Dynamics of microtubules visualized by darkfield microscopy: treadmilling and dynamic instability

Individual microtubules undergoing treadmilling in vitro were visualized by darkfield light microscopy, and the relationship between treadmilling and dynamic instability was studied as a function of microtubule-associated proteins (MAPs). In order to demonstrate treadmilling directly by real-time observation, we constructed three-block microtubules, the center-block of which was decorated with Tetrahymena dynein. The decorated block can easily be distinguished from undecorated blocks in the darkfield microscope because the decorated one appears much thicker. At steady-state conditions, the length of an undecorated block at one end increased and that at another end decreased, while the decorated center-block did not change in its length. The results from these direct observations show that calf brain 3X-microtubules exhibit a treadmilling flux of 0.9 micron/h. Using a similar microscopy technique, we previously demonstrated that phosphocellulose PC-microtubules existed in either the growing or the shortening phase and alternated quite frequently at steady-state conditions (dynamic instability). How does treadmilling relate to dynamic instability? An image recording of individual 3X-microtubules containing MAPs revealed that the microtubules undergo treadmilling and do not exhibit any dynamic instability. This evidence shows that MAPs suppress the dynamic instability of microtubules. That is, treadmilling can take place in the steady state only after microtubules have been stabilized by MAPs.     

Direct observation of microtubule treadmilling by electron microscopy

Using an immunoelectron microscopic procedure, we directly observed the concurrent addition and loss of chicken brain tubulin subunits from the opposite ends of microtubules containing erythrocyte tubulin domains. The polarity of growth of the brain tubulin on the ends of erythrocyte microtubules was determined to be similar to growth off the ends of Chlamydomonas axonemes. The flux rate for brain tubulin subunits in vitro was low, approximately 0.9 micron/h. Tubulin subunit flux did not continue through the entire microtubule as expected, but ceased when erythrocyte tubulin domains became exposed, resulting in a metastable configuration that persisted for at least several hours. We attribute this to differences in the critical concentrations of erythrocyte and brain tubulin. The exchange of tubulin subunits into the walls of preformed microtubules other than at their ends was also determined to be insignificant, the exchange rate being less than the sensitivity of the assay, or less than 0.2%/h.     

Temperature-jump studies of microtubule dynamic instability

Evidence for a slowly dissociating tubulin-GTP cap at microtubule ends was derived from observation of a delay for attaining a maximum disassembly rate, after the temperature of steady state microtubules was rapidly decreased from 36 to 34 degrees C. The possibility that the microtubules were capped by a single tubulin-GTP subunit on each subhelix was ruled out, by comparison of the disassembly kinetics following a temperature decrease and dilution. The existence of a subpopulation of microtubules that underwent irreversible or near irreversible disassembly was demonstrated by a 30-s lag for attainment of a maximum assembly rate, after steady state microtubules were shifted from 34 to 36 degrees C. A dynamic instability model predicts that a maximum assembly rate will be delayed until disappearance of a subpopulation of microtubules that disassemble before being recapped. Analysis indicates that the 30-s lag resulted because approximately 2% of the mass in the steady state microtubule population was uncapped and disassembling and not readily recapped. The half-time for recapping of disassembling microtubules, by addition of tubulin-GTP subunits to ends, was equal to or greater than 20 s. Since tubulin-GDP dissociated from microtubules at a rate of about 4500 s-1, slow recapping resulted in dramatic shortening of disassembling microtubules.     

Kinetics and mechanism of microtubule length changes by dynamic instability

Microtubules at steady state were found to undergo dramatic changes in length, with only very little change in number concentration and mean length. This result is accounted for by a mechanism in which microtubules are capped at ends by tubulin-GTP subunits; loss of the tubulin-GTP cap at one end results in disassembly of all the tubulin-GDP subunits, so that the medial edge of the distal tubulin-GTP cap is exposed; the exposed tubulin-GTP cap is sufficiently stable, so that microtubule regrowth from the cap rather than loss of the cap occurs. This mechanism predicts that a bell-shaped length distribution of sheared microtubules will be transiently bimodal, with peaks of short and moderate length microtubules, in rearranging to an exponential length distribution. We have observed the predicted transient bimodal length distribution experimentally and in a Monte Carlo simulation. Dynamic instability has recently been accounted for by assuming that microtubule ends are capped with only a single tubulin-GTP subunit at each end of the five helices that serve as elongation sites. Such a minimal tubulin-GTP cap is apparently ruled out by our observations, which require that the remnant tubulin-GTP cap generated from disassembly be able to serve as nucleating site; we do not expect that a stable nucleating site can be generated from five tubulin-GTP subunits, oriented as the five helices that serve as elongation sites.     

