Structural insight into TPX2-stimulated microtubule assembly

During mitosis and meiosis, microtubule (MT) assembly is locally upregulated by the chromatin-dependent Ran-GTP pathway. One of its key targets is the MT-associated spindle assembly factor TPX2. The molecular mechanism of how TPX2 stimulates MT assembly remains unknown because structural information about the interaction of TPX2 with MTs is lacking. Here, we determine the cryo-electron microscopy structure of a central region of TPX2 bound to the MT surface. TPX2 uses two flexibly linked elements ('ridge' and 'wedge') in a novel interaction mode to simultaneously bind across longitudinal and lateral tubulin interfaces. These MT-interacting elements overlap with the binding site of importins on TPX2. Fluorescence microscopy-based in vitro reconstitution assays reveal that this interaction mode is critical for MT binding and facilitates MT nucleation. Together, our results suggest a molecular mechanism of how the Ran-GTP gradient can regulate TPX2-dependent MT formation

Determinants of Polar versus Nematic Organization in Networks of Dynamic Microtubules and Mitotic Motors.

During cell division, mitotic motors organize microtubules in the bipolar spindle into either polar arrays at the spindle poles or a "nematic" network of aligned microtubules at the spindle center. The reasons for the distinct self-organizing capacities of dynamic microtubules and different motors are not understood. Using in vitro reconstitution experiments and computer simulations, we show that the human mitotic motors kinesin-5 KIF11 and kinesin-14 HSET, despite opposite directionalities, can both organize dynamic microtubules into either polar or nematic networks. We show that in addition to the motor properties the natural asymmetry between microtubule plus- and minus-end growth critically contributes to the organizational potential of the motors. We identify two control parameters that capture system composition and kinetic properties and predict the outcome of microtubule network organization. These results elucidate a fundamental design principle of spindle bipolarity and establish general rules for active filament network organization

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

LIS1 Clamps Dynein to the Microtubule

Cytoplasmic dynein is a motor essential for numerous mechanical processes in eukaryotic cells. How its activity is regulated is largely unknown. By using a combination of approaches including single-molecule biophysics and electron microscopy, Huang et al. in this issue uncover the regulatory mechanism by which LIS1 controls the activity of cytoplasmic dynein

Poleward transport of Eg5 by dynein–dynactin in Xenopus laevis egg extract spindles

Molecular motors are required for spindle assembly and maintenance during cell division. How motors move and interact inside spindles is unknown. Using photoactivation and photobleaching, we measure mitotic motor movement inside a dynamic spindle. We find that dynein–dynactin transports the essential motor Eg5 toward the spindle poles in Xenopus laevis egg extract spindles, revealing a direct interplay between two motors of opposite directionality. This transport occurs throughout the spindle except at the very spindle center and at the spindle poles, where Eg5 remains stationary. The variation of Eg5 dynamics with its position in the spindle is indicative of position-dependent functions of this motor protein. Our results suggest that Eg5 drives microtubule flux by antiparallel microtubule sliding in the spindle center, whereas the dynein-dependent concentration of Eg5 outside the spindle center could contribute to parallel microtubule cross-linking. These results emphasize the importance of spatially differentiated functions of motor proteins and contribute to our understanding of spindle organization

Effects of spatial dimensionality and steric interactions on microtubule-motor self-organization.

Active networks composed of filaments and motor proteins can self-organize into a variety of architectures. Computer simulations in two or three spatial dimensions and including or omitting steric interactions between filaments can be used to model active networks. Here we examine how these modelling choices affect the state space of network self-organization. We compare the networks generated by different models of a system of dynamic microtubules and microtubule-crosslinking motors. We find that a thin 3D model that includes steric interactions between filaments is the most versatile, capturing a variety of network states observed in recent experiments. In contrast, 2D models either with or without steric interactions which prohibit microtubule crossings can produce some, but not all, observed network states. Our results provide guidelines for the most appropriate choice of model for the study of different network types and elucidate mechanisms of active network organization.This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001163), the UK Medical Research Council (FC001163), and the Wellcome Trust (FC001163). F N was supported by the Centre for Modelling in the Biosciences (www.bioms.de) and EMBL. T S acknowledges support from the European Research Council (Advanced Grant, project 323042). We thank EMBL for support, in particular for its high-performance computing services

