Postpolymerization detyrosination of alpha-tubulin: a mechanism for subcellular differentiation of microtubules

Tyrosinated (Tyr) and detyrosinated (Glu) alpha-tubulin, species interconverted by posttranslational modification, are largely segregated in separate populations of microtubules in interphase cultured cells. We sought to understand how distinct Tyr and Glu microtubules are generated in vivo, by examining time-dependent alterations in Tyr and Glu tubulin levels (by immunoblots probed with antibodies specific for each species) and distributions (by immunofluorescence) after microtubule regrowth and stabilization. When microtubules were allowed to regrow after complete depolymerization by microtubule antagonists, Glu microtubules reappeared with a delay of approximately 25 min after the complete array of Tyr microtubules had regrown. In these experiments, Tyr tubulin immunofluorescence first appeared as an aster of distinct microtubules, while Glu tubulin staining first appeared as a grainy pattern that was not altered by detergent extraction, suggesting that Glu microtubules were created by detyrosination of Tyr microtubules. Treatments with taxol, azide, or vinblastine, to stabilize polymeric tubulin, all resulted in time-dependent increases in polymeric Glu tubulin levels, further supporting the hypothesis of postpolymerization detyrosination. Analysis of monomer and polymer fractions during microtubule regrowth and in microtubule stabilization experiments were also consistent with postpolymerization detyrosination; in each case, Glu polymer levels increased in the absence of detectable Glu monomer. The low level of Glu monomer in untreated or nocodazole-treated cells (we estimate that Glu tubulin comprises less than 2% of the monomer pool) also suggested that Glu tubulin entering the monomer pool is efficiently retyrosinated. Taken together these results demonstrate that microtubules are polymerized from Tyr tubulin and are then rapidly converted to Glu microtubules. When Glu microtubules depolymerize, the resulting Glu monomer is retyrosinated. This cycle generates structurally, and perhaps functionally, distinct microtubules.     

Ultrastructural colocalization of tyrosinated and detyrosinated alpha-tubulin in interphase and mitotic cells

Immunofluorescence with specific peptide antibodies has previously established that tyrosinated (Tyr) and detyrosinated (Glu) tubulin, the two species generated by posttranslational modification of the COOH-terminus of alpha-tubulin, are present in distinct, but overlapping, subsets of microtubules in cultured cells (Gundersen, G. G., M. H. Kalnoski, and J. C. Bulinski, 1984, Cell, 38:779-789). Similar results were observed by light microscopic immunogold staining in the two cell types used in this study, CV1 and PtK2 cells: most microtubules were stained with the Tyr antibody, whereas only a few were stained with the Glu antibody. We have examined immunogold-stained preparations by electron microscopy to extend these results. In general, electron microscopic localization confirmed results obtained at the light microscopic level: the majority of the microtubules in CV1 and PtK2 cells were nearly continuously labeled with the Tyr antibody, whereas only a few were heavily labeled with the Glu antibody. However, in contrast to the light microscopic staining, we found that all microtubules of interphase and mitotic CV1 and PtK2 cells contained detectable Tyr and Glu immunoreactivity at the electron microscopic level. No specific localization of either species was observed in microtubules near particular organelles (e.g., mitochondria or intermediate filaments). Quantification of the relative levels of Glu and Tyr immunoreactivity in individual interphase and metaphase microtubules showed that all classes of spindle microtubules (i.e., kinetochore, polar, and astral) contained nearly the same level of Glu immunoreactivity; this level of Glu immunoreactivity was lower than that found in all interphase microtubules. Most interphase microtubules had low levels of Glu immunoreactivity, whereas a few had relatively high levels; the latter corresponded to morphologically sinuous microtubules. Quantification of the relative levels of Tyr and Glu immunoreactivity in segments along individual microtubules suggested that the level of Tyr (or Glu) tubulin in a given microtubule was uniform along its length. Understanding how microtubules with different levels of Tyr and Glu tubulin arise will be important for understanding the role of tyrosination/detyrosination in microtubule function. Additionally, the coexistence of microtubules with different levels of the two species may have important implications for microtubule dynamics in vivo.     

