Parallel Microtubules Research Articles

Modeling Studies of the Mechanism of Context-Dependent Bidirectional Movements of Kinesin-14 Motors.

Kinesin-14s, a subfamily of the large superfamily of kinesin motor proteins, function mainly in spindle assembly and maintenance during mitosis and meiosis. KlpA from Aspergillus nidulans and GiKIN14a from Giardia intestinalis are two types of kinesin-14s. Available experimental results puzzlingly showed that while KlpA moves preferentially toward the minus end in microtubule-gliding setups and inside parallel microtubule overlaps, it moves preferentially toward the plus end on single microtubules. More puzzlingly, the insertion of an extra polypeptide linker in the central region of the neck stalk switches the motility direction of KlpA on single microtubules to the minus end. Prior experimental results showed that GiKIN14a moves preferentially toward the minus end on single microtubules in either tailless or full-length forms. The tail not only greatly enhances the processivity but also accelerates the ATPase rate and velocity of GiKIN14a. The insertion of an extra polypeptide linker in the central region of the neck stalk reduces the ATPase rate of GiKIN14a. However, the underlying mechanism of these puzzling dynamical features for KlpA and GiKIN14a is unclear. Here, to understand this mechanism, the dynamics of KlpA and GiKIN14a were studied theoretically on the basis of the proposed model, incorporating potential changes between the kinesin head and microtubule, as well as the potential between the tail and microtubule. The theoretical results quantitatively explain the available experimental results and provide predicted results. It was found that the elasticity of the neck stalk determines the directionality of KlpA on single microtubules and affects the ATPase rate and velocity of GiKIN14a on single microtubules.

Human kinesin-5 KIF11 drives the helical motion of anti-parallel and parallel microtubules around each other.

During mitosis, motor proteins and microtubule-associated protein organize the spindle apparatus by cross-linking and sliding microtubules. Kinesin-5 plays a vital role in spindle formation and maintenance, potentially inducing twist in the spindle fibers. The off-axis power stroke of kinesin-5 could generate this twist, but its implications in microtubule organization remain unclear. Here, we investigate 3D microtubule-microtubule sliding mediated by the human kinesin-5, KIF11, and found that the motor caused right-handed helical motion of anti-parallel microtubules around each other. The sidestepping ratio increased with reduced ATP concentration, indicating that forward and sideways stepping of the motor are not strictly coupled. Further, the microtubule-microtubule distance (motor extension) during sliding decreased with increasing sliding velocity. Intriguingly, parallel microtubules cross-linked by KIF11 orbited without forward motion, with nearly full motor extension. Altering the length of the neck linker increased the forward velocity and pitch of microtubules in anti-parallel overlaps. Taken together, we suggest that helical motion and orbiting of microtubules, driven by KIF11, contributes to flexible and context-dependent filament organization, as well as torque regulation within the mitotic spindle.

Chromosomal passenger complex condensates generate parallel microtubule bundles in vitro

The mitotic spindle contains many bundles of microtubules (MT) including midzones and kinetochore fibers, but little is known about how bundled structures are formed. Here, we show that the Chromosomal Passenger Complex (CPC) purified from E. coli undergoes liquid-liquid demixing (LLD) in vitro. An emergent property of the resultant condensates is to generate parallel MT bundles when incubated with free tubulin and GTP in vitro. We demonstrate MT bundles emerge from CPC droplets with protruding minus-ends that then grow into long, tapered MT structures. During this growth, we found the CPC in these condensates apparently reorganize to coat and bundle the resulting MT structures. CPC mutants attenuated for LLD or MT binding prevented the generation of parallel MT bundles in vitro and reduced the number of MTs present at spindle midzones in HeLa cells. Our data demonstrate that an in vitro biochemical activity to produce microtubule bundles emerges after the concentration of the CPC and provides models for how cells generate parallel-bundled MT structures that are important for the assembly of the mitotic spindle. Moreover, these data suggest that cells contain microtubule organizing centers that generate microtubule bundles that emerge with the opposite polarity from centrosomes.

Trim46 knockout impaired neuronal architecture and caused hypoactive behavior in rats.

