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.     

Acetylated alpha-tubulin in Physarum: immunological characterization of the isotype and its usage in particular microtubular organelles

We have used monoclonal antibodies specific for acetylated and unacetylated alpha-tubulin to characterize the acetylated alpha-tubulin isotype of Physarum polycephalum, its expression in the life cycle, and its localization in particular microtubular organelles. We have used the monoclonal antibody 6-11B-1 (Piperno, G., and M. T. Fuller, 1985, J. Cell Biol., 101:2085-2094) as the probe for acetylated alpha-tubulin and have provided a biochemical characterization of the monoclonal antibody KMP-1 as a probe for unacetylated tubulin in Physarum. Concomitant use of these two probes has allowed us to characterize the acetylated alpha-tubulin of Physarum as the alpha 3 isotype. We have detected this acetylated alpha 3 tubulin isotype in both the flagellate and in the myxameba, but not in the plasmodium. In the flagellate, acetylated tubulin is present in both the flagellar axonemes and in an extensive array of cytoplasmic microtubules. The extensive arrangement of acetylated cytoplasmic microtubules and the flagellar axonemes are elaborated during the myxameba-flagellate transformation. In the myxameba, acetylated tubulin is not present in the cytoplasmic microtubules nor in the mitotic spindle microtubules, but is associated with the two centrioles of this cell. These findings, taken together with the apparent absence of acetylated alpha-tubulin in the ephemeral microtubules of the plasmodium suggest a natural correspondence between the presence of acetylated alpha-tubulin and microtubule organelles that are intrinsically stable or cross-linked.     

Microtubules containing acetylated alpha-tubulin in mammalian cells in culture

The subcellular distribution of microtubules containing acetylated alpha-tubulin in mammalian cells in culture was analyzed with 6-11B-1, a monoclonal antibody specific for acetylated alpha-tubulin. Cultures of 3T3, HeLa, and PtK2 cells were grown on coverslips and observed by immunofluorescence microscopy after double-staining by 6-11B-1 and B-5-1-2, a monoclonal antibody specific for all alpha-tubulins. The antibody 6-11B-1 binds to primary cilia, centrioles, mitotic spindles, midbodies, and to subsets of cytoplasmic microtubules in 3T3 and HeLa cells, but not in PtK2 cells. These observations confirm that the acetylation of alpha-tubulin is a modification occurring in different microtubule structures and in a variety of eukaryotic cells. Some features of the acetylation of cytoplasmic microtubules of mammalian cells are also described here. First, acetylated alpha-tubulin is present in microtubules that, under depolymerizing conditions, are more stable than the majority of cytoplasmic microtubules. In addition to the specific microtubule frameworks already mentioned, cytoplasmic microtubules resistant to nocodazole or colchicine, but not cold-resistant microtubules, contain more acetylated alpha-tubulin than the rest of cellular microtubules. Second, the alpha-tubulin in all cytoplasmic microtubules of 3T3 and HeLa cells becomes acetylated in the presence of taxol, a drug that stabilizes microtubules. Third, acetylation and deacetylation of cytoplasmic microtubules are reversible in cells released from exposure to 0 degrees C or antimitotic drugs. Fourth, the epitope recognized by the antibody 6-11B-1 is not absolutely necessary for cell growth and division. This epitope is absent in PtK2 cells. The acetylation of alpha-tubulin could regulate the presence of microtubules in specific intracellular spaces by selective stabilization.     

Immunolocalization of tubulin isoforms and post-translational modifications in the protists Tritrichomonas foetus and Trichomonas vaginalis

In the present report we show the distribution of multiple tubulin isoforms in Trichomonas vaginalis and Tritrichomonas foetus, flagellated parasitic protists of the urogenital tracts of human and cattle, respectively, using immunofluorescence and immunoelectron microscopy. We used several monoclonal and polyclonal anti-tubulin antibodies from different sources and recognizing variant tubulin isoforms. Our results demonstrate that: (1) there is a heterogeneous distribution of the different tubulin isoforms in the main microtubular cell structures, such as axostyle, flagella, basal bodies, and mitotic spindle, (2) the axostyle-pelta junction is a structure with high affinity for glutamylated tubulin antibodies in T. foetus, (3) the spindle labeling is positive to anti-glutamylated tubulin and anti-alpha-tubulin (TAT1 and purchased from Amersham) antibodies in T. vaginalis but it is negative in T. foetus, (4) the nuclear matrix and the cytosol presented positive reaction using glutamylated and TAT1 (anti-alpha-tubulin) antibodies only in T. vaginalis, and (5) the Golgi complex exhibited staining using the glutamylated tubulin antibody. The present data corroborate with the idea of the existence of a heterogeneous population of microtubules in these protists and of a subset of intracytoplasmic microtubules. Microtubule diversity may reflect distinct tubulins, diverse microtubule-associated proteins, or a combination of both.     

