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Navigation through a dense, physically confining extracellular matrix is common in invasive cell spread and tissue reorganization but is still poorly understood. Here, we show that this migration is mediated by cyclic changes in the activity of a small GTPase RhoA, which is dependent on the oscillatory changes in the activity and abundance of the RhoA guanine nucleotide exchange factor, GEF-H1, and triggered by a persistent increase in the intracellular Ca 2+ levels. We show that the molecular clock driving these cyclic changes is mediated by two coupled negative feedback loops, dependent on the microtubule dynamics, with a frequency that can be experimentally modulated based on a predictive mathematical model. We further demonstrate that an increasing frequency of the clock translates into a faster cell migration within physically confining spaces. This work lays the foundation for a better understanding of the molecular mechanisms dynamically driving cell migration in complex environments., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Microtubule "dynamic instability," the abrupt switching from assembly to disassembly caused by the hydrolysis of GTP to GDP within the β subunit of the αβ-tubulin heterodimer, is necessary for vital cellular processes such as mitosis and migration. Despite existing high-resolution structural data, the key mechanochemical differences between the GTP and GDP states that mediate dynamic instability behavior remain unclear. Starting with a published atomic-level structure as an input, we used multiscale modeling to find that GTP hydrolysis results in both longitudinal bond weakening (~ 4 k B T) and an outward bending preference (~ 1.5 k B T) to both drive dynamic instability and give rise to the microtubule tip structures previously observed by light and electron microscopy. More generally, our study provides an example where atomic level structural information is used as the sole input to predict cellular level dynamics without parameter adjustment., (© 2021. The Author(s).)

Microtubule-targeting agents (MTAs) are widely used chemotherapy drugs capable of disrupting microtubule-dependent cellular functions, such as division and migration. We show that two clinically approved MTAs, paclitaxel and vinblastine, each suppress stiffness-sensitive migration and polarization characteristic of human glioma cells on compliant hydrogels. MTAs influence microtubule dynamics and cell traction forces by nearly opposite mechanisms, the latter of which can be explained by a combination of changes in myosin motor and adhesion clutch number. Our results support a microtubule-dependent signaling-based model for controlling traction forces through a motor-clutch mechanism, rather than microtubules directly relieving tension within F-actin and adhesions. Computational simulations of cell migration suggest that increasing protrusion number also impairs stiffness-sensitive migration, consistent with experimental MTA effects. These results provide a theoretical basis for the role of microtubules and mechanisms of MTAs in controlling cell migration., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Multidrug resistance and toxic side effects are the major challenges in cancer treatment with microtubule-targeting agents (MTAs), and thus, there is an urgent clinical need for new therapies. Chalcone, a common simple scaffold found in many natural products, is widely used as a privileged structure in medicinal chemistry. We have previously validated tubulin as the anticancer target for chalcone derivatives. In this study, an α-methyl-substituted indole-chalcone (FC77) was synthesized and found to exhibit an excellent cytotoxicity against the NCI-60 cell lines (average concentration causing 50% growth inhibition = 6 nM). More importantly, several multidrug-resistant cancer cell lines showed no resistance to FC77, and the compound demonstrated good selective toxicity against cancer cells versus normal CD34 + blood progenitor cells. A further mechanistic study demonstrated that FC77 could arrest cells that relate to the binding to tubulin and inhibit the microtubule dynamics. The National Cancer Institute COMPARE analysis and molecular modeling indicated that FC77 had a mechanism of action similar to that of colchicine. Overall, our data demonstrate that this indole-chalcone represents a novel MTA template for further development of potential drug candidates for the treatment of multidrug-resistant cancers.

Microtubules are dynamic polymers required for a number of processes, including chromosome movement in mitosis. While regulators of microtubule dynamics have been well characterized, we lack a convenient way to predict how the measured dynamic parameters shape the entire microtubule system within a cell, or how the system responds when specific parameters change in response to internal or external signals. Here we describe a Monte Carlo model to simulate an array of dynamic microtubules from parameters including the cell radius, total tubulin concentration, microtubule nucleation rate from the centrosome, and plus end dynamic instability. The algorithm also allows dynamic instability or position of the cell edge to vary during the simulation. Outputs from simulations include free tubulin concentration, average microtubule lengths, length distributions, and individual length changes over time. Using this platform and reported parameters measured in interphase LLCPK1 epithelial cells, we predict that sequestering ~ 15-20% of total tubulin results in fewer microtubules, but promotes dynamic instability of those remaining. Simulations also predict that lowering nucleation rate will increase the stability and average length of the remaining microtubules. Allowing the position of the cell's edge to vary over time changed the average length but not the number of microtubules and generated length distributions consistent with experimental measurements. Simulating the switch from interphase to prophase demonstrated that decreased rescue frequency at prophase is the critical factor needed to rapidly clear the cell of interphase microtubules prior to mitotic spindle assembly. Finally, consistent with several previous simulations, our results demonstrate that microtubule nucleation and dynamic instability in a confined space determines the partitioning of tubulin between monomer and polymer pools. The model and simulations will be useful for predicting changes to the entire microtubule array after modification to one or more parameters, including predicting the effects of tubulin-targeted chemotherapies., Competing Interests: The authors have declared that no competing interests exist.

