Efficient formation of bipolar microtubule bundles requires microtubule-bound gamma-tubulin complexes

The mechanism for forming linear microtubule (MT) arrays in cells such as neurons, polarized epithelial cells, and myotubes is not well understood. A simpler bipolar linear array is the fission yeast interphase MT bundle, which in its basic form contains two MTs that are bundled at their minus ends. Here, we characterize mto2p as a novel fission yeast protein required for MT nucleation from noncentrosomal gamma-tubulin complexes (gamma-TuCs). In interphase mto2Delta cells, MT nucleation was strongly inhibited, and MT bundling occurred infrequently and only when two MTs met by chance in the cytoplasm. In wild-type 2, we observed MT nucleation from gamma-TuCs bound along the length of existing MTs. We propose a model on how these nucleation events can more efficiently drive the formation of bipolar MT bundles in interphase. Key to the model is our observation of selective antiparallel binding of MTs, which can both explain the generation and spatial separation of multiple bipolar bundles.     

Stabilization of overlapping microtubules by fission yeast CLASP

Many microtubule (MT) structures contain dynamic MTs that are bundled and stabilized in overlapping arrays. CLASPs are conserved MT-binding proteins implicated in the regulation of MT plus ends. Here, we show that the Schizosaccharomyces pombe CLASP, cls1p/peg1p, mediates the stabilization of overlapping MTs within the mitotic spindle and interphase bundles. cls1p localizes to these regions but not to interphase MT plus ends. Inactivation of cls1p leads to the rapid depolymerization of spindle midzone MTs. cls1p also stabilizes a subset of MTs within interphase bundles. cls1p prevents disassembly of the entire microtubule, while still allowing for plus-end growth. It has no measurable effects on MT nucleation, polymerization, catastrophe, or bundling. A direct interaction with ase1p (PRC1/MAP65) targets cls1p to regions of antiparallel MT overlap. These findings show how a MT-stabilizing factor attached to specific sites on MTs can help to generate MT structures that have both dynamic and stable components.     

Slide-and-cluster models for spindle assembly

BACKGROUND: Mitotic and meiotic spindles are assemblies of microtubules (MTs) that form during cell division to physically separate sister chromosomes. How the various components of spindles act together to establish and maintain the dynamic bipolar structure of spindles is not understood. Interactions between MTs and motors have been studied both experimentally and theoretically in many contexts, including the self-organization of arrays of MTs by motors and the competition between different classes of motors to move a single load. This work demonstrates how the interplay between two types of motors together with continual nucleation of MTs by chromosomes could organize the MTs into spindles. RESULTS: We propose a slide-and-cluster model based on four known molecular activities: MT nucleation near chromosomes, the sliding of MTs by a plus-end-directed motor, the clustering of their minus ends by a minus-end-directed motor, and the loss of MTs by dynamic instability. Our model applies to overlapping, nonkinetochore MTs in anastral spindles, and perhaps also to interpolar MTs in astral spindles. We show mathematically that the slide-and-cluster mechanism robustly forms bipolar spindles with sharp poles and a stable steady-state length. This model accounts for several experimental observations that were difficult to explain with existing models. Three new predictions of the model were tested and verified in Xenopus egg extracts. CONCLUSIONS: We show that a simple two-motor model could create stable, bipolar spindles under a wide range of physical parameters. Our model is the first self-contained model for anastral spindle assembly and MT sliding (known as poleward flux). Our experimental results support the slide-and-cluster scenario; most significantly, we find that MT sliding slows near spindle poles, confirming the model's primary prediction.     

Bundling of microtubules by motor and tail domains of a kinesin-like calmodulin-binding protein from Arabidopsis: regulation by Ca(2+)/Calmodulin

