Dynamic Behavior Of Microtubules Research Articles

Spatiotemporal relationships between growth and microtubule orientation as revealed in living root cells of Arabidopsis thaliana transformed with green-fluorescent-protein gene construct GFP-MBD.

Arabidopsis thaliana plants were transformed with GFP-MBD (J. Marc et al., Plant Cell 10: 1927-1939, 1998) under the control of a constitutive (35S) or copper-inducible promoter. GFP-specific fluorescence distributions, levels, and persistence were determined and found to vary with age, tissue type, transgenic line, and individual plant. With the exception of an increased frequency of abnormal roots of 35S GFP-MBD plants grown on kanamycin-containing media, expression of GFP-MBD does not appear to affect plant phenotype. The number of leaves, branches, bolts, and siliques as well as overall height, leaf size, and seed set are similar between wild-type and transgenic plants as is the rate of root growth. Thus, we conclude that the transgenic plants can serve as a living model system in which the dynamic behavior of microtubules can be visualized. Confocal microscopy was used to simultaneously monitor growth and microtubule behavior within individual cells as they passed through the elongation zone of the Arabidopsis root. Generally, microtubules reoriented from transverse to oblique or longitudinal orientations as growth declined. Microtubule reorientation initiated at the ends of the cell did not necessarily occur simultaneously in adjacent neighboring cells and did not involve complete disintegration and repolymerization of microtubule arrays. Although growth rates correlated with microtubule reorientation, the two processes were not tightly coupled in terms of their temporal relationships, suggesting that other factor(s) may be involved in regulating both events. Additionally, microtubule orientation was more defined in cells whose growth was accelerating and less stringent in cells whose growth was decelerating, indicating that microtubule-orienting factor(s) may be sensitive to growth acceleration, rather than growth per se.

Influence of MPP+ on the state of tubulin polymerisation in NGF-differentiated PC12 cells.

Cytoskeletal proteins have been reported as constituents of cytoplasmic inclusions typical of degenerated neurones in Parkinson's disease and, in addition, the involvement of cytoskeleton in the mechanism of action of the parkinsonism-producing neurotoxin MPP+ is emerging. Here we investigate the influence of MPP+ on the dynamic behaviour of microtubules. Neurone-like cells derived from a rat pheochromocytoma cell line (PC12) and differentiated with nerve growth factor are used as a model system. We found that sublethal doses of the neurotoxin markedly affect the state of tubulin polymerisation: polymerised tubulins significantly decreased, whereas an increase of unpolymerised alpha-tubulin was observed. Since the concentration of unassembled tubulin directly regulates tubulin synthesis by a feedback mechanism, we studied alpha- and beta-tubulin synthesis by metabolic labelling of PC12 cells with [35S] methionine and following immunoprecipitations. The results showed the significant decrease of labelling in both the microtubule subunits in cells exposed to the neurotoxin. We suggest that the MPP+-induced imbalance of tubulin polymerisation and synthesis represents a novel early step in the mechanism of action of the neurotoxin.

Microtubules and microscopes: how the development of light microscopic imaging technologies has contributed to discoveries about microtubule dynamics in living cells.

