Microtubule Curvature Research Articles

Ivermectin affects Arabidopsis thaliana microtubules through predicted binding site of β-tubulin

The ivermectin is a potent nematocide and insecticide, which has low toxicity for humans and domestic animals, but due to low biotransformation, it can be dangerous for non-target organisms. The recent determination of ivermectin absorption and accumulation in tissues of higher plants and multiple shreds of evidence of its negative impact on plant physiology provide a basis for the search for ivermectin's molecular targets and mechanisms of action in plant cells. In this research, for the first time, the ivermectin effect on microtubules of Arabidopsis thaliana cells was studied. It was revealed that ivermectin (250 μg mL−1) disrupts the microtubule network, induces the loss of microtubule orientation, leads to microtubule curvature and shrinkage, and their longitudinal and cross-linked bundling in various cells of A. thaliana primary roots. Further, the previously proposed binding of ivermectin to the β1-tubulin taxane site was developed and confirmed using molecular dynamics simulations of ivermectin complexes with Haemonchus contortus and A. thaliana β1-tubulins. It was predicted that similar to other microtubule stabilizing agents ivermectin binding causes M-loop stabilization in both H. contortus and A. thaliana β-tubulin, which leads to the enhancement of lateral contacts between subunits of adjacent protofilaments preventing microtubule depolymerization.

The microtubule-severing protein UNC-45A preferentially binds to curved microtubules and counteracts the microtubule-straightening effects of Taxol

Uncoordinated protein 45A (UNC-45A) is the only known ATP-independent microtubule (MT)-severing protein. Thus, it severs MTs via a novel mechanism. Invitro and in cells, UNC-45A-mediated MT severing is preceded by the appearance of MT bends. While MTs are stiff biological polymers, in cells, they often curve, and the result of this curving can be breaking off. The contribution of MT-severing proteins on MT lattice curvature is largely undefined. Here, we show that UNC-45A curves MTs. Using invitro biophysical reconstitution and total internal fluorescence microscopy analysis, we show that UNC-45A is enriched in the areas where MTs are curved versus the areas where MTs are straight. In cells, we show that UNC-45A overexpression increases MT curvature and its depletion has the opposite effect. We also show that this effect occurs is independent of actomyosin contractility. Lastly, we show for the first time that in cells, Paclitaxel straightens MTs, and that UNC-45A can counteracts the MT-straightening effects of the drug.

Nanoscale characterization of drug-induced microtubule filament dysfunction using super-resolution microscopy

BackgroundThe integrity of microtubule filament networks is essential for the roles in diverse cellular functions, and disruption of its structure or dynamics has been explored as a therapeutic approach to tackle diseases such as cancer. Microtubule-interacting drugs, sometimes referred to as antimitotics, are used in cancer therapy to target and disrupt microtubules. However, due to associated side effects on healthy cells, there is a need to develop safer drug regimens that still retain clinical efficacy. Currently, many questions remain open regarding the extent of effects on cellular physiology of microtubule-interacting drugs at clinically relevant and low doses. Here, we use super-resolution microscopies (single-molecule localization and optical fluctuation based) to reveal the initial microtubule dysfunctions caused by nanomolar concentrations of colcemid.ResultsWe identify previously undetected microtubule (MT) damage caused by clinically relevant doses of colcemid. Short exposure to 30–80 nM colcemid results in aberrant microtubule curvature, with a trend of increased curvature associated to increased doses, and curvatures greater than 2 rad/μm, a value associated with MT breakage. Microtubule fragmentation was detected upon treatment with ≥ 100 nM colcemid. Remarkably, lower doses (< 20 nM after 5 h) led to subtle but significant microtubule architecture remodelling characterized by increased curvature and suppression of microtubule dynamics.ConclusionsOur results support the emerging hypothesis that microtubule-interacting drugs induce non-mitotic effects in cells, and establish a multi-modal imaging assay for detecting and measuring nanoscale microtubule dysfunction. The sub-diffraction visualization of these less severe precursor perturbations compared to the established antimitotic effects of microtubule-interacting drugs offers potential for improved understanding and design of anticancer agents.