Microtubules are stabilized in confluent epithelial cells but not in fibroblasts

Rhodamine-tagged tubulin was microinjected into epithelial cells (MDCK) and fibroblasts (Vero) to characterize the dynamic properties of labeled microtubules in sparse and confluent cells. Fringe pattern fluorescence photobleaching revealed two components with distinct dynamic properties. About one-third of the injected tubulin diffused rapidly in the cytoplasm with a diffusion coefficient of 1.3-1.6 x 10(- 8) cm2/s. This pool of soluble cytoplasmic tubulin was increased to greater than 80% when cells were treated with nocodazole, or reduced to approximately 20% upon treatment of cells with taxol. Fluorescence recovery of the remaining two-thirds of labeled tubulin occurred with an average half-time (t1/2) of 9-11 min. This pool corresponds to labeled tubulin associated with microtubules, since it was sensitive to treatment of cells with nocodazole and since taxol increased its average t1/2 to greater than 22 min. Movement of photobleached microtubules in the cytoplasm with rates of several micrometers per minute was shown using very small interfringe distances. A significant change in the dynamic properties of microtubules occurred when MDCK cells reached confluency. On a cell average, microtubule half-life was increased about twofold to approximately 16 min. In fact, two populations of cells were detected with respect to their microtubule turnover rates, one with a t1/2 of approximately 9 min and one with a t1/2 of greater than 25 min. Correspondingly, the rate of incorporation of microinjected tubulin into interphase microtubules was reduced about twofold in confluent MDCK cells. In contrast to the MDCK cells, no difference in microtubule dynamics was observed in sparse and confluent populations of Vero fibroblasts, where the average microtubule half- life was approximately 10 min. Thus, microtubules are significantly stabilized in epithelial but not fibroblastic cells grown to confluency.     

Microtubule dynamics in fish melanophores

We have studied the dynamics of microtubules in black tetra (Gymnocorymbus ternetzi) melanophores to test the possible correlation of microtubule stability and intracellular particle transport. X- rhodamine-or caged fluorescein-conjugated tubulin were microinjected and visualized by fluorescence digital imaging using a cooled charge coupled device and videomicroscopy. Microtubule dynamics were evaluated by determining the time course of tubulin incorporation after pulse injection, by time lapse observation, and by quantitation of fluorescence redistribution after photobleaching and photoactivation. The time course experiments showed that the kinetics of incorporation of labeled tubulin into microtubules were similar for cells with aggregated or dispersed pigment with most microtubules becoming fully labeled within 15-20 min after injection. Quantitation by fluorescence redistribution after photobleaching and photoactivation confirmed that microtubule turnover was rapid in both states, t1/2 = 3.5 +/- 1.5 and 6.1 +/- 3.0 min for cells with aggregated and dispersed pigment, respectively. In addition, immunostaining with antibodies specific to posttranslationally modified alpha-tubulin, which is usually enriched in stable microtubules, showed that microtubules composed exclusively of detyrosinated tubulin were absent and microtubules containing acetylated tubulin were sparse. We conclude that the microtubules of melanophores are very dynamic, that their dynamic properties do not depend critically on the state of pigment distribution, and that their stabilization is not a prerequisite for intracellular transport.     

Spindle microtubule dynamics: modulation by metabolic inhibitors

Recent experiments have shown that spindle microtubules are exceedingly dynamic. Measurements of fluorescence recovery after photobleaching (FRAP), in cells previously microinjected with fluorescent tubulin, provide quantitative information concerning the rate of turnover, or exchange, of tubulin subunits with the population of microtubules in living cells at steady state. In an effort to elucidate the pathways and factors that regulate tubulin exchange with microtubules in living cells, we have investigated the energy requirements for tubulin turnover as measured by FRAP. Spindle morphology was not detectably altered in cells incubated with 5 mM sodium azide and 1 mM 2-deoxyglucose (Az/DOG) for 5 minutes, as assayed by polarized light microscopy and antitubulin immunofluorescence. In FRAP experiments on these ATP-depleted cells, the average rate of recovery and the average percent of bleached fluorescence recovered were reduced to 37% and 30% of controls, respectively. When the inhibitors were removed, cells continued through mitosis, and rapid FRAP was restored. In the presence of azide and glucose, the rate of recovery and percent of fluorescence recovered were only slightly reduced, demonstrating that energy production via glycolysis can support microtubule turnover. Longer incubations with Az/DOG altered the microtubule organization in mitotic cells: astral microtubules lengthened and spindle fibers shortened. Furthermore, both astral and spindle microtubules became resistant to nocodazole-induced disassembly under these conditions. Together these observations indicate that microtubule dynamics require ATP and suggest a relationship between microtubule organization and turnover.     

