Taxol-stabilized Microtubules Research Articles

The rate of microtubule breaking increases exponentially with curvature

Microtubules, cylindrical assemblies of tubulin proteins with a 25 nm diameter and micrometer lengths, are a central part of the cytoskeleton and also serve as building blocks for nanobiodevices. Microtubule breaking can result from the activity of severing enzymes and mechanical stress. Breaking can lead to a loss of structural integrity, or an increase in the numbers of microtubules. We observed breaking of taxol-stabilized microtubules in a gliding motility assay where microtubules are propelled by surface-adhered kinesin-1 motor proteins. We find that over 95% of all breaking events are associated with the strong bending following pinning events (where the leading tip of the microtubule becomes stuck). Furthermore, the breaking rate increased exponentially with increasing curvature. These observations are explained by a model accounting for the complex mechanochemistry of a microtubule. The presence of severing enzymes is not required to observe breaking at rates comparable to those measured previously in cells.

Cypin binds to tubulin heterodimers and microtubule protofilaments and regulates microtubule spacing in developing hippocampal neurons

Cytosolic PSD-95 interactor (cypin) is a multifunctional, guanine deaminase that plays a major role in shaping the morphology of the dendritic arbor of hippocampal and cortical neurons. Cypin catalyzes the Zn2+-dependent deamination of guanine to xanthine, which is then metabolized to uric acid by xanthine oxidase. Cypin binds to tubulin heterodimers via its carboxyl terminal region (amino acids (aa) 350–454), which contains a collapsin response mediator protein (CRMP) homology domain (aa 350–403). Moreover, this region alone is not sufficient to facilitate microtubule polymerization; therefore, additional cypin regions must be involved in this process. Here, we asked whether cypin binds to fully formed microtubules and how overexpression of cypin regulates the microtubule cytoskeleton in dendrites of cultured hippocampal neurons. Protein-protein docking strategies confirm that the cypin homodimer binds to tubulin heterodimers via amino acids within aa 350–454. Biochemical pull-down data suggest that aa 1–220 are necessary for cypin binding to soluble tubulin heterodimers and to taxol-stabilized microtubules. Molecular docking of the cypin homodimer to soluble tubulin heterodimers reveals a consistently observed docking pose using aa 47–71, 113–118, 174–178, and 411–418, which is consistent with our biochemical data. Additionally, overexpression of cypin in hippocampal neurons results in decreased spacing between microtubules. Our results suggest that several protein domains facilitate cypin-mediated polymerization of tubulin heterodimers into microtubules, possibly through a mechanism whereby cypin dimers bind to multiple tubulin heterodimers.

Microtubule-binding core of the tau protein.

The protein tau associates with microtubules to maintain neuronal health. Posttranslational modifications of tau interfere with this binding, leading to tau aggregation in neurodegenerative disorders. Here, we use solid-state nuclear magnetic resonance (NMR) to investigate the structure of the microtubule-binding domain of tau. Wild-type tau that contains four microtubule-binding repeats and a pseudorepeat R′ is studied. Complexed with taxol-stabilized microtubules, the immobilized residues exhibit well-resolved two-dimensional spectra that can be assigned to the amino-terminal region of R4 and the R′ domain. When tau coassembles with tubulin to form unstable microtubules, the R′ signals remain, whereas the R4 signals disappear, indicating that R′ remains immobilized, whereas R4 becomes more mobile. Therefore, R′ outcompetes the other four repeats to associate with microtubules. These NMR data, together with previous cryo–electron microscopy densities, indicate an extended conformation for microtubule-bound R′. R′ contains the largest number of charged residues among all repeats, suggesting that charge-charge interaction drives tau-microtubule association.

Near-Infrared Photobiomodulation of Living Cells, Tubulin, and Microtubules In Vitro