Microtubule self-assembly dynamics serve to facilitate many vital cellular functions, such as chromosome segregation during mitosis and synaptic plasticity. However, the detailed atomistic basis of assembly dynamics has remained an unresolved puzzle. A key challenge is connecting together the vast range of relevant length-time scales, events happening at time scales ranging from nanoseconds, such as tubulin molecular interactions (Å-nm), to minutes-hours, such as the cellular response to microtubule dynamics during mitotic progression (μm). At the same time, microtubule interactions with associated proteins and binding agents, such as anti-cancer drugs, can strongly affect this dynamic process through atomic-level mechanisms that remain to be elucidated. New high-resolution technologies for investigating these interactions, including cryo-electron microscopy (EM) techniques and total internal reflection fluorescence (TIRF) microscopy, are yielding important new insights. Here, we focus on recent studies of microtubule dynamics, both theoretical and experimental, and how these findings shed new light on this complex phenomenon across length-time scales, from Å to μm and from nanoseconds to minutes., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Microtubule-targeting agents (MTAs), widely used as biological probes and chemotherapeutic drugs, bind directly to tubulin subunits and "kinetically stabilize" microtubules, suppressing the characteristic self-assembly process of dynamic instability. However, the molecular-level mechanisms of kinetic stabilization are unclear, and the fundamental thermodynamic and kinetic requirements for dynamic instability and its elimination by MTAs have yet to be defined. Here we integrate a computational model for microtubule assembly with nanometer-scale fluorescence microscopy measurements to identify the kinetic and thermodynamic basis of kinetic stabilization by the MTAs paclitaxel, an assembly promoter, and vinblastine, a disassembly promoter. We identify two distinct modes of kinetic stabilization in live cells, one that truly suppresses on-off kinetics, characteristic of vinblastine, and the other a "pseudo" kinetic stabilization, characteristic of paclitaxel, that nearly eliminates the energy difference between the GTP- and GDP-tubulin thermodynamic states. By either mechanism, the main effect of both MTAs is to effectively stabilize the microtubule against disassembly in the absence of a robust GTP cap., (© 2017 Castle et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Small molecule inhibitors of microtubule dynamics are widely used as cell biology research tools and clinically as cancer chemotherapeutics. By slight modification to the chemical structure of a known microtubule inhibitor, combretastatin A-4, Borowiak et al. develop a photoswitchable derivative that can be turned "on" and "off" with low-intensity light to spatially and temporally control microtubule dynamics., (Copyright © 2015 Elsevier Inc. All rights reserved.)

During mitosis, molecular motors hydrolyze ATP to generate sliding forces between adjacent microtubules and form the bipolar mitotic spindle. Lansky et al. now show that the diffusible microtubule crosslinker Ase1p can generate sliding forces between adjacent microtubules, and it does so without ATP hydrolysis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

