A microtubule-based, dynein-dependent force induces local cell protrusions: Implications for neurite initiation

A key event in neurite initiation is the accumulation of microtubule bundles at the neuron periphery. We hypothesized that such bundled microtubules may generate a force at the plasma membrane that facilitates neurite initiation. To test this idea we observed the behavior of microtubule bundles that were induced by the microtubule-associated protein MAP2c. Endogenous MAP2c contributes to neurite initiation in primary neurons, and exogeneous MAP2c is sufficient to induce neurites in Neuro-2a cells. We performed nocodazol washout experiments in primary neurons, Neuro-2a cells and COS-7 cells to investigate the underlying mechanism. During nocodazol washout, small microtubule bundles formed rapidly in the cytoplasm and immediately began to move toward the cell periphery in a unidirectional manner. In neurons and Neuro-2a cells, neurite-like processes extended within minutes and concurrently accumulated bundles of repolymerized microtubules. Speckle microscopy in COS-7 cells indicated that bundle movement was due to transport, not treadmilling. At the periphery bundles remained under a unidirectional force and induced local cell protrusions that were further enhanced by suppression of Rho kinase activity. Surprisingly, this bundle motility was independent of classical actin- or microtubule-based tracks. It was, however, reversed by function-blocking antibodies against dynein. Suppression of dynein expression in primary neurons by RNA interference severely inhibited the generation of new neurites, but not the elongation of existing neurites formed prior to dynein knockdown. Together, these cell biological data suggest that neuronal microtubule-associated proteins induce microtubule bundles that are pushed outward by dynein and locally override inward contraction to initiate neurite-like cell protrusions. A similar force-generating mechanism might participate in spontaneous initiation of neurites in developing neurons. Electronic Supplementry Materials: Supplementary Materials are available in the online version of this article at     

Neuronal differentiation triggered by blocking cell proliferation.

Treatment of the neuroblastoma cell line SHSY5Y with nerve growth factor (NGF) resulted in limited neurite extension, but proliferation continued. However, SHSY5Y cells treated with NGF and a pulse of the DNA polymerase alpha and delta inhibitor aphidicolin showed dramatic neuronal differentiation. Few differentiated cells were observed immediately following the NGF-aphidicolin treatment; however, continued treatment of the cells with NGF in the ensuing week resulted in extension of long neurites (> 400 microns). Neurite extension was not observed for cells treated with aphidicolin alone. Hence, aphidicolin and NGF act synergistically to induce differentiation of SHSY5Y cells. If maintained in NGF, the differentiated cells were stable for at least 1 month and displayed many neuronal characteristics. They were mitotically inactive, and, in contrast to control or NGF-treated cells, the differentiated cells required NGF for survival. The cells expressed multiple microtubule-associated proteins (MAP), including MAP 1A, MAP 1B, and tau. There was expression of synaptic vesicle antigens synaptophysin and SV2, but not synapsin Ia/b or synapsin IIa/b. Both hydroxyurea and thymidine, which inhibit synthesis of nucleotides, act synergistically with NGF to induce differentiation of SHSY5Y cells. Since aphidicolin, hydroxyurea, and thymidine are chemically unrelated, we conclude that these drugs enhance NGF-induced differentiation by blocking cell proliferation and not through an unrelated side effect. The model suggested by these studies is that differentiation is triggered by two simultaneous signals: NGF and cessation of cell proliferation.     

A Nerve Growth Factor-Dependent Protein Kinase That Phosphorylates Microtubule-Associated Proteins In Vitro: Possible Involvement of Its Activity in the Outgrowth of Neurites from PC12 Cells

