Dynamic Instability Of Individual Microtubules Research Articles

Physical properties of the cytoplasm modulate the rates of microtubule polymerization and depolymerization.

The cytoplasm is a crowded, visco-elastic environment whose physical properties change according to physiological or developmental states. How the physical properties of the cytoplasm impact cellular functions invivo remains poorly understood. Here, we probe the effects of cytoplasmic concentration on microtubules by applying osmotic shifts to fission yeast, moss, and mammalian cells. We show that the rates of both microtubule polymerization and depolymerization scale linearly and inversely with cytoplasmic concentration; an increase in cytoplasmic concentration decreases the rates of microtubule polymerization and depolymerization proportionally, whereas a decrease in cytoplasmic concentration leads to the opposite. Numerous lines of evidence indicate that these effects are due to changes in cytoplasmic viscosity rather than cellular stress responses or macromolecular crowding per se. We reconstituted these effects on microtubules invitro by tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of the cytoplasm modulates the reactions that underlie microtubule dynamic behaviors.

Relationship between dynamic instability of individual microtubules and flux of subunits into and out of polymer.

Behaviors of dynamic polymers such as microtubules and actin are frequently assessed at one or both of the following scales: (a) net assembly or disassembly of bulk polymer, (b) growth and shortening of individual filaments. Previous work has derived various forms of an equation to relate the rate of change in bulk polymer mass (i.e., flux of subunits into and out of polymer, often abbreviated as "J") to individual filament behaviors. However, these versions of the "J equation" differ in the variables used to quantify individual filament behavior, which correspond to different experimental approaches. For example, some variants of the J equation use dynamic instability parameters, obtained by following particular individual filaments for long periods of time. Another form of the equation uses measurements from many individuals followed over short time steps. We use a combination of derivations and computer simulations that mimic experiments to (a) relate the various forms of the J equation to each other, (b) determine conditions under which these J equation forms are and are not equivalent, and (c) identify aspects of the measurements that can affect the accuracy of each form of the J equation. Improved understanding of the J equation and its connections to experimentally measurable quantities will contribute to efforts to build a multiscale understanding of steady-state polymer behavior.

Indibulin dampens microtubule dynamics and produces synergistic antiproliferative effect with vinblastine in MCF-7 cells: Implications in cancer chemotherapy

Indibulin, a synthetic inhibitor of tubulin assembly, has shown promising anticancer activity with a minimal neurotoxicity in preclinical animal studies and in Phase I clinical trials for cancer chemotherapy. Using time-lapse confocal microscopy, we show that indibulin dampens the dynamic instability of individual microtubules in live breast cancer cells. Indibulin treatment also perturbed the localization of end-binding proteins at the growing microtubule ends in MCF-7 cells. Indibulin reduced inter-kinetochoric tension, produced aberrant spindles, activated mitotic checkpoint proteins Mad2 and BubR1, and induced mitotic arrest in MCF-7 cells. Indibulin-treated MCF-7 cells underwent apoptosis-mediated cell death. Further, the combination of indibulin with an anticancer drug vinblastine was found to exert synergistic cytotoxic effects on MCF-7 cells. Interestingly, indibulin displayed a stronger effect on the undifferentiated neuroblastoma (SH-SY5Y) cells than the differentiated neuronal cells. Unlike indibulin, vinblastine and colchicine produced similar depolymerizing effects on microtubules in both differentiated and undifferentiated SH-SY5Y cells. The data indicated a possibility that indibulin may reduce chemotherapy-induced peripheral neuropathy in cancer patients.

Estimating the Microtubule GTP Cap Size In Vivo

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.

Curcumin suppresses the dynamic instability of microtubules, activates the mitotic checkpoint and induces apoptosis in MCF‐7 cells

In this study, curcumin, a potential anticancer agent, was found to dampen the dynamic instability of individual microtubules in living MCF-7 cells. It strongly reduced the rate and extent of shortening states, and modestly reduced the rate and extent of growing states. In addition, curcumin decreased the fraction of time microtubules spent in the growing state and strongly increased the time microtubules spent in the pause state. Brief treatment with curcumin depolymerized mitotic microtubules, perturbed microtubule-kinetochore attachment and disturbed the mitotic spindle structure. Curcumin also perturbed the localization of the kinesin protein Eg5 and induced monopolar spindle formation. Further, curcumin increased the accumulation of Mad2 and BubR1 at the kinetochores, indicating that it activated the mitotic checkpoint. In addition, curcumin treatment increased the metaphase/anaphase ratio, indicating that it can delay mitotic progression from the metaphase to anaphase. We provide evidence suggesting that the affected cells underwent apoptosis via the p53-dependent apoptotic pathway. The results support the idea that kinetic stabilization of microtubule dynamics assists in the nuclear translocation of p53. Curcumin exerted additive effects when combined with vinblastine, a microtubule depolymerizing drug, whereas the combination of curcumin with paclitaxel, a microtubule-stabilizing drug, produced an antagonistic effect on the inhibition of MCF-7 cell proliferation. The results together suggested that curcumin inhibited MCF-7 cell proliferation by inhibiting the assembly dynamics of microtubules.