During cell division, a microtubule-based mitotic spindle mediates the faithful segregation of duplicated chromosomes into daughter cells. Proper length control of the metaphase mitotic spindle is critical to this process and is thought to be achieved through a mechanism in which spindle pole separation forces from plus-end-directed motors are balanced by forces from minus-end-directed motors that pull spindle poles together. However, in contrast to this model, metaphase mitotic spindles with inactive kinesin-14 minus-end-directed motors often have shorter spindle lengths, along with poorly aligned spindle microtubules. A mechanistic explanation for this paradox is unknown. Using computational modeling, in vitro reconstitution, live-cell fluorescence microscopy, and electron microscopy, we now find that the budding yeast kinesin-14 molecular motor Kar3-Cik1 can efficiently align spindle microtubules along the spindle axis. This then allows plus-end-directed kinesin-5 motors to efficiently exert the outward microtubule sliding forces needed for proper spindle bipolarity., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Microtubules are dynamic polymers of the cytoskeleton, which play important roles in cell division, polarization, and intracellular transport. Self-assembly of microtubule polymer from αβ-tubulin heterodimers is highly variable, with stochastic switching between alternate states of net growth and net shortening, a phenomenon known as dynamic instability. Microtubule tip structures are also variable and directly influence the kinetics of assembly and vice versa. TipTracker, a semiautomated, image processing-based tool, permits high spatial and temporal resolution measurements from fluorescence microscopy images (~10-40 nm, or 1-5 dimer lengths, at 1-10 Hz) with simultaneous tip structure estimation. We provide a walkthrough of the TipTracker code to demonstrate methods used to (1) fit the coordinates of the microtubule backbone; (2) track microtubule tip position; and (3) estimate tip structure from the spatial decay of the tip fluorescence distribution, discuss possible sources of error, and include an example protocol for nanometer-scale tip tracking in living cells. Additionally, we evaluate TipTracker's accuracy on simulated digital images and fixed microtubules to estimate accuracy under realistic imaging conditions. In summary, this chapter demonstrates the use of TipTracker in making robust, high-resolution measurements of microtubule tip dynamics and structures, facilitating quantitative investigations into nanoscale/molecular control of microtubule assembly. Although our primary focus is on microtubules, these methods are, in principle, suitable for other polymer structures, such as F-actin., (© 2014 Elsevier Inc. All rights reserved.)