Suppression of microtubule dynamic instability by the +TIP protein EB1 and its modulation by the CAP-Gly domain of p150glued

The EB1+TIP protein family and its binding partners track growing plus ends of microtubules in cells and are thought to regulate their dynamics. Here we determined the effects of EB1 and the N-terminal CAP-Gly domain (p150n) of one of its major binding partners, p150Glued, both separately and together, on the dynamic instability parameters at plus ends of purified steady-state microtubules. With EB1 alone, the shortening rate, the extent of shortening, and the catastrophe frequency were suppressed in the absence of significant effects on the growth rate or rescue frequency. The effects of EB1 on dynamics were significantly different when p150n was added together with EB1. The rate and extent of shortening and the catastrophe frequency were suppressed 3-4 times more strongly than with EB1 alone. In addition, the EB1-p150n complex increased the rescue frequency and the mean length the microtubules grew, parameters that were not significantly affected by EB1 alone. Similarly, deletion of EB1's C-terminal tail, which is a crucial binding region for p150n, significantly increased the ability of EB1 to suppress shortening dynamics. EB1 by itself bound along the length of the microtubules with 1 mol of EB1 dimer bound per approximately 12 mol of tubulin dimer. Approximately twice the amount of EB1 was recruited to the microtubules in the presence of p150n. Our results indicate that inactivation of EB1's flexible C-terminal tail significantly changes EB1's ability to modulate microtubule dynamics. They further suggest that p150Glued may activate and thereby facilitate the recruitment of EB1 to the tips of microtubules to regulate their dynamics.     

Phase dynamics at microtubule ends: the coexistence of microtubule length changes and treadmilling

The length dynamics both of microtubule-associated protein (MAP)-rich and MAP-depleted bovine brain microtubules were examined at polymer mass steady state. In both preparations, the microtubules exhibited length redistributions shortly after polymer mass steady state was attained. With time, however, both populations relaxed to a state in which no further changes in length distributions could be detected. Shearing the microtubules or diluting the microtubule suspensions transiently increased the extent to which microtubule length redistributions occurred, but again the microtubules relaxed to a state in which changes in the polymer length distributions were not detected. Under steady-state conditions of constant polymer mass and stable microtubule length distribution, both MAP-rich and MAP-depleted microtubules exhibited behavior consistent with treadmilling. MAPs strongly suppressed the magnitude of length redistributions and the steady-state treadmilling rates. These data indicate that the inherent tendency of microtubules in vitro is to relax to a steady state in which net changes in the microtubule length distributions are zero. If the basis of the observed length redistributions is the spontaneous loss and regain of GTP-tubulin ("GTP caps") at microtubule ends, then in order to account for stable length distributions the microtubule ends must reside in the capped state far longer than in the uncapped state, and uncapped microtubule ends must be rapidly recapped. The data suggest that microtubules in cells may have an inherent tendency to remain in the polymerized state, and that microtubule disassembly must be induced actively.     

Real-time observations of microtubule dynamic instability in living cells

Individual microtubule dynamics were observed in real time in primary cultures of newt lung epithelium using video-enhanced differential interference contrast microscopy and digital image processing. The linear filaments observed in cells corresponded to microtubules based on three criteria: (a) small particles translocated along them; (b) the majority of them disappeared after incubation in nocodazole; (c) and the distribution observed by differential interference contrast correlated with anti-tubulin immunofluorescence staining of the same cell. Microtubules were most clearly observed at the leading edge of cells located at the periphery of the epithelial sheet. Microtubules exhibited dynamic instability behavior: individual microtubules existed in persistent phases of elongation or rapid shortening. Microtubules elongated at a velocity of 7.2 micron/min +/- 0.3 SEM (n = 42) and rapidly shortened at a velocity of 17.3 micron/min +/- 0.7 SEM (n = 35). The transitions between elongation and rapid shortening occurred abruptly and stochastically with a transition frequency of 0.014 s-1 for catastrophe and 0.044 s-1 for rescue. Approximately 70% of the rapidly shortening microtubules were rescued and resumed elongation within the 35 x 35 micron microscopic field. A portion of the microtubule population appeared differentially stable and did not display any measurable elongation or shortening during 10-15-min observations.     

