Microtubule Length Research Articles

Katanin Regulates Microtubule Dynamic Instability

Microtubule length regulation is required for proper spindle length and positioning, cilia formation, and axonal outgrowth. For correct microtubule organization to occur, microtubule associated proteins (MAPs) bind to microtubules and control the dynamics. Despite much work on stabilizing MAPs affecting microtubule dynamics, few studies have investigate the role of destabilizing MAPs on microtubules. Katanin, a known destabilizing MAP and the first-discovered microtubule severing enzyme, is a AAA+ enzyme that oligomerizes into hexamers and uses ATP hydrolysis to sever microtubules. We seek to measure the effects of katanin severing on microtubule dynamic instability in vitro, away from compounding cellular factors. We will use Total Internal Reflection Fluorescence (TIRF) microscopy on fluorescent microtubules and purified MBP-katanin to reveal that katanin is unable to sever dynamic microtubules, and instead modulates microtubules by changing the rates of growth and depolymerization. Our experiments also indicate that katanin does not modify the catastrophe rate of microtubules. Further work is required to disclose how katanin may work with stabilizing MAPs to control microtubule length.

Cut7-Driven Microtubule Sliding Reverses Direction Depending on Motor Density

Cut 7, the only kinesin-5 in S. pombe, is essential for mitosis. We have found that full length Cut7 slides microtubules (MTs) bidirectionally, with MT sliding velocity and direction dependent on motor crowding and ionic strength but not MT length. By contrast, truncated Cut7 monomers drive only plus end directed MT sliding, indicating that plus end directed strokes are the basal activity of the Cut7 motor head and that directional reversal is an emergent property of interacting head-pairs. We propose a possible mechanism for directional reversal, in which minus ended strokes are inhibited by motor crowding, causing the basal plus end directed activity to dominate.

γ-Tubulin Ring Complexes and EB1 play antagonistic roles in microtubule dynamics and spindle positioning

γ-Tubulin is critical for microtubule (MT) assembly and organization. In metazoa, this protein acts in multiprotein complexes called γ-Tubulin Ring Complexes (γ-TuRCs). While the subunits that constitute γ-Tubulin Small Complexes (γ-TuSCs), the core of the MT nucleation machinery, are essential, mutation of γ-TuRC-specific proteins in Drosophila causes sterility and morphological abnormalities via hitherto unidentified mechanisms. Here, we demonstrate a role of γ-TuRCs in controlling spindle orientation independent of MT nucleation activity, both in cultured cells and in vivo, and examine a potential function for γ-TuRCs on astral MTs. γ-TuRCs locate along the length of astral MTs, and depletion of γ-TuRC-specific proteins increases MT dynamics and causes the plus-end tracking protein EB1 to redistribute along MTs. Moreover, suppression of MT dynamics through drug treatment or EB1 down-regulation rescues spindle orientation defects induced by γ-TuRC depletion. Therefore, we propose a role for γ-TuRCs in regulating spindle positioning by controlling the stability of astral MTs.

Measurements of Single Fluorescent Motor Proteins: The Right Way

Cytoskeletal motor proteins are required in many cellular processes, such as intracellular transport and mitosis. Therefore, the biophysical characterization of motor protein movement along their filamentous tracks is essential. Commonly, stepping motility assays are used to determine the stepping and detachment rates of various molecular motor proteins by measuring their speed, run length and interaction time. However, comparison of these results proved to be difficult because the experimental setup (e.g. bead assay vs. single-molecule fluorescence assay), the experimental conditions (e.g. temperature, buffer or filament preparation) and data analysis (e.g. normal vs. exponential distribution) can influence the results. Here, we describe a method to evaluate traces of fluorescent motor proteins and propose an algorithm to correct the measurements for photobleaching and the limited length of the filaments. Additionally, bootstrapping is used to estimate statistical errors of the evaluation method. The method was tested with numerical simulations as well as with experimental data from kinesin-1 stepping experiments to show that the run length of kinesin-1 is independent of the microtubule length distribution. Our work will not only improve the evaluation of experimental data, but will also allow for better statistical comparison of two or more populations of motor proteins (e.g. motors with distinct mutations or motors linked to different cargos).

