Preparation of Polarity-Marked Microtubules Using a Plus-End Capping DARPin.

The eukaryotic cytoskeleton is formed in part by microtubules, which are relatively rigid filaments with inherent structural polarity. One consequence of this polarity is that the two ends of a microtubule have different properties with important consequences for their cellular roles. These differences are often challenging to probe within the crowded environment of the cell.

Transition of human γ-tubulin ring complex into a closed conformation during microtubule nucleation.

Microtubules are essential for intracellular organization and chromosome segregation. They are nucleated by the γ-tubulin ring complex (γTuRC). However, isolated vertebrate γTuRC adopts an open conformation that deviates from the microtubule structure, raising the question of the nucleation mechanism.

Branched microtubule nucleation and dynein transport organize RanGTP asters in egg extract.

Chromosome segregation relies on the correct assembly of a bipolar spindle. Spindle pole self-organization requires dynein-dependent microtubule (MT) transport along other MTs. However, during M-phase RanGTP triggers MT nucleation and branching generating polarized arrays with nonastral organization in which MT minus ends are linked to the sides of other MTs.

The minus-end depolymerase KIF2A drives flux-like treadmilling of γTuRC-uncapped microtubules.

During mitosis, microtubules in the spindle turn over continuously. At spindle poles, where microtubule minus ends are concentrated, microtubule nucleation and depolymerization, the latter required for poleward microtubule flux, happen side by side. How these seemingly antagonistic processes of nucleation and depolymerization are coordinated is not understood.

Microtubule binding of the human augmin complex is directly controlled by importins and Ran-GTP.

Mitotic spindle assembly during cell division is a highly regulated process. Ran-GTP produced around chromosomes controls the activity of a multitude of spindle assembly factors by releasing them from inhibitory interaction with importins. A major consequence of Ran-GTP regulation is the local stimulation of branched microtubule nucleation around chromosomes, which is mediated by the augmin complex (composed of the eight subunits HAUS1-HAUS8), a process that is crucially important for correct spindle assembly.

Effects of microtubule length and crowding on active microtubule network organization.

Active filament networks can organize into various dynamic architectures driven by cross-linking motors. Densities and kinetic properties of motors and microtubules have been shown previously to determine active microtubule network self-organization, but the effects of other control parameters are less understood. Using computer simulations, we study here how microtubule lengths and crowding effects determine active network architecture and dynamics.

Cross-linker design determines microtubule network organization by opposing motors.

During cell division, cross-linking motors determine the architecture of the spindle, a dynamic microtubule network that segregates the chromosomes in eukaryotes. It is unclear how motors with opposite directionality coordinate to drive both contractile and extensile behaviors in the spindle. Particularly, the impact of different cross-linker designs on network self-organization is not understood, limiting our understanding of self-organizing structures in cells but also our ability to engineer new active materials.

Real-Time Imaging of Single γTuRC-Mediated Microtubule Nucleation Events In Vitro by TIRF Microscopy.

The γ-tubulin ring complex (γTuRC) is the major microtubule nucleator in cells. How γTuRC nucleates microtubules, and how nucleation is regulated is not understood. To gain an understanding of γTuRC activity and regulation at the molecular level, it is important to measure quantitatively how γTuRC interacts with tubulin and potential regulators in space and time.

Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules.

Microtubules (MTs) are polymers of αβ-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is believed that GTP hydrolysis within the MT lattice is accompanied by destabilizing conformational changes and that MT stability depends on a transiently existing GTP cap at the growing MT end.

Gradual compaction of the central spindle decreases its dynamicity in PRC1 and EB1 gene-edited cells.

During mitosis, the spindle undergoes morphological and dynamic changes. It reorganizes at the onset of the anaphase when the antiparallel bundler PRC1 accumulates and recruits central spindle proteins to the midzone. Little is known about how the dynamic properties of the central spindle change during its morphological changes in human cells.

Quantifying the Monomer-Dimer Equilibrium of Tubulin with Mass Photometry.

The αβ-tubulin heterodimer is the fundamental building block of microtubules, making it central to several cellular processes. Despite the apparent simplicity of heterodimerisation, the associated energetics and kinetics remain disputed, largely due to experimental challenges associated with quantifying affinities in the <µM range. We use mass photometry to observe tubulin monomers and heterodimers in solution simultaneously, thereby quantifying the αβ-tubulin dissociation constant (8.

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.

The speed of GTP hydrolysis determines GTP cap size and controls microtubule stability.

Microtubules are cytoskeletal polymers whose function depends on their property to switch between states of growth and shrinkage. Growing microtubules are thought to be stabilized by a GTP cap at their ends. The nature of this cap, however, is still poorly understood.

Self-Organization of Minimal Anaphase Spindle Midzone Bundles.

In anaphase spindles, antiparallel microtubules associate to form tight midzone bundles, as required for functional spindle architecture and correct chromosome segregation. Several proteins selectively bind to these overlaps to control cytokinesis. How midzone bundles assemble is poorly understood.

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.

Selection and Characterization of Artificial Proteins Targeting the Tubulin α Subunit.

Microtubules are cytoskeletal filaments of eukaryotic cells made of αβ-tubulin heterodimers. Structural studies of non-microtubular tubulin rely mainly on molecules that prevent its self-assembly and are used as crystallization chaperones. Here we identified artificial proteins from an αRep library that are specific to α-tubulin.

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.

Spherical network contraction forms microtubule asters in confinement.

Microtubules and motor proteins form active filament networks that are critical for a variety of functions in living cells. Network topology and dynamics are the result of a self-organisation process that takes place within the boundaries of the cell. Previous biochemical in vitro studies with biomimetic systems consisting of purified motors and microtubules have demonstrated that confinement has an important effect on the outcome of the self-organisation process.

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.

Ensembles of Bidirectional Kinesin Cin8 Produce Additive Forces in Both Directions of Movement.

Most kinesin motors move in only one direction along microtubules. Members of the kinesin-5 subfamily were initially described as unidirectional plus-end-directed motors and shown to produce piconewton forces. However, some fungal kinesin-5 motors are bidirectional.

Combinatorial regulation of the balance between dynein microtubule end accumulation and initiation of directed motility.

Cytoplasmic dynein is involved in a multitude of essential cellular functions. Dynein's activity is controlled by the combinatorial action of several regulatory proteins. The molecular mechanism of this regulation is still poorly understood.

Microtubule nucleation: beyond the template.

Microtubules are cytoskeletal filaments central to a wide range of essential cellular functions in eukaryotic cells. Consequently, cells need to exert tight control over when, where and how many microtubules are being made. Whereas the regulation of microtubule dynamics is well studied, the molecular mechanisms of microtubule nucleation are still poorly understood.

Purification and characterisation of the fission yeast Ndc80 complex.

The Ndc80 complex is a conserved outer kinetochore protein complex consisting of Ndc80 (Hec1), Nuf2, Spc24 and Spc25. This complex comprises a major, if not the sole, platform with which the plus ends of the spindle microtubules directly interact. In fission yeast, several studies indicate that multiple microtubule-associated proteins including the Dis1/chTOG microtubule polymerase and the Mal3/EB1 microtubule plus-end tracking protein directly or indirectly bind Ndc80, thereby ensuring stable kinetochore-microtubule attachment.

Steady-state EB cap size fluctuations are determined by stochastic microtubule growth and maturation.

Growing microtubules are protected from depolymerization by the presence of a GTP or GDP/Pi cap. End-binding proteins of the EB1 family bind to the stabilizing cap, allowing monitoring of its size in real time. The cap size has been shown to correlate with instantaneous microtubule stability.