Sites of Tubulin Polymerization in PC 12 Cells

The site at which tubulin enters into polymer in the neuritic process is a very important datum in terms of our understanding of the mechanism of transport of the microtubular cytoskeleton out the axon. If the form of tubulin being transported out the axon is the microtubule, then assembly of tubulin into microtubules should occur at or near the cell body; if, however, the form of tubulin transported is free tubulin dimer, then assembly can occur at any free microtubule end out the neurite. We have injected a fluorescent analog of tubulin into differentiated PC 12 cells and used differential extraction protocols to extract free dimer but not microtubules. We have imaged these cells before and after extraction by low-light-level video fluorescence microscopy and have used image analysis to examine the sites of tubulin incorporation into polymer or other unextracted components as a function of time. We find that tubulin in the distal reaches of the neurite is found initially as monomer and that its appearance in the unextracted component occurs later. This pattern of appearance of fluorescent tubulin initially in the soluble fraction and later in the unextractable component is qualitatively similar to that reported by other workers for biotinylated tubulin, but we see a larger gap between the rates of appearance in soluble fraction and in polymer. Quantitative analysis of fluorescence intensities in the two compartments with distance out the neurite reveals substantial variation between different neurites: In some neurites, the pattern of variation of unextracted/total tubulin suggests that tubulin enters into the unextracted component primarily near the cell body and that this unextracted component moves out the neurite with time, and in other neurites it suggest that monomer adds into microtubule ends staggered out the neurite. In no case do we see a pattern suggesting that distal addition predominates. These analyses of fluorescence intensities in extracted and unextracted neurites suggest that both transport of polymerized microtubules and monomer addition onto staggered microtubule ends occur in PC12 neurites and that in individual neurites one or the other of these two behaviors may predominate.     

Sites of microtubule assembly and disassembly in the mitotic spindle

We have microinjected biotinylated tubulin into mitotic fibroblast cells to identify the sites in the spindle at which new subunits are incorporated into microtubules (MTs). Labeled subunits were visualized in the electron microscope using an antibody to biotin followed by a secondary antibody coupled to colloidal gold. Astral MTs incorporate labeled subunits very rapidly by elongation of existing MTs and by new nucleation from the centrosome. At a slower rate, kinetochore MTs incorporate subunits at the kinetochore progressively during metaphase, suggesting a slow poleward flux of subunits in the kinetochore fiber. When cells injected in metaphase were examined in anaphase, a significant fraction of kinetochore MTs was unlabeled, suggesting that depolymerization had occurred at the kinetochore concomitant with chromosome to pole movement. The existence of opposite fluxes at the kinetochore during metaphase and anaphase suggests that two separate forces are responsible for chromosome congression and anaphase movement.     

神经营养因子诱导分化的神经元样PC12细胞分裂的研究

神经营养因子（nerve growth factor,NGF）诱导PC12细胞分化产生的神经元样细胞一直被认为属于分裂后的细胞,没有分裂能力。然而在本研究中,我们观察了一些已经发生分化的PC12细胞,这些细胞长有很长的神经突起,在形态上属于神经元样细胞。在这些细胞中,我们不仅检测到DNA合成,而且观察到这些细胞的分裂现象。更令人感兴趣的是,除了胞体发生分裂外,位于胞体分裂位置的突起也一分为二,分别分配给两个子细胞。这些结果说明,形态发生分化的神经元样PC12细胞仍有分裂能力。本研究首次报道神经元样PC12细胞及其突起能发生分裂。     

Changes in the organization of the neuritic cytoskeleton during nerve growth factor-activated differentiation of PC12 cells: a serial electron microscopic study of the development and control of neurite shape

After exposure to nerve growth factor, PC12 cells differentiate within a period of only a few days into cholinergic sympathetic neurons. Using computer-assisted three-dimensional serial electron microscopic reconstruction, we describe the progressive cytoskeletal and structural changes of PC12 neurites at different stages in their differentiation. Developmental changes in these neurites can be characterized by two major transitions. First, microtubules (MTs), which define the longitudinal axis of the neurite, increase in number leading to a more cylindrical and uniform neurite shape. Second, there are major changes in the relative numbers of other organelle types, which reflect the functional specialization of the neurite. These changes do not in themselves seriously affect shape change of the neurite during development, however the presence of these organelles and their associated obligatory volumes (volumes surrounding organelle) account for well over 50% of the neurite's volume at all stages of development. The MT-MT distances and obligatory volumes associated with the organelles remain constant throughout development. Thus, we can conclude that many of the observed changes seen in developing PC12 neurites are due simply to the production of a greater number of MTs in the cell, and that many of the other important parameters that can be measured and contribute to neurite shape remain constant during development.     

