controls overlap size by overlap length-dependent microtubule growth inhibition. Our results mechanistically explain how the two conserved, essential midzone proteins PRC1 and Xklp1 cooperate to constitute a minimal protein module capable of dynamically organizing the core structure of the central anaphase spindle.

GTPgammaS microtubules mimic the growing microtubule end structure recognized by end-binding proteins (EBs)

Microtubule plus-end-tracking proteins (+TIPs) localize to growing microtubule plus ends to regulate a multitude of essential microtubule functions. End-binding proteins (EBs) form the core of this network by recognizing a distinct structural feature transiently existing in an extended region at growing microtubule ends and by recruiting other +TIPs to this region. The nature of the conformational difference allowing EBs to discriminate between tubulins in this region and other potential tubulin binding sites farther away from the microtubule end is unknown. By combining in vitro reconstitution, multicolor total internal reflection fluorescence microscopy, and electron microscopy, we demonstrate here that a closed microtubule B lattice with incorporated GTPγS, a slowly hydrolyzable GTP analog, can mimic the natural EB protein binding site. Our findings indicate that the guanine nucleotide γ-phosphate binding site is crucial for determining the affinity of EBs for lattice-incorporated tubulin. This defines the molecular mechanism by which EBs recognize growing microtubule ends.

Development and Biological Evaluation of Potent and Specific Inhibitors of Mitotic Kinesin Eg5

Anticancer drugs that perturb mitosis, for example, vinca alkaloids, taxol and epothilone, play a major role in the therapy of malignant diseases. One of their major drawbacks is that they are all directed against the same protein, tubulin—the microtubule subunit which forms the mitotic spindle. 2] However, microtubules are also involved in many other cellular processes such as maintenance of organelles and cell shape, cell motility, synaptic vesicles and intracellular transport phenomena. 4, 5] Interference with their formation or depolymerisation often leads to dose-limiting side effects like, for example, neurotoxicity. The discovery of a new class of proteins, the mitotic kinesins, presents a novel approach to cancer treatment. These proteins are exclusively involved in the formation and function of the mitotic spindle and some of them are only expressed in proliferating cells. 8] Their inhibition leads to cell cycle arrest and ultimately to apoptosis without interfering with other microtubule-dependent processes. The mitotic kinesin, Eg5 (also called kinesin-5 or kinesin spindle protein, KSP) plays an important role in the early stages of mitosis. It mediates centrosome separation and formation of the bipolar mitotic spindle. Inhibition of Eg5 leads to cellcycle arrest during mitosis and cells with a monopolar spindle, so-called monoasters. In 1999 the screening of a large library of synthetic compounds identified racemic monastrol, a 4-aryl3,4-dihydropyrimidin-2(1H)-thione derivative, as the first smallmolecule inhibitor of Eg5. Monastrol is a moderate allosteric inhibitor (IC50 = 30 mm) of Eg5 [12] which binds some 12 away from the nucleotide binding site of the protein. In doing so, it triggers both local and distal structural changes throughout the motor domain. In the meantime, four other inhibitors of Eg5 have been published: terpendole E (IC50 = 14.6 mm), S-trityl-l-cysteine (IC50 = 1.0 mm), HR22C16 (IC50 = 0.8 mm) and CK0106023 (IC50 = 12 nm) [14–17] . According to recent investigations monastrol does not display any neurotoxicity in fact, short-term treatment with monastrol has been reported to enhance axonal growth—in contrast to anticancer drugs such as the taxanes which are highly deleterious to axonal formation and growth. There is no indication of any kind of toxicity caused by monastrol to cultures of sympathetic neurons over longer exposure times. For the other Eg5 inhibitors mentioned above such studies have not been reported. For these reasons we focused on the development of potent, specific and cell permeable monastrol analogues. Here we describe the discovery of such derivatives and their inhibitory activity against Eg5 as well as their ability to inhibit cell division. The monastrol analogues 1–4 (Scheme 1 A, B) were synthesized as racemic mixtures by using the Biginelli reaction. Either the appropriate aldehyde, urea and ethyl acetoacetate, were heated or the appropriate aldehyde, urea or thiourea and the 1,3-dicarbonyl compound were irradiation together with polyphosphate ester (PPE) in a domestic microwave oven (Scheme 1 A, B and Table 1). Alcohol 5 was synthesized from 4 a by selective Luche reduction of the 5-carbonyl function and used as a 3:1 diastereomeric mixture—every diastereomer as enantiomeric mixture (Scheme 1 C). Subsequently we screened 40 compounds for their ability to inhibit Eg5 by using an in vitro steady-state ATPase assay. We found that most of the synthesized compounds were less potent Eg5 inhibitors as compared to 36 % inhibition by monastrol (2 c). Nine of the compounds showed less than 5 % inhibition of Eg5 activity under the assay conditions (Table 2). Three compounds, however, were significantly more potent than monastrol (Figure 1 A, Table 2). The assay showed that enhancement of inhibition cannot be achieved by variation of the aromatic substitution pattern of the 4-aryl moiety in 4-aryl3,4-dihydropyrimidin-2(1H)-ones or -thiones, compared to monastrol (2 c). Furthermore, the use of sterically demanding [a] Dipl.-Chem. M. Gartner, N. Sunder-Plassmann, Prof. Dr. A. Giannis University of Leipzig, Institute for Organic Chemistry Johannisallee 29, 04103 Leipzig (Germany) Fax: (+ 49) 41-0341-973-6599 E-mail : giannis@chemie.uni-leipzig.de [b] J. Seiler, M. Utz, Dr. I. Vernos, Dr. T. Surrey European Molecular Biology Laboratory Cell Biology and Biophysics Programme Meyerhofstrasse 1, 69117 Heidelberg (Germany) Supporting information for this article is available on the WWW under http ://www.chembiochem.org or from the author.