Stable, detyrosinated microtubules function to localize vimentin intermediate filaments in fibroblasts

Separate populations of microtubules (MTs) distinguishable by their level of posttranslationally modified tubulin subunits and by their stability in vivo have been described. In polarized 3T3 cells at the edge of an in vitro wound, we have found a striking preferential coalignment of vimentin intermediate filaments (IFs) with detyrosinated MTs (Glu MTs) rather than with the bulk of the MTs, which were tyrosinated MTs (Tyr MTs). Vimentin IFs were not stabilizing the Glu MTs since collapse of the IF network to a perinuclear location, induced by microinjection of monoclonal anti-IF antibody, had no noticeable effect on the array of Glu MTs. To test whether Glu MTs may affect the organization of IFs we regrew MTs in cells that had been treated with nocodazole to depolymerize all the MTs and to collapse IFs; the reextension of IFs into the lamella lagged behind the rapid regrowth of Tyr MTs, but was correlated with the slower reformation of Glu MTs. Similar realignment of IFs with newly formed Glu MTs was observed in serum-starved cells treated with either serum or taxol to induce the formation of Glu MTs. Next, we microinjected affinity purified antibodies specific for Glu tubulin (polyclonal SG and monoclonal 4B8) and specific for Tyr tubulin (polyclonal W2 and monoclonal YL1/2) into 3T3 cells. Both injected SG and 4B8 antibodies labeled the subset of endogenous Glu MTs; W2 and YL1/2 antibodies labeled virtually all of the cytoplasmic MTs. Injection of SG or 4B8 resulted in the collapse of IFs to a perinuclear region. This collapse was comparable to that observed after complete MT depolymerization by nocodazole. Injection of W2, YL1/2, or nonspecific control IgGs did not result in collapse of the IFs. Taken together, these results show that Glu MTs localize IFs in migrating 3T3 fibroblasts and suggest that detyrosination of tubulin acts as a signal for the recruitment of vimentin IFs to MTs.     

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.     

How microtubules get fluorescent speckles.

The dynamics of microtubules in living cells can be seen by fluorescence microscopy when fluorescently labeled tubulin is microinjected into cells, mixing with the cellular tubulin pool and incorporating into microtubules. The subsequent fluorescence distribution along microtubules can appear "speckled" in high-resolution images obtained with a cooled CCD camera (Waterman-Storer and Salmon, 1997. J. Cell Biol. 139:417-434). In this paper we investigate the origins of these fluorescent speckles. In vivo microtubules exhibited a random pattern of speckles for different microtubules and different regions of an individual microtubule. The speckle pattern changed only after microtubule shortening and regrowth. Microtubules assembled from mixtures of labeled and unlabeled pure tubulin in vitro also exhibited fluorescent speckles, demonstrating that cellular factors or organelles do not contribute to the speckle pattern. Speckle contrast (measured as the standard deviation of fluorescence intensity along the microtubule divided by the mean fluorescence intensity) decreased as the fraction of labeled tubulin increased, and it was not altered by the binding of purified brain microtubule-associated proteins. Computer simulation of microtubule assembly with labeled and unlabeled tubulin showed that the speckle patterns can be explained solely by the stochastic nature of tubulin dimer association with a growing end. Speckle patterns can provide fiduciary marks in the microtubule lattice for motility studies or can be used to determine the fraction of labeled tubulin microinjected into living cells.     

Tubulin subunit sorting: Analysis of the mechanisms involved in the segregation of unique tubulin isotypes within microtubule copolymers

Summary Vertebrate cells contain biochemical and genetic isotypes of tubulin which are expressed in unique combinations in different tissues and cell types. To determine if mixtures of tubulin isotypes assemblein vitro to form different classes of microtubules, we analyzed the composition of microtubule copolymers assembled from mixtures of chicken brain and erythrocyte tubulin. During microtubule elongation brain tubulin assembled onto the ends of microtubules faster than erythrocyte tubulin, resulting in copolymers with continually changing ratios of isotypes along their lengths. Unlike examples of microtubule assembly where the rate of polymerization depends on the association rate constant (k⁺) and the subunit concentration, the rate and extent of sorting in copolymers appear to depend on the dissociation rate constant (k^–), which governs the rate at which subunits are released from tubulin oligomers and microtubules and thereby made available for reassembly into copolymers. The type of microtubule seed used to initiate elongation was also found to influence the composition of copolymers, indicating that polymerization favors association of subunits of the same isotype.     