A novel acetylation of β-tubulin by San modulates microtubule polymerization via down-regulating tubulin incorporation

Dynamic instability is a critical property of microtubules (MTs). By regulating the rate of tubulin polymerization and depolymerization, cells organize the MT cytoskeleton to accommodate their specific functions. Among many processes, posttranslational modifications of tubulin are implicated in regulating MT functions. Here we report a novel tubulin acetylation catalyzed by acetyltransferase San at lysine 252 (K252) of β-tubulin. This acetylation, which is also detected in vivo, is added to soluble tubulin heterodimers but not tubulins in MTs. The acetylation-mimicking K252A/Q mutants were incorporated into the MT cytoskeleton in HeLa cells without causing any obvious MT defect. However, after cold-induced catastrophe, MT regrowth is accelerated in San-siRNA cells while the incorporation of acetylation-mimicking mutant tubulins is severely impeded. K252 of β-tubulin localizes at the interface of α-/β-tubulins and interacts with the phosphate group of the α-tubulin-bound GTP. We propose that the acetylation slows down tubulin incorporation into MTs by neutralizing the positive charge on K252 and allowing tubulin heterodimers to adopt a conformation that disfavors tubulin incorporation.     

RGS2 promotes formation of neurites by stimulating microtubule polymerization

Regulator of G-protein signaling (RGS) proteins interact with subunits of heterotrimeric G-proteins via the RGS domain and attenuate their activity by accelerating GTPase activity. RGS2, a member of the RGS family, regulates synaptic development via hereto unknown mechanism. In this study, we found that RGS2 directly interacted with tubulin via a short region at the N-terminus: amino acids 41–60. RGS2 enhanced microtubule polymerization in vitro, and the tubulin binding region was necessary and sufficient for this activity. In Vero cells, polymerization of microtubule was stimulated when peptides containing the tubulin binding region were microinjected. Immunocytochemical analysis showed that endogenous RGS2 was localized at the termini of neurites in differentiated PC12 cells. Over-expression of RGS2 enhanced the nerve growth factor-induced neurite outgrowth in PC12 cells, while specific knock-down of endogenous RGS2 suppressed the neurite outgrowth. These findings demonstrate that RGS2 contributes to the neuronal cell differentiation via regulation of microtubule dynamics.     

The cytoskeleton of neurites after microtubule depolymerization

We previously reported a positive correlation between the number of cold-stable microtubules (MTs) remaining after cold treatment of cat sympathetic nerve and the extent to which the original uniform polarity orientation of axonal MTs was recapitulated after rewarming (J cell biol 99 (1984) 1289). We interpreted these data to indicate that cold-stable fragments, part of larger, generally labile MTs, could act as seeds to organize MT assembly in axons. We report here a direct investigation of the form of cold-stable MTs in neurites of PC-12 cells using two-dimensional reconstruction of serial thin sections. Our data provides strong support for our previous interpretation. The number of MTs in cold-treated neurites was 2-3 times as great while the total length of polymer was approximately half that in control neurites. The average length of MTs in cold-treated neurites was 7-10 times lower than in control neurites. We observed that treatments that depolymerize axonal microtubules cause a marked increase in the number of membranous elements within the axoplasm. This may, however, be a non-specific result of an insult to the axon, since such changes have also been observed in severed, regenerating nerve fibres. We observed that neuroblastoma neurites respond to MT-depolymerization stimuli by developing lateral filopodia similar to those observed in chick dorsal root ganglion cells. Ultrastructural observation of detergent-lysed, whole mounted neuroblastoma (Neuro 2A) cells indicated that the cytoskeleton remaining after MT depolymerization splayed out perpendicular to the long axis of the neurite. That is, we were able to observe many more cytoskeletal 'ends' after MT depolymerization. The concomitant production of filopodia and the splaying of the cytoskeleton after MT depolymerization supports the hypothesis put forward by Wessels et al. (Exp cell res 117 (1978) 335) that the presence or absence of cytoskeletal ends regulates which region of the cell surface is involved in motile behaviour.     

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)     

Differential sorting of beta tubulin isotypes into colchicine-stable microtubules during neuronal and muscle differentiation of embryonal carcinoma cells.