Tripartite motif (TRIM46) is a relatively novel protein that belongs to tripartite motif family. TRIM46 organizes parallel microtubule arrays on the axons, which are important for neuronal polarity and axonal function. TRIM46 is highly expressed in the brain, but its biological function in adults has not yet been determined. Trim46 knockout (KO) rat line was established using CRISPR/cas9. Trim46 KO rats had smaller hippocampus sizes, fewer neuronal dendritic arbors and dendritic spines, and shorter and more distant axon initial segment. Furthermore, the protein interaction between endogenous TRIM46 and FK506 binding protein 5 (FKBP5) in brain tissues was determined; Trim46 KO increased hippocampal FKBP5 protein levels and decreased hippocampal protein kinase B (Akt) phosphorylation, gamma-aminobutyric acid type A receptor subunit alpha1 (GABRA1) and glutamate ionotropic receptor NMDA type subunit 1 (NMDAR1) protein levels. Trim46 KO rats exhibited hypoactive behavioral changes such as reduced spontaneous activity, social interaction, sucrose preference, impaired prepulse inhibition (PPI), and short-term reference memory. These results demonstrate the significant impact of Trim46 KO on brain structure and behavioral function. This study revealed a novel potential association of TRIM46 with dendritic development and neuropsychiatric behavior, providing new insights into the role of TRIM46 in the brain.

The midbody component Prc1-like is required for microtubule reorganization during cytokinesis and dorsal determinant segregation in the early zebrafish embryo.

We show that the zebrafish maternal-effect mutation too much information (tmi) corresponds to zebrafish prc1-like (prc1l), which encodes a member of the MAP65/Ase1/PRC1 family of microtubule-associated proteins. Embryos from tmi homozygous mutant mothers display cytokinesis defects in meiotic and mitotic divisions in the early embryo, indicating that Prc1l has a role in midbody formation during cell division at the egg-to-embryo transition. Unexpectedly, maternal Prc1l function is also essential for the reorganization of vegetal pole microtubules required for the segregation of dorsal determinants. Whereas Prc1 is widely regarded to crosslink microtubules in an antiparallel conformation, our studies provide evidence for an additional function of Prc1l in the bundling of parallel microtubules in the vegetal cortex of the early embryo during cortical rotation and prior to mitotic cycling. These findings highlight common yet distinct aspects of microtubule reorganization that occur during the egg-to-embryo transition, driven by maternal product for the midbody component Prc1l and required for embryonic cell division and pattern formation.

ARK2 stabilizes the plus-end of microtubules and promotes microtubule bundling in Arabidopsis.

Microtubule dynamics and organization are important for plant cell morphogenesis and development. The microtubule-based motor protein kinesins are mainly responsible for the transport of some organelles and vesicles, although several have also been shown to regulate microtubule organization. The ARMADILLO REPEAT KINESIN (ARK) family is a plant-specific motor protein subfamily that consists of three members (ARK1, ARK2, and ARK3) in Arabidopsis thaliana. ARK2 has been shown to participate in root epidermal cell morphogenesis. However, whether and how ARK2 associates with microtubules needs further elucidation. Here, we demonstrated that ARK2 co-localizes with microtubules and facilitates microtubule bundling in vitro and in vivo. Pharmacological assays and microtubule dynamics analyses indicated that ARK2 stabilizes cortical microtubules. Live-cell imaging revealed that ARK2 moves along cortical microtubules in a processive mode and localizes both at the plus-end and the sidewall of microtubules. ARK2 therefore tracks and stabilizes the growing plus-ends of microtubules, which facilitates the formation of parallel microtubule bundles.