Mechanisms of inhibition of endothelial cell migration by taxanes

Several antiangiogenic mechanisms have been proposed for the widely-used cancer chemotherapeutic drugs taxotere (docetaxel) and taxol (paclitaxel), but none has been definitively identified. We analyzed their effects at a range of concentrations on migration and mitosis of human umbilical vein endothelial cells (HUVECs) on microtubule and focal adhesion morphology and microtubule dynamic instability during migration. Both taxanes inhibited migration by inhibiting both maintenance of the forward direction of the cell and by slowing migration over the entire contorted path length. At low (but not all) taxane concentrations that inhibit HUVEC migration, the shortening rates and shortening lengths of microtubules at the leading edge were strongly inhibited; peripheral microtubules were reduced in number and fewer targeted focal adhesions; focal adhesions doubled in length and became ring-shaped, elongate, and reduced in number; and an increase in stabilized microtubules occurred centrally. HUVEC migration was 13-19-fold more sensitive to taxanes than was mitosis confirming that taxanes exhibit significant effects in addition to mitotic arrest that may contribute to their overall antitumor effects. No relationship was detected between centrosome position and migration characteristics. The data suggest that taxanes inhibit migration, at least in part, by inhibiting the dynamic instability of microtubules that possibly interact with guanine nucleotide exchange factors and thus with the focal adhesion-associated G-proteins that then lead to disruption of the regulated formation and turnover of focal adhesions. A mechanism is presented by which subcytotoxic concentrations of taxanes might stabilize dynamic instability of a few microtubules and thereby inhibit migration and angiogenesis.

Microtubule-regulating kinesins

Microtubule-regulating kinesins

Aurora B and Kif2A control microtubule length for assembly of a functional central spindle during anaphase

The central spindle is built during anaphase by coupling antiparallel microtubules (MTs) at a central overlap zone, which provides a signaling scaffold for the regulation of cytokinesis. The mechanisms underlying central spindle morphogenesis are still poorly understood. In this paper, we show that the MT depolymerase Kif2A controls the length and alignment of central spindle MTs through depolymerization at their minus ends. The distribution of Kif2A was limited to the distal ends of the central spindle through Aurora B-dependent phosphorylation and exclusion from the spindle midzone. Overactivation or inhibition of Kif2A affected interchromosomal MT length and disorganized the central spindle, resulting in uncoordinated cell division. Experimental data and model simulations suggest that the steady-state length of the central spindle and its symmetric position between segregating chromosomes are predominantly determined by the Aurora B activity gradient. On the basis of these results, we propose a robust self-organization mechanism for central spindle formation.

Aurora B suppresses microtubule dynamics and limits central spindle size by locally activating KIF4A

Anaphase central spindle formation is controlled by the microtubule-stabilizing factor PRC1 and the kinesin KIF4A. We show that an MKlp2-dependent pool of Aurora B at the central spindle, rather than global Aurora B activity, regulates KIF4A accumulation at the central spindle. KIF4A phosphorylation by Aurora B stimulates the maximal microtubule-dependent ATPase activity of KIF4A and promotes its interaction with PRC1. In the presence of phosphorylated KIF4A, microtubules grew more slowly and showed long pauses in growth, resulting in the generation of shorter PRC1-stabilized microtubule overlaps in vitro. Cells expressing only mutant forms of KIF4A lacking the Aurora B phosphorylation site overextended the anaphase central spindle, demonstrating that this regulation is crucial for microtubule length control in vivo. Aurora B therefore ensures that suppression of microtubule dynamic instability by KIF4A is restricted to a specific subset of microtubules and thereby contributes to central spindle size control in anaphase.

Localized buckling of a microtubule surrounded by randomly distributed cross linkers

Microtubules supported by surrounding cross linkers in eukaryotic cells can bear a much higher compressive force than free-standing microtubules. Different from some previous studies, which treated the surroundings as a continuum elastic foundation or elastic medium, the present paper develops a micromechanics numerical model to examine the role of randomly distributed discrete cross linkers in the buckling of compressed microtubules. First, the proposed numerical approach is validated by reproducing the uniform multiwave buckling mode predicted by the existing elastic-foundation model. For more realistic buckling of microtubules surrounded by randomly distributed cross linkers, the present numerical model predicts that the buckling mode is localized at one end in agreement with some known experimental observations. In particular, the critical force for localized buckling, predicted by the present model, is insensitive to microtubule length and can be about 1 order of magnitude lower than those given by the elastic-foundation model, which suggests that the elastic-foundation model may have overestimated the critical force for buckling of microtubules in vivo. In addition, unlike the elastic-foundation model, the present model can capture the effect of end conditions on the critical force and wavelength of localized buckling. Based on the known data of spacing and elastic constants of cross linkers available in literature, the critical force and wavelength of the localized buckling mode, predicted by the present model, are compared to some experimental data with reasonable agreement. Finally, two empirical formulas are proposed for the critical force and wavelength of the localized buckling of microtubules surrounded by cross linkers.