We report the results of experimental investigations involving photobiomodulation (PBM) of living cells, tubulin, and microtubules in buffer solutions exposed to near-infrared (NIR) light emitted from an 810 nm LED with a power density of 25 mW/cm2 pulsed at a frequency of 10 Hz. In the first group of experiments, we measured changes in the alternating current (AC) ionic conductivity in the 50–100 kHz range of HeLa and U251 cancer cell lines as living cells exposed to PBM for 60 min, and an increased resistance compared to the control cells was observed. In the second group of experiments, we investigated the stability and polymerization of microtubules under exposure to PBM. The protein buffer solution used was a mixture of Britton-Robinson buffer (BRB aka PEM) and microtubule cushion buffer. Exposure of Taxol-stabilized microtubules (~2 μM tubulin) to the LED for 120 min resulted in gradual disassembly of microtubules observed in fluorescence microscopy images. These results were compared to controls where microtubules remained stable. In the third group of experiments, we performed turbidity measurements throughout the tubulin polymerization process to quantify the rate and amount of polymerization for PBM-exposed tubulin vs. unexposed tubulin samples, using tubulin resuspended to final concentrations of ~ 22.7 μM and ~ 45.5 μM in the same buffer solution as before. Compared to the unexposed control samples, absorbance measurement results demonstrated a slower rate and reduced overall amount of polymerization in the less concentrated tubulin samples exposed to PBM for 30 min with the parameters mentioned above. Paradoxically, the opposite effect was observed in the 45.5 μM tubulin samples, demonstrating a remarkable increase in the polymerization rates and total polymer mass achieved after exposure to PBM. These results on the effects of PBM on living cells, tubulin, and microtubules are novel, further validating the modulating effects of PBM and contributing to designing more effective PBM parameters. Finally, potential consequences for the use of PBM in the context of neurodegenerative diseases are discussed.

Minireview - Microtubules and Tubulin Oligomers: Shape Transitions and Assembly by Intrinsically Disordered Protein Tau and Cationic Biomolecules.

In this minireview, which is part of a special issue in honor of Jacob N. Israelachvili's remarkable research career on intermolecular forces and interfacial science, we present studies of structures, phase behavior, and forces in reaction mixtures of microtubules (MTs) and tubulin oligomers with either intrinsically disordered protein (IDP) Tau, cationic vesicles, or the polyamine spermine (4+). Bare MTs consist of 13 protofilaments (PFs), on average, where each PF is made of a linear stack of αβ-tubulin dimers (i.e., tubulin oligomers). We begin with a series of experiments which demonstrate the flexibility of PFs toward shape changes in response to local environmental cues. First, studies show that MT-associated protein (MAP) Tau controls the diameter of microtubules upon binding to the outer surface, implying a shape change in the cross-sectional area of PFs forming the MT perimeter. The diameter of a MT may also be controlled by the charge density of a lipid bilayer membrane that coats the outer surface. We further describe an experimental study where it is unexpectedly found that the biologically relevant polyamine spermine (+4e) is able to depolymerize taxol-stabilized microtubules with efficiency that increases with decreasing temperature. This MT destabilization drives a dynamical structural transition where inside-out curving of PFs, during the depolymerization peeling process, is followed by reassembly of ring-like curved PF building blocks into an array of helical inverted tubulin tubules. We finally turn to a very recent study on pressure-distance measurements in bundles of MTs employing the small-angle X-ray scattering (SAXS)-osmotic pressure technique, which complements the surface-forces-apparatus technique developed by Jacob N. Israelachvili. These latter studies are among the very few which are beginning to shed light on the precise nature of the interactions between MTs mediated by MAP Tau in 37 °C reaction mixtures containing GTP and lacking taxol.

Tubulin Acetylation Mediates Bisphenol A Effects on the Microtubule Arrays of Allium cepa and Triticum turgidum.

The effects of bisphenol A (BPA), a prevalent endocrine disruptor, on both interphase and mitotic microtubule array organization was examined by immunofluorescence microscopy in meristematic root cells of Triticum turgidum (durum wheat) and Allium cepa (onion). In interphase cells of A. cepa, BPA treatment resulted in substitution of cortical microtubules by annular/spiral tubulin structures, while in T. turgidum BPA induced cortical microtubule fragmentation. Immunolocalization of acetylated α-tubulin revealed that cortical microtubules of T. turgidum were highly acetylated, unlike those of A. cepa. In addition, elevation of tubulin acetylation by trichostatin A in A. cepa resulted in microtubule disruption similar to that observed in T. turgidum. BPA also disrupted all mitotic microtubule arrays in both species. It is also worth noting that mitotic microtubule arrays were acetylated in both plants. As assessed by BPA removal, its effects are reversible. Furthermore, taxol-stabilized microtubules were resistant to BPA, while recovery from oryzalin treatment in BPA solution resulted in the formation of ring-like tubulin conformations. Overall, these findings indicate the following: (1) BPA affects plant mitosis/cytokinesis by disrupting microtubule organization. (2) Microtubule disassembly probably results from impairment of free tubulin subunit polymerization. (3) The differences in cortical microtubule responses to BPA among the species studied are correlated to the degree of tubulin acetylation.