Injectable and ultra-compressible shape-memory mushroom: Highly aligned microtubules for ultra-fast blood absorption and hemostasis

Noncompressible bleeding is associated with a significant source of traumatic fatality. Due to its inaccessibility to the pressure dressing, patients admitted with internal bleeding mainly rely on blood transfusion, coming with a high cost and immunological side effects. Injectable materials have emerged as a strategy; however, they have limitations on either large mechanical compressibilty or blood clotting speed and blood absorbing capacity. Here, we report that the mushroom's (Agaricus bisporus) stalk has aligned microtubular passages, which shows exceptionally fast capillary effects to draw liquid. After decellularization, the foam has primary component of chitin and maintains the highly parallel microtubules, which endorse exceptionally compressible (90% strain) and injectable to the deep wound, swiftly absorb the blood, elicit clotting by activating blood cells and platelets to promote blood clot formation. The foams' negatively charged surface and fast absorption channels can activate the intrinsic coagulation pathway and non-clotting factor dependent pathway to promote immediate fibrin growth. The hydrophilicity improves the adhesion of the platelets and blood cells, forming firm clotting. The channelled and high compressible foams show great hemostatic performances in vitro blood and in vivo livers and hearts.

Oscillatory movement of a dynein-microtubule complex crosslinked with DNA origami.

Bending of cilia and flagella occurs when axonemal dynein molecules on one side of the axoneme produce force and move toward the microtubule (MT) minus end. These dyneins are then pulled back when the axoneme bends in the other direction, meaning oscillatory back and forth movement of dynein during repetitive bending of cilia/flagella. There are various factors that may regulate the dynein activity, e.g. the nexin-dynein regulatory complex, radial spokes, and central apparatus. In order to understand the basic mechanism of dynein's oscillatory movement, we constructed a simple model system composed of MTs, outer-arm dyneins, and crosslinks between the MTs made of DNA origami. Electron microscopy (EM) showed pairs of parallel MTs crossbridged by patches of regularly arranged dynein molecules bound in two different orientations, depending on which of the MTs their tails bind to. The oppositely oriented dyneins are expected to produce opposing forces when the pair of MTs have the same polarity. Optical trapping experiments showed that the dynein-MT-DNA-origami complex actually oscillates back and forth after photolysis of caged ATP. Intriguingly, the complex, when held at one end, showed repetitive bending motions. The results show that a simple system composed of ensembles of oppositely oriented dyneins, MTs, and inter-MT crosslinkers, without any additional regulatory structures, has an intrinsic ability to cause oscillation and repetitive bending motions.

Alleles of unc-33/CRMP exhibit defects during Caenorhabditis elegans epidermal morphogenesis.

Microtubule-associated proteins regulate the dynamics, organization, and function of microtubules, impacting a number of vital cellular processes. CRMPs have been shown to control microtubule assembly and axon outgrowth during neuronal differentiation. While many microtubule-associated proteins have been linked to roles in cell division and neuronal development, it is still unclear the complement that control the formation of parallel microtubule arrays in epithelial cells. Here we show through time-lapse DIC microscopy that Caenorhabditis elegans embryos homozygous for the weak loss-of-function allele unc-33(e204) progress more slowly through epidermal morphogenesis, while animals homozygous for strong loss-of-function alleles exhibit more embryonic lethality. Identification of two novel missense mutations in unc-33(e572), Val476Gly, and Ser731Thr, lead to computational approaches to determine the potential effects of these changes on UNC-33/CRMP structure. Molecular dynamics simulations show that for Asp389Asn and Arg502His, two other known missense mutations, local changes in protein-protein hydrogen bonding affect the stability of the protein. However, the Val476Gly/Ser731Thr combination does not alter the structure or energetics of UNC-33 drastically when compared to the wild-type protein. These results support a novel role for UNC-33/CRMP in C. elegans epidermal development and shed light on how individual amino acid changes cause a loss-of-function in UNC-33.

Tau, XMAP215/Msps and Eb1 co-operate interdependently to regulate microtubule polymerisation and bundle formation in axons.