Kinesin-8 Is a Low-Force Motor Protein with a Weakly Bound Slip State

During the cell cycle, kinesin-8s control the length of microtubules by interacting with their plus ends. To reach these ends, the motors have to be able to take many steps without dissociating. However, the underlying mechanism for this high processivity and how stepping is affected by force are unclear. Here, we tracked the motion of yeast (Kip3) and human (Kif18A) kinesin-8s with high precision under varying loads using optical tweezers. Surprisingly, both kinesin-8 motors were much weaker compared with other kinesins. Furthermore, we discovered a force-induced stick-slip motion: the motor frequently slipped, recovered from this state, and then resumed normal stepping motility without detaching from the microtubule. The low forces are consistent with kinesin-8s being regulators of microtubule dynamics rather than cargo transporters. The weakly bound slip state, reminiscent of a molecular safety leash, may be an adaptation for high processivity.

Single-Molecule Motility: Statistical Analysis and the Effects of Track Length on Quantification of Processive Motion

In vitro, single-molecule motility assays allow for the direct characterization of molecular motor properties including stepping velocity and characteristic run length. Although application of these techniques in vivo is feasible, the challenges involved in sample preparation, as well as the added complexity of the cell and its systems, result in a reduced ability to collect large datasets, as well as difficulty in simultaneous observation of the components of the motility system, namely motor and track. To address these challenges, we have developed simulations to characterize motility datasets as a function of sample size, processive run length of the motor, and distribution of track lengths. We introduce the use of a simple bootstrapping technique that allows for the quantification of measurement uncertainty and a Monte Carlo permutation resampling scheme for the measurement of statistical significance and the estimation of required sample size. In addition, we have found that, despite conventional wisdom, the measured characteristic run length is directly coupled to the characteristic track length that describes the microtubule length distribution. To be able to make comparisons between motility experiments performed on different track populations as well as make measurements of motility when motors and tracks cannot be simultaneously resolved, we have developed a theoretical framework for the determination of the effect that track length has on observed characteristic run lengths. This shows good agreement with in vitro motility experiments on two kinesin constructs walking on microtubule populations of different characteristic track lengths.

Mitotic Rounding Alters Cell Geometry to Ensure Efficient Bipolar Spindle Formation

Accurate animal cell division requires precise coordination of changes in the structure of the microtubule-based spindle and the actin-based cell cortex. Here, we use a series of perturbation experiments to dissect the relative roles of actin, cortical mechanics, and cell shape in spindle formation. We find that, whereas the actin cortex is largely dispensable for rounding and timely mitotic progression in isolated cells, it is needed to drive rounding to enable unperturbed spindle morphogenesis under conditions of confinement. Using different methods to limit mitotic cell height, we show that a failure to round up causes defects in spindle assembly, pole splitting, and a delay in mitotic progression. These defects can be rescued by increasing microtubule lengths and therefore appear to be a direct consequence of thelimited reach of mitotic centrosome-nucleated microtubules. These findings help to explain why most animal cells round up as they enter mitosis.

Biophysics of filament length regulation by molecular motors

Regulating physical size is an essential problem that biological organisms must solve from the subcellular to the organismal scales, but it is not well understood what physical principles and mechanisms organisms use to sense and regulate their size. Any biophysical size-regulation scheme operates in a noisy environment and must be robust to other cellular dynamics and fluctuations. This work develops theory of filament length regulation inspired by recent experiments on kinesin-8 motor proteins, which move with directional bias on microtubule filaments and alter microtubule dynamics. Purified kinesin-8 motors can depolymerize chemically-stabilized microtubules. In the length-dependent depolymerization model, the rate of depolymerization tends to increase with filament length, because long filaments accumulate more motors at their tips and therefore shorten more quickly. When balanced with a constant filament growth rate, this mechanism can lead to a fixed polymer length. However, the mechanism by which kinesin-8 motors affect the length of dynamic microtubules in cells is less clear. We study the more biologically realistic problem of microtubule dynamic instability modulated by a motor-dependent increase in the filament catastrophe frequency. This leads to a significant decrease in the mean filament length and a narrowing of the filament length distribution. The results improve our understanding of the biophysics of length regulation in cells.