Microtubules are dynamic components of the cell cytoskeleton that participate in many important cellular processes, including mitosis, cell motility, morphogenesis, and organelle transport (reviewed in Desai and Mitchison, 1997 ). Microtubules are made up of α/β-tubulin heterodimers that assemble into ∼25 nm cylindrical polymers. Immunofluorescent localization of tubulin has shown that microtubules in interphase tissue cells are for the most part arranged in a radial array with the “minus” end oriented toward a central microtubule organizing center, the centrosome, and the “plus” end radiating toward the cell periphery. Biochemical studies in the 1970s and early 1980s on the assembly–disassembly dynamics of tubulin culminated in the demonstration that microtubules exhibit an unusual form of assembly behavior known as dynamic instability, defined as the coexistence of microtubules in growing and shrinking populations that interconvert infrequently and stochastically (reviewed in Waterman-Storer and Salmon, 1997a ). However, corroboration of these studies with real-time microscopic data was difficult, because 25-nm microtubules are ∼10 times below the resolution limit of the light microscope. This Video Essay provides a review of the ingenious methods that have been developed and applied over the past 10–15 years to the study of microtubule dynamic behavior in living cells. I have not included the many unique microscopic approaches that have been used for the study of microtubule behavior in vitro (for review, see Scholey 1993 ). Furthermore, this review intends by no means to be exhaustive but summarizes a few highlights in the field of which primary data was generously made available to me from the original authors for digitization and conversion into QuickTime movies. Finally, important advances such as the expression of green fluorescent protein–coupled proteins to follow microtubule dynamics in the ∼6-μm-diameter budding yeast Saccharomyces cerevisiae cell (Shaw et al., 1998 ) and the use of polarized light microscopy for monitoring microtubule dynamics during mitosis (Inoue and Oldenbourg, 1998 ) have been omitted from this review, because these topics have been dealt with in detail in recent Video Essays in this series.

Detection of GTP and Pi in wild-type and mutated yeast microtubules: implications for the role of the GTP/GDP-Pi cap in microtubule dynamics.

Microtubule dynamics are believed to be controlled by a stabilizing cap of tubulin dimers at microtubule ends that contain either GTP or GDP and Pi in the exchangeable nucleotide site (E-site) of the beta-subunit. However, it has been difficult to obtain convincing evidence to support this hypothesis because the quantity of GTP and Pi in the E-site of assembled brain tubulin (the tubulin used in most studies thus far) is extremely low. In this study, we have measured the amount of GTP and Pi in the E-site of wild-type and mutated yeast assembled tubulins. In contrast to brain microtubules, 6% of the tubulin in a wild-type yeast microtubule contains a combination of E-site GTP and Pi. This result indicates that GTP hydrolysis and Pi release are not coupled to dimer addition to the end of the microtubule and supports the hypothesis that microtubules contain a cap of tubulin dimers with GTP or Pi in their E-sites. In addition, we have measured the E-site content of GTP and Pi in microtubules assembled from two yeast tubulins that had been mutated at residues T107 and T143 in beta-tubulin, sites thought to interact with the nucleotide bound in the E-site. Previous studies have shown that microtubules containing these mutated tubulins have modified dynamic behavior in vitro. The results from these experiments indicate that the GTP or GDP-Pi cap model does not adequately explain yeast microtubule dynamic behavior.

Stabilization and functional modulation of microtubules by microtubule-associated protein 4.

Microtubules (MTs) are hollow cytoplasmic fibers that are composed of a dimeric protein called tubulin, as well as several MT-associated proteins (MAPS) bound along their surface. MTs are believed to play important roles in a variety of cellular processes, including mitosis, cell motility, and intracellular vesicle transport. Both in vitro and in vivo, individual MTs are dynamic; that is, they undergo alternating periods of polymerization and depolymerization from their ends, a process known as dynamic instability. The dynamic behavior of MTs is thought to play an important role both in cell cycle events and in cell differentiation. MAPS have been postulated to function as in vivo regulators of the dynamics and functions of MTs. Based on in vitro studies, several MAPS have been classified as

Functional assays to identify and characterize regulators of microtubule behavior.