The formation and maintenance of microtubules requires their polymerisation, but little is known about how this polymerisation is regulated in cells. Focussing on the essential microtubule bundles in axons of Drosophila and Xenopus neurons, we show that the plus-end scaffold Eb1, the polymerase XMAP215/Msps and the lattice-binder Tau co-operate interdependently to promote microtubule polymerisation and bundle organisation during axon development and maintenance. Eb1 and XMAP215/Msps promote each other's localisation at polymerising microtubule plus-ends. Tau outcompetes Eb1-binding along microtubule lattices, thus preventing depletion of Eb1 tip pools. The three factors genetically interact and show shared mutant phenotypes: reductions in axon growth, comet sizes, comet numbers and comet velocities, as well as prominent deterioration of parallel microtubule bundles into disorganised curled conformations. This microtubule curling is caused by Eb1 plus-end depletion which impairs spectraplakin-mediated guidance of extending microtubules into parallel bundles. Our demonstration that Eb1, XMAP215/Msps and Tau co-operate during the regulation of microtubule polymerisation and bundle organisation, offers new conceptual explanations for developmental and degenerative axon pathologies.

Trim9 and Klp61F promote polymerization of new dendritic microtubules along parallel microtubules.

Axons and dendrites are distinguished by microtubule polarity. In Drosophila, dendrites are dominated by minus-end-out microtubules, whereas axons contain plus-end-out microtubules. Local nucleation in dendrites generates microtubules in both orientations. To understand why dendritic nucleation does not disrupt polarity, we used live imaging to analyze the fate of microtubules generated at branch points. We found that they had different rates of success exiting the branch based on orientation: correctly oriented minus-end-out microtubules succeeded in leaving about twice as often as incorrectly oriented microtubules. Increased success relied on other microtubules in a parallel orientation. From a candidate screen, we identified Trim9 and kinesin-5 (Klp61F) as machinery that promoted growth of new microtubules. In S2 cells, Eb1 recruited Trim9 to microtubules. Klp61F promoted microtubule growth in vitro and in vivo, and could recruit Trim9 in S2 cells. In summary, the data argue that Trim9 and kinesin-5 act together at microtubule plus ends to help polymerizing microtubules parallel to pre-existing ones resist catastrophe.

Insights into the Morphology of Haplozoan Parasites (Dinoflagellata) using Confocal Laser Scanning Microscopy.

We describe new insights into the morphology and life history of the bizarre parasite Haplozoon axiothellae (Dinoflagellata) using light microscopy (LM), scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). Trophonts were isolated from the intestines of host maldanid polychaetes, Axiothella rubrocincta, collected from San Juan Island, Washington, USA. LM and SEM confirmed features previously observed, such as amphiesmal projections, mature and immature junctions between the nucleated compartments of the vermiform syncytium and visible polygonal alveoli. CLSM of adult trophonts fluorescently stained for DNA, tubulin, centrin, and plasma membrane demonstrated several new ultrastructural traits: (1) an extensive basket of parallel microtubules within the trophomere used for host attachment, (2) two physically separated MTOCs (i.e. putative pairs of basal bodies) beneath pores on the ventral side of each compartment, (3) robust mitotic and/or meiotic spindles associated with one to four nuclei in each compartment, (4) spindles with polar bodies that are disconnected from the MTOCs, (5) a centrin-stained fibril within the trophomeres that potentially functions to retract the motile stylet, and (6) cytokinesis in the posterior-most compartments. This study renames haplozoan compartments using the suffix "-mere" rather than "-cyte" (i.e. trophomere, gonomere, sporomere) to reflect their status within a single syncytium.

Trim46 contributes to the midbrain development via Sonic Hedgehog signaling pathway in zebrafish embryos

ABSTRACT TRIM46 is a RING finger E3 ligase which belongs to TRIM (tripartite motif-containing) protein family. TRIM46 is required for neuronal polarity and axon specification by driving the formation of parallel microtubule arrays, whereas its embryological functions remain to be determined yet. Expression patterns and biological functions of trim46a, a zebrafish homologue of TRIM46, were studied in zebrafish embryo. First, maternal transcripts of trim46a were present at 1 cell stage whereas zygotic messages were abundant in the eyes, MHB (Midbrain-Hindbrain Boundary) and hindbrain at 24 hpf (hours post fertilization). Second, transcriptional regulatory region of trim46a contains cis-acting elements binding a transcriptional factor Foxa2. Transcription of foxa2 is positively regulated by Sonic Hedgehog (SHH), and treatment of cyclopamine, an SHH inhibitor, represses transcription of foxa2 in 4 hpf through 24 hpf embryos. Third, the transcriptional repression of foxa2 inhibited transcription of trim46a to cause developmental defects in the midbrain and MHB. Finally, spatiotemporal expression patterns of a midbrain marker otx2b in the developmental defects confirmed inhibition of SHH by cyclopamine caused underdevelopment of the midbrain and MHB at 24 hpf. We propose a signaling network where trim46a contributes to development of the midbrain and MHB via Foxa2, a downstream element of SHH signaling in zebrafish embryogenesis.