On the dynamics of a curved microtubule-associated proteins by considering viscoelastic properties of the living biological cells

Over the last few years, some novel researches in the field of medical science made a tendency to have therapy without any complications or side-effects of the disease with the aid of prognosis about the behaviors of the microtubules. Regarding this issue, the stability/instability analysis of curved microtubule-associated protein in axons with attention to different size effect parameters based on an exact continuum method is presented. The real property of the living biological cells is presented as the Kelvin-Voight viscoelastic properties. Considering length scale parameter (l/R = 0.2) in modified couple stress theory (MCST) leads to a better agreement with experimental results in comparison by other theories that in the results section is presented, in detail. Based on presented exact results, the effect of parameter on the relative frequency changes of the microtubules is hardly depended to the value of the external forced load that should be attention to this value. Another important consequence is that the influence of the microtubule curvature parameter on the relative frequency changes of the living substructure is hardly depended on the value of the time-dependent viscoelastic property, that researchers in the analysis of the microtubule should be attention to this important issue. Communicated by Ramaswamy H. Sarma

Integrated 3D Tomography and Computational Modeling to Study Forces in Metaphase Spindles

Chromosome segregation during mitosis and meiosis are fundamental processes of life. During its course a bipolar spindle assembles and initially aligns the chromosomes on a metaphase plate before they are segregated to the two arising daughter cells during anaphase. The different cellular components of mitotic and meiotic spindles: microtubules, chromosomes, motor proteins and also membranes, such as the cell cortex or nuclear envelope, must interact with each other during spindle assembly, chromosome alignment and segregation.While we have achieved a very good understanding of signaling cascades and checkpoints that monitor and control mitosis, we are lacking an understanding on how the nano‐scale components of a spindle physically interact and generate the forces needed to faithfully align and segregate chromosomes. We have very little knowledge about the mechanics of spindles. In order to overcome the current limitations, we are generating a holistic description of mitotic and meiotic spindles in C. elegans embryos and mouse oocytes. This ambitious task will be achieved by a combination of 3D electron tomography, light microscopy and computational simulations. 3D tomography provides unprecedented detailed information about every individual microtubule within the spindle. We can measure the length, position and interactions of single microtubules with subcellular targets. In addition, we have developed tools to analyze the curvature of microtubules. Curvature originates from forces experienced by the microtubule and provides a read out of spindle intrinsic forces. The source of those forces can be determined by a combined approach of mutant analysis and computational simulations. To complement the static tomography data, we perform state‐of‐the‐art light microscopy. In conjunction with the light and electron microscopy we develop hypothesis and mechanical models of chromosome congression, alignment and segregation and test those in computational simulations and by selective depletion of proteins. We are using this approach to develop a (near) true‐to‐life model of microtubule, chromosome and membrane interactions and forces during mitosis.3D reconstruction of a meiotic metaphase spindle in the C. elegans embryoThis reconstruction is generated using 3D electron tomography of high‐pressure frozen samples and subsequent image processing and segmentation. The resolution of 2.3nm provides single microtubule resolution.Figure 1

Modulation of Microtubule Curvature under Different Cellular Conditions Revealed by In-Cell Ceryo-Electron Tomography

With persistence lengths in the range of 1–6 mm, microtubules (MTs) are the most rigid polymer of all cytoskeletal elements. However, inside cells they often exhibit highly curved shapes. Thus, it is tempting to speculate that the curvature of MTs in vivo are modulated by cellular factors resulting in bending at micrometer length scales. In order to better characterize the curvature of MTs and its potential causes in vivo, we quantified the curvature of cellular MTs in their native state from frozen-hydrated HeLa cells in interphase and mitosis as model systems using cryo-electron tomography (cryo-ET). We show that cellular MTs are highly curved beyond the limit posed by the polymer's intrinsic material's property. In fact, the apparent persistence lengths measured from the curved MTs are ∼100-1000 times lower than the persistence length obtained from thermal fluctuations alone, indicating prevalence of high magnitude of nonthermal forces in vivo. By using specific inhibitors, we pinpoint the sources of these forces that predominantly include actin-mediated compressive forces, ATP-dependent motor activities and microtubule polymerization. MT curvature was also found to vary depending on the cellular state, with mitotic MTs being relatively less curved implying that the magnitude of nonthermal forces have different regimes across cellular states in vivo. Notably, in-cell cryo-ET provides a glimpse into the native organization of MTs intertwined within the composite network of other cytoskeletal filaments and reveals differences in their organization across different cell cycle stages and cell types. We demonstrate that the presence of a heterogeneous elastic actin matrix and its organization is one of the main factors that influence the MT curvature in vivo.