Previous experiments have clearly demonstrated that microtubule dynamic instability is regulated in living cells, but the molecular mechanisms that are responsible for this regulation are not well understood. We describe two rapid, functional assays that can be used to screen cell extracts for regulators of microtubule dynamic instability behavior. In both assays, highly purified tubulin is used to assemble microtubules from Tetrahymena axonemes. In the immunofluorescence assay, microtubules are visualized by fixation and staining with anti-tubulin antibodies. Alternatively, microtubule assembly has been visualized by the addition of rhodamine-labeled tubulin to axonemes, followed by low-light-level fluorescence microscopy. In either case, polymerization is quantified by measuring polymer length, total polymer and the number of microtubules per axoneme. In these assays, addition of brain microtubule-associated proteins (MAPs) results in a 2-fold-3-fold increase in average microtubule length, and addition of vinblastine results in a 50%-75% decrease in average microtubule length. The number of microtubules per axoneme was significantly increased by the addition of MAPs and significantly decreased by the addition of vinblastine. These functional assays can detect molecules that stimulate or suppress net microtubule assembly and provide a useful initial screen to isolate regulators of microtubule dynamic behavior.

Microtubule dynamics in serum‐starved and serum‐stimulated swiss 3T3 mouse fibroblasts: Implications for the relationship between serum‐induced contractility and microtubules

It has been established that cell contractility can be stimulated with low or depolymerizing doses of microtubule (MT) poisons. In addition, low doses of nocodazole and vinblastine have recently been shown to decrease MT dynamics in vivo. In this study, investigated whether there is a direct, or reciprocal feedback-type relationship between contractility and microtubule dynamics, by examining MT dynamic behavior in live cells under conditions where contractility is known to be altered. Quiescent, serum-starved Swiss 3T3 mouse fibroblasts have been shown to be weakened in their contractility; serum stimulation increases cell contractility and causes the formation of stress fibers and adhesion plaques. Growing (control), quiescent (Go), and serum-stimulated cells were injected with rhodamine-tubulin, and MT dynamics were determined by analysis of MT length changes obtained from digitized images of the extreme periphery of the cells, where the MT ends were readily apparent. The MTs in quiescent cells were less dynamic than those in control cells: the growth and shortening rates were reduced by 30% and 45%, respectively. Dynamicity decreased by 47%, and the MTs spent more time in pause. After serum stimulation, MT growth rate, dynamicity, and time spent in pause returned to control cell levels. Although the shortening rate increased by 28%, it remained significantly lower than in control cells. In this system, the serum-induced increase in contractility was accompanied by an increase in MT dynamics. However, increased contractility stimulated with low doses of MT poisons is known to be accompanied by a decrease in MT dynamics. These results suggest that the relationship between MT dynamics and contractility is an indirect one. Cell Motil. Cytoskeleton 40:1–12, 1998. © 1998 Wiley-Liss, Inc.

Microtubule dynamic turnover is suppressed during polarization and stimulated in hepatocyte growth factor scattered Madin-Darby canine kidney epithelial cells.

The dynamic behavior of microtubules has been measured in non-polarized, polarized, and hepatocyte growth factor treated Madin-Darby canine kidney epithelial cells. In a nocodazole disassembly assay, microtubules in polarized cells were more resistant to depolymerization than microtubules in non-polarized cells; microtubules in scattered cells were nearly completely disassembled. Analysis of fluorescent microtubules in living cells further revealed that individual microtubules in polarized cells were kinetically stabilized and microtubules in scattered cells were highly dynamic. Individual microtubule behavior in polarized cells was characterized by a suppression of the average rate of shortening, an increase in the average duration of pause, a decrease in the frequency of catastrophe transitions, and an increase in the frequency of rescue transitions, when compared with microtubules in non-polarized cells. In contrast, microtubule behavior in epithelial cells treated with hepatocyte growth factor was characterized by increase in the average rates of microtubule growth and shortening, a decrease in the frequency of rescue transitions, and an increase in the frequency of catastrophe transitions, when compared with polarized cells. Dynamicity, a measure of the gain and loss of subunits from microtubule plus ends, was 2.7 microns/min in polarized cells and 11.1 microns/min in scattered cells. These results demonstrate that individual microtubule dynamic behavior is markedly suppressed in polarized epithelial cells. Our results further demonstrate that in addition to its previously characterized effects on cell locomotion, hepatocyte growth factor stimulates microtubule dynamic turnover in lamellar regions of living cells.