Okadaic acid induces interphase to mitotic-like microtubule dynamic instability by inactivating rescue

We used high-resolution video microscopy to visualize microtubule dynamic instability in extracts of interphase sea urchin eggs and to analyze the changes that occur upon addition of 0.8-2.5 microM okadaic acid, an inhibitor of phosphatase 1 and 2A (PP1, PP2a) (Bialojan, D., and A. Takai. 1988. Biochem. J. 256:283-290). Microtubule plus-ends in these extracts oscillated between the elongation and shortening phases of dynamic instability at frequencies typical for interphase cells. Switching from elongation to shortening (catastrophe) was frequent, but microtubules persisted and grew long because of frequent switching back to elongation (rescue). Addition of okadaic acid to the extract induced rapid (< 5 min) conversion to short, dynamic microtubules typical of mitosis. The frequency of catastrophe doubled and the velocities of elongation and shortening increased slightly; however, the major change was an elimination of rescue. Thus, modulation of the rescue frequency by phosphorylation-dependent mechanisms may be a major regulatory pathway for selectively controlling microtubule dynamics without dramatically changing velocities of microtubule elongation and shortening.     

Alteration of microtubule dynamic instability during preprophase band formation revealed by yellow fluorescent protein-CLIP170 microtubule plus-end labeling

At the onset of mitosis, plant cells form a microtubular preprophase band that defines the plane of cell division, but the mechanism of its formation remains a mystery. Here, we describe the use of mammalian yellow fluorescent protein-tagged CLIP170 to visualize the dynamic plus ends of plant microtubules in transfected cowpea protoplasts and in stably transformed and dividing tobacco Bright Yellow 2 cells. Using plus-end labeling, we observed dynamic instability in different microtubular conformations in live plant cells. The interphase plant microtubules grow at 5 micro m/min, shrink at 20 micro m/min, and display catastrophe and rescue frequencies of 0.02 and 0.08 events/s, respectively, exhibiting faster turnover than their mammalian counterparts. Strikingly, during preprophase band formation, the growth rate and catastrophe frequency of plant microtubules double, whereas the shrinkage rate and rescue frequency remain unchanged, making microtubules shorter and more dynamic. Using these novel insights and four-dimensional time-lapse imaging data, we propose a model that can explain the mechanism by which changes in microtubule dynamic instability drive the dramatic rearrangements of microtubules during preprophase band and spindle formation in plant cells.     

Compartment volume influences microtubule dynamic instability: a model study

Microtubules (MTs) are cytoskeletal polymers that exhibit dynamic instability, the random alternation between growth and shrinkage. MT dynamic instability plays an essential role in cell development, division, and motility. To investigate dynamic instability, simulation models have been widely used. However, conditions under which the concentration of free tubulin fluctuates as a result of growing or shrinking MTs have not been studied before. Such conditions can arise, for example, in small compartments, such as neuronal growth cones. Here we investigate by means of computational modeling how concentration fluctuations caused by growing and shrinking MTs affect dynamic instability. We show that these fluctuations shorten MT growth and shrinkage times and change their distributions from exponential to non-exponential, gamma-like. Gamma-like distributions of MT growth and shrinkage times, which allow optimal stochastic searching by MTs, have been observed in various cell types and are believed to require structural changes in the MT during growth or shrinkage. Our results, however, show that these distributions can already arise as a result of fluctuations in the concentration of free tubulin due to growing and shrinking MTs. Such fluctuations are possible not only in small compartments but also when tubulin diffusion is slow or when many MTs (de)polymerize synchronously. Volume and all other factors that influence these fluctuations can affect MT dynamic instability and, consequently, the processes that depend on it, such as neuronal growth cone behavior and cell motility in general.     