Griseofulvin stabilizes microtubule dynamics, activates p53 and inhibits the proliferation of MCF-7 cells synergistically with vinblastine

BackgroundGriseofulvin, an antifungal drug, has recently been shown to inhibit proliferation of various types of cancer cells and to inhibit tumor growth in athymic mice. Due to its low toxicity, griseofulvin has drawn considerable attention for its potential use in cancer chemotherapy. This work aims to understand how griseofulvin suppresses microtubule dynamics in living cells and sought to elucidate the antimitotic and antiproliferative action of the drug.MethodsThe effects of griseofulvin on the dynamics of individual microtubules in live MCF-7 cells were measured by confocal microscopy. Immunofluorescence microscopy, western blotting and flow cytometry were used to analyze the effects of griseofulvin on spindle microtubule organization, cell cycle progression and apoptosis. Further, interactions of purified tubulin with griseofulvin were studied in vitro by spectrophotometry and spectrofluorimetry. Docking analysis was performed using autodock4 and LigandFit module of Discovery Studio 2.1.ResultsGriseofulvin strongly suppressed the dynamic instability of individual microtubules in live MCF-7 cells by reducing the rate and extent of the growing and shortening phases. At or near half-maximal proliferation inhibitory concentration, griseofulvin dampened the dynamicity of microtubules in MCF-7 cells without significantly disrupting the microtubule network. Griseofulvin-induced mitotic arrest was associated with several mitotic abnormalities like misaligned chromosomes, multipolar spindles, misegregated chromosomes resulting in cells containing fragmented nuclei. These fragmented nuclei were found to contain increased concentration of p53. Using both computational and experimental approaches, we provided evidence suggesting that griseofulvin binds to tubulin in two different sites; one site overlaps with the paclitaxel binding site while the second site is located at the αβ intra-dimer interface. In combination studies, griseofulvin and vinblastine were found to exert synergistic effects against MCF-7 cell proliferation.ConclusionsThe study provided evidence suggesting that griseofulvin shares its binding site in tubulin with paclitaxel and kinetically suppresses microtubule dynamics in a similar manner. The results revealed the antimitotic mechanism of action of griseofulvin and provided evidence suggesting that griseofulvin alone and/or in combination with vinblastine may have promising role in breast cancer chemotherapy.

History-Dependent Catastrophes Regulate Axonal Microtubule Behavior

In Chinese hamster ovary cells, microtubules originate at the microtubule organizing center (MTOC) and grow persistently toward the cell edge, where they undergo catastrophe. In axons, microtubule dynamics must be regulated differently because microtubules grow parallel to the plasma membrane and there is no MTOC. GFP-tagged microtubule plus end tracking proteins (+TIPs) mark the ends of growing neuronal microtubules. Their fluorescent "comet-like" pattern reflects turnover of +TIP binding sites. Using GFP-tagged +TIPs and fluorescence-based segmentation and tracking tools, we show that axonal microtubules grow with a constant average velocity and that they undergo catastrophes at random positions, yet in a programmed fashion. Using protein depletion approaches, we find that the +TIPs CLIP-115 and CLIP-170 affect average microtubule growth rate and growth distance in neurons but not the duration of a microtubule growth event. In N1E-115 neuroblastoma cells, we find that EB1, the core +TIP, regulates microtubule growth rate, growth distance, and duration, consistent with in vitro data. Combined, our data suggest that CLIPs influence the axonal microtubule/tubulin ratio, whereas EB1 stimulates microtubule growth and structural transitions at microtubule ends, thereby regulating microtubule catastrophes and the turnover of +TIP binding sites.

Kinetic Stabilization of Microtubule Dynamics by Estramustine Is Associated with Tubulin Acetylation, Spindle Abnormalities, and Mitotic Arrest

Estramustine (EM) alone or in combination with other anticancer agents is clinically used for the treatment of hormone refractory prostate cancer. Furthermore, EM has been shown to potently inhibit the proliferation of different types of cancer cells in culture apparently by targeting microtubules; however, the antiproliferative mechanism of action of EM is not clear. In this work, we have shown that EM strongly suppressed the dynamic instability of individual microtubules in MCF-7 cells by reducing the rates of growing and shortening excursions and increasing the time microtubule spent in the pause state. At its half maximal proliferation inhibitory concentration (IC(50)), EM exerted strong suppressive effects on the dynamics of microtubules in MCF-7 cells without detectably affecting either the organization or the polymerized mass of microtubules. At relatively high concentrations (5 x IC(50)), EM significantly depolymerized microtubules in the cells. Furthermore, the microtubules were found highly acetylated, supporting the conclusion that they were stabilized by the drug. EM treatment induced spindle abnormalities in MCF-7 cells, and a major population of the arrested mitotic cells was multipolar. EM also perturbed the microtubule-kinetochore interaction, thereby activating the spindle assembly checkpoint and leading to apoptotic cell death.