Curvature-Sensitive Kinesin Binding Can Explain Microtubule Ring Formation and Reveals Chaotic Dynamics in a Mathematical Model

Microtubules are filamentous tubular protein polymers which are essential for a range of cellular behaviour, and are generally straight over micron length scales. However, in some gliding assays, where microtubules move over a carpet of molecular motors, individual microtubules can also form tight arcs or rings, even in the absence of crosslinking proteins. Understanding this phenomenon may provide important explanations for similar highly curved microtubules which can be found in nerve cells undergoing neurodegeneration. We propose a model for gliding assays where the kinesins moving the microtubules over the surface induce ring formation through differential binding, substantiated by recent findings that a mutant version of the motor protein kinesin applied in solution is able to lock-in microtubule curvature. For certain parameter regimes, our model predicts that both straight and curved microtubules can exist simultaneously as stable steady states, as has been seen experimentally. Additionally, unsteady solutions are found, where a wave of differential binding propagates down the microtubule as it glides across the surface, which can lead to chaotic motion. Whilst this model explains two-dimensional microtubule behaviour in an experimental gliding assay, it has the potential to be adapted to explain pathological curling in nerve cells.

Erratum for the Research Article: "The synthetic diazonamide DZ-2384 has distinct effects on microtubule curvature and dynamics without neurotoxicity" by M. Wieczorek, J. Tcherkezian, C. Bernier, A. E. Prota, S. Chaaban, Y. Rolland, C. Godbout, M. A. Hancock, J. C. Arezzo, O. Ocal, C. Rocha, N. Olieric, A. Hall, H. Ding, A. Bramoullé, M. G. Annis, G. Zogopoulos, P. G.

Erratum for the Research Article: "The synthetic diazonamide DZ-2384 has distinct effects on microtubule curvature and dynamics without neurotoxicity" by M. Wieczorek, J. Tcherkezian, C. Bernier, A. E. Prota, S. Chaaban, Y. Rolland, C. Godbout, M. A. Hancock, J. C. Arezzo, O. Ocal, C. Rocha, N. Olieric, A. Hall, H. Ding, A. Bramoullé, M. G. Annis, G. Zogopoulos, P. G.

Calcium-axonemal microtubuli interactions underlie mechanism(s) of primary cilia morphological changes.

We have used cell culture of astrocytes aligned within microchannels to investigate calcium effects on primary cilia morphology. In the absence of calcium and in the presence of flow of media (10μL.s-1) the majority (90%) of primary cilia showed reversible bending with an average curvature of 2.1±0.9×10-4nm-1. When 1.0mM calcium was present, 90% of cilia underwent bending. Forty percent of these cilia demonstrated strong irreversible bending, resulting in a final average curvature of 3.9±1×10-4nm-1, while 50% of cilia underwent bending similar to that observed during calcium-free flow. The average length of cilia was shifted toward shorter values (3.67±0.34μm) when exposed to excess calcium (1.0mM), compared to media devoid of calcium (3.96±0.26μm). The number of primary cilia that became curved after calcium application was reduced when the cell culture was pre-incubated with 15μM of the microtubule stabilizer, taxol, for 60min prior to calcium application. Calcium caused single microtubules to curve at a concentration ≈1.0mM in vitro, but at higher concentration (≈1.5mM) multiple microtubule curving occurred. Additionally, calcium causes microtubule-associated protein-2 conformational changes and its dislocation from the microtubule wall at the location of microtubule curvature. A very small amount of calcium, that is 1.45×1011 times lower than the maximal capacity of TRPPs calcium channels, may cause gross morphological changes (curving) of primary cilia, while global cytosol calcium levels are expected to remain unchanged. These findings reflect the non-linear manner in which primary cilia may respond to calcium signaling, which in turn may influence the course of development of ciliopathies and cancer.