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.     

Distribution of acetylated alpha-tubulin in retina and in vitro-assembled microtubules

We have used the mouse monoclonal antibody 6-11 B-1, specific for acetylated alpha-tubulin, to determine the distribution of acetylated alpha-tubulin in in vitro-assembled microtubules and retinal tissue. Analysis by immunoblots revealed that microtubules assembled from bovine brain extracts contain both acetylated and nonacetylated alpha-tubulin. Immunofluorescence, using 6-11 B-1 and antitubulin B-5-1-2, a monoclonal antibody specific for alpha-tubulin, demonstrated the colocalization of both alpha-tubulin species in neurons of the retina and that acetylated microtubules are relatively abundant in neurons. However, analysis at higher resolution revealed that rod photoreceptors contain spatially distinct microtubule arrays which differ in content of acetylated alpha-tubulin and differ in stability. Acetylated microtubules which composed those of the rod outer segment and connecting cilium were resistant to depolymerization in nocodazole or colchicine. In contrast, the nonacetylated microtubules which composed those of the rod-inner segment were depolymerized in nocodazole or colchicine. Therefore, these acetylated microtubules are more resistant to depolymerization than non-acetylated microtubules.     

Tubulins in Trichomonas vaginalis: molecular characterization of alpha-tubulin genes, posttranslational modifications, and homology modeling of the tubulin dimer

We have isolated and analysed an alpha-tubulin-encoding gene (atub1) in an early-diverging eukaryote, Trichomonas vaginalis. The complete atub1 open reading frame included 1.356 bp encoding a polypeptide of 452 amino-acyl residues. A second alpha-tubulin gene (atub2) was amplified by PCR using primers derived from consensus alpha-tubulin amino acid sequences. Both T. vaginalis alpha-tubulin sequences showed high identity to those described in other parabasalids (94.4%-97.3%), and exhibited a high degree of similarity to sequences from Metazoa (such as pig brain) and diplomonads (such as Giardia). Despite large evolutionary distances previously observed between trichomonads and mammals, the three-dimensional model of the T. vaginalis tubulin dimer was very similar to that of pig brain. Possible correlations between alpha-tubulin sequences and posttranslational modifications (PTMs) were examined. Our observations corroborated previous data obtained in T. vaginalis using specific anti-PTMs antibodies. As described in the related species Tritrichomonas mobilensis, microtubules are likely acetylated, non-tyrosinated, glutamylated, and non-glycylated in T. vaginalis. Evolutionary considerations concerning the time of appearance of these tubulin PTMs are also discussed since trichomonads are potentially one of the earliest diverging eukaryotic lineages.     

Cytoplasmic microtubules containing acetylated alpha-tubulin in Chlamydomonas reinhardtii: spatial arrangement and properties

A monoclonal antibody, 6-11B-1, specific for acetylated alpha-tubulin (Piperno, G., and M. T. Fuller, 1985, J. Cell Biol., 101:2085-2094) was used to study the distribution of this molecule in interphase cells of Chlamydomonas reinhardtii. Double-label immunofluorescence was performed using 6-11B-1, and 3A5, an antibody specific for all alpha-tubulin isoforms. It was found that acetylated alpha-tubulin is not restricted to the axonemes, but is also present in basal bodies and in a subset of cytoplasmic microtubules that radiate from the basal bodies just beneath the plasma membrane. Immunoblotting experiments of basal body polypeptide components using 6-11B-1 as a probe confirmed that basal bodies contain acetylated alpha-tubulin. In the cell body, 6-11B-1 stained an average of 2.2 microtubules/cell, while 3A5 stained an average of 6.5 microtubules. Although exposure to 0 degrees C depolymerized both types of cytoplasmic microtubules, exposure to various concentrations of colchicine or nocodazole showed that the acetylated microtubules are much more resistant to drug-induced depolymerization than nonacetylated microtubules. Axonemes and basal bodies are already known to be colchicine-resistant. All acetylated microtubules appear, therefore, to be more drug-resistant than nonacetylated microtubules. The acetylation of alpha-tubulin may be part of a mechanism that stabilizes microtubules.     