Microtubule Polymerization: One Step at a Time

The dynamic assembly of microtubules is a key factor in many of their functions in the cell and recent experiments give new insight into this process at the molecular level.

FTDP-17 Mutations Compromise the Ability of Tau to Regulate Microtubule Dynamics in Cells

The neural microtubule-associated protein Tau binds directly to microtubules and regulates their dynamic behavior. In addition to being required for normal development, maintenance, and function of the nervous system, Tau is associated with several neurodegenerative diseases, including Alzheimer disease. One group of neurodegenerative dementias known as FTDP-17 (fronto-temporal dementia with Parkinsonism linked to chromosome 17) is directly linked genetically to mutations in the tau gene, demonstrating that Tau misfunction can cause neuronal cell death and dementia. These mutations result either in amino acid substitutions in Tau or in altered Tau mRNA splicing that skews the expression ratio of wild-type 3-repeat and 4-repeat Tau isoforms. Because wild-type Tau regulates microtubule dynamics, one possible mechanism underlying Tau-mediated neurodegeneration is aberrant regulation of microtubule behavior. In this study, we microinjected normal and mutated Tau protein into cultured cells expressing fluorescent tubulin and measured the effects on the dynamic instability of individual microtubules. We found that the FTDP-17 amino acid substitutions G272V (in both 3-repeat and 4-repeat Tau contexts), DeltaK280, and P301L all exhibited markedly reduced abilities to regulate dynamic instability relative to wild-type Tau. In contrast, the FTDP-17 R406W mutation (which maps in a regulatory region outside the microtubule binding domain of Tau) did not significantly alter the ability of 3-repeat or 4-repeat Tau to regulate microtubule dynamics. Overall, these data are consistent with a loss-of-function model in which both amino acid substitutions and altered mRNA splicing in Tau lead to neurodegeneration by diminishing the ability of Tau to properly regulate microtubule dynamics.

Recombinant microtubule-associated protein 2c reduces the dynamic instability of individual microtubules.

The effects of purified recombinant microtubule-associated protein 2c (rMAP2c) on the dynamic instability of microtubules were examined by direct observation of individual microtubules in vitro by video-enhanced differential interference contrast light microscopy. Microtubules were grown in the absence or presence of varying concentrations of rMAP2c and were analyzed to determine growth rates, shortening rates, and the frequencies of conversion between growing and shortening phases. We found rMAP2c to stabilize microtubules dramatically. The most notable effect is a reduction in both the frequency of catastrophes (transitions from growth to shortening) and the mean length of shortening events: no microtubule catastrophes were observed at concentrations of rMAP2c as low as 1.06 microM in a solution of 10 microM tubulin. Even at lower rMAP2c concentrations, there is a marked stabilizing effect. As the concentration of rMAP2c increases, average growth rates increase slightly, shortening rates decrease, and the frequency of rescues (transitions from shortening to growth) increases significantly. Together, these changes in parameters produce a population of extremely stable microtubules in the presence of rMAP2c. This stabilization is consistent with a structural role for MAP2c during early postnatal neural development.

Buffer conditions and non-tubulin factors critically affect the microtubule dynamic instability of sea urchin egg tubulin.

The dynamic instability of individual microtubules (Mts) in cytoplasmic extracts or assembled from highly purified sea urchin egg tubulin was examined using video-enhanced, differential-interference contrast (VE-DIC) light microscopy. Extract Mts (endogenous tubulin = 12.1 microM) displayed only plus-ended growth. The elongation velocity was 7.8 microns/min for an average duration of 1.3 min before switching (catastrophe) to rapid shortening, which occurred at 13.0 microns/min for an average duration of 0.5 min before switching (rescue) back to the elongation phase. These parameters are typical of interphase Mt dynamic instability. Surprisingly, Mts assembled from purified urchin egg tubulin in standard buffers were less dynamic that those reported for purified brain tubulin or Mts in the extract. Buffer parameters were changed in an attempt to mimic the extract Mt results. The pH buffer itself, Hepes or Pipes, drastically altered Mt dynamics but could not achieve high elongation velocity with high catastrophe frequencies. Calcium at 1 microM had negligible effects, while increasing pH from 6.9 to 7.2 stimulated elongation velocity. Finally, Mt dynamics of purified egg tubulin (11.9 microM) were assayed in ultrafiltrates (MW cut-off less than 30 kD) of the cytoplasmic extracts. Mts elongated slowly at 1.2 microns/min for 26 min before a catastrophe and rapid shortening at 11.8 microns/min. Rescue was less frequent than unfiltered extracts, minus-ended growth was observed, and self-assembly occurred at slightly higher tubulin concentrations. Therefore, the egg extracts and cytoplasm must contain non-buffer factors which stimulate elongation velocity by 6.5-fold without self-assembly, increase catastrophe frequency by 20-fold, and block minus-ended growth.

