The plus ends of stable microtubules are the exclusive nucleating structures for microtubules in the axon.

Microtubules (MTs) in the axon have a uniform polarity orientation that is recapitulated during recovery from episodes of MT depolymerization (Heidemann, S. R., M. A. Hamborg, S. J. Thomas, B. Song, S. Lindley, and D. Chu. 1984. J. Cell Biol. 99:1289-1295). This tight regulation of their organization indicates that axonal MTs are spatially regulated by discrete nucleating structures comparable in function to the centrosome. Several authors have proposed that an especially stable class of MTs in the axon may serve as these nucleating structures. In a previous report (Baas, P. W., and M. M. Black. 1990. J. Cell Biol. 111:495-509), we determined that the axons of cultured sympathetic neurons contain two classes of MT polymer, stable and labile, that differ in their sensitivity to nocodazole by roughly 35-fold. The stable and labile polymer represent long-lived and recently assembled polymer, respectively. We also determined that these two classes of polymer can be visually distinguished at the immunoelectron microscopic level based on their content of tyrosinated alpha-tubulin: the labile polymer stains densely, while the stable polymer does not stain. In the present study, we have taken advantage of these observations to directly identify MT nucleating structures in the axon. Neuron cultures were treated with nocodazole for 6 h to completely depolymerize the labile polymer in the axon, and substantially shorten the stable polymer. The cultures were then rinsed free of the drug, permitted to reassemble polymer for various periods of time, and prepared for immunoelectron microscopic localization of tyrosinated alpha-tubulin. Serial reconstruction of consecutive thin sections was undertaken to determine the spatial relationship between the stable MTs and the newly assembled polymer. All of the new polymer assembled in direct continuity with the plus ends of stable MTs, indicating that these ends are assembly competent, and hence capable of acting as nucleating structures. Our results further indicate that no self-assembly of MTs occurs in the axon, nor do any MT nucleating structures exist in the axon other than the plus ends of stable MTs. Thus the plus ends of stable MTs are the exclusive nucleating structures for MTs in the axon.     

Disruption of the Golgi apparatus with brefeldin A does not destabilize the associated detyrosinated microtubule network.

Stable subsets of microtubules (MTs) are often enriched in detyrosinated alpha-tubulin. Recently it has been found that the Golgi apparatus is associated with a subset of relatively stable MTs and that detyrosinated MTs colocalize spatially and temporally with the Golgi apparatus in several cell lines. To determine whether the Golgi apparatus actively stabilizes associated MTs and thus allows their time-dependent detyrosination, we have used the drug brefeldin A (BFA) to disrupt the Golgi apparatus and have monitored changes in the Golgi apparatus and MT populations using simultaneous immunofluorescence and fluorescent lectin microscopy. We found that although BFA caused the Golgi apparatus to completely redistribute to the endoplasmic reticulum (ER), the detyrosinated MTs were not disrupted and remained in a juxtanuclear region. By Western blot analysis we found that even after 6 h of continuous exposure of cells to BFA, there was no detectable reduction in the level of detyrosinated alpha-tubulin. Simultaneous treatment with nocodazole and BFA led to a complete disruption of all MTs and normal Golgi structure/organization. Upon removal of nocodazole in the continued presence of BFA, we found that the detyrosinated MTs reformed in a compact juxtanuclear location in the absence of an intact Golgi complex. Finally, we found that the detyrosinated MTs colocalized precisely with a BFA-resistant structure that binds to the lectin, wheat germ agglutinin. We conclude that the juxtanuclear detyrosinated MTs are not actively stabilized by association with BFA-sensitive Golgi membranes. However, another closely associated structure which binds wheat germ agglutinin may serve to stabilize the juxtanuclear MTs. Alternatively, the MT organizing center (MTOC) and/or MT-associated proteins (MAPs) may organize and stabilize the juxtanuclear detyrosinated MTs.     