Kinesin-like calmodulin-binding protein (KCBP), a novel kinesin-like protein from plants, is unique among kinesins and kinesin-like proteins in having a calmodulin-binding domain adjacent to its motor domain. KCBP localizes to mitotic microtubule (MT) arrays including the preprophase band, the spindle apparatus, and the phragmoplast, suggesting a role for KCBP in establishing these MT arrays by bundling MTs. To determine if KCBP bundles MTs, we expressed C-terminal motor and N-terminal tail domains of KCBP, and used the purified proteins in MT bundling assays. The 1.5 C protein with the motor and calmodulin-binding domains induced MT bundling. The 1.5 C-induced bundles were dissociated in the presence of Ca(2+)/calmodulin. Similar results were obtained with a 1.4 C protein, which lacks much of the coiled-coil region present in 1.5 C protein and does not form dimers. The N-terminal tail of KCBP, which contains an ATP-independent MT binding site, is also capable of bundling MTs. These results, together with the KCBP localization data, suggest the involvement of KCBP in establishing mitotic MT arrays during different stages of cell division and that Ca(2+)/calmodulin regulates the formation of these MT arrays.     

Single-molecule analysis of the microtubule cross-linking protein MAP65-1 reveals a molecular mechanism for contact-angle-dependent microtubule bundling

Bundling of microtubules (MTs) is critical for the formation of complex MT arrays. In land plants, the interphase cortical MTs form bundles specifically following shallow-angle encounters between them. To investigate how cells select particular MT contact angles for bundling, we used an in vitro reconstitution approach consisting of dynamic MTs and the MT-cross-linking protein MAP65-1. We found that MAP65-1 binds to MTs as monomers and inherently targets antiparallel MTs for bundling. Dwell-time analysis showed that the affinity of MAP65-1 for antiparallel overlapping MTs is about three times higher than its affinity for single MTs and parallel overlapping MTs. We also found that purified MAP65-1 exclusively selects shallow-angle MT encounters for bundling, indicating that this activity is an intrinsic property of MAP65-1. Reconstitution experiments with mutant MAP65-1 proteins with different numbers of spectrin repeats within the N-terminal rod domain showed that the length of the rod domain is a major determinant of the range of MT bundling angles. The length of the rod domain also determined the distance between MTs within a bundle. Together, our data show that the rod domain of MAP65-1 acts both as a spacer and as a structural element that specifies the MT encounter angles that are conducive for bundling.     

Microtubule Patterning in the Presence of Stationary Motor Distributions

In this paper, we construct a novel nonlocal transport model that describes the evolution of microtubules (MTs) as they interact with stationary distributions of motor proteins. An advection term accounts for directed MT transport (sliding due to motor protein action), and an integral term accounts for reorientation of MTs due to their interactions with cross-linking motor proteins. Simulations of our model show how MT patterns depend on boundary constraints, as well as model parameters that represent motor speed, cross-linking capability (motor activity), and directionality. In large domains, and using motor parameter values consistent with experimentally-derived values, we find that patterns such as asters, vortices, and bundles are able to persist. In vivo, MTs take on aster patterns during interphase and they form bundles in neurons and polarized epithelial cells. Vortex patterns have not been observed in vivo, however, are found in in vitro experiments. In constrained domains, we find that similar patterns form (asters, bundles, and vortices). However, we also find that when two opposing motors are present, anti-parallel bundles are able to form, resembling the mitotic spindle during cell division. This model demonstrates how MT sliding and MT reorientation are sufficient to produce experimentally observed patterns.     

A computational model predicts Xenopus meiotic spindle organization

The metaphase spindle is a dynamic bipolar structure crucial for proper chromosome segregation, but how microtubules (MTs) are organized within the bipolar architecture remains controversial. To explore MT organization along the pole-to-pole axis, we simulated meiotic spindle assembly in two dimensions using dynamic MTs, a MT cross-linking force, and a kinesin-5-like motor. The bipolar structures that form consist of antiparallel fluxing MTs, but spindle pole formation requires the addition of a NuMA-like minus-end cross-linker and directed transport of MT depolymerization activity toward minus ends. Dynamic instability and minus-end depolymerization generate realistic MT lifetimes and a truncated exponential MT length distribution. Keeping the number of MTs in the simulation constant, we explored the influence of two different MT nucleation pathways on spindle organization. When nucleation occurs throughout the spindle, the simulation quantitatively reproduces features of meiotic spindles assembled in Xenopus egg extracts.     