EB1 Accelerates Two Conformational Transitions Important for Microtubule Maturation and Dynamics

Summary Background The dynamic properties of microtubules depend on complex nanoscale structural rearrangements in their end regions. Members of the EB1 and XMAP215 protein families interact autonomously with microtubule ends. EB1 recruits several other proteins to growing microtubule ends and has seemingly antagonistic effects on microtubule dynamics: it induces catastrophes, and it increases growth velocity, as does the polymerase XMAP215. Results Using a combination of in vitro reconstitution, time-lapse fluorescence microscopy, and subpixel-precision image analysis and convolved model fitting, we have studied the effects of EB1 on conformational transitions in growing microtubule ends and on the time course of catastrophes. EB1 density distributions at growing microtubule ends reveal two consecutive conformational transitions in the microtubule end region, which have growth-velocity-independent kinetics. EB1 binds to the microtubule after the first and before the second conformational transition has occurred, positioning it several tens of nanometers behind XMAP215, which binds to the extreme microtubule end. EB1 binding accelerates conformational maturation in the microtubule, most likely by promoting lateral protofilament interactions and by accelerating reactions of the guanosine triphosphate (GTP) hydrolysis cycle. The microtubule maturation time is directly linked to the duration of a growth pause just before microtubule depolymerization, indicating an important role of the maturation time for the control of dynamic instability. Conclusions These activities establish EB1 as a microtubule maturation factor and provide a mechanistic explanation for its effects on microtubule growth and catastrophe frequency, which cause microtubules to be more dynamic.

Microtubule organization by the antagonistic mitotic motors kinesin-5 and kinesin-14

Interpolar microtubules are sorted by the directional instability resulting from antagonistic molecular motors, not a stable balance of force.

Directional Switching of the Kinesin Cin8 Through Motor Coupling

A molecular motor switches direction upon interacting with individual microtubules or antiparallel microtubules. Kinesin motor proteins are thought to move exclusively in either one or the other direction along microtubules. Proteins of the kinesin-5 family are tetrameric microtubule cross-linking motors important for cell division and differentiation in various organisms. Kinesin-5 motors are considered to be plus-end–directed. However, here we found that purified kinesin-5 Cin8 from budding yeast could behave as a bidirectional kinesin. On individual microtubules, single Cin8 motors were minus-end–directed motors, whereas they switched to plus-end–directed motility when working in a team of motors sliding antiparallel microtubules apart. This kinesin can thus change directionality of movement depending on whether it acts alone or in an ensemble.