Pluripotent P19 embryonal carcinoma (EC) cells were differentiated along the neuronal and muscle pathways. Comparisons of class I, II, III, and IV beta tubulin isotypes in total and colchicine-stable microtubule (MT) arrays from uncommitted EC, neuronal, and muscle cells were made by immunoblotting and by indirect immunofluorescence microscopy. In undifferentiated EC cells the relative amounts of these four isotypes are the same in both the total and stable MT populations. Subcellular sorting of beta tubulin isotypes was demonstrated in both neuronal and muscle differentiated cells. During neuronal differentiation, class II beta tubulin is preferentially incorporated into the colchicine-stable MTs while class III beta tubulin is preferentially found in the colchicine-labile MTs. The subcellular sorting of class II into stable MTs correlates with the increased staining of MAP 1B, and with the expression of MAP 2C and tau. Although muscle differentiated cells express class II beta tubulin, stable MTs in these cells do not preferentially incorporate this isotype but instead show increased incorporation of class IV beta tubulin. Muscle cells do not show high levels of MAP 1B and do not express MAP 2C or tau. These results are consistent with the hypothesis that a subcellular sorting of tubulin isotypes is the result of a complex interaction between tubulin isotypes and MT-associated proteins.     

Microinjection of fluorescent tubulin into plant cells provides a representative picture of the cortical microtubule array

By microinjecting rhodamine-labelled tubulin into living plant cells, it is possible to observe microtubules (MTs) directly and to see how the cortical array reorganizes itself. The validity of the conclusions drawn from such observations depends upon the assumption that most, if not all, of the native MTs are dynamic and incorporate labelled tubulin. However, if arrays also contain MTs that are not exchanging tubulin subunits, such MTs will remain unlabelled, and the labelled MT population will be under-representative of the whole array. To address this potential problem, we microinjected pea epidermal cells with rhodamine-labelled tubulin, then fixed the cells and used fluorescein-conjugated antibodies against tubulin to detect the entire MT array. The two fluorescent patterns corresponded well, confirming that the MTs labelled with exogenous tubulin were evenly distributed throughout the entire array. Also, by comparing the MT image before and after aldehyde fixation, we observed that, although some of the MTs were lost in the procedure, the fixation was able to preserve the arrangement of MTs seen in the living cell. We conclude that fluorescence analogue cytochemistry provides a valid representation of the entire cortical MT array.     

The polymeric immunoglobulin receptor accumulates in specialized endosomes but not synaptic vesicles within the neurites of transfected neuroendocrine PC12 cells

We have expressed in neuroendocrine PC12 cells the polymeric immunoglobulin receptor (pIgR), which is normally targeted from the basolateral to the apical surface of epithelial cells. In the presence of nerve growth factor, PC12 cells extend neurites which contain synaptic vesicle-like structures and regulated secretory granules. By immunofluorescence microscopy, pIgR, like the synaptic vesicle protein synaptophysin, accumulates in both the cell body and the neurites. On the other hand, the transferrin receptor, which normally recycles at the basolateral surface in epithelial cells, and the cation-independent mannose 6-phosphate receptor, a marker of late endosomes, are largely restricted to the cell body. pIgR internalizes ligand into endosomes within the cell body and the neurites, while uptake of ligand by the low density lipoprotein receptor occurs primarily into endosomes within the cell body. We conclude that transport of membrane proteins to PC12 neurites as well as to specialized endosomes within these processes is selective and appears to be governed by similar mechanisms that dictate sorting in epithelial cells. Additionally, two types of endosomes can be identified in polarized PC12 cells by the differential uptake of ligand, a housekeeping type in the cell bodies and a specialized endosome in the neurites. Recent findings suggest that specialized axonal endosomes in neurons are likely to give rise to synaptic vesicles (Mundigl, O., M. Matteoli, L. Daniell, A. Thomas-Reetz, A. Metcalf, R. Jahn, and P. De Camilli. 1993. J. Cell Biol. 122:1207- 1221). Although pIgR reaches the specialized endosomes in the neurites of PC12 cells, we find by subcellular fractionation that under a variety of conditions it is efficiently excluded from synaptic vesicle- like structures as well as from secretory granules.     