Rapid assembly and collective behavior of microtubule bundles in the presence of polyamines

Microtubules (MTs) are cylindrical cytoskeleton polymers composed of α-β tubulin heterodimers whose dynamic properties are essential to fulfill their numerous cellular functions. In response to spatial confinement, dynamic MTs, even in the absence of protein partners, were shown to self-organize into higher order structures (spindle or striped structures) which lead to interesting dynamical properties (MT oscillations). In this study, we considered the assembly and sensitivity of dynamic MTs when in bundles. To perform this study, spermine, a natural tetravalent polyamine present at high concentrations in all eukaryote cells, was used to trigger MT bundling while preserving MT dynamics. Interestingly, we first show that, near physiological ionic strengths, spermine promotes the bundling of MTs whereas it does not lead to aggregation of free tubulin, which would have been detrimental to MT polymerization. Experimental and theoretical results also indicate that, to obtain a high rate of bundle assembly, bundling should take place at the beginning of assembly when rapid rotational movements of short and newly nucleated MTs are still possible. On the other hand, the bundling process is significantly slowed down for long MTs. Finally, we found that short MT bundles exhibit a higher sensitivity to cold exposure than do isolated MTs. To account for this phenomenon, we suggest that a collective behavior takes place within MT bundles because an MT entering into a phase of shortening could increase the probability of the other MTs in the same bundle to enter into shortening phase due to their close proximity. We then elaborate on some putative applications of our findings to in vivo conditions including neurons.     

Straighten up and fly right: microtubule dynamics and organization of non-centrosomal arrays in higher plants

Live cell imaging and genetic studies are demonstrating that cortical microtubule arrays in plant cells are dynamic structures in which microtubule (MT) bundles play a key role in creating array organization and function. Steps important for creating and organizing these arrays include recruitment of nucleation complexes to the cell cortex and to the lattices of previously established MTs, association of newly created MTs to the cell cortex, release of MTs from sites of nucleation, transport of released MTs by polymer treadmilling, and subsequent interactions between treadmilling MTs. The results of MT interactions include induced catastrophe, severing, and the capture and reorientation of growing polymer ends by bundling interactions. Together, these properties predict a capacity for self-ordering that is likely to play an important role in establishing the parallel organization of the arrays.     

Mechanical Regulation of Neurite Polarization and Growth: A Computational Study

The densely packed microtubule (MT) array found in neuronal cell projections (neurites) serves two fundamental functions simultaneously: it provides a mechanically stable track for molecular motor-based transport and produces forces that drive neurite growth. The local pattern of MT polarity along the neurite shaft has been found to differ between axons and dendrites. In axons, the neurons’ dominating long projections, roughly 90% of the MTs orient with their rapidly growing plus end away from the cell body, whereas in vertebrate dendrites, their orientations are locally mixed. Molecular motors are known to be responsible for cytoskeletal ordering and force generation, but their collective function in the dense MT cytoskeleton of neurites remains elusive. We here hypothesized that both the polarity pattern of MTs along the neurite shaft and the shaft’s global extension are simultaneously driven by molecular motor forces and should thus be regulated by the mechanical load acting on the MT array as a whole. To investigate this, we simulated cylindrical bundles of MTs that are cross-linked and powered by molecular motors by iteratively solving a set of force-balance equations. The bundles were subjected to a fixed load arising from actively generated tension in the actomyosin cortex enveloping the MTs. The magnitude of the load and the level of motor-induced connectivity between the MTs have been varied systematically. With an increasing load and decreasing motor-induced connectivity between MTs, the bundles became wider in cross section and extended more slowly, and the local MT orientational order was reduced. These results reveal two, to our knowledge, novel mechanical factors that may underlie the distinctive development of the MT cytoskeleton in axons and dendrites: the cross-linking level of MTs by motors and the load acting on this cytoskeleton during growth.     