The synthetic diazonamide DZ-2384 has distinct effects on microtubule curvature and dynamics without neurotoxicity.

Microtubule-targeting agents (MTAs) are widely used anticancer agents, but toxicities such as neuropathy limit their clinical use. MTAs bind to and alter the stability of microtubules, causing cell death in mitosis. We describe DZ-2384, a preclinical compound that exhibits potent antitumor activity in models of multiple cancer types. It has an unusually high safety margin and lacks neurotoxicity in rats at effective plasma concentrations. DZ-2384 binds the vinca domain of tubulin in a distinct way, imparting structurally and functionally different effects on microtubule dynamics compared to other vinca-binding compounds. X-ray crystallography and electron microscopy studies demonstrate that DZ-2384 causes straightening of curved protofilaments, an effect proposed to favor polymerization of tubulin. Both DZ-2384 and the vinca alkaloid vinorelbine inhibit microtubule growth rate; however, DZ-2384 increases the rescue frequency and preserves the microtubule network in nonmitotic cells and in primary neurons. This differential modulation of tubulin results in a potent MTA therapeutic with enhanced safety.

Microtubule Recognition: A Curvy Attraction

Summary While many proteins specifically associate with microtubule ends, the mechanisms underlying these associations remain largely undetermined. A new study demonstrates that doublecortin may localize to microtubule tips through preferential binding to regions of microtubule curvature.

3-D Reconstruction of Microtubules From Multi-Angle Total Internal Reflection Fluorescence Microscopy Using Bayesian Framework

Total internal reflection fluorescence (TIRF) microscopy excites a thin evanescent field which theoretically decays exponentially. Each TIRF image is actually the projection of a 3-D volume and hence cannot alone produce an accurate localization of structures in the z-dimension, however, it provides greatly improved axial resolution for biological samples. Multiple angle-TIRF microscopy allows controlled variation of the incident angle of the illuminating laser beam, thus generating a set of images of different penetration depths with the potential to reconstruct the 3-D volume of the sample. With the ultimate goal to quantify important biological parameters of microtubules, we present a method to reconstruct 3-D position and orientation of microtubules based on multi-angle TIRF data, as well as experimental calibration of the actual decay function of the evanescent field at each angle. We validate our method using computer simulations, by creating a phantom simulating the curvilinear characteristics of microtubules and project the artificially constructed volume into a set of TIRF image for different penetration depth. The reconstructed depth information for the phantom data is shown to be accurate and robust to noise. We apply our method to microtubule TIRF images of PtK(2) cells in vivo. By comparing microtubule curvatures of the reconstruction results and several electron microscopy (EM) images of vertically sliced sample of microtubules, we find that the curvature statistics of our reconstruction agree well with the ground truth (EM data). Quantifying the distribution of microtubule curvature reveals an interesting discovery that microtubules can buckle and form local bendings of considerably small radius of curvature which is also visually spotted on the EM images, while microtubule bendings on a larger scale generally have a much larger radius and cannot bear the stress of a large curvature. The presented method has the potential to provide a reliable tool for 3-D reconstruction and tracking of microtubules.

Measuring the number and spacing of molecular motors propelling a gliding microtubule

The molecular motor gliding assay, in which a microtubule or other filament moves across a surface coated with motors, has provided much insight into how molecular motors work. The kinesin-microtubule system is also a strong candidate for the job of nanoparticle transporter in nanotechnology devices. In most cases, several motors transport each filament. Each motor serves both to bind the microtubule to a stationary surface and to propel the microtubule along the surface. By applying a uniform transverse force of 4-19 pN to a superparamagnetic bead attached to the trailing end of the microtubule, we have measured the distance d between binding points (motors). The average value of d was determined as a function of motor surface density σ. The measurements agree well with the scaling model of Duke, Holy, and Liebler, which predicts that (d)~σ(-2/5) if 0.05≤σ≤20 μm(-2) [Phys. Rev. Lett. 74, 330 (1995)]. The distribution of d fits an extension of the model. The radius of curvature of a microtubule bent at a binding point by the force of the magnetic bead was ≈1 μm, 5000-fold smaller than the radius of curvature of microtubules subjected only to thermal forces. This is evidence that at these points of high bending stress, generated by the force on the magnetic bead, the microtubule is in the more flexible state of a two-state model of microtubule bending proposed by Heussinger, Schüller, and Frey [Phys. Rev. E 81, 021904 (2010)].