We have established a subline of PC12 cells (PC12D) that extend neurites very quickly in response not only to nerve growth factor (NGF) but also to cyclic AMP (cAMP) in the same way as primed PC12 cells (NGF-pretreated cells). When phosphorylation of brain microtubule proteins by extracts of these cells was monitored, two distinct kinase activities were found to be increased [from three- to eightfold in terms of phosphorylation of microtubule-associated protein (MAP) 2] by a brief exposure of cells to NGF or to dibutyryl cAMP(dbcAMP). The effect of the combined stimulation with both NGF and dbcAMP was additive in terms of the phosphorylation of MAP2. The apparent molecular mass of the kinase activated by dbcAMP was 40 kDa, and this kinase appears to be cAMP-dependent protein kinase. The molecular mass of the kinase activated by NGF was 50 kDa. The latter was activated to a measurable extent after 5 min of exposure of cells to NGF; it required Mg²⁺ for activity but not Mn²⁺ or Ca²⁺. This kinase appears to be distinct from previously reported kinases in PC12 cells, and it has been designated as NGF-dependent MAP kinase, although its physiological substrates are not known at present. An inhibitor of protein kinases, K-252a, selectively inhibited the outgrowth of neurites from PC12D cells in response to NGF but not to dbcAMP. When this inhibitor was added to the incubation medium of cells exposed simultaneously to NGF or dbcAMP, the increase in activity of the NGF-dependent MAP kinase was selectively abolished. We isolated several mutant clones of PC12D cells that were deficient in the ability to induce neurites in response to either of the two stimulators. In these variant cells, the activity of the relevant protein kinase was decreased, in parallel with the deficiency in the neurite response to NGF or dbcAMP. These observations suggest that the NGF-dependent MAP kinase may play an important role in the outgrowth of neurites from PC12 cells in response to NGF.     

Microtubule-associated proteins characteristic of embryonic brain are found in the adult mammalian retina

We have used monoclonal antibodies against each of the major mammalian brain microtubule-associated proteins (MAPs), MAP1, MAP2, MAP3, MAP5, and tau, to study the timing of appearance and the cytological distribution of these proteins during the development of the rat retina. Western blots of adult rat retina reveal MAPs that are characteristic of embryonic brain, i.e., MAP5 and the low-molecular-weight forms of MAP2 (MAP2c) and tau (juvenile tau). At the onset of neuronal differentiation within the embryonic retina, MAP5, MAP3, MAP2c, and tau are found in the perikarya or extending axons of ganglion cells. High-molecular-weight MAP2, a dendrite marker, does not appear in the retina until the second day of postnatal development, when ganglion cell dendrites ramify within the inner plexiform layer. MAP1, which is characteristic of adult brain, does not appear in the retina until 1 week after birth, and is limited to ganglion cells and their processes. In the adult retina, MAP5 and MAP2c are concentrated within the inner segments and cell bodies of photosensitive cells, whereas tau is found in horizontal cells and more internal cell layers. Since photosensitive cells are unique among retinal neurons in their constant regeneration of their primary processes, the photoreceptive outer segments, both MAP5 and MAP2c appear not only to be involved in events associated with the embryonic differentiation and growth of neurites, but also in process regeneration in adult neurons that maintain some embryonic characteristics.     

Nerve growth factor-induced neurite outgrowth in PC12 cells involves the coordinate induction of microtubule assembly and assembly-promoting factors

Nerve growth factor (NGF) regulates the microtubule-dependent extension and maintenance of axons by some peripheral neurons. We show here that one effect of NGF is to promote microtubule assembly during neurite outgrowth in PC12 cells. Though NGF causes an increase in total tubulin levels, the formation of neurites and the assembly of microtubules follow a time course completely distinct from that of the tubulin induction. The increases in microtubule mass and neurite extension closely parallel 10- and 20-fold inductions of tau and MAP1, proteins shown previously to promote microtubule assembly in vitro. When NGF is removed from PC12 cells, neurites disappear, microtubule mass decreases, and both microtubule-associated proteins return to undifferentiated levels. These data suggest that the induction of tau and MAP1 in response to NGF promotes microtubule assembly and that these factors are therefore key regulators of neurite outgrowth.     

Regulation of microtubule composition and stability during nerve growth factor-promoted neurite outgrowth