Vinblastine suppresses dynamics of individual microtubules in living interphase cells.

We have characterized the effects of vinblastine on the dynamic instability behavior of individual microtubules in living BS-C-1 cells microinjected with rhodamine-labeled tubulin and have found that at low concentrations (3-64 nM), vinblastine potently suppresses dynamic instability without causing net microtubule depolymerization. Vinblastine suppressed the rates of microtubule growth and shortening, and decreased the frequency of transitions from growth or pause to shortening, also called catastrophe. In vinblastine-treated cells, both the average duration of a pause (a state of attenuated dynamics where neither growth nor shortening could be detected) and the percentage of total time spent in pause were significantly increased. Vinblastine potently decreased dynamicity, a measure of the overall dynamic activity of microtubules, reducing this parameter by 75% at 32 nM. The present work, consistent with earlier in vitro studies, demonstrates that vinblastine kinetically caps the ends of microtubules in living cells and supports the hypothesis that the potent chemotherapeutic action of vinblastine as an antitumor drug is suppression of mitotic spindle microtubule dynamics. Further, the results indicate that molecules that bind to microtubule ends can regulate microtubule dynamic behavior in living cells and suggest that endogenous regulators of microtubule dynamics that work by similar mechanisms may exist in living cells.

Beta-Tubulin mutation suppresses microtubule dynamics in vitro and slows mitosis in vivo.

Microtubule (MT) dynamics vary both spatially and temporally within cells and are thought to be important for proper MT cellular function. Because MT dynamics appear to be closely tied to the guanosine triphosphatase (GTPase) activity of beta-tubulin subunits, we examined the importance of MT dynamics in the budding yeast S. cerevisiae by introducing a T107K point mutation into a region of the single beta-tubulin gene, TUB2, known to affect the assembly-dependent GTPase activity of MTs in vitro. Analysis of MT dynamic behavior by video-enhanced differential interference contrast microscopy, revealed that T107K subunits slowed both the growth rates and catastrophic disassembly rates of individual MTs in vitro. In haploid cells tub2-T107K is lethal; but in tub2-T107K/tub2-590 heterozygotes the mutation is viable, dominant, and slows cell-cycle progression through mitosis, without causing wholesale disruption of cellular MTs. The correlation between the slower growing and shortening rates of MTs in vitro, and the slower mitosis in vivo suggests that MT dynamics are important in budding yeast and may regulate the rate of nuclear movement and segregation. The slower mitosis in mutant cells did not result in premature cytokinesis and cell death, further suggesting that cell-cycle control mechanisms "sense" the mitotic slowdown, possibly by monitoring MT dynamics directly.

The conversion of tubulin carboxyl groups to amides has a stabilizing effect on microtubules.

In a recent study, we demonstrated that the conversion of carboxyl residues in the C-termini of tubulin to neutral amides with glycine ethyl ester enhanced the ability of the protein to assemble into microtubules and decreased its interaction with microtubule-associated proteins (MAPs). In this work, we investigated the effects of carboxyl modification on the dynamic behavior of microtubules at polymer mass steady state. After steady state, microtubules assembled from unmodified tubulin were sheared, and the mean polymer lengths decreased to 5 microns and then increased to 29 microns within 130 min. In contrast, lengths of sheared microtubules polymerized from tubulin containing 23 modified carboxyl groups increased by only 2-fold. Stabilization of polymer lengths was also observed directly by video-enhanced light microscopy of microtubules grown off of axonemes. Rapid shortening was seen in microtubules composed of unmodified but not modified tubulin. Further evidence for the less dynamic behavior of microtubules as a result of carboxyl modification was obtained from kinetic studies of the elongation phase during assembly which showed a 3-fold lower off-rate constant, k-, for modified microtubules. Another effect of the modification was a 12-fold reduction in the steady-state rate constant for GTP hydrolysis (165 s-1 for unmodified and 14 s-1 for modified). These results suggest that reduction of the negative charges in the C-termini by modification of the acidic residues stabilizes microtubules against depolymerization. MAPs may stabilize microtubules in an analogous manner.