TTLL7 is a mammalian beta-tubulin polyglutamylase required for growth of MAP2-positive neurites

Microtubules form a cytoskeletal framework that influences cell shape and provides structural support for the cell. Microtubules in the nervous system undergo a unique post-translational modification, polyglutamylation of the C termini of their tubulin subunits. The mammalian enzymes that perform beta-tubulin polyglutamylation as well as their physiological functions in the neuronal tissue remain elusive. We report identification of a mammalian polyglutamylase with specificity for beta-tubulin as well as its distribution and function in neurite growth. To identify putative tubulin polyglutamylases, we searched tubulin tyrosine ligase-like (TTLL) proteins for those predominantly expressed in the nervous system. Of 13 TTLL proteins, TTLL7 was transcribed at the highest level in the nervous system. Recombinant TTLL7 catalyzed tubulin polyglutamylation with high preference to beta-tubulin in vitro. When expressed in HEK293T cells, TTLL7 demonstrated specificity for beta-tubulin and not for alpha-tubulin or nucleosome assembly protein 1. Consistent with these findings, knockdown of TTLL7 in a primary culture of superior cervical ganglion neurons caused a loss of polyglutamylated beta-tubulin. Following stimulation of PC12 cells with nerve growth factor to differentiate, the level of TTLL7 increased concomitantly with polyglutamylation of beta-tubulin. Short interference RNA-mediated knockdown of TTLL7 repressed nerve growth factor-stimulated MAP (microtubule-associated protein) 2-positive neurite growth in PC12 cells. Consistent with having a role in the growth of MAP2-positive neurites, TTLL7 accumulated within a MAP2-enriched somatodendritic portion of superior cervical ganglion, as did polyglutamylated beta-tubulin. Anti-TTLL7 antibody revealed that TTLL7 was distributed in a somatodendritic compartment in the mouse brain. These findings indicate that TTLL7 is a beta-tubulin polyglutamylase and is required for the growth of MAP2-positive neurites in PC12 cells.     

Microtubule dynamics in interphase cells

The sites of microtubule growth and the kinetics of elongation have been studied in vivo by microinjection of biotin-labeled tubulin and subsequent visualization with immunocytochemical probes. Immunofluorescence and immunoelectron microscopy demonstrate that injected biotin-labeled subunits are incorporated into new segments of growth which are contiguous with unlabeled microtubules. Rapid incorporation occurs by elongation of existing microtubules and new nucleation off the centrosome. The growth rate is 3.6 micron/min and is independent of the concentration of injected labeled tubulin. This rate of incorporation together with turnover of existing microtubules leads to approximately 80% exchange in 15 min. The observed kinetics and pattern of microtubule turnover allow for an evaluation of the relevance of several in vitro models for steady-state dynamics to the in vivo situation. We have also observed a substantial population of quasi-stable microtubules that does not exchange subunits as rapidly as the majority of microtubules and may have specialized functions in the cell.     

Pb/Cu effects on the organization of microtubule cytoskeleton in interphase and mitotic cells of Allium sativum L.

The effects of lead and copper on the arrangement of microtubule (MT) cytoskeleton in root tip cells of Allium sativum L. were investigated. Batch cultures of garlic were carried out under defined conditions in the presence 10⁻⁴ M Pb/Cu of various duration treatments. With tubulin immunolabelling and transmission electron microscopy (TEM), we found four different types of MT structures depending on the cell cycle stage: the interphase array, preprophase band, mitotic spindle and phragmoplast were typical for the control cells. Pb/Cu affected the mechanisms controlling the organization of MT cytoskeleton, and induces the following aberrations in interphase and mitotic cells. (1) Pb/Cu induced the formation of atypical MT arrays in the cortical cytoplasm of the interphase cells, consisting of skewed, wavy MT bundles, MT fragments and ring-like tubulin aggregations. (2) Pb/Cu disordered the chromosome movements carried out by the mitotic spindle. The outcome was chromosome aberrations, for example, chromosome bridges and chromosome stickiness, as well as inhibition of cells from entering mitosis. (3) Depending on the time of exposure, MTs disintegrated into shorter fragments or they completely disappeared, indicating MT depolymerization. (4) Different metals had different effects on MT organization. MTs were more sensitive to the pressure of Cu ions than Pb. Moreover, TEM observations showed that the MTs were relatively short and in some places wavy when exposed to 10⁻⁴ M Pb/Cu solutions for 1–2 h. In many sections MTs were no longer visible with increasing duration of treatment (>4 h). Based on these results, we suggested that MT cytoskeleton is primarily responsible for Pb/Cu-associated toxicity and tolerance in plants.     