Microtubule catastrophe under force: mathematical and computational results from a Brownian ratchet model

In the intracellular environment, the intrinsic dynamics of microtubule filaments is often hindered by the presence of barriers of various kind, such as kinetochore complexes and cell cortex, which impact their polymerisation force and dynamical properties such as catastrophe frequency. We present a theoretical study of the effect of a forced barrier, also subjected to thermal noise, on the statistics of catastrophe events in a single microtubule as well as a ‘bundle’ of two parallel microtubules. For microtubule dynamics, which includes growth, detachment, hydrolysis and the consequent dynamic instability, we employ a one-dimensional discrete stochastic model. The dynamics of the barrier is captured by over-damped Langevin equation, while its interaction with a growing filament is assumed to be hard-core repulsion. A unified treatment of the continuum dynamics of the barrier and the discrete dynamics of the filament is realized using a hybrid Fokker–Planck equation. An explicit mathematical formula for the force-dependent catastrophe frequency of a single microtubule is obtained by solving the above equation, under some assumptions. The prediction agrees well with results of numerical simulations in the appropriate parameter regime. More general situations are studied via numerical simulations. To investigate the extent of ‘load-sharing’ in a microtubule bundle, and its impact on the frequency of catastrophes, the dynamics of a two-filament bundle is also studied. Here, two parallel, non-interacting microtubules interact with a common, forced barrier. The equations for the two-filament model, when solved using a mean-field assumption, predicts equal sharing of load between the filaments. However, numerical results indicate the existence of a wide spectrum of load-sharing behaviour, which is characterized using a dimensionless parameter.

Intrinsically Disordered Domain of Kinesin-3 Kif14 Enables Unique Functional Diversity

In addition to their force-generating motor domains, kinesin motor proteins feature various accessory domains enabling them to fulfill a variety of functions in the cell. Human kinesin-3, Kif14, localizes to the midbody of the mitotic spindle and is involved in the progression of cytokinesis. The specific motor properties enabling Kif14's cellular functions, however, remain unknown. Here, we show invitro that the intrinsically disordered N-terminal domain of Kif14 enables unique functional diversity of the kinesin. Using single molecule TIRF microscopy, we found that Kif14 exists either as a diffusible monomer or as processive dimer and that the disordered domain (1) enables diffusibility of the monomeric Kif14, (2) renders the dimeric Kif14 super-processive and enables the kinesin to pass through highly crowded areas, (3) enables robust, autonomous Kif14 tracking of growing microtubule tips, independent of microtubule end-binding (EB) proteins, and (4) is sufficient to enable crosslinking of parallel microtubules and necessary to enable Kif14-driven sliding of antiparallel ones. We explain these features of Kif14 by the observed diffusible interaction of the disordered domain with the microtubule lattice and the observed increased affinity of the disordered domain for GTP-bound tubulin. We suggest that the disordered domain tethers the motor domain to the microtubule providing a diffusible foothold and a regulatory hub, tuning the kinesin's interaction with microtubules. Our findings thus exemplify pliable protein tethering as a fundamental mechanism of molecular motor regulation.