We have used the nerve growth factor (NGF)-responsive line of PC12 pheochromocytoma cells as a model system to study microtubule specializations associated with neurite outgrowth. PC12 cells treated with NGF cease proliferating and extend neurites. Long-term NGF treatment results in a two- to threefold increase in the proportion of total cellular tubulin that is polymerized in PC12 cells. The increase in this parameter first becomes apparent at 2-4 d with NGF and increases steadily thereafter. Several changes in microtubule-associated proteins (MAPs) of PC12 cells also occur after exposure to NGF. In immunoprecipitation assays, we observed the levels of MAP-2 to increase by at least several-fold after treatment with NGF. We also found that the compositions of three MAP classes with apparent Mr of 64K, 67K, and 80K are altered by NGF treatment. These MAPs, recently designated "chartins," are biochemically and immunologically distinct from the similarly-sized tau MAPs (Peng et al., 1985 Brain Res. 361: 200; Magendantz and Solomon, 1985 Proc. Natl. Acad. Sci. 82: 6581). In two-dimensional isoelectric focusing x SDS polyacrylamide gels, each chartin MAP class resolves into a set of proteins of similar apparent Mr but distinct pI. Peptide mapping analyses confirm that the isoelectric variants comprising each chartin MAP class are closely related in primary structure. Several striking differences in the composition of the chartin MAPs of PC12 cells grown with or without NGF were consistently observed. In particular, following longterm NGF treatment, the abundances of the more acidic variants of each chartin MAP class were markedly enhanced relative to the more basic members. This occurs without substantial changes in the abundance of each MAP class as a whole relative to total cell protein. The combined results of in vivo phosphorylation and peptide mapping experiments indicate that the NGF-inducible chartin MAP species are not primary translation products, but are generated posttranslationally, apparently by differential phosphorylation of other chartin MAPs. These observations suggest that NGF treatment of PC12 cells leads to changes in the posttranslational processing of the chartin MAPs. The time course of these changes closely resembles that for the increase in the proportion of cellular tubulin that is polymerized and for neurite outgrowth. One of the important events in the growth and stabilization of neurites appears to be the formation of microtubule bundles that extend from the cell body to the tips of the neurites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Alzheimer's Disease Neurofibrillary Tangles Contain Mitosis-Specific Phosphoepitopes

Abstract: Paired helical filaments (PHFs) are the major components of neurofibrillary lesions present in Alzheimer's disease (AD). PHFs are composed of the microtubule-associated protein (MAP) τ, which is abnormally phosphorylated in AD. Normal fetal τ is also phosphorylated and shares certain phosphoepitopes with PHF-τ. The abnormal phosphorylation of PHF-τ is considered to be involved in the formation of PHFs and subsequent degeneration of AD neurons. We have previously shown that other neuronal MAPs, such as MAP1B, contain mitosis-specific phosphoepitopes. In addition to mitotic cells, these epitopes are also expressed in fetal brain and PC12 cells during differentiation and neurite outgrowth. One hypothesis regarding the etiology of AD involves the reactivation of a fetal-like state and mitotic conditions in selected neurons. To determine if similar mitosis-associated phosphoepitopes appeared in AD, sections of hippocampal tissue were stained for immunoreactivity with antibodies recognizing both τ and mitotic phosphoepitopes. Both the MPM2 mitotic phosphoepitope antibody and the AT8 PHF-τ antibody stained neurofibrillary lesions and colocalized to pyramidal neurons in AD samples. In addition, PHFs isolated from an AD brain reacted with both antibodies. The MPM2 antibody specifically reacted with τ in the isolated PHF fraction but not normal adult τ. In addition, MPM2 failed to react with normal fetal or adult τ obtained from rat brains. The MPM2 antibody also recognized human MAP1B; however, MAP1B was not present in the PHF fraction. Our results indicate that MPM2 recognized a phosphoepitope present on PHF-τ. Because normal fetal or adult rat brain τ did not express the MPM2 epitope, it is likely that this phosphoepitope is specific for the disease state.     

Activation of m1 Muscarinic Acetylcholine Receptor Regulates τ Phosphorylation in Transfected PC12 Cells

Abstract: Hyperphosphorylated τ proteins are the principal fibrous component of the neurofibrillary tangle pathology in Alzheimer's disease. The possibility that τ phosphorylation is controlled by cell surface neurotransmitter receptors was examined in PC12 cells transfected with the gene for the rat m₁ muscarinic acetylcholine receptor. Stimulation of m₁ receptor in these cells with two acetylcholine agonists, carbachol and AF102B, decreased τ phosphorylation, as indicated by specific τ monoclonal antibodies that recognize phosphorylation-dependent epitopes and by alkaline phosphatase treatment. The muscarinic effect was both time and dose dependent. In addition, a synergistic effect on τ phosphorylation was found between treatments with muscarinic agonists and nerve growth factor. These studies provide the first evidence for a link between the cholinergic signal transduction system and the neuronal cytoskeleton that can be mediated by regulated phosphorylation of τ microtubule-associated protein.     