Detection of microtubules of the flagellate Trichomonas vaginalis by monoclonal antibodies specific for beta-tubulin

Monoclonal antibodies specific for mammalian beta-tubulin recognized the microtubule cytoskeleton of the flagellated protozoon Trichomonas vaginalis. Of seven antibodies, two demonstrated the axostyle, costa, recurrent flagellum, and anterior flagella by indirect immunofluorescence microscopy. The remaining five stained a hazy reticular pattern in the cytoplasm of formaldehydefixed, detergent-extracted organisms. Western immunoblots of whole T. vaginalis extracts treated with protease inhibitors and electrophoresed on polyacrylamide gels containing sodium dodecyl sulfate showed a major band at molecular weight 50,000 when probed with only one of the antibodies which stained the axial cytoskeleton. The antibodies which stained only the cytoplasm showed a different western blot pattern with a major doublet band at MW 58,000-60,000. Another antibody, which stained both the axial cytoskeleton and the reticular cytoplasmic pattern showed major bands at MW 58,000-60,000 and also at MW 40,000-42,000. The recognition of microtubule populations in T. vaginalis by these monoclonal antibodies was different than we found earlier with Leishmania donovani and Toxoplasma gondii, where all seven antibodies recognize cytoskeletal microtubules and produce western blots characteristic of tubulin. Only one of these seven antibodies recognizes tubulin in T. vaginalis by immunoblot. The microtubules of T. vaginalis do not demonstrate all epitopes recognized by monoclonal antibodies specific for mammalian beta-tubulin; one of the antibodies appears to recognize an epitope which is morphologically associated with microtubules but does not have the characteristic MW of tubulin.     

Monoclonal antibodies specific for an acetylated form of alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms

Seven monoclonal antibodies raised against tubulin from the axonemes of sea urchin sperm flagella recognize an acetylated form of alpha-tubulin present in the axoneme of a variety of organisms. The antigen was not detected among soluble, cytoplasmic alpha-tubulin isoforms from a variety of cells. The specificity of the antibodies was determined by in vitro acetylation of sea urchin and Chlamydomonas cytoplasmic tubulins in crude extracts. Of all the acetylated polypeptides in the extracts, only alpha-tubulin became antigenic. Among Chlamydomonas tubulin isoforms, the antibodies recognize only the axonemal alpha-tubulin isoform acetylated in vivo on the epsilon-amino group of lysine(s) (L'Hernault, S.W., and J.L. Rosenbaum, 1985, Biochemistry, 24:473-478). The antibodies do not recognize unmodified axonemal alpha-tubulin, unassembled alpha-tubulin present in a flagellar matrix-plus-membrane fraction, or soluble, cytoplasmic alpha-tubulin from Chlamydomonas cell bodies. The antigen was found in protein fractions that contained axonemal microtubules from a variety of sources, including cilia from sea urchin blastulae and Tetrahymena, sperm and testis from Drosophila, and human sperm. In contrast, the antigen was not detected in preparations of soluble, cytoplasmic tubulin, which would not have contained tubulin from stable microtubule arrays such as centrioles, from unfertilized sea urchin eggs, Drosophila embryos, and HeLa cells. Although the acetylated alpha-tubulin recognized by the antibodies is present in axonemes from a variety of sources and may be necessary for axoneme formation, it is not found exclusively in any one subset of morphologically distinct axonemal microtubules. The antigen was found in similar proportions in fractions from sea urchin sperm axonemes enriched for central pair or outer doublet B or outer doublet A microtubules. Therefore the acetylation of alpha-tubulin does not provide the mechanism that specifies the structure of any one class of axonemal microtubules. Preliminary evidence indicates that acetylated alpha-tubulin is not restricted to the axoneme. The antibodies described in this report may allow us to deduce the role of tubulin acetylation in the structure and function of microtubules in vivo.     