Cyclical Interactions between Two Outer Doublet Microtubules in Split Flagellar Axonemes

The beating of cilia and flagella is based on the localized sliding between adjacent outer doublet microtubules; however, the mechanism that produces oscillatory bending is unclear. To elucidate this mechanism, we examined the behavior of frayed axonemes of Chlamydomonas by using high-speed video recording. A pair of doublet microtubules frequently displayed association and dissociation cycles in the presence of ATP. In many instances, the dissociation of two microtubules was not accompanied by noticeable bending, suggesting that the dynein-microtubule interaction is not necessarily regulated by the microtubule curvature. On rare occasions, association and dissociation occurred simultaneously in the same interacting pair, resulting in a tip-directed movement of a stretch of gap between the pair. Based on these observations, we propose a model for cyclical bend propagation in the axoneme.

Opposing views on tensegrity as a structural framework for understanding cell mechanics.

Donald E. Ingber: Important new theories in science often ignite heated debates. If they do not, they are probably of little significance. Thus a strong argument in support of the importance of the tensegrity model of cell and tissue architecture first proposed almost 20 years ago ([23][1], [24][2

The geometric curvature of microtubules may play a part in information processing

The effect of general anaesthetics has been physically linked to the inhibition of the transmission of waves of electrical polarization along microtubules, as a physiological correlate to the effect on consciousness. Evidence for this is provided by the effects of general anaesthetics, where the biophysical mode of action is known. In this work, we consider the nature of wave propagation along quantum waveguides with curvature as a simple physical model for microtubules, which are of similar physical dimensions. We suggest that the geometrical configuration of the cytoskeleton may well play a role in cytoskeletal information processing, and provide further evidence for quantum coherence effects in the cytoarchitecture. Finally, we attempt to formulate some simple experimental tests involving anaesthetics, designed to test the hypothesis that microtubular curvature is involved in information processing at the cytoskeletal level.

Effect of metabolic inhibitors on sucrose-induced metaphase spindle elongation and spindle recovery.

Hyperosmotic sucrose treatment of metaphase PtK-1 cells has been shown to produce a reversible concentration-dependent effect on spindle elongation linked to a functional alteration in the connection of the chromosome to the spindle (Pover et al.: European Journal of Cell Biology 39:366-372, 1985). Spindle elongation, similar to that which occurs at anaphase B, is thought to be driven by the compression stored in the form of microtubule curvature in the nonkinetochore (nkMT) population of microtubules at metaphase (Snyder et al.: European Journal of Cell Biology 35:62-69, 1984 and 39:373-379, 1985). Addition of metabolic inhibitors to Ham's F-12 salts with deoxyglucose (D/F-12 medium) containing 0.4 M sucrose and 1 mM DNP does not within statistical error affect the rate and extent of sucrose-induced spindle elongation; rates and extents are 60-75% of normal anaphase B motions. Electron microscopic analysis of metaphase cells treated with D/F-12 medium and 0.4 M sucrose with 1 mM DNP demonstrates that spindle microtubules lose curvature and become straight in appearance, typical of microtubule organization in untreated anaphase cells. Sucrose-treated cells released into D/F-12 medium show a rapid reduction in spindle length; however, cells treated with either 0.4 M sucrose or 0.4 M sucrose and 1 mM DNP-containing D/F-12 medium and released into DNP-containing D/F-12 medium do not exhibit a significant reduction in spindle length. Electron microscopic analysis links changes in spindle length with microtubule/kinetochore associations. These data suggest that energy required for the initial phases of spindle elongation during anaphase is preloaded into the mitotic spindle by metaphase and does not require additional energy to be expressed as examined by sucrose-induced spindle elongation in the presence of metabolic inhibitors. Second, energy is required to make or maintain (or both) functional chromosome associations with the spindle as measured by reduction in spindle length following sucrose removal.