The von Hippel-Lindau tumor suppressor protein influences microtubule dynamics at the cell periphery

The von Hippel-Lindau (VHL) protein protects microtubules (MTs) from destabilization by nocodazole treatment. Based on this fixed-cell assay with static end points, VHL has been reported to directly stabilize the MT cytoskeleton. To investigate the dynamic changes in MTs induced by VHL in living cells, we measured the influence of VHL on tubulin turnover using fluorescence recovery after photobleaching (FRAP). To this end, we engineered VHL-deficient renal cell carcinoma cells to constitutively incorporate fluorescently labeled tubulin and to inducibly express VHL. Induction of VHL in these cells resulted in a decrease of tubulin turnover as measured by FRAP at the cell periphery, while minimally influencing MT dynamics around the centrosome. Our data indicates that VHL changes the behavior of MTs dependent on their subcellular localization implying a role for VHL in cellular processes such as migration, polarization, and cell-cell interactions. Here we propose a complementary method to directly measure VHL-induced subcellular changes in microtubule dynamics, which may serve as a tool to study the effect of MT binding proteins such as VHL.     

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.     

Respective roles of neurofilaments, microtubules, MAP1B, and tau in neurite outgrowth and stabilization.

The respective roles of neurofilaments (NFs), microtubules (MTs), and the microtubule-associated proteins (MAPs) MAP 1B and tau on neurite outgrowth and stabilization were probed by the intracellular delivery of specific antisera into transiently permeabilized NB2a/d1 cells during treatment with dbcAMP. Intracellular delivery of antisera specific for the low (NF-L), middle (NF-M), or extensively phosphorylated high (NF-H) molecular weight subunits did not prevent initial neurite elaboration, nor did it induce retraction of existing neurites elaborated by cells that had been previously treated for 1 d with dbcAMP. By contrast, intracellular delivery of antisera directed against tubulin reduced the percentage of cells with neurites at both these time points. Intracellular delivery of anti-NF-L and anti-NF-M antisera did not induce retraction in cells treated with dbcAMP for 3 d. However, intracellular delivery of antisera directed against extensively phosphorylated NF-H, MAP1B, tau, or tubulin induced similar levels of neurite retraction at this time. Intracellular delivery of monoclonal antibodies (RT97 or SMI-31) directed against phosphorylated NF-H induced neurite retraction in cell treated with dbcAMP for 3 d; a monoclonal antibody (SMI-32) directed against nonphosphorylated NF-H did not induce neurite retraction at this time. By contrast, none of the above antisera induced retraction of neurites in cells treated with dbcAMP for 7 d. Neurites develop resistance to retraction by colchicine, first detectable in some neurites after 3 d and in the majority of neurites after 7 d of dbcAMP treatment. We therefore examined whether or not colchicine resistance was compromised by intracellular delivery of the above antisera. Colchicine treatment resulted in rapid neurite retraction after intracellular delivery of antisera directed against extensively phosphorylated NF-H, MAP1B, or tau into cells that had previously been treated with dbcAMP for 7 d. By contrast, colchicine resistance was not compromised by the intracellular delivery of antisera directed against NF-L, NF-M, or tubulin. These findings support previous studies indicating that MT polymerization mediates certain aspects of axonal neurite outgrowth and suggest that NFs do not directly participate in these events. These findings further suggest that NFs function in stabilization of the axonal cytoskeleton, apparently by interactions among NFs and MTs that are mediated by NF-H and MAPs.     

Structural and functional differences between porcine brain and budding yeast microtubules

The cytoskeleton of eukaryotic cells relies on microtubules to perform many essential functions. We have previously shown that, in spite of the overall conservation in sequence and structure of tubulin subunits across species, there are differences between mammalian and budding yeast microtubules with likely functional consequences for the cell. Here we expand our structural and function comparison of yeast and porcine microtubules to show different distribution of protofilament number in microtubules assembled in vitro from these two species. The different geometry at lateral contacts between protofilaments is likely due to a more polar interface in yeast. We also find that yeast tubulin forms longer and less curved oligomers in solution, suggesting stronger tubulin:tubulin interactions along the protofilament. Finally, we observed species-specific plus-end tracking activity for EB proteins: yeast Bim1 tracked yeast but not mammalian MTs, and human EB1 tracked mammalian but not yeast MTs. These findings further demonstrate that subtle sequence differences in tubulin sequence can have significant structural and functional consequences in microtubule structure and behavior.     

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.