Dynamics of microtubules bundled by microtubule associated protein 2C (MAP2C)

MAP2C is a microtubule-associated protein abundant in immature nerve cells. We isolated a cDNA clone encoding whole mouse MAP2C of 467 amino acid residues. In fibroblasts transiently transfected with cDNA of MAP2C, interphase microtubule networks were reorganized into microtubule bundles. To reveal the dynamic properties of microtubule bundles, we analyzed the incorporation sites of exogenously introduced tubulin by microinjection of biotin-labeled tubulin and the turnover rate of microtubule bundles by photoactivation of caged fluorescein- labeled tubulin. The injected biotin-labeled tubulin was rapidly incorporated into distal ends of preexisting microtubule bundles, suggesting a concentration of the available ends of microtubules at this region. Although homogenous staining of microtubule bundles with antibiotin antibody was observed 2 h after injection, the photoactivation study indicated that turnover of microtubule bundles was extremely suppressed and < 10% of tubulin molecules would be exchanged within 1 h. Multiple photoactivation experiments provided evidence that neither catastrophic disassembly at the distal ends of bundles nor concerted disassembly due to treadmilling at the proximal ends could explain the observed rapid incorporation of exogenously introduced tubulin molecules. We conclude that microtubules bundled by MAP2C molecules are very stable while the abrupt increase of free tubulin molecules by microinjection results in rapid assembly from the distal ends within the bundles as well as free nucleation of small microtubules which are progressively associated laterally with preexisting microtubule bundles. This is the first detailed study of the function of MAPs on the dynamics of microtubules in vivo.     

Cell cycle-dependent changes in the dynamics of MAP 2 and MAP 4 in cultured cells

To examine the behavior of microtubule-associated proteins (MAPs) in living cells, MAP 4 and MAP 2 have been derivatized with 6-iodoacetamido-fluorescein, and the distribution of microinjected MAP has been analyzed using a low light level video system and fluorescence redistribution after photobleaching. Within 1 min following microinjection of fluoresceinated MAP 4 or MAP 2, fluorescent microtubule arrays were visible in interphase or mitotic PtK1 cells. After cold treatment of fluorescent MAP 2-containing cells (3 h, 4 degrees C), microtubule fluorescence disappeared, and the only fluorescence above background was located at the centrosomes; microtubule patterns returned upon warming. Loss of microtubule immunofluorescence after nocodozole treatment was similar in MAP-injected and control cells, suggesting that injected fluorescein-labeled MAP 2 did not stabilize microtubules. The dynamics of the MAPs were examined further by FRAP. FRAP analysis of interphase cells demonstrated that MAP 2 redistributed with half-times slightly longer (60 +/- 25 s) than those for MAP 4 (44 +/- 20 s), but both types of MAPs bound to microtubules in vivo exchanged with soluble MAPs at rates exceeding the rate of tubulin turnover. These data imply that microtubules in interphase cells are assembled with constantly exchanging populations of MAP. Metaphase cells at 37 degrees C or 26 degrees C showed similar mean redistribution half-times for both MAP 2 and MAP 4; these were 3-4 fold faster than the interphase rates (MAP 2, t1/2 = 14 +/- 6 s; MAP 4, t1/2 = 17 +/- 5 s). The extent of recovery of spindle fluorescence in MAP-injected cells was to 84-94% at either 26 or 37 degrees C. Although most metaphase tubulin, like the MAPs, turns over rapidly and completely under physiologic conditions, published work shows either reduced rates or extents of turnover at 26 degrees C, suggesting that the fast mitotic MAP exchange is not simply because of fast tubulin turnover. Exchange of MAP 4 bound to telophase midbodies occurred with dynamics comparable to those seen in metaphase spindles (t1/2 = approximately 27 s) whereas midbody tubulin exchange was slow (greater than 300 s). These data demonstrate that the rate of MAP exchange on microtubules is a function of time in the cell cycle.     