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.     

Heterogeneity of microtubules recognized by monoclonal antibodies to alpha-tubulin

A set of four monoclonal antibodies against tubulin (TU-01, TU-02, TU-03, and TU-04) were produced using pig brain microtubule protein as antigen. Their characterization shows that all recognize antigenic determinants located on the tubulin alpha-subunit. However, peptide mapping of isolated alpha-tubulin, followed by immunoblotting with the monoclonal antibodies, shows that the antigenic determinants are located on different peptide fragments in at least three cases. The immunoreactivity with tubulins from different cells and tissues, ranging from eukaryotic microorganisms to man, was studied by immunoblotting and immunofluorescence. The antigenic determinants recognized by the antibodies are not uniformly distributed but, in some instances, are absent from tubulins of lower eukaryotic cells. These antibodies also make it possible to distinguish between different sets of microtubules within individual cells. Antigenically different microtubules are particularly evident in mouse spermatozoa and in some protozoa (T. vaginalis, H. muscarum, L. tropica, N. gruberi) possessing different sets of microtubules with different functions. These monoclonal antibodies can clearly identify the heterogeneity of tubulin or microtubules both from different organisms and within the same cell.     

The appearance of acetylated alpha-tubulin during early development and cellular differentiation in Xenopus

Early development in Xenopus is characterized by dramatic changes in the organization of the microtubule cytoskeleton. We have used whole-mount immunocytochemistry to follow the expression of the acetylated form of alpha-tubulin during early Xenopus development. In the egg and early embryo, the monoclonal anti-acetylated tubulin antibody 6-11B-1 stained meiotic and mitotic spindles, midbody microtubules, and what appears to be the central region of the sperm aster; the antibody did not stain the sperm aster itself or the cortical microtubule system associated with the rotation of the fertilized egg. Following gastrulation, acetylated tubulin disappeared from all but mitotic midbody microtubules. During the course of neurulation high levels of acetylated tubulin reappeared in the precursors of the ciliated epidermal cells (stage 15), transiently in neural folds (stage 16/17), in neuronal processes (stage 18/19), and in somas (stage 21). The changing pattern of anti-acetylated tubulin staining during Xenopus development raises intriguing questions as to the physiological significance of tubulin acetylation.     

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.     

Acetylation of lysine 40 in alpha-tubulin is not essential in Tetrahymena thermophila

In Tetrahymena, at least 17 distinct microtubule structures are assembled from a single primary sequence type of alpha- and beta- tubulin heterodimer, precluding distinctions among microtubular systems based on tubulin primary sequence isotypes. Tetrahymena tubulins also are modified by several types of posttranslational reactions including acetylation of alpha-tubulin at lysine 40, a modification found in most eukaryotes. In Tetrahymena, axonemal alpha-tubulin and numerous other microtubules are acetylated. We completely replaced the single type of alpha-tubulin gene in the macronucleus with a version encoding arginine instead of lysine 40 and therefore cannot be acetylated at this position. No acetylated tubulin was detectable in these transformants using a monoclonal antibody specific for acetylated lysine 40. Surprisingly, mutants lacking detectable acetylated tubulin are indistinguishable from wild-type cells. Thus, acetylation of alpha- tubulin at lysine 40 is non-essential in Tetrahymena. In addition, isoelectric focusing gel analysis of axonemal tubulin from cells unable to acetylate alpha-tubulin leads us to conclude that: (a) most or all ciliary alpha-tubulin is acetylated, (b) other lysines cannot be acetylated to compensate for loss of acetylation at lysine 40, and (c) acetylated alpha-tubulin molecules in wild-type cells contain one or more additional charge-altering modifications.     