The structure and free energy of multistranded linear polymer ends evolves as individual subunits are added and lost. Thus, the energetic state of the polymer end is not constant, as assembly theory has assumed. Here we utilize a Brownian dynamics approach to simulate the addition and loss of individual subunits at the polymer tip. Using the microtubule as a primary example, we examined how the structure of the polymer tip dictates the rate at which units are added to and lost from individual protofilaments. We find that freely diffusing subunits arrive less frequently to lagging protofilaments but bind more efficiently, such that there is no kinetic difference between leading and lagging protofilaments within a tapered tip. However, local structure at the nanoscale has up to an order-of-magnitude effect on the rate of addition. Thus, the kinetic on-rate constant, integrated across the microtubule tip (kon,MT), is an ensemble average of the varying individual protofilament on-rate constants (kon,PF). Our findings have implications for both catastrophe and rescue of the dynamic microtubule end, and provide a subnanoscale framework for understanding the mechanism of action of microtubule-associated proteins and microtubule-directed drugs. Although we utilize the specific example of the microtubule here, the findings are applicable to multistranded polymers generally., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Microtubules are key structural and transport elements in cells. The dynamics at microtubule ends are characterized by periods of slow growth, followed by stochastic switching events termed "catastrophes," in which microtubules suddenly undergo rapid shortening. Growing microtubules are thought to be protected from catastrophe by a GTP-tubulin "cap": GTP-tubulin subunits add to the tips of growing microtubules but are subsequently hydrolyzed to GDP-tubulin subunits once they are incorporated into the microtubule lattice. Loss of the GTP-tubulin cap exposes GDP-tubulin subunits at the microtubule tip, resulting in a catastrophe event. However, the mechanistic basis for sudden loss of the GTP cap, leading to catastrophe, is not known. To investigate microtubule catastrophe events, we performed 3D mechanochemical simulations that account for interactions between neighboring protofilaments. We found that there are two separate factors that contribute to catastrophe events in the 3D simulation: the GTP-tubulin cap size, which settles into a steady-state value that depends on the free tubulin concentration during microtubule growth, and the structure of the microtubule tip. Importantly, 3D simulations predict, and both fluorescence and electron microscopy experiments confirm, that microtubule tips become more tapered as the microtubule grows. This effect destabilizes the tip and ultimately contributes to microtubule catastrophe. Thus, the likelihood of a catastrophe event may be intimately linked to the aging physical structure of the growing microtubule tip. These results have important consequences for catastrophe regulation in cells, as microtubule-associated proteins could promote catastrophe events in part by modifying microtubule tip structures., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In everyday life, gravity and inertial forces often dominate our movements; in the cell, these forces pale in comparison to thermal forces. The violent, collisional world of the cell, where water moves faster than a jet airliner, can be difficult to imagine. To develop our intuitive understanding of cellular and molecular processes, we are exploring the concept of 'bodystorming', where human 'movers' act as molecules that diffuse, undergo reactions, and generate/absorb forces., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Microtubules (MTs) polymerize via net addition of GTP-tubulin subunits to the MT plus end, which subsequently hydrolyze to GDP-tubulin in the MT lattice. Relatively stable GTP-tubulin subunits create a "GTP cap" at the growing MT plus end that suppresses catastrophe. To understand MT assembly regulation, we need to understand GTP hydrolysis reaction kinetics and the GTP cap size. In vitro, the GTP cap has been estimated to be as small as one layer (13 subunits) or as large as 100-200 subunits. GTP cap size estimates in vivo have not yet been reported. Using EB1-EGFP as a marker for GTP-tubulin in epithelial cells, we find on average (1) 270 EB1 dimers bound to growing MT plus ends, and (2) a GTP cap size of ∼750 tubulin subunits. Thus, in vivo, the GTP cap is far larger than previous estimates in vitro, and ∼60-fold larger than a single layer cap. We also find that the tail of a large GTP cap promotes MT rescue and suppresses shortening. We speculate that a large GTP cap provides a locally concentrated scaffold for tip-tracking proteins and confers persistence to assembly in the face of physical barriers such as the cell cortex., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Microtubules undergo alternating periods of growth and shortening, known as dynamic instability. These dynamics allow microtubule plus ends to explore cellular space. The "search and capture" model posits that selective anchoring of microtubule plus ends at the cell cortex may contribute to cell polarization, spindle orientation, or targeted trafficking to specific cellular domains. Whereas cytoplasmic dynein is primarily known as a minus-end-directed microtubule motor for organelle transport, cortically localized dynein has been shown to capture and tether microtubules at the cell periphery in both dividing and interphase cells. To explore the mechanism involved, we developed a minimal in vitro system, with dynein-bound beads positioned near microtubule plus ends using an optical trap. Dynein induced a significant reduction in the lateral diffusion of microtubule ends, distinct from the effects of other microtubule-associated proteins such as kinesin-1 and EB1. In assays with dynamic microtubules, dynein delayed barrier-induced catastrophe of microtubules. This effect was ATP dependent, indicating that dynein motor activity was required. Computational modeling suggests that dynein delays catastrophe by exerting tension on individual protofilaments, leading to microtubule stabilization. Thus, dynein-mediated capture and tethering of microtubules at the cortex can lead to enhanced stability of dynamic plus ends., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Microtubule assembly is vital for many fundamental cellular processes. Current models for microtubule assembly kinetics assume that the subunit dissociation rate from a microtubule tip is independent of free subunit concentration. Total-Internal-Reflection-Fluorescence (TIRF) microscopy experiments and data from a laser tweezers assay that measures in vitro microtubule assembly with nanometer resolution, provides evidence that the subunit dissociation rate from a microtubule tip increases as the free subunit concentration increases. These data are consistent with a two-dimensional model for microtubule assembly, and are explained by a shift in microtubule tip structure from a relatively blunt shape at low free concentrations to relatively tapered at high free concentrations. We find that because both the association and the dissociation rates increase at higher free subunit concentrations, the kinetics of microtubule assembly are an order-of-magnitude higher than currently estimated in the literature., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Breaking cell symmetry, known as polarization, requires dynamic reorganization of microtubules (MTs) and is essential to many cellular processes, including axon formation in neurons. A critical step in polarization is believed to be the "selective stabilization" of MTs, which hypothesizes a spatial and/or temporal shift toward net MT assembly in a preferred direction of growth., Results: We now find that a simpler "length-dependent" model, in which MT assembly parameters are spatially and temporally constant, predicts MT accumulation in the direction of growth because of longer mean first passage times in the longer direction. We experimentally tested both models by tracking MT assembly dynamics in polarizing embryonic chick forebrain neurons, and we confirmed that assembly is spatially and temporally constant during axon formation., Conclusion: Cell polarization occurs most simply through cell-length-dependent accumulation of MTs without MT stabilization or capture. In this way, F-actin-mediated cell shape and size changes can be read out by dynamic MTs undergoing simple dynamic instability to ultimately break cell symmetry., (2010 Elsevier Ltd. All rights reserved.)