Acetylated and detyrosinated alpha-tubulins are co-localized in stable microtubules in rat meningeal fibroblasts

We have examined the distribution of acetylated alpha-tubulin using immunofluorescence microscopy in fibroblastic cells of rat brain meninges. Meningeal fibroblasts showed heterogeneous staining patterns with a monoclonal antibody against acetylated alpha-tubulin ranging from staining of primary cilia or microtubule-organising centers (MTOCs) alone to extensive microtubule networks. Staining with a broad spectrum anti-alpha-tubulin monoclonal indicated that all cells possessed cytoplasmic microtubule networks. From double-labeling experiments using an antibody against acetylated alpha-tubulin (6-11B-1) and antibodies against either tyrosinated or detyrosinated alpha-tubulin, it was found that acetylated alpha-tubulin and tyrosinated alpha-tubulin were often segregated to different microtubules. The microtubules containing acetylated but not tyrosinated alpha-tubulin were cold stable. Therefore, it appeared that in general meningeal cells possessed two subset of microtubules: One subset contained detyrosinated and acetylated alpha-tubulin and was cold stable, and the other contained tyrosinated alpha-tubulin and was cold labile. These results are consistent with the idea that acetylation and detyrosination of alpha-tubulin are involved in the specification of stable microtubules.     

Distribution of microtubules containing post-translationally modified alpha-tubulin during Drosophila embryogenesis

The distribution of microtubules (MTs) enriched in detyrosinated alpha-tubulin (Glu-tubulin) was studied in Drosophila embryos by immunofluorescence microscopy by using a monoclonal antibody (ID5) which was raised against a 14-residue synthetic peptide spanning the carboxyterminal sequence of Glu-tubulin (Wehland and Weber: J. Cell Sci. 88:185-203, 1987). While all MT arrays contained tyrosinated alpha-tubulin (Tyr-tubulin), MTs rich in Glu-tubulin were not found during early stages of development even by using an image intensification camera. Elevated levels of microtubular Glu-tubulin were first detected after CNS condensation in neurone processes. In addition, sperm tails, which remained remarkably stable inside the embryo until late stages of development, were decorated by ID5. This was in marked contrast to the distribution of microtubule arrays containing acetylated alpha-tubulin, which could already be detected during the cellular blastoderm stage. Additional experiments with taxol suggested that the absence of MTs rich in Glu-tubulin during early stages of development was not due to the rapid turnover rate of MTs, which would be too fast for alpha-tubulin to be detyrosinated. The possible significance of the differential detyrosination and acetylation of microtubules during development is discussed.     

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.     

Newly assembled microtubules are concentrated in the proximal and distal regions of growing axons

We have investigated the sites of microtubule (MT) assembly in neurons during axon growth by taking advantage of the relationship between the proportion of tyrosinated alpha-tubulin (tyr-tubulin) in MTs and their age. Specifically, young (newly assembled) MTs contain more tyr-tubulin than older (more long-lived) MTs. To quantify the relative proportion of tyr-tubulin in MTs, cultured rat sympathetic neurons were permeabilized under conditions that stabilize existing MTs and remove unassembled tubulin. The MTs were then double-stained with antibodies to tyr-tubulin (as a measure of the amount of tyr-tubulin in MTs) and to beta-tubulin (as a measure of total MT mass), using immunofluorescence procedures. Cells were imaged with a cooled charge-coupled device camera and the relative proportion of tyr-tubulin in the MTs was quantified by computing the ratio of the tyr-tubulin fluorescence to the beta-tubulin fluorescence using a novel application of digital image processing and analysis techniques. The amount of tyr-tubulin in the MTs was highest in the cell body and at the growth cone; peak ratios in these two regions were approximately 10-fold higher than for the axon shaft. Moving out from the cell body into the axon, the tyr-tubulin content declined over an average distance of 40 microns to reach a constant low value within the axon shaft and then rose again more distally, over an average distance of 110 microns, to reach a peak at the growth cone (average axon length = 358 microns). These observations indicate that newly assembled MTs are concentrated in the proximal and distal regions of growing axons and therefore that the cell body and growth cone are the most active sites of MT assembly dynamics in neurons that are actively extending axons.     