The formin Drosophila homologue of Diaphanous2 (Diaph2) controls microtubule dynamics in colorectal cancer cells independent of its FH2-domain

In this study, we analyzed the functional role of the formin Drosophila Homologue of Diaphanous2 (Diaph2) in colorectal cancer cells. We show that stable down-regulation of Diaph2 expression in HT29 cells decreased chromosome alignment and the velocity of chromosome movement during M-phase, thus reducing the proliferation rate and colony formation. In interphase cells, Diaph2 was diffusely distributed in the cytosol, while in metaphase cells the protein was located to spindle microtubules (MTs). Diaph2-depletion increased the concentration of stable spindle MTs, showing that the formin is required to control spindle MT-dynamics. Our cellular data indicate that Diaph2-controls spindle MT-dynamics independent of Cdc42 activity and our in vitro results reveal that bacterially produced full-length (FL) Diaph2 strongly altered MT-dynamics in absence of Cdc42, where its actin-nucleating activity is auto-inhibited. FL-Diaph2 mediates a 10-fold increase in MT-polymerization compared to the Diaph2-FH2-domain. Interestingly, a Diaph2-mutant lacking the FH2-domain (ΔFH2) increased MT-polymerization to a similar extent as the FH2-domain, indicating the existence of a second MT-binding domain. However, in contrast to FL-Diaph2 and the FH2-domain, ΔFH2 did not alter the density of taxol-stabilized MTs. Thus, the FH2-domain and the second Diaph2-binding domain appear to control MT-dynamics by different mechanisms. In summary, our data indicate that Diaph2 controls M-phase progression under basal conditions by regulating spindle MT-dynamics. In addition, a region outside of the canonical MT-regulating FH2-domain is involved in Diaph2-mediated control of MT-dynamics.

UNC-45A Is a Novel Microtubule-Associated Protein and Regulator of Paclitaxel Sensitivity in Ovarian Cancer Cells.

UNC-45A, a highly conserved member of the UCS (UNC45A/CRO1/SHE4P) protein family of cochaperones, plays an important role in regulating cytoskeletal-associated functions in invertebrates and mammalian cells, including cytokinesis, exocytosis, cell motility, and neuronal development. Here, for the first time, UNC-45A is demonstrated to function as a mitotic spindle-associated protein that destabilizes microtubules (MT) activity. Using in vitro biophysical reconstitution and total internal reflection fluorescence microscopy analysis, we reveal that UNC-45A directly binds to taxol-stabilized MTs in the absence of any additional cellular cofactors or other MT-associated proteins and acts as an ATP-independent MT destabilizer. In cells, UNC-45A binds to and destabilizes mitotic spindles, and its depletion causes severe defects in chromosome congression and segregation. UNC-45A is overexpressed in human clinical specimens from chemoresistant ovarian cancer and that UNC-45A-overexpressing cells resist chromosome missegregation and aneuploidy when treated with clinically relevant concentrations of paclitaxel. Lastly, UNC-45A depletion exacerbates paclitaxel-mediated stabilizing effects on mitotic spindles and restores sensitivity to paclitaxel. IMPLICATIONS: These findings reveal novel and significant roles for UNC-45A in regulation of cytoskeletal dynamics, broadening our understanding of the basic mechanisms regulating MT stability and human cancer susceptibility to paclitaxel, one of the most widely used chemotherapy agents for the treatment of human cancers.

Structure of Dynamic, Taxol-Stabilized, and GMPPCP-Stabilized Microtubule.