Polarity orientation and assembly process of microtubule bundles in nocodazole-treated, MAP2c-transfected COS cells.

Microtubule bundles reminiscent of those found in neuronal processes are formed in fibroblasts and Sf9 cells that are transfected with the microtubule-associated proteins tau, MAP2, or MAP2c. To analyze the assembly process of these bundles and its relation to the microtubule polarity, we depolymerized the bundles formed in MAP2c-transfected COS cells using nocodazole, and observed the process of assembly of microtubule bundles after removal of the drug in cells microinjected with rhodamine-labeled tubulin. Within minutes of its removal, numerous short microtubule fragments were observed throughout the cytoplasm. These short fragments were randomly oriented and were already bundled. Somewhat longer, but still short bundles, were then found in the peripheral cytoplasm. These bundles became the primordium of the larger bundles, and gradually grew in length and width. The polarity orientation of microtubules in the reformed bundle as determined by "hook" procedure using electron microscope was uniform with the plus end distal to the cell nucleus. The results suggest that some mechanism(s) exists to orient the polarity of microtubules, which are not in direct continuity with the centrosome, during the formation of large bundles. The observed process presents a useful model system for studying the organization of microtubules that are not directly associated with the centrosomes, such as those observed in axons.     

Nerve growth factor and fibroblast growth factor selectively activate a protein kinase that phosphorylates high molecular weight microtubule-associated proteins. Detection, partial purification, and characterization in PC12 cells

A cell-free assay has been developed to detect and characterize a nerve growth factor (NGF)-stimulated protein kinase activity in PC12 cells that phosphorylates high molecular weight microtubule-associated proteins (HMW-MAPs). The activity was partially purified and separated from other endogenous nonregulated HMW-MAP kinase activities by chromatography on heparin-Sepharose and Mono-Q resin. Characterization of the NGF-activated kinase (designated HMK) revealed the following features. 1) Both MAP1 and MAP2 are phosphorylated with approximately equal efficiencies. 2) Activation reaches a plateau within 3 min of NGF treatment and persists for approximately 60 min; subsequently, a substantial decline occurs by 5 h. 3) Maximal activation reaches 15-20-fold; activation is nearly as high with fibroblast growth factor, an agent that mimics NGF in promoting PC12 cell neuronal differentiation. 4) Epidermal growth factor and depolarizing levels of K+ stimulate HMK activity by only 2-4-fold; additional agents without PC12 cell differentiation activity (insulin, phorbol ester, and a permeant cAMP analogue) do not stimulate HMK activity. 5) The divalent cation requirement shows a preference for Mn2+ over Mg2+. 6) There is inhibition by 10 mM 2-aminopurine but not by 6-thioguanine, heparin, or NaF. 7) HMW-MAPs and myelin basic protein are effective substrates while histones IIIs and H1, dephospho-beta-casein, and S6 protein are not phosphorylated by HMK. These and other features appear to distinguish HMK from a variety of other well-characterized protein kinases as well as from other previously described NGF-activated kinases. The properties of HMK indicate that it could play a role in the signaling pathway for growth-factor-promoted neuronal differentiation.     

Nerve growth factor regulates both the phosphorylation and steady-state levels of microtubule-associated protein 1.2 (MAP1.2)

This study characterizes effects of nerve growth factor (NGF) on the steady-state level and phosphorylation of a high molecular mass microtubule-associated protein in PC12 rat pheochromocytoma cells. Past work showed that NGF significantly raises the relative levels of this phosphoprotein, designated MAP1.2, with a time course similar to that of neurite outgrowth. To study this in greater detail, MAP1.2 in PC12 cell lysates was resolved by SDS-PAGE in gels containing 3.25% acrylamide/4 M urea and identified by comigration with material immunoprecipitated from the lysates by MAP1 antibodies. Quantification by metabolic radiolabeling with [35S]methionine or by silver staining revealed a 3.0-3.5-fold increase in MAP1.2 levels relative to total cell protein after NGF treatment for 2 wk or longer. A partial increase was detectable after 3 d, but not after 2 h of NGF exposure. As measured by incorporation of [32P]phosphate, NGF had a dual effect on MAP1.2. Within 15 min to 2 h, NGF enhanced the incorporation of phosphate into MAP1.2 by two- to threefold relative to total cell phosphoproteins. This value slowly increased thereafter so that by 2 wk or more of NGF exposure, the average enhancement of phosphate incorporation per MAP1.2 molecule was over fourfold. The rapid action of NGF on MAP1.2 could not be mimicked by either epidermal growth factor, a permeant cAMP derivative, phorbol ester, or elevated K+, each of which alters phosphorylation of other PC12 cell proteins. SDS-PAGE revealed multiple forms of MAP1.2 which, based on the effects of alkaline phosphatase on their electrophoretic mobilities, differ, at least in part, in extent of phosphorylation. Before NGF treatment, most PC12 cell MAP1.2 is in more rapidly migrating, relatively poorly phosphorylated forms. After long-term NGF exposure, most is in more slowly migrating, more highly phosphorylated forms. The effects of NGF on the rapid phosphorylation of MAP1.2 and on the long-term large increase in highly phosphorylated MAP1.2 forms could play major functional roles in NGF-mediated neuronal differentiation. Such roles may include effects on microtubule assembly, stability, and cross-linking and, possibly for the rapid effects, nuclear signaling.     