Detyrosination of tubulin regulates the interaction of intermediate filaments with microtubules in vivo via a kinesin-dependent mechanism

Posttranslationally modified forms of tubulin accumulate in the subset of stabilized microtubules (MTs) in cells but are not themselves involved in generating MT stability. We showed previously that stabilized, detyrosinated (Glu) MTs function to localize vimentin intermediate filaments (IFs) in fibroblasts. To determine whether tubulin detyrosination or MT stability is the critical element in the preferential association of IFs with Glu MTs, we microinjected nonpolymerizable Glu tubulin into cells. If detyrosination is critical, then soluble Glu tubulin should be a competitive inhibitor of the IF-MT interaction. Before microinjection, Glu tubulin was rendered nonpolymerizable and nontyrosinatable by treatment with iodoacetamide (IAA). Microinjected IAA-Glu tubulin disrupted the interaction of IFs with MTs, as assayed by the collapse of IFs to a perinuclear location, and had no detectable effect on the array of Glu or tyrosinated MTs in cells. Conversely, neither IAA-tyrosinated tubulin nor untreated Glu tubulin, which assembled into MTs, caused collapse of IFs when microinjected. The epitope on Glu tubulin responsible for interfering with the Glu MT-IF interaction was mapped by microinjecting tubulin fragments of alpha-tubulin. The 14-kDa C-terminal fragment of Glu tubulin (alpha-C Glu) induced IF collapse, whereas the 36-kDa N-terminal fragment of alpha-tubulin did not alter the IF array. The epitope required more than the detyrosination site at the C terminus, because a short peptide (a 7-mer) mimicking the C terminus of Glu tubulin did not disrupt the IF distribution. We previously showed that kinesin may mediate the interaction of Glu MTs and IFs. In this study we found that kinesin binding to MTs in vitro was inhibited by the same reagents (i.e., IAA-Glu tubulin and alpha-C Glu) that disrupted the IF-Glu MT interaction in vivo. These results demonstrate for the first time that tubulin detyrosination functions as a signal for the recruitment of IFs to MTs via a mechanism that is likely to involve kinesin.     

Microtubule dynamics in the chromosomal spindle fiber: analysis by fluorescence and high-resolution polarization microscopy

We describe preliminary results from two studies exploring the dynamics of microtubule assembly and organization within chromosomal spindle fibers. In the first study, we microinjected fluorescently labeled tubulin into mitotic PtK1 cells and measured fluorescence redistribution after photobleaching (FRAP) to determine the assembly dynamics of the microtubules within the chromosomal fibers in metaphase cells depleted of nonkinetochore microtubules by cooling to 23-24 degrees C. FRAP measurements showed that the tubulin throughout at least 72% of the microtubules within the chromosomal fibers exchanges with the cellular tubulin pool with a half-time of 77 sec. There was no observable poleward flux of subunits. If the assembly of the kinetochore microtubules is governed by dynamic instability, our results indicate that the half-life of microtubule attachment to the kinetochore is less than several min at 23-24 degrees C. In the second study, we used high-resolution polarization microscopy to observe microtubule dynamics during mitosis in newt lung epithelial cells. We obtained evidence from 150-nm-thick optical sections that microtubules throughout the spindle laterally associate for several sec into "rods" composed of a few microtubules. These transient lateral associations between microtubules appeared to produce the clustering of nonkinetochore and kinetochore microtubules into the chromosomal fibers. Our results indicate that the chromosomal fiber is a dynamic structure, because microtubule assembly is transient, lateral interactions between microtubules are transient, and the attachment of the kinetochores to microtubules may also be transient.     

Tau protein function in living cells

Tau protein from mammalian brain promotes microtubule polymerization in vitro and is induced during nerve cell differentiation. However, the effects of tau or any other microtubule-associated protein on tubulin assembly within cells are presently unknown. We have tested tau protein activity in vivo by microinjection into a cell type that has no endogenous tau protein. Immunofluorescence shows that tau protein microinjected into fibroblast cells associates specifically with microtubules. The injected tau protein increases tubulin polymerization and stabilizes microtubules against depolymerization. This increased polymerization does not, however, cause major changes in cell morphology or microtubule arrangement. Thus, tau protein acts in vivo primarily to induce tubulin assembly and stabilize microtubules, activities that may be necessary, but not sufficient, for neuronal morphogenesis.     