Ultrastructure of the spermatozoon of Lecithostaphylus parexocoeti (Digenea, Microphalloidea, Zoogonidae) parasite of the flying fish Cheilopogon pinnatibarbatus (Teleostei, Exocoetidae) off Senegal and their implication on the phylogenetic relationships in Microphalloidea

This study shows the ultrastructure of the mature spermatozoon of Lecithostaphylus parexocoeti (Digenea, Microphalloidea, Zoogonidae) parasite of the flying fish Cheilopogon pinnatibarbatus (Exocoetidae) captured in the Atlantic Ocean near Dakar (Senegal). This is the first report of L. parexocoeti from Senegal and from this host species. The mature spermatozoon is filiform and exhibits two axonemes of the 9+“1” pattern of the Trepaxonemata, an external ornamentation associated with spine-like bodies, parallel cortical microtubules, a nucleus, two mitochondria and granules of glycogen. The particularities of the spermatozoon of L. parexocoeti are the location of the first mitochondrion and the morphology of its anterior extremity with only cortical microtubules and external ornamentations of the plasma membrane. To our knowledge, this type of anterior extremity has never been described in the superfamily Microphalloidea. However, the ultrastructure of the posterior extremity of the spermatozoon corresponds to the fasciolidean type that was previously described in the Zoogonidae.

Complementary Superresolution Visualization of Composite Plant Microtubule Organization and Dynamics.

Microtubule bundling is an essential mechanism underlying the biased organization of interphase and mitotic microtubular systems of eukaryotes in ordered arrays. Microtubule bundle formation can be exemplified in plants, where the formation of parallel microtubule systems in the cell cortex or the spindle midzone is largely owing to the microtubule crosslinking activity of a family of microtubule associated proteins, designated as MAP65s. Among the nine members of this family in Arabidopsis thaliana, MAP65-1 and MAP65-2 are ubiquitous and functionally redundant. Crosslinked microtubules can form high-order arrays, which are difficult to track using widefield or confocal laser scanning microscopy approaches. Here, we followed spatiotemporal patterns of MAP65-2 localization in hypocotyl cells of Arabidopsis stably expressing fluorescent protein fusions of MAP65-2 and tubulin. To circumvent imaging difficulties arising from the density of cortical microtubule bundles, we use different superresolution approaches including Airyscan confocal laser scanning microscopy (ACLSM), structured illumination microscopy (SIM), total internal reflection SIM (TIRF-SIM), and photoactivation localization microscopy (PALM). We provide insights into spatiotemporal relations between microtubules and MAP65-2 crossbridges by combining SIM and ACLSM. We obtain further details on MAP65-2 distribution by single molecule localization microscopy (SMLM) imaging of either mEos3.2-MAP65-2 stochastic photoconversion, or eGFP-MAP65-2 stochastic emission fluctuations under specific illumination conditions. Time-dependent dynamics of MAP65-2 were tracked at variable time resolution using SIM, TIRF-SIM, and ACLSM and post-acquisition kymograph analysis. ACLSM imaging further allowed to track end-wise dynamics of microtubules labeled with TUA6-GFP and to correlate them with concomitant fluctuations of MAP65-2 tagged with tagRFP. All different microscopy modules examined herein are accompanied by restrictions in either the spatial resolution achieved, or in the frame rates of image acquisition. PALM imaging is compromised by speed of acquisition. This limitation was partially compensated by exploiting emission fluctuations of eGFP which allowed much higher photon counts at substantially smaller time series compared to mEos3.2. SIM, TIRF-SIM, and ACLSM were the methods of choice to follow the dynamics of MAP65-2 in bundles of different complexity. Conclusively, the combination of different superresolution methods allowed for inferences on the distribution and dynamics of MAP65-2 within microtubule bundles of living A. thaliana cells.

Sperm characteristics in the digenean Diplodiscus amphichrus (Paramphistomoidea, Diplodiscidae), a parasite of the Chinese edible frog Hoplobatrachus rugulosus

The ultrastructural characteristics of the mature spermatozoon of Diplodiscus amphichrus (Digenea, Paramphistomoidea, Diplodiscidae) and their ultrastructural organisation were examined by means of transmission electron microscopy. Live digeneans were collected from the Chinese edible frog Hoplobatrachus rugulosus in Thailand. The male gamete of D. amphichrus is a filiform cell with two axonemes of the 9 + ’1’ trepaxonematan pattern, nucleus, one mitochondrion, parallel cortical microtubules, a well-developed lateral expansion, external ornamentation of the plasma membrane, spine-like bodies, and granules of glycogen. These ultrastructural characteristics have already been described in other paramphistomoids studied so far except for the cladorchiids, which present some differences. Two characteristics of the male gamete of D. amphichrus were found for the first time in a digenean: (i) the appearance of the initial part of the lateral expansion before the axonemes in the anterior extremity and (ii) the separation between the second axoneme and the nucleus in the posterior tip. Our results are compared with the available data in the Digenea and particularly with other paramphistomoids.