Microtubules (MTs) have been proposed to act mechanically as compressive struts that resist both actomyosin contractile forces and their own polymerization forces to mechanically stabilize cell shape. To identify the origin of MT bending, we directly observed MT bending and F-actin transport dynamics in the periphery of LLC-PK1 epithelial cells. We found that F-actin is nearly stationary in these cells even as MTs are deformed, demonstrating that MT bending is not driven by actomyosin contractility. Furthermore, the inhibition of myosin II activity through the use of blebbistatin results in microtubules that are still dynamically bending. In addition, as determined by fluorescent speckle microscopy, MT polymerization rarely results, if ever, in bending. We suppressed dynamic instability using nocodazole, and we observed no qualitative change in the MT bending dynamics. Bending most often results from anterograde transport of proximal portions of the MT toward a nearly stationary distal tip. Interestingly, we found that in an in vitro kinesin-MT gliding assay, MTs buckle in a similar manner. To make quantitative comparisons, we measured curvature distributions of observed MTs and found that the in vivo and in vitro curvature distributions agree quantitatively. In addition, the measured MT curvature distribution is not Gaussian, as expected for a thermally driven semiflexible polymer, indicating that thermal forces play a minor role in MT bending. We conclude that many of the known mechanisms of MT deformation, such as polymerization and acto-myosin contractility, play an inconsequential role in mediating MT bending in LLC-PK1 cells and that MT-based molecular motors likely generate most of the strain energy stored in the MT lattice. The results argue against models in which MTs play a major mechanical role in LLC-PK1 cells and instead favor a model in which mechanical forces control the spatial distribution of the MT array.

During mitosis, sister chromatids congress to the spindle equator and are subsequently segregated via attachment to dynamic kinetochore microtubule (kMT) plus ends. A major question is how kMT plus-end assembly is spatially regulated to achieve chromosome congression. Here we find in budding yeast that the widely conserved kinesin-5 sliding motor proteins, Cin8p and Kip1p, mediate chromosome congression by suppressing kMT plus-end assembly of longer kMTs. Of the two, Cin8p is the major effector and its activity requires a functional motor domain. In contrast, the depolymerizing kinesin-8 motor Kip3p plays a minor role in spatial regulation of yeast kMT assembly. Our analysis identified a model where kinesin-5 motors bind to kMTs, move to kMT plus ends, and upon arrival at a growing plus end promote net kMT plus-end disassembly. In conclusion, we find that length-dependent control of net kMT assembly by kinesin-5 motors yields a simple and stable self-organizing mechanism for chromosome congression.

Recent studies suggest that the human Kinesin-8 molecular motor Kif18A has a role in chromosome congression. Specifically, these studies find that Kif18A promotes chromosome congression by attenuating chromosome oscillation magnitudes. Together with recent modeling work, in vitro studies, and the analysis of in vivo yeast data, these reports reveal how Kinesin-8 molecular motors might control chromosome oscillation amplitudes by spatially regulating the dynamic instability of microtubule plus-ends within the mitotic spindle.

Kinetochores maintain a mechanical grip on disassembling microtubule plus ends, possibly through a 16-member Dam1 ring that acts as a sliding clamp. It turns out, however, that a ring is not required for maintaining grip: individual Dam1 complexes in vitro can diffuse on the microtubule lattice and track shortening microtubule tips.

Although the dynamic self-assembly behavior of microtubule ends has been well characterized at the spatial resolution of light microscopy (~200 nm), the single-molecule events that lead to these dynamics are less clear. Recently, a number of in vitro studies used novel approaches combining laser tweezers, microfabricated chambers, and high-resolution tracking of microtubule-bound beads to characterize mechanochemical aspects of MT dynamics at nanometer scale resolution. In addition, computational modeling is providing a framework for integrating these experimental results into physically plausible models of molecular scale microtubule dynamics. These nanoscale studies are providing new fundamental insights about microtubule assembly, and will be important for advancing our understanding of how microtubule dynamic instability is regulated in vivo via microtubule-associated proteins, therapeutic agents, and mechanical forces.

Background: The labile nature of microtubules is critical for establishing cellular morphology and motility, yet the molecular basis of assembly remains unclear. Here we use optical tweezers to track microtubule polymerization against microfabricated barriers, permitting unprecedented spatial resolution., Results: We find that microtubules exhibit extensive nanometer-scale variability in growth rate and often undergo shortening excursions, in some cases exceeding five tubulin layers, during periods of overall net growth. This result indicates that the guanosine triphosphate (GTP) cap does not exist as a single layer as previously proposed. We also find that length increments (over 100 ms time intervals, n = 16,762) are small, 0.81 +/- 6.60 nm (mean +/- standard deviation), and very rarely exceed 16 nm (about two dimer lengths), indicating that assembly occurs almost exclusively via single-subunit addition rather than via oligomers as was recently suggested. Finally, the assembly rate depends only weakly on load, with the average growth rate decreasing only 2-fold as the force increases 7-fold from 0.4 pN to 2.8 pN., Conclusions: The data are consistent with a mechanochemical model in which a spatially extended GTP cap allows substantial shortening on the nanoscale, while still preventing complete catastrophe in most cases.