Generation of microtubule stability subclasses by microtubule- associated proteins: implications for the microtubule "dynamic instability" model

We have developed a method to distinguish microtubule associated protein (MAP)-containing regions from MAP-free regions within a microtubule, or within microtubule sub-populations. In this method, we measure the MAP-dependent stabilization of microtubule regions to dilution-induced disassembly of the polymer. The appropriate microtubule regions are identified by assembly in the presence of [3H]GTP, and assayed by filter trapping and quantitation of microtubule regions that contain label. We find that MAPs bind very rapidly to polymer binding sites and that they do not exchange from these sites measurably once bound. Also, very low concentrations of MAPs yield measurable stabilization of local microtubule regions. Unlike the stable tubule only polypeptide (STOP) proteins, MAPs do not exhibit any sliding behavior under our assay conditions. These results predict the presence of different stability subclasses of microtubules when MAPs are present in less than saturating amounts. The data can readily account for the observed "dynamic instability" of microtubules through unequal MAP distributions. Further, we report that MAP dependent stabilization is quantitatively reversed by MAP phosphorylation, but that calmodulin, in large excess, has no specific influence on MAP protein activity when MAPs are on microtubules.     

The microtubule-destabilizing kinesin XKCM1 regulates microtubule dynamic instability in cells

The dynamic activities of cellular microtubules (MTs) are tightly regulated by a balance between MT-stabilizing and -destabilizing proteins. Studies in Xenopus egg extracts have shown that the major MT destabilizer during interphase and mitosis is the kinesin-related protein XKCM1, which depolymerizes MT ends in an ATP-dependent manner. Herein, we examine the effects of both overexpression and inhibition of XKCM1 on the regulation of MT dynamics in vertebrate somatic cells. We found that XKCM1 is a MT-destabilizing enzyme in PtK2 cells and that XKCM1 modulates cellular MT dynamics. Our results indicate that perturbation of XKCM1 levels alters the catastrophe frequency and the rescue frequency of cellular MTs. In addition, we found that overexpression of XKCM1 or inhibition of KCM1 during mitosis leads to the formation of aberrant spindles and a mitotic delay. The predominant spindle defects from excess XKCM1 included monoastral and monopolar spindles, as well as small prometaphase-like spindles with improper chromosomal attachments. Inhibition of KCM1 during mitosis led to prometaphase spindles with excessively long MTs and spindles with partially separated poles and a radial MT array. These results show that KCM1 plays a critical role in regulating both interphase and mitotic MT dynamics in mammalian cells.     

The contributions of microtubule stability and dynamic instability to adenovirus nuclear localization efficiency

Adenoviruses (Ads) utilize host cell microtubules to traverse the intracellular space and reach the nucleus in a highly efficient manner. Previous studies have shown that Ad infection promotes the formation of stable, posttranslationally modified microtubules by a RhoA-dependent mechanism. Ad infection also shifts key parameters of microtubule dynamic instability by a Rac1-dependent mechanism, resulting in microtubules with lower catastrophe frequencies, persistent growth phases, and a bias toward net growth compared to microtubules in uninfected cells. Until now it was unclear whether changes in RhoGTPase activity or microtubule dynamics had a direct impact on the efficiency of Ad microtubule-dependent nuclear localization. Here we have performed synchronous Ad infections and utilized confocal microscopy to analyze the individual contributions of RhoA activation, Rac1 activation, microtubule stability, dynamic behavior, and posttranslational modifications on Ad nuclear localization efficiency (NLE). We found that drug-induced suppression of microtubule dynamics impaired Ad NLE by disrupting the radial organization of the microtubule array. When the microtubule array was maintained, the suppression or enhancement of microtubule turnover did not significantly affect Ad NLE. Furthermore, RhoA activation or the formation of acetylated microtubules did not enhance Ad NLE. In contrast, active Rac1 was required for efficient Ad nuclear localization. Because Rac1 mediates persistent growth of microtubules to the lamellar regions of cells, we propose that Ad-induced activation of Rac1 enhances the ability of microtubules to "search and capture" incoming virus particles.     