Potential implication of IL-24 in lymphangiogenesis of human breast cancer

Lymphangiogenesis is involved in the dissemination of malignant cells from solid tumours to regional lymph nodes and possibly to various distant sites. Lymphangiogenesis is regulated by vascular endothelial growth factor (VEGF)-C and VEGF-D. Interleukin (IL)-24 is known as a cytokine with potent antitumour and tumour-suppressive activity which functions through its receptor (IL-22R). Expression of IL-24 has been shown to be reduced in breast cancer, and the reduced expression is associated with lymphatic metastases and a poor prognosis. However, the involvement of IL-24 in lymphangiogenesis during lymphatic metastasis remains unclear. The aim of the present study was to determine whether there is an association between IL-24, IL-22R and lymphangiogenic factors and markers in breast cancer. Analysis of IL-24, IL-22R and lymphangiogenic factors in malignant breast tissue samples (n=127) revealed a correlation between increased expression of lymphangiogenic markers (podoplanin, Prox-1 and LYVE-1) and reduced levels of IL-24 and IL-22R. Samples stained with a high degree of positivity for lymphangiogenic factors and markers whereas staining for IL-24 was weak. In vitro assays showed that the average perimeter length of microtubules formed by endothelial cells treated with IL-24 was significantly reduced compared to the control. The growth of endothelial cells was significantly reduced when exposed to a high concentration of IL-24 (250 ng/ml). Treatment of HECV cells with IL-24 resulted in significantly reduced expression of VEGF-C (P<0.05) and VEGF-D (P<0.001). In conclusion, reduced expression of IL-24 and IL-22R in breast cancer is correlated with increased expression of specific lymphangiogenic markers. IL-24 suppressed in vitro growth and microtubule formation of endothelial cells. IL-24 may downregulate the expression of lymphangiogenic markers and factors although further research is required. This suggests that IL-24 plays a profound role in suppressing tumour lymphangiogenesis, thereby, reducing the likelihood of cancer metastasis via the lymphatic route.

Atomic water channel controlling remarkable properties of a single brain microtubule: Correlating single protein to its supramolecular assembly

Microtubule nanotubes are found in every living eukaryotic cells; these are formed by reversible polymerization of the tubulin protein, and their hollow fibers are filled with uniquely arranged water molecules. Here we measure single tubulin molecule and single brain-neuron extracted microtubule nanowire with and without water channel inside to unravel their unique electronic and optical properties for the first time. We demonstrate that the energy levels of a single tubulin protein and single microtubule made of 40,000 tubulin dimers are identical unlike conventional materials. Moreover, the transmitted ac power and the transient fluorescence decay (single photon count) are independent of the microtubule length. Even more remarkable is the fact that the microtubule nanowire is more conducting than a single protein molecule that constitutes the nanowire. Microtubule's vibrational peaks condense to a single mode that controls the emergence of size independent electronic/optical properties, and automated noise alleviation, which disappear when the atomic water core is released from the inner cylinder. We have carried out several tricky state-of-the-art experiments and identified the electromagnetic resonance peaks of single microtubule reliably. The resonant vibrations established that the condensation of energy levels and periodic oscillation of unique energy fringes on the microtubule surface, emerge as the atomic water core resonantly integrates all proteins around it such that the nanotube irrespective of its size functions like a single protein molecule. Thus, a monomolecular water channel residing inside the protein-cylinder displays an unprecedented control in governing the tantalizing electronic and optical properties of microtubule.

Analysis of a single soybean microtubule’s persistence length

In this study, we have reported for the first time the persistence length of a single soybean microtubule in vitro. The measurement is based on analysis of thermal bending of a single soybean microtubule. Similar to mammalian microtubules, the length dependency of the persistence length was also observed for this species of plant microtubule. The identification of two intrinsic aspects of plant microtubules: the persistence length and the compositional structure in terms of variety of beta tubulin isotypes, and the potential correlation between these two factors, provides evidence for the functionality mechanism of plant microtubules. In this work, these two fundamental factors have been further discussed for our case study: single soybean microtubule.

Thermal Noise Imaging Indicates a New Regime of Length-Dependent Persistence Length in Microtubules

Microtubules perform a diverse set of essential functions within the cell. Most of these functions are mechanical and, therefore, their mechanical properties and the extent to which these properties are tunable are of wide interest. It is known that the persistence length of microtubules depends on factors, such as type and concentration of associated proteins, binding of stabilizers, and their length. We present a new method for measuring microtubule persistence length using a recently developed technique known as three-dimensional thermal noise imaging. Thermal noise imaging is a three-dimensional scanning probe technique capable of probing soft matter at physiological conditions on the nanometer scale. Transverse fluctuations of microtubules have been imaged with thermal noise imaging, and subsequent analysis reveals mechanical properties of the filaments. A novel assay that allows for the isolation of single, grafted microtubules provides a method for measuring the persistence length of microtubules for contour lengths as small as 500 nm. Such a length regime has proven difficult to study using other techniques. We present data that indicates a length regime of microtubules in which the persistence length is significantly smaller than has been measured in longer filaments.