The microtubule cytoskeleton in living cells generate and resist mechanical forces to mediate fundamental cell processes, including cell division and migration. Recent advances in digital fluorescence microscopy have enabled the direct observation of bending of individual microtubules in living cells, which has enabled quantitative estimation of the mechanical state of the microtubule array. Although a variety of mechanisms have been proposed, the precise origins of microtubule deformation in living cells remain largely obscure. To investigate these mechanisms and their relative importance in cellular processes, a method is needed to accurately quantify microtubule bending within living cells. Here we describe a method for quantification of bending, using digital fluorescence microscope images to estimate the distribution of curvature in the microtubule. Digital images of individual microtubules can be used to obtain a set of discrete x-y coordinates along the microtubule contour, which is then used to estimate the curvature distribution. Due to system noise and digitization error, the estimate will be inaccurate to some degree. To quantify the inaccuracy, a computational model is used to simulate both the bending of thermally driven microtubules and their observation by digital fluorescence microscopy. This allows for direct comparison between experimental and simulated images, a method which we call model convolution microscopy. We assess the accuracy of various methods and present a suitable method for estimating the curvature distribution for thermally driven semiflexible polymers. Finally, we discuss extensions of the method to quantify microtubule curvature in living cells.

A new study shows that an antagonistic force model can explain a number of complex mitotic spindle movements in the first mitosis of the Caenorhabditis elegans embryo by simply assuming that cortical force generators become increasingly persistent in their interaction with microtubules during mitosis.

A computational model for the budding yeast mitotic spindle predicts a spatial gradient in tubulin turnover that is produced by kinetochore-attached microtubule (kMT) plus-end polymerization and depolymerization dynamics. However, kMTs in yeast are often much shorter than the resolution limit of the light microscope, making visualization of this gradient difficult. To overcome this limitation, we combined digital imaging of fluorescence redistribution after photobleaching (FRAP) with model convolution methods to compare computer simulations at nanometer scale resolution to microscopic data. We measured a gradient in microtubule dynamics in yeast spindles at approximately 65-nm spatial intervals. Tubulin turnover is greatest near kinetochores and lowest near the spindle poles. A beta-tubulin mutant with decreased plus-end dynamics preserves the spatial gradient in tubulin turnover at a slower time scale, increases average kinetochore microtubule length approximately 14%, and decreases tension at kinetochores. The beta-tubulin mutant cells have an increased frequency of chromosome loss, suggesting that the accuracy of chromosome segregation is linked to robust kMT plus-end dynamics.

Background: Duplicated chromosomes are equally segregated to daughter cells by a bipolar mitotic spindle during cell division. By metaphase, sister chromatids are coupled to microtubule (MT) plus ends from opposite poles of the bipolar spindle via kinetochores. Here we describe a phosphorylation event that promotes the coupling of kinetochores to microtubule plus ends., Results: Dam1 is a kinetochore component that directly binds to microtubules. We identified DAM1-765, a dominant allele of DAM1, in a genetic screen for mutations that increase stress on the spindle pole body (SPB) in Saccharomyces cerevisiae. DAM1-765 contains the single mutation S221F. We show that S221 is one of six Dam1 serines (S13, S49, S217, S218, S221, and S232) phosphorylated by Mps1 in vitro. In cells with single mutations S221F, S218A, or S221A, kinetochores in the metaphase spindle form tight clusters that are closer to the SPBs than in a wild-type cell. Five lines of experimental evidence, including localization of spindle components by fluorescence microscopy, measurement of microtubule dynamics by fluorescence redistribution after photobleaching, and reconstructions of three-dimensional structure by electron tomography, combined with computational modeling of microtubule behavior strongly indicate that, unlike wild-type kinetochores, Dam1-765 kinetochores do not colocalize with an equal number of plus ends. Despite the uncoupling of the kinetochores from the plus ends of MTs, the DAM1-765 cells are viable, complete the cell cycle with the same kinetics as wild-type cells, and biorient their chromosomes as efficiently as wild-type cells., Conclusions: We conclude that phosphorylation of Dam1 residues S218 and S221 by Mps1 is required for efficient coupling of kinetochores to MT plus ends. We find that efficient plus-end coupling is not required for (1) maintenance of chromosome biorientation, (2) maintenance of tension between sister kinetochores, or (3) chromosome segregation.