The effect of solution composition on microtubule dynamic instability

The exchange of tubulin dimer into steady-state microtubules was studied over a range of solution conditions, in order to assess the effects of various common buffer components on the dynamic instability of microtubules. In comparison with standard buffer conditions (100 mM-Pipes buffer, pH 6.5, containing 0.1 mM-EGTA, 1.8 mM-MgC12 and 1 M-glycerol), the rate and extent of exchange, and thus of dynamic instability, are suppressed by increasing the concentration of glycerol above 2 M. Exchange is enhanced by the addition of further Mg2+ (up to 17 mM) or by the addition of Ca2+ (up to 0.4 mM). Phosphate ion (150 mM) has relatively little effect on the dynamic behaviour of microtubules, as judged by the exchange method. The findings are interpreted within the framework of the Lateral Cap model for microtubule dynamic instability, in terms of the effects of these changes on the intrinsic rate constants of the system. By contrast, the extent of tubulin exchange depends selectively on the value of the dissociation rate constant for tubulin-GDP. A decrease in the extent of exchange, and hence in dynamic activity, is associated with a decreased value for this rate constant, and vice versa. The results also show good agreement of predictions of the model in treating the observed variations in the dynamic properties of individual microtubules, induced by different solution conditions.

Inhibition of spindle elongation by taxol

During anaphase B spindle elongation, interzonal microtubules lengthen to accomplish pole-pole separation, while at the same time remaining highly dynamic [Shelden and Wadsworth, J. Cell Sci. 97:273-281, 1990]. To further examine the role of microtubule polymerization and dynamics during spindle elongation, cells have been treated with taxol, which induces microtubule polymerization and stabilizes microtubules. Taxol was added to PtK1 cells 3 minutes after initial chromatid separation, so that the effect on anaphase B could be observed with minimal disruption to anaphase A movement. In 20 microM taxol, the rate and extent of pole-pole separation, measured from time-lapse video records, are reduced to 4% and 9.5% of controls, respectively. The organization of microtubules in taxol treated cells was examined using tubulin immunofluorescence and confocal fluorescence microscopy. Taxol induces a dramatic reorganization of interzonal microtubules resulting in a narrow gap, which is nearly completely lacking in MTs, across the center of the interzone. Furthermore, microtubules in taxol treated cells are resistant to nocodazole induced microtubule disassembly. Our results reveal that taxol rapidly inhibits anaphase B spindle elongation; inhibition is accompanied by a depletion of interdigitated interzonal microtubules and a reduction in microtubule dynamic behavior.

Fluorescent microtubules break up under illumination

The dynamic behavior of microtubules (MTs) in living cells has recently been elucidated by the use of tubulin tagged with a fluorescent molecule and then injected into cultured cells. The dynamics of the labelled tubulin are then followed either by watching the incorporation of the fluorescence immediately after injection, or by monitoring fluorescence redistribution after photobleaching (FRAP).Until recently, the best fluorescent analogue of tubulin appeared to be dichlorotriazinyl-amino-fluorescein tubulin (DTAF-tubulin). We have now made fluorescein, rhodamine and X-rhodamine n-hydroxy-succinimidyl derivatives of tubulin. These analogues all have a higher fluorescence to protein (f-to-p) ratio and better in-vitro assembly characteristics than the DTAF-tubulin previously used. They have been injected into cultured PtKl cells, where they incorporate well into the microtubule cytoskeltons of the cells. However, while characterising the properties of these four analogues we discovered that they all suffer from a major problem: Microtubules formed from them, either in vitro or in vivo, are destroyed as the bound fluorophore becomes photobleached. We have characterised the photolability of the fluorescent microtubules both in vivo, using epifluorescence microscopy, and in vitro with DIC microscopy.