Microtubule (MT) is made of αβ-tubulin heterodimers that dynamically assemble into a hollow nanotube composed of straight protofilaments. MT dynamics is facilitated by hydrolysis of guanosine-5'-triphosphate (GTP) and can be inhibited by either anticancer agents like taxol or the nonhydrolyzable GTP analogues like GMPPCP. Using high-resolution synchrotron X-ray scattering, we have measured and analyzed the scattering curves from solutions of dynamic MT (in other words, in the presence of excess GTP and free of dynamic-inhibiting agents) and examined the effect of two MT stabilizers: taxol and GMPPCP. Previously, we have analyzed the structure of dynamic MT by docking the atomic model of tubulin dimer onto a 3-start left handed helical lattice, derived from the PDB ID 3J6F . 3J6F corresponds to a MT with 14 protofilaments. In this paper, we took into account the possibility of having MT structures containing between 12 and 15 protofilaments. MTs with 12 protofilaments were never observed. We determined the radii, the pitch, and the distribution of protofilament number that best fit the scattering data from dynamic MT or stabilized MT by taxol or GMPPCP. We found that the protofilament number distribution shifted when the MT was stabilized. Taxol increased the mass fraction of MT with 13 protofilaments and decreased the mass fraction of MT with 14 protofilaments. GMPPCP reduced the mass fraction of MT with 15 protofilaments and increased the mass fraction of MT with 14 protofilaments. The pitch, however, remained unchanged regardless of whether the MT was dynamic or stabilized. Higher tubulin concentrations increased the fraction of dynamic MT with 14 protofilaments.

Commentary: Transformation of Taxol-Stabilized Microtubules into Inverted Tubulin Tubules Triggered by a Tubulin Conformation Switch

Commentary: Transformation of Taxol-Stabilized Microtubules into Inverted Tubulin Tubules Triggered by a Tubulin Conformation Switch

Kinesin-1 motors can increase the lifetime of taxol-stabilized microtubules.

Kinesin-1 motors can increase the lifetime of taxol-stabilized microtubules.

Sulfo-SMCC Prevents Annealing of Taxol-Stabilized Microtubules In Vitro

Microtubule structure and functions have been widely studied in vitro and in cells. Research has shown that cysteines on tubulin play a crucial role in the polymerization of microtubules. Here, we show that blocking sulfhydryl groups of cysteines in taxol-stabilized polymerized microtubules with a commonly used chemical crosslinker prevents temporal end-to-end annealing of microtubules in vitro. This can dramatically affect the length distribution of the microtubules. The crosslinker sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, sulfo-SMCC, consists of a maleimide and an N-hydroxysuccinimide ester group to bind to sulfhydryl groups and primary amines, respectively. Interestingly, addition of a maleimide dye alone does not show the same interference with annealing in stabilized microtubules. This study shows that the sulfhydryl groups of cysteines of tubulin that are vital for the polymerization are also important for the subsequent annealing of microtubules.

Effects of Spermine on Microtubule Structures

Spermine is a tetravalent polyamine present at millimolar concentration in all eukaryotic cells. The adsorption of cationic spermine on anionic microtubules (MTs) surface may have a potential regulatory role on the complex dynamics of MTs. Experiments have shown that polyamines such as spermine can facilitate the polymerization of tubulin at physiological concentration, including the formation of nuclei or growth of MTs. The underlying molecular mechanism is thought to be based on the bridging interaction of spermine with the highly negative C-terminal tail on the tubulin surface that helps bind tubulin together. Recent experiments also show that bundles of taxol-stabilized MTs undergo a shape transformation to bundles of inverted tubulin tubules (ITTs) at high concentrations of spermine, where the outside surface of an ITT corresponds to the inner surface of a regular MT. Furthermore, the longitudinal (lateral) bonds in a regular MT along its axis (circumference) are transformed into lateral (longitudinal) bonds in an ITT along its circumference (axis). However, the molecular mechanism responsible for the shape transformation is unclear. We performed all atom molecular dynamics simulation on taxol-stabilized MT sheets containing two protofilaments with and without spermine in an explicit water solvent. Our simulations have identified important regions on the MT surface where spermine binds and the influence of spermine binding on the structure of tubulin proteins and tubulin-tubulin contacts in MTs. In contrast to Taxol, spermine-binding seems to decrease the flexibility of tubulin proteins, resulting in weaker tubulin-tubulin contacts. Our results point to a possible molecular mechanism of how spermine disrupts the contact between protofilaments and facilitates the bending of protofilaments into inverted rings and eventually inverted tubules.

Is Microtubule Rigidity Proportional to Protofilament Number?