Effects of magnesium on the dynamic instability of individual microtubules

We investigated the effect of magnesium ion (Mg) on the parameters of dynamic instability of individual porcine brain microtubules. Rates of elongation and rapid shortening were measured by using video-enhanced DIC light microscopy and evaluated by using computer-generated plots of microtubule length vs time. Increasing [Mg] from 0.25 to 6 mM increased the second-order association rate constant for elongation about 25% at each end. At plus ends, this resulted in a 1.5-2-fold increase in elongation rates over the tubulin concentrations explored. Rapid shortening rates were more dramatically affected by Mg. As [Mg] was increased from 0.25 to 6 mM, the average rate of rapid shortening increased about 3-fold at plus ends and 4-5-fold at minus ends. The ends had roughly equivalent average rates at low [Mg], of 30-45 microns/min. At any Mg concentration, rates of disassembly varied from one microtubule to another, and often an individual microtubule would exhibit more than one rate during a single shortening phase. Individual rates at 6 mM Mg varied from 12 to 250 microns/min. Over the concentration range explored, Mg affected the frequencies of transition from elongation to shortening and back only at minus ends. Minus ends were relatively stable at low [Mg], having 4 times the frequency of rescue than at high [Mg], and a lower frequency of catastrophe (particularly evident at low tubulin concentrations). Plus ends, surprisingly, were highly unstable at all Mg concentrations investigated, having about the same transition frequencies as did the least stable (high Mg) minus ends. Our results have implications for models of the GTP cap, again emphasizing that GTP caps cannot build up in proportion to elongation rate, and must be constrained to the tips of growing microtubules.

Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies.

We have developed video microscopy methods to visualize the assembly and disassembly of individual microtubules at 33-ms intervals. Porcine brain tubulin, free of microtubule-associated proteins, was assembled onto axoneme fragments at 37 degrees C, and the dynamic behavior of the plus and minus ends of microtubules was analyzed for tubulin concentrations between 7 and 15.5 microM. Elongation and rapid shortening were distinctly different phases. At each end, the elongation phase was characterized by a second order association and a substantial first order dissociation reaction. Association rate constants were 8.9 and 4.3 microM-1 s-1 for the plus and minus ends, respectively; and the corresponding dissociation rate constants were 44 and 23 s-1. For both ends, the rate of tubulin dissociation equaled the rate of tubulin association at 5 microM. The rate of rapid shortening was similar at the two ends (plus = 733 s-1; minus = 915 s-1), and did not vary with tubulin concentration. Transitions between phases were abrupt and stochastic. As the tubulin concentration was increased, catastrophe frequency decreased at both ends, and rescue frequency increased dramatically at the minus end. This resulted in fewer rapid shortening phases at higher tubulin concentrations for both ends and shorter rapid shortening phases at the minus end. At each concentration, the frequency of catastrophe was slightly greater at the plus end, and the frequency of rescue was greater at the minus end. Our data demonstrate that microtubules assembled from pure tubulin undergo dynamic instability over a twofold range of tubulin concentrations, and that the dynamic instability of the plus and minus ends of microtubules can be significantly different. Our analysis indicates that this difference could produce treadmilling, and establishes general limits on the effectiveness of length redistribution as a measure of dynamic instability. Our results are consistent with the existence of a GTP cap during elongation, but are not consistent with existing GTP cap models.

Visualization of the dynamic instability of individual microtubules by dark-field microscopy.

It has previously been shown that two populations of microtubules coexist in a dynamically unstable manner in vitro: those in one population elongate while those in the other shorten and finally disappear. This conclusion was based on changes in the number and length distribution of microtubules after dilution of the microtubule solution. Here, we demonstrate directly that growing and shortening populations coexist in steady-state conditions, by visualization of the dynamic behaviour of individual microtubules in vitro by dark-field microscopy. Real-time video recording reveals that both ends of a microtubule exist in either the growing or the shortening phase and alternate quite frequently between the two phases in a stochastic manner. Moreover, growing and shortening ends can coexist on a single microtubule, one end continuing to grow simultaneously with shortening at the other end. We find no correlation in the phase conversion either among individual microtubules or between the two ends of a single microtubule. The two ends of any given microtubule have remarkably different characteristics; the active end grows faster, alternates in phase more frequently and fluctuates in length to a greater extent than the inactive end. Microtubule-associated proteins (MAPs) suppress the phase conversion and stabilize microtubules in the growing phase.