Differential localisation of tyrosinated, detyrosinated, and acetylated alpha-tubulins in neurites and growth cones of dorsal root ganglion neurons

The comparative distribution of tyrosinated, detyrosinated, and acetylated alpha-tubulins was examined in neurites of rat dorsal root ganglion neurones in culture using immunofluorescence microscopy. Phase contrast observations of single neurones revealed that the neurites were actively motile, and rhodamine phalloidin staining of actin filaments showed the extent of lamellopodia and microspike projections from the growth cones. From double-labelling experiments using antibodies against tyrosinated, detryrosinated, or acetylated alpha-tubulin, it was found that the three different isoforms were differentially localised in neurites and growth cones. Detyrosinated and acetylated forms of alpha-tubulin were in the main restricted to the neurites extending no further than the base of the growth cones. Tyrosinated alpha-tubulin was, however, distributed throughout the body of the growth cone and into the base of some microspikes. Following treatment with taxol to promote microtubule assembly, detyrosinated and acetylated alpha-tubulins were found to be colocalised with tyrosinated alpha-tubulins throughout the growth cones of all cells examined. These results would be consistent with axonal transport of tyrosinated alpha-tubulin followed by assembly in the growth cone and subsequent detyrosination and acetylation. In addition the presence of unmodified alpha-tubulin in the growth cone may be necessary for the provision of labile microtubules for growth cone motility and extension.     

Polyglutamylated alpha-tubulin can enter the tyrosination/detyrosination cycle.

We have previously identified a major modification of neuronal alpha-tubulin which consists of the posttranslational addition of a varying number of glutamyl units on the gamma-carboxyl group of glutamate residue 445. This modification, called polyglutamylation, was initially found associated with detyrosinated alpha-tubulin [Eddé, B., Rossier, J., Le Caer, J.P., Desbruyères, E., Gros, F., & Denoulet, P. (1990) Science 247, 83-85]. In this report we show that a lateral chain of glutamyl units can also be present on tyrosinated alpha-tubulin. Incubation of cultured mouse brain neurons with radioactive tyrosine, in the presence of cycloheximide, resulted in a posttranslational labeling of six alpha-tubulin isoelectric variants. Because both tyrosination and polyglutamylation occur in the C-terminal region of alpha-tubulin, the structure of this region was investigated. [3H]tyrosinated tubulin was mixed with a large excess of unlabeled mouse brain tubulin and digested with thermolysin. Five peptides, detected by their radioactivity, were purified by high-performance liquid chromatography. Amino acid sequencing and mass spectrometry showed that one of these peptides corresponds to the native C-terminal part of alpha-tubulin 440VEGEGEEEGEEY451 and that the remainders bear a varying number of glutamyl units linked to glutamate residue 445, which explains the observed heterogeneity of tyrosinated alpha-tubulin. A quantitative analysis showed that the different tyrosinated forms of alpha-tubulin represent a minor (13%) fraction of the total alpha-tubulin present in the brain and that most (80%) of these tyrosinated forms are polyglutamylated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Posttranslational modifications of alpha-tubulin: acetylated and detyrosinated forms in axons of rat cerebellum

The distribution of acetylated alpha-tubulin in rat cerebellum was examined and compared with that of total alpha-tubulin and tyrosinated alpha-tubulin. From immunoperoxidase-stained vibratome sections of rat cerebellum it was found that acetylated alpha-tubulin, detectable with monoclonal 6-11B-1, was preferentially enriched in axons compared with dendrites. Parallel fiber axons, in particular, were labeled with 6-11B-1 yet unstained by an antibody recognizing tyrosinated alpha-tubulin, indicating that parallel fibers contain alpha-tubulin that is acetylated and detyrosinated. Axonal microtubules are known to be highly stable and the distribution of acetylated alpha-tubulin in other classes of stable microtubules suggests that acetylation and possibly detyrosination may play a role in the maintenance of stable populations of microtubules.     

Motor-dependent microtubule disassembly driven by tubulin tyrosination

In cells, stable microtubules (MTs) are covalently modified by a carboxypeptidase, which removes the C-terminal Tyr residue of α-tubulin. The significance of this selective detyrosination of MTs is not understood. In this study, we report that tubulin detyrosination in fibroblasts inhibits MT disassembly. This inhibition is relieved by overexpression of the depolymerizing motor mitotic centromere-associated kinesin (MCAK). Conversely, suppression of MCAK expression prevents disassembly of normal tyrosinated MTs in fibroblasts. Detyrosination of MTs suppresses the activity of MCAK in vitro, apparently as the result of a decreased affinity of the adenosine diphosphate (ADP)–inorganic phosphate- and ADP-bound forms of MCAK for the MT lattice. Detyrosination also impairs MT disassembly in neurons and inhibits the activity of the neuronal depolymerizing motor KIF2A in vitro. These results indicate that MT depolymerizing motors are directly inhibited by the detyrosination of tubulin, resulting in the stabilization of cellular MTs. Detyrosination of transiently stabilized MTs may give rise to persistent subpopulations of disassembly-resistant polymers to sustain subcellular cytoskeletal differentiation.     