Microtubules are cytoskeletal filaments that participate in key cellular processes by providing structural support, intracellular transportation, and cell division. Despite having been measured for the past 20 years, there are still open questions regarding the mechanical stiffness of microtubules and their persistence length, Lp. The persistence length is a measure of stiffness, proportional to the flexural rigidity, EI, defined as the elastic modulus and the second moment of area. In vitro, GMPCPP (a slowly hydrolyzable form of GTP) microtubules produce 14 protofilaments, taxol-stabilized microtubules produce 12-13 protofilaments, and microtubules polymerized in high-sodium (580 mM NaCl) concentration produce 9-10 protofilaments and lattice shifts resulting in numerous “seams.”Persistence lengths of single microtubule filaments were measured. Microtubules were formed in respective buffers and stabilized with the chemotherapeutic drug Taxol. After formation, microtubules were confined to oscillate within a thin, 2-D chamber (≤ 3 μm). Fourier mode analysis was used to fit images of fluorescently labeled microtubules. From each individual fit, a respective persistence length was determined. Bootstrapping statistics was then applied to produce a more accurate measure of filament rigidity. Our prior measurements for GMPCPP microtubules have lognormal distributions with average Lp of 1.8 ± 0.5 mm and 1.9 ± 0.7 mm (with taxol) respectively. Similarly, Taxol stabilized microtubules have a lognormal distribution but with a smaller Lp of 0.65 ± 0.1 mm. Preliminary results show high-sodium polymerized microtubules with an Lp of 0.67 ± 0.1 mm.

Self protein-protein interactions are involved in TPPP/p25 mediated microtubule bundling

TPPP/p25 is a microtubule-associated protein, detected in protein inclusions associated with various neurodegenerative diseases. Deletion analysis data show that TPPP/p25 has two microtubule binding sites, both located in intrinsically disordered domains, one at the N-terminal and the other in the C-terminal domain. In copolymerization assays the full-length protein exhibits microtubule stimulation and bundling activity. In contrast, at the same ratio relative to tubulin, truncated forms of TPPP/p25 exhibit either lower or no microtubule stimulation and no bundling activity, suggesting a cooperative phenomenon which is enhanced by the presence of the two binding sites. The binding characteristics of the N- and C-terminally truncated proteins to taxol-stabilized microtubules are similar to the full-length protein. However, the C-terminally truncated TPPP/p25 shows a lower Bmax for microtubule binding, suggesting that it may bind to a site of tubulin that is masked in microtubules. Bimolecular fluorescent complementation assays in cells expressing combinations of various TPPP/p25 fragments, but not that of the central folded domain, resulted in the generation of a fluorescence signal colocalized with perinuclear microtubule bundles insensitive to microtubule inhibitors. The data suggest that the central folded domain of TPPP/p25 following binding to microtubules can drive s homotypic protein-protein interactions leading to bundled microtubules.

Inter-domain Cooperation in INCENP Promotes Aurora B Relocation from Centromeres to Microtubules.

The chromosomal passenger complex is essential for error-free chromosome segregation and proper execution of cytokinesis. To coordinate nuclear division with cytoplasmic division, its enzymatic subunit, Aurora B, relocalizes from centromeres in metaphase to the spindle midzone in anaphase. In budding yeast, this requires dephosphorylation of the microtubule-binding (MTB) domain of the INCENP analog Sli15. The mechanistic basis for this relocalization in metazoans is incompletely understood. We demonstrate that the putative coiled-coil domain within INCENP drives midzone localization of Aurora B via a direct, electrostatic interaction with microtubules. Furthermore, we provide evidence that the CPC multimerizes via INCENP's centromere-targeting domain (CEN box), which increases the MTB affinity of INCENP. In (pro)metaphase, the MTB affinity of INCENP is outcompeted by the affinity of its CEN box for centromeres, while at anaphase onset—when the histone mark H2AT120 is dephosphorylated—INCENP and Aurora B switch from centromere to microtubule localization.

The Determination of Young's Modulus for Microtubules Stabilized with Taxol and Analysis of Vibrational Modes

We present here the effects of Taxol, a cancer drug, on the intracellular mechanisms associated with dynamic instability of microtubules. Since Taxol affects the stability of microtubules, the general process of microtubule polymerization and depolymerization will be studied closely throughout this research project. By analyzing the dynamic instability of microtubules, the effects of Taxol on the microtubules can be used to elucidate the complex functioning of Taxol within the cell. The observations and discoveries of the research can have revolutionary effects in the fields of life science and medicine.The short-term goal of this project involves analysis of the thermal fluctuations of a single Taxol-stabilized microtubule. From our measurement of the elastic properties for Taxol-stabilized microtubules we can gain insight on vibrational modes of the microtubules. We propose that the vibrational modes of microtubules will vary based on the Taxol concentration at which they are grown and diluted. Then, by comparing the vibrational modes to the resonant frequency of the Taxol-stabilized microtubules, we will relate the dynamic instability of the microtubules to the change in vibrational mode. Therefore, the ultimate goal of the research is to acquire more knowledge about the function of Taxol in order to discover more effective cancer treatment methods. These findings will elucidate the confounding enigma that plagues humanity (cancer) and will lead to further advancements in cancer therapy research. Based on current findings, the microtubules grown with higher Taxol concentrations have lower Young's Modulus values.