Stabilization of tubulin by deuterium oxide

Tubulin is an unstable protein when stored in solution and loses its ability to form microtubules rapidly. We have found that D2O stabilizes the protein against inactivation at both 4 and 37 degrees C. In H2O-based buffer, tubulin was completely inactivated after 40 h at 4 degrees C, but in buffer prepared in D2O, no activity was lost after 54 h. Tubulin was completely inactivated at 37 degrees C in 8 h in H2O buffer, but only 20% of the activity was lost in D2O buffer. Tubulin also lost its colchicine binding activity at a slower rate in D2O. The deuterated solvent retarded an aggregation process that occurs during incubation at both temperatures. Inactivation in H2O buffer was partially reversed by transferring the protein to D2O buffer; however, aggregation was not reversed. The level of binding of BisANS, a probe of exposed hydrophobic sites in proteins, increases during the inactivation of tubulin. In D2O, the rate of this increase is slowed somewhat. We propose that D2O has its stabilizing effect on a conformational step or steps that involve the disruption of hydrophobic forces. The conformational change is followed by an aggregation process that cannot be reversed by D2O. As reported previously [Ito, T., and Sato, H. (1984) Biochim. Biophys. Acta 800, 21-27], we found that D2O stimulates the formation of microtubules from tubulin. We also observed that the products of assembly in D2O/8% DMSO consisted of a high percentage of ribbon structures and incompletely folded microtubules. When these polymers were disassembled and reassembled in H2O/8% DMSO, the products were microtubules. We suggest that the combination of D2O and DMSO, both stimulators of tubulin assembly, leads to the rapid production of nuclei that lead to the formation of ribbon structures rather than microtubules.     

Nanomolar concentrations of nocodazole alter microtubule dynamic instability in vivo and in vitro.

Previous studies demonstrated that nanomolar concentrations of nocodazole can block cells in mitosis without net microtubule disassembly and resulted in the hypothesis that this block was due to a nocodazole-induced stabilization of microtubules. We tested this hypothesis by examining the effects of nanomolar concentrations of nocodazole on microtubule dynamic instability in interphase cells and in vitro with purified brain tubulin. Newt lung epithelial cell microtubules were visualized by video-enhanced differential interference contrast microscopy and cells were perfused with solutions of nocodazole ranging in concentration from 4 to 400 nM. Microtubules showed a loss of the two-state behavior typical of dynamic instability as evidenced by the addition of a third state where they exhibited little net change in length (a paused state). Nocodazole perfusion also resulted in slower elongation and shortening velocities, increased catastrophe, and an overall decrease in microtubule turnover. Experiments performed on BSC-1 cells that were microinjected with rhodamine-labeled tubulin, incubated in nocodazole for 1 h, and visualized by using low-light-level fluorescence microscopy showed similar results except that nocodazole-treated BSC-1 cells showed a decrease in catastrophe. To gain insight into possible mechanisms responsible for changes in dynamic instability, we examined the effects of 4 nM to 12 microM nocodazole on the assembly of purified tubulin from axoneme seeds. At both microtubule plus and minus ends, perfusion with nocodazole resulted in a dose-dependent decrease in elongation and shortening velocities, increase in pause duration and catastrophe frequency, and decrease in rescue frequency. These effects, which result in an overall decrease in microtubule turnover after nocodazole treatment, suggest that the mitotic block observed is due to a reduction in microtubule dynamic turnover. In addition, the in vitro results are similar to the effects of increasing concentrations of GDP-tubulin (TuD) subunits on microtubule assembly. Given that nocodazole increases tubulin GTPase activity, we propose that nocodazole acts by generating TuD subunits that then alter dynamic instability.     