Acetylated alpha-tubulin in the pollen tube microtubules.

Three monoclonal alpha-tubulin antibodies YL 1/2 (Kilmartin et al., 1982), 6-11B-1 (Piperno and Fuller, 1985) and DM1A (Blose et al., 1984) were used in indirect immunofluorescence (IIF) microscopy of the microtubule (MT) cytoskeleton in tobacco (Nicotiana tabacum) pollen tubes. The majority of pollen tube MTs contain tyrosinated alpha-tubulin recognized by YL 1/2. Acetylated alpha-tubulin revealed by 6-11B-1 was detected in the generative cell and in the kinetochore fibers, in polar spindle regions, and in the cell plate of the phragmoplast during generative cell division. In addition, small fragments of acetylated microtubules were seen in the older parts of the pollen tube grown on a taxol medium. The interaction of pollen tube MTs with mAb 6-11B-1 suggested that acetylation of alpha-tubulin correlates well with the putative arrays of stable MTs.     

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.     

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.     

Microtubules containing detyrosinated tubulin are less dynamic

Peptide antibodies specific for tyrosinated (tyr-tubulin) or detyrosinated alpha-tubulin (glu-tubulin) have been generated for studying the relative stability of microtubules enriched in either form of alpha-tubulin. Treatment of Vero cells with nocodazole has revealed that interphase microtubules rich in glu-tubulin (glu-microtubules) are resistant to higher concentrations of the microtubule-disrupting drug than the microtubules containing only tyr-tubulin (tyr-microtubules). Glu-tubulin is enriched in centrioles and mid-bodies, but absent from the first interphase microtubules that have repolymerized in late telophase. Tubulin (including both forms) has been labeled with rhodamine (rh-tubulin) and microinjected into Vero cells to study in vivo the dynamic properties and incorporation rates of tubulin into microtubules rich in either glu- or tyr-tubulin. Tyr-microtubules are significantly more rapidly labeled by the microinjected rh-tubulin than glu-microtubules. Ten minutes after injection, rh-tubulin is present in virtually all tyr-microtubules. The half-time of turnover of glu-microtubules is approximately 1 h. Even several hours after microinjection, some of the glu-microtubules have consistently not incorporated visible amounts of rh-tubulin. These results suggest that tyr- and glu-microtubules respectively represent relatively dynamic and stable subclasses of interphase microtubules.     

Tubulin tyrosination is a major factor affecting the recruitment of CAP-Gly proteins at microtubule plus ends

Tubulin-tyrosine ligase (TTL), the enzyme that catalyzes the addition of a C-terminal tyrosine residue to alpha-tubulin in the tubulin tyrosination cycle, is involved in tumor progression and has a vital role in neuronal organization. We show that in mammalian fibroblasts, cytoplasmic linker protein (CLIP) 170 and other microtubule plus-end tracking proteins comprising a cytoskeleton-associated protein glycine-rich (CAP-Gly) microtubule binding domain such as CLIP-115 and p150 Glued, localize to the ends of tyrosinated microtubules but not to the ends of detyrosinated microtubules. In vitro, the head domains of CLIP-170 and of p150 Glued bind more efficiently to tyrosinated microtubules than to detyrosinated polymers. In TTL-null fibroblasts, tubulin detyrosination and CAP-Gly protein mislocalization correlate with defects in both spindle positioning during mitosis and cell morphology during interphase. These results indicate that tubulin tyrosination regulates microtubule interactions with CAP-Gly microtubule plus-end tracking proteins and provide explanations for the involvement of TTL in tumor progression and in neuronal organization.     

Regulation of cytoplasmic tubulin carboxypeptidase activity during neural and muscle differentiation: characterization using a microtubule-based assay.