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

Phosphorylation by Cdk1 Increases the Binding of Eg5 to Microtubules In Vitro and in Xenopus Egg Extract Spindles

BACKGROUND:Motor proteins from the kinesin-5 subfamily play an essential role in spindle assembly during cell division of most organisms. These motors crosslink and slide microtubules in the spindle. Kinesin-5 motors are phosphorylated at a conserved site by Cyclin-dependent kinase 1 (Cdk1) during mitosis. Xenopus laevis kinesin-5 has also been reported to be phosphorylated by Aurora A in vitro. METHODOLOGY/PRINCIPAL FINDINGS:We investigate here the effect of these phosphorylations on kinesin-5 from Xenopus laevis, called Eg5. We find that phosphorylation at threonine 937 in the C-terminal tail of Eg5 by Cdk1 does not affect the velocity of Eg5, but strongly increases its binding to microtubules assembled in buffer. Likewise, this phosphorylation promotes binding of Eg5 to microtubules in Xenopus egg extract spindles. This enhancement of binding elevates the amount of Eg5 in spindles above a critical level required for bipolar spindle formation. We find furthermore that phosphorylation of Xenopus laevis Eg5 by Aurora A at serine 543 in the stalk is not required for spindle formation. CONCLUSIONS/SIGNIFICANCE:These results show that phosphorylation of Eg5 by Cdk1 has a direct effect on the interaction of this motor with microtubules. In egg extract, phosphorylation of Eg5 by Cdk1 ensures that the amount of Eg5 in the spindle is above a level that is required for spindle formation. This enhanced targeting to the spindle appears therefore to be, at least in part, a direct consequence of the enhanced binding of Eg5 to microtubules upon phosphorylation by Cdk1. These findings advance our understanding of the regulation of this essential mitotic motor protein

Microtubule nucleation properties of single human γTuRCs explained by their Cryo-EM structure

The γ-tubulin ring complex (γTuRC) is the major microtubule nucleator in cells. The mechanism of its regulation is not understood. We purified human γTuRC and measured its nucleation properties in a total internal reflection fluorescence (TIRF) microscopy-based real-time nucleation assay. We find that γTuRC stably caps the minus ends of microtubules that it nucleates stochastically. Nucleation is inefficient compared with microtubule elongation. The 4 Å resolution cryoelectron microscopy (cryo-EM) structure of γTuRC, combined with crosslinking mass spectrometry analysis, reveals an asymmetric conformation with only part of the complex in a "closed" conformation matching the microtubule geometry. Actin in the core of the complex, and MZT2 at the outer perimeter of the closed part of γTuRC appear to stabilize the closed conformation. The opposite side of γTuRC is in an "open," nucleation-incompetent conformation, leading to a structural asymmetry explaining the low nucleation efficiency of purified human γTuRC. Our data suggest possible regulatory mechanisms for microtubule nucleation by γTuRC closure.This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001163 and FC0010065), the UK Medical Research Council (FC001163 and FC0010065), and the Wellcome Trust (FC001163 and FC0010065) to T.S. and A.C. The Wellcome Centre for Cell Biology is supported by core funding from the Wellcome Trust (203149). J. Rappsilber is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy – EXC 2008 – 390540038 – UniSysCat and 329673113. J. Roostalu. was supported by a Sir Henry Wellcome Postdoctoral Fellowship (100145/Z/12/Z) and M.A.C. is supported by a Marie Sk1odowska-Curie Postdoctoral Fellowship (agreement no. 845939). T.S. acknowledges support from the European Research Council (Advanced Grant, project 323042). A.C. receives funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 820102). T.C., J.A., J.W.M., and T.S. acknowledge also the support of the Spanish Ministry of Economy, Industry and Competitiveness to the CRG-EMBL partnership, the Centro de Excelencia Severo Ochoa and the CERCA Programme of the Generalitat de Cataluny