Microtubule arrays in differentiated cells contain elevated levels of a post-translationally modified form of tubulin

Tyrosinated (Tyr) and detyrosinated (Glu) alpha-tubulins are post-translationally modified species that differ by a single amino acid at their respective C-termini. We have examined the distribution of these two species by immunofluorescence in proliferating and differentiated cells using antisera specifically reactive with each of the forms. In proliferating PtK1 cells, Tyr tubulin was the predominant form in almost every cytoplasmic microtubule (MT); only a few MTs contained detectable Glu tubulin. In contrast, staining of centrioles and primary cilia of PtK1 cells suggested that Glu tubulin was the predominant form in these stable assemblies of MTs. An examination of the distribution (by immunofluorescence) and relative amount (by immunoblot analysis) of the two forms of tubulin in the stable assemblies of MTs present in cultured neuronal cells (neurites), sperm and tracheal cells (axonemes and basal bodies), and platelets and erythrocytes (marginal bands) revealed that, in general, the MTs in these arrays contained substantially elevated levels of Glu tubulin in comparison with the levels in MTs of cultured cells. The one exception, the marginal band of toad erythrocytes, which contained only Tyr tubulin, demonstrates that an elevated level of Glu tubulin is not an obligate feature of a stable array of MTs. Nonetheless, an elevated level of Glu tubulin may be a useful indicator of stable MTs in differentiated cells. It is important to note that commonly used sources of tubulin (e.g., brain or flagella) necessarily yield tubulin that differs strikingly from tubulin of proliferating cells in its content of Glu tubulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exposure of lumenal microtubule sites after mild fixation

High-resolution analysis of tubulin structure and docking the structure of tubulin dimer into a map of microtubules led to a prediction that sites for tubulin acetylation are in the interior of microtubules. This is somehow difficult to reconcile with their susceptibility to proteases and acetylation in assembled microtubules. To assess the availability of acetylated alpha-tubulin for antibodies, immunofluorescence on detergent-extracted cells, on cells fixed under various conditions and in microinjected cells was performed with monoclonal antibodies of known epitope locations. The presented data indicate that acetylated alpha:Lys40 is not exposed on unfixed microtubules but that this region of lumenal microtubule surface becomes easily exposed under mild fixation conditions.     

Distribution of tyrosinated and nontyrosinated alpha-tubulin during mitosis

The C-terminus of alpha-tubulin undergoes a reversible posttranslational tyrosination/detyrosination. The distributions of the tyrosinated (Tyr) and nontyrosinated (Glu) species during mitosis of cultured cells have been investigated by immunofluorescence using antibodies directed against the C-terminus of either Tyr or Glu tubulin. The distribution of Tyr tubulin differed from that of Glu tubulin at each stage of mitosis; in general, the distribution of Tyr tubulin was similar to that of total tubulin, whereas Glu tubulin had a more restricted distribution. The Glu species was found in half-spindle fibers but was not detected in astral fibers at any stage and was seen in the interzone only during telophase. These results were confirmed by a direct comparison of the distributions of Tyr and Glu tubulin in cells double-labeled with the two antibodies. Evidence for the occurrence of Tyr and Glu tubulin in each class of half-spindle fibers (kinetochore and polar) was obtained from the staining patterns of the two antibodies in cold-treated cells. Immunoblots of extracts prepared from synchronous mitotic cells showed that Glu tubulin was a minor species of the total tubulin in the spindle; no changes in the amount of either Tyr or Glu tubulin were detected at any stage of mitosis. These results show that Tyr tubulin is the major species in the mitotic spindle and is found in all classes of spindle fibers, whereas Glu tubulin is present in small amounts and shows a more restricted distribution. The presence of two biochemically distinct forms of alpha-tubulin in the spindle may be important for spindle function.     

Microtubules of the kinetochore fiber turn over in metaphase but not in anaphase

In previous work we injected mitotic cells with fluorescent tubulin and photobleached them to mark domains on the spindle microtubules. We concluded that chromosomes move poleward along kinetochore fiber microtubules that remain stationary with respect to the pole while depolymerizing at the kinetochore. In those experiments, bleached zones in anaphase spindles showed some recovery of fluorescence with time. We wished to determine the nature of this recovery. Was it due to turnover of kinetochore fiber microtubules or of nonkinetochore microtubules or both? We also wished to investigate the question of turnover of kinetochore microtubules in metaphase. We microinjected cells with x- rhodamine tubulin (x-rh tubulin) and photobleached spindles in anaphase and metaphase. At various times after photobleaching, cells were detergent lysed in a cold buffer containing 80 microM calcium, conditions that led to the disassembly of almost all nonkinetochore microtubules. Quantitative analysis with a charge coupled device image sensor revealed that the bleached zones in anaphase cells showed no fluorescence recovery, suggesting that these kinetochore fiber microtubules do not turn over. Thus, the partial fluorescence recovery seen in our earlier anaphase experiments was likely due to turnover of nonkinetochore microtubules. In contrast fluorescence in metaphase cells recovered to approximately 70% the control level within 7 min suggesting that many, but perhaps not all, kinetochore fiber microtubules of metaphase cells do turn over. Analysis of the movements of metaphase bleached zones suggested that a slow poleward translocation of kinetochore microtubules occurred. However, within the variation of the data (0.12 +/- 0.24 micron/min), it could not be determined whether the apparent movement was real or artifactual.     