Analyzing the Frequency of Thermally Fluctuating Segments of Microtubules

Taxol is a drug used to treat cancer by stabilizing microtubules. The purpose of this research is to understand and explain how Taxol stabilizes microtubules and build a foundation upon which new discoveries involving cancer research can be made. We analyzed if Taxol affects the vibrational modes of microtubules by determining a frequency of Taxol-stabilized microtubules. Microtubules are grown, imaged, and analyzed by measuring the change of angle in radians of the end segments at 83ms intervals. The results depicted a sinusoidal movement of the end segment of the microtubule. From this, we found the resonant frequency by taking the Fourier Transform of the data and analyzing where the maximum peak occurred. The smaller peaks in the transform may be a result of the surrounding solution or internal fluctuations of the microtubule. We interpreted a 10.2 µm microtubule to have a frequency of 0.96 Hz. The process is repeated with microtubules of similar lengths, incubated with Taxol. We compared the resonant frequencies of the various lengths of microtubules and observed that there is a relationship between the length of a microtubule and its fundamental resonant frequency. The trend shows that as length of a microtubule increases, the fundamental frequency decreases.

Molecular Dynamics Study of the Effect of Polyamine on Microtubule Conformations

Microtubules (MTs) play important roles in cell division, intracellular protein transport, and cell structure and movement in eukaryotic cells. MTs are hollow supramolecular polymers made of alpha and beta tubulin heterodimers. Microtubule assembly in cells require a proper orientation of tubulin molecules to the MTs end. Experiments have shown that polyamines such as spermine can facilitate the diffusion of tubulin dimers in the process of nuclei growth or MT growth. The mechanism of this aggregation is the bridging interaction of polyamines with highly negative C-terminal tail of tubulin surface which helps tubulin together. Recent experiments also show that with a high concentration of spermine, bundles of taxol-stabilized MTs undergo a shape transformation to bundles of inverted tubulin tubules. However, the molecular mechanism underlying the transformation is unclear. We performed all atom molecular dynamics simulation of tubulin clusters in presence of spermine in an explicit water solvent to study the structural dynamics of alpha/beta tubulin and microtubule protofilament. We discuss our results on the interaction between spermine and tubulin proteins and the effect of spermine on conformational dynamics of MT protofilaments.

Kinesin-5 Acts as a Microtubule Stabilizer, Polymerase and Plus-Tip Tracker

Kinesin-5 slides antiparallel microtubules apart during mitosis and is necessary for bipolar spindle formation. Besides its unique homotetrameric configuration, determined by the coiled-coil domain, kinesin-5 motor domains also possess specific properties optimal for their spindle organizing function. To study properties intrinsic to the kinesin-5 motor domain, we generated functional kinesin-5 dimers by fusing the kinesin-5 head and 18-residue neck linker to the coiled-coil rod of kinesin-1. In ATP this kinesin-5 dimer decorated plus-ends of taxol-stabilized microtubules and slowed depolymerization of GMPCPP microtubules. On dynamic microtubules, kinesin-5 dimer increased the microtubule growth rate by more than a factor of two and reduced the catastrophe frequency three-fold. These findings are consistent with kinesin-5 acting as a microtubule polymerase. To understand this polymerase mechanism, TIRF microscopy was used to visualize individual GFP-labeled kinesin-5 dimers on immobilized microtubules. Motors walked to the ends of microtubules and remained bound there for 7 seconds, much longer than the 0.1 s motor step time. We hypothesize that kinesin-5 promotes microtubule polymerization by stabilizing longitudinal interactions between tubulin subunits on a single protofilament. Consistent with this protofilament stabilization hypothesis, fluorescence analysis suggests that microtubule plus-ends are more tapered in the presence of kinesin-5. Furthermore, curved and looped ends were observed, which occasionally resolved into straight microtubules, consistent with kinesin-5 stabilizing long protofilament bundles. In cells, these end-binding and polymerase activities should enhance the ability of kinesin-5 to establish and maintain spindle pole separation during mitosis.