Microtubule-associated proteins in higher plants

A variety of microtubule-associated proteins (MAPs) have been reported in higher plants. Microtubule (MT) polymerization starts from the γ-tubulin complex (γTuC), a component of the MT nucleation site. MAP200/MOR1 and katanin regulate the length of the MT by promoting the dynamic instability of MTs and cutting MTs, respectively. In construction of different MT structures, MTs are bundled or are associated with other components—actin filaments, the plasma membrane, and organelles. The MAP65 family and some of kinesin family are important in bundling MTs. MT plus-end-tracking proteins (+TIPs) including end-binding protein 1 (EB1), Arabidopsis thaliana kinesin 5 (ATK5), and SPIRAL 1 (SPR1) localize to the plus end of MTs. It has been suggested that +TIPs are involved in binding of MT to other structures. Phospholipase D (PLD) is a possible candidate responsible for binding of MTs to the plasma membrane. Many candidates have been reported as actin-binding MAPs, for example calponin-homology domain (KCH) family kinesin, kinesin-like calmodulin-binding protein (KCBP), and MAP190. RNA distribution and translation depends on MT structures, and several RNA-related MAPs have been reported. This article gives an overview of predicted roles of these MAPs in higher plants.     

Microtubule motor Ncd induces sliding of microtubules in vivo

The mitotic spindle is a microtubule (MT)-based molecular machine that serves for equal segregation of chromosomes during cell division. The formation of the mitotic spindle requires the activity of MT motors, including members of the kinesin-14 family. Although evidence suggests that kinesins-14 act by driving the sliding of MT bundles in different areas of the spindle, such sliding activity had never been demonstrated directly. To test the hypothesis that kinesins-14 can induce MT sliding in living cells, we developed an in vivo assay, which involves overexpression of the kinesin-14 family member Drosophila Ncd in interphase mammalian fibroblasts. We found that green fluorescent protein (GFP)-Ncd colocalized with cytoplasmic MTs, whose distribution was determined by microinjection of Cy3 tubulin into GFP-transfected cells. Ncd overexpression resulted in the formation of MT bundles that exhibited dynamic "looping" behavior never observed in control cells. Photobleaching studies and fluorescence speckle microscopy analysis demonstrated that neighboring MTs in bundles could slide against each other with velocities of 0.1 microm/s, corresponding to the velocities of movement of the recombinant Ncd in in vitro motility assays. Our data, for the first time, demonstrate generation of sliding forces between adjacent MTs by Ncd, and they confirm the proposed roles of kinesins-14 in the mitotic spindle morphogenesis.     

The Ncd tail domain promotes microtubule assembly and stability

Non-claret disjunctional (Ncd) is a Drosophila kinesin-like motor required for spindle assembly and maintenance in oocytes and early embryos. Ncd has an ATP-independent microtubule binding site in the N-terminal tail domain as well as an ATP-dependent microtubule binding site in the C-terminal motor domain. The Ncd tail domain shares many properties with the microtubule-associated proteins that regulate microtubule assembly, including microtubule binding and bundling activity and an abundance of basic and proline residues. Given these similarities, we examined the ability of Ncd tail domain proteins to promote MT assembly and stability. The results indicate that the Ncd tail domain can promote MT assembly and stabilize MTs against conditions that induce MT disassembly, and suggest that Ncd may influence MT dynamics within the spindle.     

Thermal drift is enough to drive a single microtubule along its axis even in the absence of motor proteins.

One-dimensional diffusion of microtubules (MTs), a back-and-forth motion of MTs due to thermal diffusion, was reported in dynein motility assay. The interaction between MTs and dynein that allows such motion was implicated in its importance in the force generating cycle of dynein ATPase cycle. However, it was not known whether the phenomenon is special to motor proteins. Here we show two independent examples of one-dimensional diffusion of MTs in the absence of motor proteins. Dynamin, a MT-activated GTPase, causes a nucleotide dependent back-and-forth movement of single MT up to 1 micron along the longitudinal axes, although the MT never showed unidirectional consistent movement. Quantitative analysis of the motion and its nucleotide condition indicates that the motion is due to a thermal driven diffusion, restricted to one dimension, under the weak interaction between MT and dynamin. However, specific protein-protein interaction is not essential for the motion, because similar back-and-forth movement of MT was achieved on coverslips coated with only 0.8% methylcellulose. Both cases demonstrate that thermal diffusion could provide a considerable sliding of MTs only if MTs are restricted on the surface appropriately.     