Quantitative image analysis identifies pVHL as a key regulator of microtubule dynamic instability

Von Hippel-Lindau (VHL) tumor suppressor gene mutations predispose carriers to kidney cancer. The protein pVHL has been shown to interact with microtubules (MTs), which is critical to cilia maintenance and mitotic spindle orientation. However, the function for pVHL in the regulation of MT dynamics is unknown. We tracked MT growth via the plus end marker EB3 (end-binding protein 3)-GFP and inferred additional parameters of MT dynamics indirectly by spatiotemporal grouping of growth tracks from live cell imaging. Our data establish pVHL as a near-optimal MT-stabilizing protein: it attenuates tubulin turnover, both during MT growth and shrinkage, inhibits catastrophe, and enhances rescue frequencies. These functions are mediated, in part, by inhibition of tubulin guanosine triphosphatase activity in vitro and at MT plus ends and along the MT lattice in vivo. Mutants connected to the VHL cancer syndrome are differentially compromised in these activities. Thus, single cell–level analysis of pVHL MT regulatory function allows new predictions for genotype to phenotype associations that deviate from the coarser clinically defined mutant classifications.     

BetaIII-tubulin induces paclitaxel resistance in association with reduced effects on microtubule dynamic instability

The development of resistance to paclitaxel in tumors is one of the most significant obstacles to successful therapy. Overexpression of the betaIII-tubulin isotype has been associated with paclitaxel resistance in a number of cancer cell lines and in tumors, but the mechanism of resistance has remained unclear. Paclitaxel inhibits cancer cell proliferation by binding to the beta-subunit of tubulin in microtubules and suppressing microtubule dynamic instability, leading to mitotic arrest and cell death. We hypothesized that betaIII-tubulin overexpression induces resistance to paclitaxel either by constitutively enhancing microtubule dynamic instability in resistant cells or by rendering the microtubules less sensitive to the suppression of dynamics by paclitaxel. Using Chinese hamster ovary cells that inducibly overexpress either betaI- or betaIII-tubulin, we analyzed microtubule dynamic instability during interphase by microinjection of rhodamine-labeled tubulin and time-lapse fluorescence microscopy. In the absence of paclitaxel, there were no differences in any aspect of dynamic instability between the two beta-tubulin-overexpressing cell types. However, in the presence of 150 nm paclitaxel, dynamic instability was suppressed to a significantly lesser extent (suppressed only 12%) in cells overexpressing betaIII-tubulin than in cells overexpressing similar levels of betaI-tubulin (suppressed 47%). The results suggest that overexpression of betaIII-tubulin induces paclitaxel resistance by reducing the ability of paclitaxel to suppress microtubule dynamics. The results also suggest that endogenous regulators of microtubule dynamics may differentially interact with individual tubulin isotypes, supporting the idea that differential expression of tubulin isotypes has functional consequences in cells.     

Self-organization of anastral spindles by synergy of dynamic instability, autocatalytic microtubule production, and a spatial signaling gradient

Assembly of the mitotic spindle is a classic example of macromolecular self-organization. During spindle assembly, microtubules (MTs) accumulate around chromatin. In centrosomal spindles, centrosomes at the spindle poles are the dominating source of MT production. However, many systems assemble anastral spindles, i.e., spindles without centrosomes at the poles. How anastral spindles produce and maintain a high concentration of MTs in the absence of centrosome-catalyzed MT production is unknown. With a combined biochemistry-computer simulation approach, we show that the concerted activity of three components can efficiently concentrate microtubules (MTs) at chromatin: (1) an external stimulus in form of a RanGTP gradient centered on chromatin, (2) a feed-back loop where MTs induce production of new MTs, and (3) continuous re-organization of MT structures by dynamic instability. The mechanism proposed here can generate and maintain a dissipative MT super-structure within a RanGTP gradient.     

Protein localization by actin treadmilling and molecular motors regulates stereocilia shape and treadmilling rate

We present a physical model that describes the active localization of actin-regulating proteins inside stereocilia during steady-state conditions. The mechanism of localization is through the interplay of free diffusion and directed motion, which is driven by coupling to the treadmilling actin filaments and to myosin motors that move along the actin filaments. The resulting localization of both the molecular motors and their cargo is calculated, and is found to have an exponential (or steeper) profile. This localization can be at the base (driven by actin retrograde flow and minus-end myosin motors), or at the stereocilia tip (driven by plus-end myosin motors). The localization of proteins that influence the actin depolymerization and polymerization rates allow us to describe the narrow shape of the stereocilia base, and the observed increase of the actin polymerization rate with the stereocilia height.