Microtubule self-assembly is largely governed by the chemical kinetics and thermodynamics of tubulin-tubulin interactions. An important aspect of microtubule assembly is that hydrolysis of the beta-tubulin-associated GTP promotes protofilament curling. Protofilament curling presumably drives the transition from tip structures associated with growth (sheetlike projections and blunt ends) to those associated with shortening (rams' horns and frayed ends), and transitions between these structures have been proposed to be important for growth-shortening transitions. However, previous models for microtubule dynamic instability have not considered such structures or mechanics explicitly. Here we present a three-dimensional model that explicitly incorporates mechanical stress and strain within the microtubule lattice. First, we found that the model recapitulates three-dimensional tip structures and rates of assembly and disassembly for microtubules grown under standard conditions, and we propose that taxol may stabilize microtubule growth by reducing flexural rigidity. Second, in contrast to recent suggestions, it was determined that sheetlike tips are more likely to undergo catastrophe than blunt tips. Third, partial uncapping of the tubulin-GTP cap provides a possible mechanism for microtubule pause events. Finally, simulations of the binding and structural effects of XMAP215 produced the experimentally observed growth and shortening rates, and tip structure.

During metaphase in budding yeast mitosis, sister kinetochores are tethered to opposite poles and separated, stretching their intervening chromatin, by singly attached kinetochore microtubules (kMTs). Kinetochore movements are coupled to single microtubule plus-end polymerization/depolymerization at kinetochore attachment sites. Here, we use computer modeling to test possible mechanisms controlling chromosome alignment during yeast metaphase by simulating experiments that determine the 1) mean positions of kinetochore Cse4-GFP, 2) extent of oscillation of kinetochores during metaphase as measured by fluorescence recovery after photobleaching (FRAP) of kinetochore Cse4-GFP, 3) dynamics of kMTs as measured by FRAP of GFP-tubulin, and 4) mean positions of unreplicated chromosome kinetochores that lack pulling forces from a sister kinetochore. We rule out a number of possible models and find the best fit between theory and experiment when it is assumed that kinetochores sense both a spatial gradient that suppresses kMT catastrophe near the poles and attachment site tension that promotes kMT rescue at higher amounts of chromatin stretch.

To form a proper mitotic spindle, all kinetochores must capture at least one microtubule plus end. A new computational modeling study shows that a search-and-capture mechanism based on spatially unbiased microtubule dynamic instability is too slow to account for the experimentally observed rate of chromosome capture.

The microtubule-severing enzyme katanin uses ATP hydrolysis to disrupt noncovalent bonds between tubulin dimers within the microtubule lattice. Although its microtubule severing activity is likely important for fundamental processes including mitosis and axonal outgrowth, its mechanism of action is poorly understood. To better understand this activity, an in vitro assay was developed to enable the real-time observation of katanin-mediated severing of individual, mechanically unconstrained microtubules. To interpret the experimental observations, a number of theoretical models were developed and compared quantitatively to the experimental data via Monte Carlo simulation. Models that assumed that katanin acts on a uniform microtubule lattice were incompatible with the in vitro data, whereas a model that assumed that katanin acts preferentially on spatially infrequent microtubule lattice defects was found to correctly predict the experimentally observed breaking rates, number and spatial frequency of severing events, final levels of severing, and sensitivity to katanin concentration over the range 6-300 nM. As a result of our analysis, we propose that defects in the microtubule lattice, which are known to exist but previously not known to have any biological function, serve as sites for katanin activity.

We developed a stochastic model of microtubule (MT) assembly dynamics that estimates tubulin-tubulin bond energies, mechanical energy stored in the lattice dimers, and the size of the tubulin-GTP cap at MT tips. First, a simple assembly/disassembly state model was used to screen possible combinations of lateral bond energy (DeltaG(Lat)) and longitudinal bond energy (DeltaG(Long)) plus the free energy of immobilizing a dimer in the MT lattice (DeltaG(S)) for rates of MT growth and shortening measured experimentally. This analysis predicts DeltaG(Lat) in the range of -3.2 to -5.7 k(B)T and DeltaG(Long) plus DeltaG(S) in the range of -6.8 to -9.4 k(B)T. Based on these estimates, the energy of conformational stress for a single tubulin-GDP dimer in the lattice is 2.1-2.5 k(B)T. Second, we studied how tubulin-GTP cap size fluctuates with different hydrolysis rules and show that a mechanism of directly coupling subunit addition to hydrolysis fails to support MT growth, whereas a finite hydrolysis rate allows growth. By adding rules to mimic the mechanical constraints present at the MT tip, the model generates tubulin-GTP caps similar in size to experimental estimates. Finally, by combining assembly/disassembly and cap dynamics, we generate MT dynamic instability with rates and transition frequencies similar to those measured experimentally. Our model serves as a platform to examine GTP-cap dynamics and allows predictions of how MT-associated proteins and other effectors alter the energetics of MT assembly.