Detyrosination of alpha tubulin does not stabilize microtubules in vivo [published erratum appears in J Cell Biol 1990 Sep;111(3):1325-6]

The relationship between alpha tubulin detyrosination and microtubule (MT) stability was examined directly in cultured fibroblasts by experimentally converting the predominantly tyrosinated MT array to a detyrosinated (Glu) array and then assaying MT stability. MTs in mouse Swiss 3T3 cells displayed an increase in Glu immunostaining fluorescence approximately 1 h after microinjecting antibodies to the tyrosinating enzyme, tubulin tyrosine ligase. Detyrosination progressed to virtual completion after 12 h and persisted for 30-35 h before tyrosinated subunits within MTs were again detected. The stability of these experimentally detyrosinated MTs was tested by first injecting either biotinylated or Xrhodamine-labeled tubulin and then measuring bulk turnover by hapten-mediated immunocytochemistry or fluorescence recovery after photobleaching, respectively. By both methods, turnover was found to be similarly rapid, possessing a half time of approximately 3 min. As a final test of MT stability, the level of acetylated tubulin staining in antibody-injected cells was compared with that observed in adjacent, uninjected cells and also with the staining observed in cells whose MTs had been stabilized with taxol. Although intense Glu staining was observed in both injected and taxol-treated cells, increased acetylated tubulin staining was observed only in the taxol-stabilized MTs, indicating that the MTs were not stabilized by detyrosination. Together, these results demonstrated clearly that detyrosination does not directly confer stability on MTs. Therefore, the stable MTs observed in these and other cell lines must have arisen by another mechanism, and may have become posttranslationally modified after their stabilization.     

Generation of a stable, posttranslationally modified microtubule array is an early event in myogenic differentiation

Microtubules (MTs) have been implicated to function in the change of cell shape and intracellular organization that occurs during myogenesis. However, the mechanism by which MTs are involved in these morphogenetic events is unclear. As a first step in elucidating the role of MTs in myogenesis, we have examined the accumulation and subcellular distribution of posttranslationally modified forms of tubulin in differentiating rat L6 muscle cells, using antibodies specific for tyrosinated (Tyr), detyrosinated (Glu), and acetylated (Ac) tubulin. Both Glu and Ac tubulin are components of stable MTs, whereas Tyr tubulin is the predominant constituent of dynamic MTs. In proliferating L6 myoblasts, as in other types of proliferating cells, the level of Glu tubulin was very low when compared with the level of Tyr tubulin. However, when we shifted proliferating L6 cells to differentiation media, we observed a rapid accumulation of Glu tubulin in cellular MTs. By immunofluorescence, the increase in Glu tubulin was first detected in MTs of prefusion myoblasts and was specifically localized to MTs that were associated with elongating portions of the cell. MTs in the multinucleated myotubes observed at later stages of differentiation maintained the elevated level of Glu tubulin that was observed in the prefusion myoblasts. When cells at early stages of differentiation (less than 1 d after switching the culture medium) were immunostained for Glu tubulin and the muscle-specific marker, muscle myosin, we found that the increase in Glu tubulin preceded the accumulation of muscle myosin. Thus, the elaboration of Glu MTs is one of the very early events in myogenesis. Ac tubulin also increased during L6 myogenesis; however, the increase in acetylation occurred later in myogenesis, after fusion had already occurred. Because detyrosination was temporally correlated with early events of myogenesis, we examined the mechanism responsible for the accumulation of Glu tubulin in the MTs of prefusion myoblasts. We found that an increase in the stability of L6 cell MTs occurred at the onset of differentiation, suggesting that the early increase in detyrosination that we observed is a manifestation of a decrease in MT dynamics in elongating myoblasts. We conclude that the establishment of an oriented array of microtubules heightened in its stability and its level of posttranslationally modified subunits may be involved in the subcellular remodeling that occurs during myogenesis.