Bistability and oscillations in cooperative microtubule and kinetochore dynamics in the mitotic spindle

In the mitotic spindle microtubules attach to kinetochores via catch bonds during metaphase, and microtubule depolymerization forces give rise to stochastic chromosome oscillations. We investigate the cooperative stochastic microtubule dynamics in spindle models consisting of ensembles of parallel microtubules, which attach to a kinetochore via elastic linkers. We include the dynamic instability of microtubules and forces on microtubules and kinetochores from elastic linkers. A one-sided model, where an external force acts on the kinetochore is solved analytically employing a mean-field approach based on Fokker–Planck equations. The solution establishes a bistable force–velocity relation of the microtubule ensemble in agreement with stochastic simulations. We derive constraints on linker stiffness and microtubule number for bistability. The bistable force–velocity relation of the one-sided spindle model gives rise to oscillations in the two-sided model, which can explain stochastic chromosome oscillations in metaphase (directional instability). We derive constraints on linker stiffness and microtubule number for metaphase chromosome oscillations. Including poleward microtubule flux into the model we can provide an explanation for the experimentally observed suppression of chromosome oscillations in cells with high poleward flux velocities. Chromosome oscillations persist in the presence of polar ejection forces, however, with a reduced amplitude and a phase shift between sister kinetochores. Moreover, polar ejection forces are necessary to align the chromosomes at the spindle equator and stabilize an alternating oscillation pattern of the two kinetochores. Finally, we modify the model such that microtubules can only exert tensile forces on the kinetochore resulting in a tug-of-war between the two microtubule ensembles. Then, induced microtubule catastrophes after reaching the kinetochore are necessary to stimulate oscillations. The model can reproduce experimental results for kinetochore oscillations in PtK1 cells quantitatively.

The Tail of XCTK2 Contains Two Distinct Microtubule Binding Domains

Molecular motors organize the microtubules (MTs) that make up the mitotic spindle, and defects in motor function can cause improper spindle assembly that leads to chromosome mis-segregation. The Kinesin-14 family of molecular motors facilitates spindle pole focusing by cross-linking and sliding anti-parallel and parallel MTs through their motor and tail domains. How the tail contributes to these functions is unclear. Kinesin-14 tail MT binding is inhibited by importin α/β association through a nuclear localization signal (NLS). To understand how importin α/β association regulates the cross-linking and sliding activity of Kinesin-14s, we visualized Xenopus XCTK2 cross-linking and sliding in vitro. Surprisingly, importin α/β preferentially inhibited anti-parallel cross-linking and sliding. These results suggest that the tail may contain two different MT binding domains (MBDs) that recognize the anti-parallel and parallel MT orientations. To test this hypothesis, we created a deletion series of proteins from the N-terminus and C-terminus of the tail and tested them for MT binding. We identified two MBDs, which we call MBD1 and MBD2, that had MT binding abilities similar to the full-length tail. YPet-tagged MBD1, which contains the NLS, displayed robust FRET with importin α-CyPet and importin β, indicating tight association; whereas, MBD2 did not. In the presence of importin α/β, MBD1 was also completely inhibited from binding to MTs. These results are consistent with the model that MBD1 recognizes the anti-parallel conformation, and MBD2 recognizes the parallel conformation. We are currently generating site-specific mutations within MBD1 and MBD2 to inhibit MT binding so that we can demonstrate separation of cross-linking activities in the full-length protein. Once we identify the mutations that disrupt anti-parallel or parallel MT cross-linking, we will test how these modes of cross-linking and sliding contribute to spindle assembly.