Protein concentration gradients organize cells and tissues and commonly form through diffusion away from a local source of protein. Interestingly, during the asymmetric division of the Caenorhabditis elegans zygote, the RNA-binding proteins MEX- 5 and PIE-1 form opposing concentration gradients in the absence of a local source. In this study, we use near-total internal reflection fluorescence (TIRF) imaging and single-particle tracking to characterize the reaction/diffusion dynamics that maintain the MEX- 5 and PIE-1 gradients. Our findings suggest that both proteins interconvert between fast-diffusing and slow-diffusing states on timescales that are much shorter (seconds) than the timescale of gradient formation (minutes). The kinetics of diffusion-state switching are strongly polarized along the anterior/posterior (A/ P) axis by the PAR polarity system such that fast-diffusing MEX- 5 and PIE-1 particles are approximately symmetrically distributed, whereas slow-diffusing particles are highly enriched in the anterior and posterior cytoplasm, respectively. Using mathematical modeling, we show that local differences in the kinetics of diffusion-state switching can rapidly generate stable concentration gradients over a broad range of spatial and temporal scales. [ABSTRACT FROM AUTHOR]

Introduction: The microtubule motor protein kinesin-5 is well known to establish the bipolar spindle by outward sliding of antiparallel interpolar microtubules. In yeast, kinesin-5 also facilitates chromosome alignment 'congression' at the spindle equator by preferentially depolymerizing long kinetochore microtubules (kMTs). The motor protein kinesin-8 has also been linked to chromosome congression. Therefore, we sought to determine whether kinesin-5 or kinesin-8 facilitates chromosome congression in insect spindles. Methods: RNAi of the kinesin-5 Klp61F and kinesin-8 Klp67A were performed separately in Drosophila melanogaster S2 cells to test for inhibited chromosome congression. Klp61F RNAi, Klp67A RNAi, and control metaphase mitotic spindles expressing fluorescent tubulin and fluorescent Cid were imaged, and their fluorescence distributions were compared. Results: RNAi of Klp61F with a weak Klp61F knockdown resulted in longer kMTs and less congressed kinetochores compared to control over a range of conditions, consistent with kinesin-5 length-dependent depolymerase activity. RNAi of the kinesin-8 Klp67A revealed that kMTs relative to the spindle lengths were not longer compared to control, but rather that the spindles were longer, indicating that Klp67A acts preferentially as a length-dependent depolymerase on interpolar microtubules without significantly affecting kMT length and chromosome congression. Conclusions: This study demonstrates that in addition to establishing the bipolar spindle, kinesin-5 regulates kMT length to facilitate chromosome congression in insect spindles. It expands on previous yeast studies, and it expands the role of kinesin-5 to include kMT assembly regulation in eukaryotic mitosis. [ABSTRACT FROM AUTHOR]

Abstract: Computational modeling can be extremely useful in interpreting experimental results. Here we describe how a relatively sophisticated stochastic model for microtubule dynamic instability in the mitotic spindle can be developed starting with straightforward rules and simple programming code. Once this model is developed, the method for comparing simulation results to experimental data must be carefully considered. The ultimate utility of any computational model relies on its predictive power and the ability to assist in designing new experiments. We describe how “deconstructing” the model through the use of quantitative animations contributes to a better qualitative understanding of model behavior. By extracting key qualitative elements of the model in this fashion, model predictions and new experiments can be more easily extracted from model results. [Copyright &y& Elsevier]

In budding yeast, the mitotic spindle is comprised of 32 kinetochore microtubules (kMTs) and ∼8 interpolar MTs (ipMTs). Upon anaphase onset, kMTs shorten to the pole, whereas ipMTs increase in length. Overlapping MTs are responsible for the maintenance of spindle integrity during anaphase. To dissect the requirements for anaphase spindle stability, we introduced a conditionally functional dicentric chromosome into yeast. When centromeres from the same sister chromatid attach to opposite poles, anaphase spindle elongation is delayed and a DNA breakage-fusion-bridge cycle ensues that is dependent on DNA repair proteins. We find that cell survival after dicentric chromosome activation requires the MT-binding proteins Kar3p, Bim1p, and Ase1p. In their absence, anaphase spindles are prone to collapse and buckle in the presence of a dicentric chromosome. Our analysis reveals the importance of Bim1p in maintaining a stable ipMT overlap zone by promoting polymerization of ipMTs during anaphase, whereas Kar3p contributes to spindle stability by cross-linking spindle MTs. [ABSTRACT FROM AUTHOR]