Effects of {gamma}-tubulin complex proteins on microtubule nucleation and catastrophe in fission yeast

Although gamma-tubulin complexes (gamma-TuCs) are known as microtubule (MT) nucleators, their function in vivo is still poorly defined. Mto1p (also known as mbo1p or mod20p) is a gamma-TuC-associated protein that recruits gamma-TuCs specifically to cytoplasmic MT organizing centers (MTOCs) and interphase MTs. Here, we investigated gamma-TuC function by analyzing MT behavior in mto1Delta and alp4 (GCP2 homologue) mutants. These cells have free, extra-long interphase MTs that exhibit abnormal behaviors such as cycles of growth and breakage, MT sliding, treadmilling, and hyperstability. The plus ends of interphase and spindle MTs grow continuously, exhibiting catastrophe defects that are dependent on the CLIP170 tip1p. The minus ends of interphase MTs exhibit shrinkage and pauses. As mto1Delta mutants lack cytoplasmic MTOCs, cytoplasmic MTs arise from spindle or other intranuclear MTs that exit the nucleus. Our findings show that mto1p and gamma-TuCs affect multiple properties of MTs including nucleation, nuclear attachment, plus-end catastrophe, and minus-end shrinkage.     

Feo, the Drosophila homolog of PRC1, is required for central-spindle formation and cytokinesis

We performed a functional analysis of fascetto (feo), a Drosophila gene that encodes a protein homologous to the Ase1p/PRC1/MAP65 conserved family of microtubule-associated proteins (MAPs). These MAPs are enriched at the spindle midzone in yeast and mammals and at the fragmoplast in plants, and are essential for the organization and function of these microtubule arrays. Here we show that the Feo protein is specifically enriched at the central-spindle midzone and that its depletion either by mutation or by RNAi results in aberrant central spindles. In Feo-depleted cells, late anaphases showed normal overlap of the antiparallel MTs at the cell equator, but telophases displayed thin MT bundles of uniform width instead of robust hourglass-shaped central spindles. These thin central spindles exhibited diffuse localizations of both the Pav and Asp proteins, suggesting that these spindles comprise improperly oriented MTs. Feo-depleted cells also displayed defects in the contractile apparatus that correlated with those in the central spindle; late anaphase cells formed regular contractile structures, but these structures did not constrict during telophase, leading to failures in cytokinesis. The phenotype of Feo-depleted telophases suggests that Feo interacts with the plus ends of central spindle MTs so as to maintain their precise interdigitation during anaphase-telophase MT elongation and antiparallel sliding.     

Mechanism for Anaphase B: Evaluation of “Slide-and-Cluster” versus “Slide-and-Flux-or-Elongate” Models

Elongation of the mitotic spindle during anaphase B contributes to chromosome segregation in many cells. Here, we quantitatively test the ability of two models for spindle length control to describe the dynamics of anaphase B spindle elongation using experimental data from Drosophila embryos. In the slide-and-flux-or-elongate (SAFE) model, kinesin-5 motors persistently slide apart antiparallel interpolar microtubules (ipMTs). During pre-anaphase B, this outward sliding of ipMTs is balanced by depolymerization of their minus ends at the poles, producing poleward flux, while the spindle maintains a constant length. Following cyclin B degradation, ipMT depolymerization ceases so the sliding ipMTs can push the poles apart. The competing slide-and-cluster (SAC) model proposes that MTs nucleated at the equator are slid outward by the cooperative actions of the bipolar kinesin-5 and a minus-end-directed motor, which then pulls the sliding MTs inward and clusters them at the poles. In assessing both models, we assume that kinesin-5 preferentially cross-links and slides apart antiparallel MTs while the MT plus ends exhibit dynamic instability. However, in the SAC model, minus-end-directed motors bind the minus ends of MTs as cargo and transport them poleward along adjacent, parallel MT tracks, whereas in the SAFE model, all MT minus ends that reach the pole are depolymerized by kinesin-13. Remarkably, the results show that within a narrow range of MT dynamic instability parameters, both models can reproduce the steady-state length and dynamics of pre-anaphase B spindles and the rate of anaphase B spindle elongation. However, only the SAFE model reproduces the change in MT dynamics observed experimentally at anaphase B onset. Thus, although both models explain many features of anaphase B in this system, our quantitative evaluation of experimental data regarding several different aspects of spindle dynamics suggests that the SAFE model provides a better fit.     