A cycle of posttranslational modification of alpha-tubulin has previously been described in higher eukaryotes, in which a C-terminal tyrosine residue is removed and replaced by two complementary cytoplasmic enzymes. The activity of the detyrosinating enzyme, tubulin carboxypeptidase (TCP), and its potential for regulating the level of detyrosinated (Glu) subunits in microtubules (MTs) is of great interest, since TCP catalyzes the primary modification of tubulin and since the level of Glu alpha-tubulin in MTs increases during a variety of differentiative and morphogenetic events. As a first step in examining the role of TCP in cellular morphogenesis, it was necessary to develop an assay for TCP with sufficient sensitivity and specificity to detect TCP activity during these events. Unlike previously described assays for TCP, ours makes use of the affinity TCP exhibits for MTs. NGF-induced neurite outgrowth in PC-12 cells was accompanied by a moderate (approximately 2-fold) increase in TCP activity, while myogenesis of L6 cells resulted in an almost insignificant decrease in activity. Measurements of TCP activity during differentiation were correlated with the level of extract Tyr tubulin, which increased (approximately 37%) during neurite outgrowth and was unchanged during myogenic differentiation. Our results suggest that TCP activity is regulated relative to its substrate, Tyr tubulin, and that changes in MT dynamics, rather than enzymatic activities, are the primary determinants of MT posttranslational modification state during differentiation. In addition, the assay we have devised for TCP and the characterization of TCP during differentiation may allow the future delineation of the mechanism(s) of regulation of TCP and the role this enzyme plays in modulating MT function during differentiation.     

Enhanced stability of microtubules enriched in detyrosinated tubulin is not a direct function of detyrosination level

Interphase cultured monkey kidney (TC-7) cells contain distinct subsets of cellular microtubules (MTs) enriched in posttranslationally detyrosinated (Glu) or tyrosinated (Tyr) alpha tubulin (Gundersen, G. G., M. H. Kalnoski, and J. C. Bulinski. 1984. Cell. 38:779-789). To determine the relative stability of these subsets of MTs, we subjected TC-7 cells to treatments that slowly depolymerized MTs. We found Glu MTs to be more resistant than Tyr MTs to depolymerization by nocodazole in living cells, and to depolymerization by dilution in detergent-permeabilized cell models. However, in cold-treated cells, Glu and Tyr MTs did not differ significantly in their stability. Digestion of permeabilized cell models with pancreatic carboxypeptidase A, to generate Glu MTs from endogenous Tyr MTs, did not significantly alter the resistance of the endogenous Tyr MTs toward dilution-induced depolymerization. Furthermore, in human fibroblasts that contained no distinct Glu MTs, we observed a population of nocodazole-resistant MTs. These data suggest that Glu MTs possess enhanced stability against end-mediated depolymerization, yet detyrosination alone appears to be insufficient to confer this enhanced stability.     

Acetylation of α-tubilin in different bovine cell types: implications for microtubule dynamics in interphase and mitosis.

Previous work on five cell types isolated from the bovine corpus luteum showed that the mass of acetylated microtubules (acet-MTs) in interphase differed. Endothelial cells, termed type 3, showed few acet-MTs, whereas the interphase cytoskeleton of granulosal-like cells, termed type 5, was rich in acet-MTs. In the present study, these cultured cells were used to determine whether the degree of α-tubulin acetylation in interphase had consequences on mitosis. To this end, the distribution of acet-MTs was determined throughout the cell cycle using a monoclonal antibody, 6-11B-1, directed against acetylated α-tubulin. For comparison, tyrosinated MTs were visualized with another monoclonal antibody, YL1/2, detecting tyrosinated α-tubulin. Although the amount of acet-MTs in interphase differed significantly between both cell types, major differences in the appearance of acet-MTs during mitosis were only apparent in prophase and during transition from late telophase to interphase. Thus, irrespective of different α-tubulin acetylation in interphase, spindle structure is uniform. Since acetylation of α-tubulin is believed to indicate the presence of relatively stable MTs, we conclude that MT dynamics is differently controlled in interphase and mitosis. Thereby interphase cells are able to carry out functions which involve stable MTs and the cells progress through mitosis in the presence of more dynamic MTs.     