Recognition of specific Physarum alpha-tubulin isotypes by a monoclonal antibody. Sequence heterogeneity around the acetylation site at lysine 40

The monoclonal antibody 6-11B-1 recognises specifically the acetylated form of alpha-tubulin. The acetylation event occurs on a unique lysine residue, lysine 40. Using 6-11B-1, acetylated alpha-tubulin was detected in myxamoebae but not plasmodia of Physarum polycephalum. Following chemical acetylation plasmodial alpha-tubulin was detected by 6-11B-1. The monoclonal antibody KMP-1 recognises certain Physarum alpha-tubulin isotypes but only in non-acetylated form. Whilst recognising all the non-acetylated fraction of myxamoebal alpha-tubulin only a proportion of plasmodial alpha-tubulin was recognised by KMP-1. Peptides were synthesised corresponding to the acetylation domains (containing lysine 40) of myxamoebal alpha-tubulin and the inferred acetylation domains of two plasmodial-specific alpha-tubulin isotypes. The only difference between the two peptides was at a single residue corresponding to amino acid 44 in the polypeptide. Tyrosine was present in myxamoebal alpha-tubulin and glycine was present in the plasmodial specific peptides; the peptides are referred to as the Tyr44 and Gly44 peptides respectively. Both peptides in acetylated form blocked 6-11B-1 reactivity towards acetylated myxamoebal alpha-tubulin. The Tyr44 but not the Gly44 peptide blocked KMP-1 reactivity towards non-acetylated myxamoebal alpha-tubulin. Tyrosine at position 44 is not found in any other known alpha-tubulin. Thus a unique antigenic determinant exists in certain Physarum alpha-tubulin isotypes, close to the acetylation site at lysine 40. This antigenic determinant forms part of the KMP-1 recognition epitope and explains the unique isotype selectivity of this monoclonal antibody.     

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.     

Distribution of acetylated alpha-tubulin in Physarum polycephalum

The expression and cytological distribution of acetylated alpha-tubulin was investigated in Physarum polycephalum. A monoclonal antibody specific for acetylated alpha-tubulin, 6-11B-1 (Piperno, G., and M. T. Fuller, 1985, J. Cell Biol., 101:2085-2094), was used to screen for this protein during three different stages of the Physarum life cycle--the amoeba, the flagellate, and the plasmodium. Western blots of two-dimensional gels of amoebal and flagellate proteins reveal that this antibody recognizes the alpha 3 tubulin isotype, which was previously shown to be formed by posttranslational modification (Green, L. L., and W. F. Dove, 1984, Mol. Cell. Biol., 4:1706-1711). Double-label immunofluorescence demonstrates that, in the flagellate, acetylated alpha-tubulin is localized in the flagella and flagellar cone. Similar experiments with amoebae interestingly reveal that only within the microtubule organizing center (MTOC) are there detectable amounts of acetylated alpha-tubulin. In contrast, the plasmodial stage gives no evidence for acetylated alpha-tubulin by Western blotting or by immunofluorescence.     

The initiation of neurite outgrowth by sympathetic neurons grown in vitro does not depend on assembly of microtubules [published erratum appears in J Cell Biol 1995 Feb;128(3):443]

Neurite formation by dissociated chick sympathetic neurons in vitro begins when one of the many filopodia that emanate from the cell body of a neuron is invaded by cytoplasm containing microtubules and other components of axoplasm (Smith, 1994). This study was undertaken to determine whether this process depends on assembly of microtubules. To inhibit microtubule assembly, neurons were grown in medium containing nocodazole or colchicine. In one series of experiments, neurons first were exposed to the microtubule-stabilizing drug, taxol, so that existing microtubules would remain intact while assembly of new microtubules was inhibited. The ability of neurons to form neurites was assessed by time-lapse video microscopy. Neurons subsequently were stained with antibodies against the tyrosinated and acetylated forms of alpha-tubulin and examined by laser confocal microscopy to visualize microtubules. Neurons were able to form short processes despite inhibition of microtubule assembly and they did so in a way that closely resembled process formation in control medium. Processes formed by neurons that had not been pretreated with taxol were devoid of microtubules. However, microtubules were present in processes of taxol- pretreated neurons. These microtubules contained acetylated alpha- tubulin, as is typical of stable microtubules, but not tyrosinated alpha-tubulin, the form present in recently assembled microtubules. These findings show that the initial steps in neurite formation do not depend on microtubule assembly and suggest that microtubules assembled in the cell body can be translocated into developing neurites as they emerge. The results are compatible with models of neurite formation which postulate that cytoplasm from the cell body is transported into filopodia by actomyosin-based motility mechanisms.