Nuclear congression is driven by cytoplasmic microtubule plus end interactions in S. cerevisiae

Nuclear movement before karyogamy in eukaryotes is known as pronuclear migration or as nuclear congression in Saccharomyces cerevisiae. In this study, S. cerevisiae is used as a model system to study microtubule (MT)-dependent nuclear movements during mating. We find that nuclear congression occurs through the interaction of MT plus ends rather than sliding and extensive MT overlap. Furthermore, the orientation and attachment of MTs to the shmoo tip before cell wall breakdown is not required for nuclear congression. The MT plus end-binding proteins Kar3p, a class 14 COOH-terminal kinesin, and Bik1p, the CLIP-170 orthologue, localize to plus ends in the shmoo tip and initiate MT interactions and depolymerization after cell wall breakdown. These data support a model in which nuclear congression in budding yeast occurs by plus end MT capture and depolymerization, generating forces sufficient to move nuclei through the cytoplasm. This is the first evidence that MT plus end interactions from oppositely oriented organizing centers can provide the force for organelle transport in vivo.     

Geometrical nonlinear elasticity of axon under tension: A coarse-grained computational study

Axon bundles cross-linked by microtubule (MT) associate proteins and bounded by a shell skeleton are critical for normal function of neurons. Understanding effects of the complexly geometrical parameters on their mechanical properties can help gain a biomechanical perspective on the neurological functions of axons and thus brain disorders caused by the structural failure of axons. Here, the tensile mechanical properties of MT bundles cross-linked by tau proteins are investigated by systematically tuning MT length, axonal cross-section radius, and tau protein spacing in a bead-spring coarse-grained model. Our results indicate that the stress-strain curves of axons can be divided into two regimes, a nonlinear elastic regime dominated by rigid-body like inter-MT sliding, and a linear elastic regime dominated by affine deformation of both tau proteins and MTs. From the energetic analyses, first, the tau proteins dominate the mechanical performance of axons under tension. In the nonlinear regime, tau proteins undergo a rigid-body like rotating motion rather than elongating, whereas in the nonlinear elastic regime, tau proteins undergo a flexible elongating deformation along the MT axis. Second, as the average spacing between adjacent tau proteins along the MT axial direction increases from 25 to 125 nm, the Young’s modulus of axon experiences a linear decrease whereas with the average space varying from 125 to 175 nm, and later reaches a plateau value with a stable fluctuation. Third, the increment of the cross-section radius of the MT bundle leads to a decrease in Young’s modulus of axon, which is possibly attributed to the decrease in MT numbers per cross section. Overall, our research findings offer a new perspective into understanding the effects of geometrical parameters on the mechanics of MT bundles as well as serving as a theoretical basis for the development of artificial MT complexes potentially toward medical applications.     

Characterization of the Arabidopsis augmin complex uncovers its critical function in the assembly of the acentrosomal spindle and phragmoplast microtubule arrays

Plant cells assemble the bipolar spindle and phragmoplast microtubule (MT) arrays in the absence of the centrosome structure. Our recent findings in Arabidopsis thaliana indicated that AUGMIN subunit3 (AUG3), a homolog of animal dim γ-tubulin 3, plays a critical role in γ-tubulin-dependent MT nucleation and amplification during mitosis. Here, we report the isolation of the entire plant augmin complex that contains eight subunits. Among them, AUG1 to AUG6 share low sequence similarity with their animal counterparts, but AUG7 and AUG8 share homology only with proteins of plant origin. Genetic analyses indicate that the AUG1, AUG2, AUG4, and AUG5 genes are essential, as stable mutations in these genes could only be transmitted to heterozygous plants. The sterile aug7-1 homozygous mutant in which AUG7 expression is significantly reduced exhibited pleiotropic phenotypes of seriously retarded vegetative and reproductive growth. The aug7-1 mutation caused delocalization of γ-tubulin in the mitotic spindle and phragmoplast. Consequently, spindles were abnormally elongated, and their poles failed to converge, as MTs were splayed to discrete positions rendering deformed arrays. In addition, the mutant phragmoplasts often had disorganized MT bundles with uneven edges. We conclude that assembly of MT arrays during plant mitosis depends on the augmin complex, which includes two plant-specific subunits.