Microtubule reassembly from nucleating fragments during the regrowth of amputated neurites

We have proposed that stable microtubule (MT) fragments that resist depolymerization may serve as nucleating elements for the local control of MT dynamics in the axon (Heidemann, S. R., M. A. Hamborg, S. J. Thomas, B. Song, S. Lindley, and D. Chu, 1984, J. Cell Biol., 99:1289-1295). Here we report evidence that supports this proposal in studies on the role of MTs in the regrowth of neurites from the distal segments of amputated chick sensory neurites. Amputated neurites collapse to "beads" of axoplasm that rapidly regrow (Shaw, G., and D. Bray, 1977, Exp. Cell Res., 104:55-62). We examined both unarrested regrowth and regrowth after MT disassembly by either cold (-5 degrees C for 2 h) or nocodazole (0.1 microgram/ml for 15-20 min). In all these cases regrowth occurred at 3.5-4.5 micron/min with no delay times other than the times to reach 37 degrees C or rinse out the nocodazole. Electron micrographs of untreated beads show many MTs of varying lengths, while those of cold- and nocodazole-treated beads show markedly shorter MTs. The robust regrowth of neurites from beads containing only very short MTs argues against unfurling of intact MTs from the bead into the growing neurite. Electron micrographs of cold-treated beads lysed under conditions that cause substantial MT depolymerization in untreated intact neurites show persistent MT fragments similar to those in unlysed cold-treated beads. We interpret this as evidence that the MT fragments in cold-treated beads are somehow distinct from the majority of the MT mass that had depolymerized. Collapsed neurites treated with a higher dose of nocodazole (1.0 microgram/ml for 15-20 min) were completely devoid of MTs and regrew only after a 15-20 min delay in two cases but never regrew in 11 other cases. We found that MTs did not return in beads treated with 1.0 microgram/ml nocodazole even 30 min after removal of the drug. It was unlikely that the inability of these beads to reassemble MTs was due to incomplete removal of nocodazole in that a much higher dose (20 micrograms/ml nocodazole) could be quickly rinsed from intact neurites. Beads treated with 1.0 microgram/ml nocodazole could, however, be stimulated to reassemble MTs and regrow neurites by treatment with taxol. We conclude that the immediate, robust regrowth of neurites from collapsed beads of axoplasm requires MT nucleation sites to support MT reassembly.(ABSTRACT TRUNCATED AT 400 WORDS)     

Extracellular matrix allows PC12 neurite elongation in the absence of microtubules

Several groups have shown that PC12 will extend microtubule-containing neurites on extracellular matrix (ECM) with no lag period in the absence of nerve growth factor. This is in contrast to nerve growth factor (NGF)-induced neurite outgrowth that occurs with a lag period of several days. During this lag period, increased synthesis or activation of assembly-promoting microtubule-associated proteins (MAPs) occurs and is apparently required for neurite extension. We investigated the growth and microtubule (MT) content of PC12 neurites grown on ECM in the presence or absence of inhibitors of neurite outgrowth. On ECM, neurites of cells with or without prior exposure to NGF contain a normal density of MTs, but frequently contain unusual loops of MTs in their termini that may indicate increased MT assembly. On ECM, neurites extend from PC12 cells in the presence of 10 microM LiCl at significantly higher frequency than on polylysine. On other substrates, LiCl inhibits neurite outgrowth, apparently by inhibiting phosphorylation of particular MAPs (Burstein, D. E., P. J. Seeley, and L. A. Greene. 1985. J. Cell Biol. 101:862-870). Although 35-45% of 60 Li(+)-neurites examined were found to contain a normal array of MTs, 25-30% were found to have a MT density approximately 15% of normal. The remaining 30% of these neurites were found to be nearly devoid of MTs, containing only occasional, ambiguous, short tubular elements. We also found that neurites would extend on ECM in the presence of the microtubule depolymerizing drug, nocodazole. At 0.1 micrograms/ml nocodazole, cells on ECM produce neurites that contain a normal density of MTs. This is in contrast to the lack of neurite outgrowth and retraction of extant neurites that this dose produces in cells grown on polylysine. At 0.2 microgram/ml nocodazole, neurites again grew out in substantial number and four of five neurites examined ultrastructurally were found to be completely devoid of microtubules. We interpret these results by postulating that growth on ECM relieves the need for MTs to serve as compressive supports for neurite tension (Dennerll, T. J., H. C. Joshi, U. L. Steel, R. E. Buxbaum, and S. R. Heidemann. 1988. J. Cell Biol. 107:665). Because compression destabilizes MTs and favors disassembly, this would tend to increase MT assembly relative to other conditions, as we found. Additionally, if MTs are not needed as compressive supports, neurites could grow out in their absence, as we also observed.