The Effects of Nanomolar Concentrations of Taxol on Microtubule Polymerization

Microtubules are intracellular polymers that assemble from heterodimeric αβ-tubulin subunits. Polymerizing microtubules exhibit dynamic instability, a GTP-hydrolysis-driven phenomenon in which they alternate abruptly between phases of growth and relatively rapid shortening. The kinetics of tubulin subunit exchange at the microtubule tip, which are crucial to processes such as mitosis, are affected by the chemotherapeutic drug taxol, but the precise mechanism of action at therapeutic concentrations remains unclear. An understanding of how taxol alters exchange kinetics may assist in the development of new chemotherapeutic drugs.We observe microtubules polymerized at 4.5 micromolar tubulin and at 1-480 nanomolar taxol using total internal reflection fluorescence microscopy (TIRFM), achieving improvements in spatial and temporal resolution of, respectively, 1 and 2 orders of magnitude compared to previous taxol studies. We measure microtubule length changes to within 25 nm at 8 Hz, and we detect changes in the structure of the microtubule tip. We find that taxol potently affects microtubule growth at concentrations as low as 10 nM. At these concentrations we observed higher variability in growth rate than seen under control conditions, especially on long timescales (on the order of 100 seconds). In some cases, microtubules switched between normal growth and periods of very slow growth. Further, we have found that 10 nM taxol almost completely suppresses rapid shortening, and beginning at 100 nM taxol, the tubulin on and off rates gradually decrease, though the microtubule net growth rate (excluding periods of very slow growth in taxol-microtubules) remains constant.

Estimating Microtubule Distributions from 2D Immunofluorescence Microscopy Images Reveals Differences among Human Cultured Cell Lines

Microtubules are filamentous structures that are involved in several important cellular processes, including cell division, cellular structure and mechanics, and intracellular transportation. Little is known about potential differences in microtubule distributions within and across cell lines. Here we describe a method to estimate information pertaining to 3D microtubule distributions from 2D fluorescence images. Our method allows for quantitative comparisons of microtubule distribution parameters (number of microtubules, mean length) between different cell lines. Among eleven cell lines compared, some showed differences that could be accounted for by differences in the total amount of tubulin per cell while others showed statistically significant differences in the balance between number and length of microtubules. We also observed that some cell lines that visually appear different in their microtubule distributions are quite similar when the model parameters are considered. The method is expected to be generally useful for comparing microtubule distributions between cell lines and for a given cell line after various perturbations. The results are also expected to enable analysis of the differences in gene expression underlying the observed differences in microtubule distributions among cell types.

Dynamics and length distribution of microtubules under force and confinement

We investigate the microtubule polymerization dynamics with catastrophe and rescue events for three different confinement scenarios, which mimic typical cellular environments: (i) The microtubule is confined by rigid and fixed walls, (ii) it grows under constant force, and (iii) it grows against an elastic obstacle with a linearly increasing force. We use realistic catastrophe models and analyze the microtubule dynamics, the resulting microtubule length distributions, and force generation by stochastic and mean field calculations; in addition, we perform stochastic simulations. Freely growing microtubules exhibit a phase of bounded growth with finite microtubule length and a phase of unbounded growth. The main results for the three confinement scenarios are as follows: (i) In confinement by fixed rigid walls, we find exponentially decreasing or increasing stationary microtubule length distributions instead of bounded or unbounded phases, respectively. We introduce a realistic model for wall-induced catastrophes and investigate the behavior of the average length as a function of microtubule growth parameters. (ii) Under a constant force, the boundary between bounded and unbounded growth is shifted to higher tubulin concentrations and rescue rates. The critical force f(c) for the transition from unbounded to bounded growth increases logarithmically with tubulin concentration and the rescue rate, and it is smaller than the stall force. (iii) For microtubule growth against an elastic obstacle, the microtubule length and polymerization force can be regulated by microtubule growth parameters. For zero rescue rate, we find that the average polymerization force depends logarithmically on the tubulin concentration and is always smaller than the stall force in the absence of catastrophes and rescues. For a nonzero rescue rate, we find a sharply peaked steady-state length distribution, which is tightly controlled by microtubule growth parameters. The corresponding average microtubule length self-organizes such that the average polymerization force equals the critical force f(c) for the transition from unbounded to bounded growth. We also investigate the force dynamics if growth parameters are perturbed in dilution experiments. Finally, we show the robustness of our results against changes of catastrophe models and load distribution factors.