Chromatographic Resolution and Characterization of a Nerve Growth Factor-Dependent Kinase That Phosphorylates Microtubule-Associated Proteins 1 and 2 in PC12 Cells

When the supernatant fractions from extracts of control and nerve growth factor (NGF)- or dibutyryl cyclic AMP-treated PC12D cells were applied to DEAE-Sepharose columns and proteins were eluted with a gradient of NaCl, three separate peaks of kinase activity that phosphorylated microtubule-associated proteins (MAPs) were recovered. Enhancement of the kinase activity in peak 1 was noted in the case of dibutyryl cyclic AMP-treated cells. In contrast, the kinase activity in the third peak was markedly elevated, in terms of the ability to phosphorylate MAP1 and MAP2, in the case of the extract from NGF-treated cells. This activity was designated previously as NGF-dependent MAP kinase. The apparent molecular mass of the active kinase was 45-50 kDa. The apparent Km value was 35 microM for ATP with either MAP1 or MAP2 as substrate. When the kinase activity in the fractions from the DEAE-Sepharose column was assayed in the presence of Mn2+ instead of Mg2+, another NGF-stimulated kinase activity was detected in the fractions eluted by a lower concentration of NaCl than that which eluted the Mg(2+)-activated kinase. Other growth factors, namely, epidermal growth factor and basic fibroblast growth factor, also stimulated the activity of NGF-dependent MAP kinase. Possible involvement of the kinase in the outgrowth of neurites has been suggested. The NGF-induced activation of NGF-dependent MAP kinase was blocked by the presence of K-252a. In contrast, the activation of NGF-dependent MAP kinase by basic fibroblast growth factor and by epidermal growth factor was not blocked, but actually stimulated by K-252a, a result that correlates well with the analogous actions of the drug on the outgrowth of neurites that is induced by these growth factors. The latter observation strengthens the possibility of a close relationship between the outgrowth of neurites and the activation of NGF-dependent MAP kinase.     

Bundling of microtubules in transfected cells does not involve an autonomous dimerization site on the MAP2 molecule.

We have searched for putative dimerization sites in microtubule-associated protein 2 (MAP2) that may be involved in the bundling of microtubules. An overlapping series of fragments of the embryonic form MAP2c were created and immunologically "tagged" with an 11 amino acid sequence from human c-myc. Nonneuronal cells were transfected simultaneously with one of these myc-tagged fragments and with full-length native MAP2c. Immunolabeling with site-specific antibodies allowed the two transgene products to be located independently within the cytoplasm of a single double-transfected cell. All transfected cells contained bundled microtubules to which the full-length native MAP2 was bound. The distribution of the tagged MAP2 fragment relative to these MAP2-induced bundles was determined by the anti-myc staining. None of the fragments tested, representing all of the MAP2c sequence in overlapping pieces, were associated with MAP2-induced microtubule bundles. These results suggest that MAP2-induced bundle formation in cells does not involve an autonomous dimerization site within the MAP2 sequence.     

Ca2+, calmodulin-dependent regulation of microtubule formation via phosphorylation of microtubule-associated protein 2, tau factor, and tubulin, and comparison with the cyclic AMP-dependent phosphorylation

Abstract: Isolated microtubule-associated protein 2 (MAP2), τ factor, and tubulin were phosphorylated by a purified Ca²⁺, calmodulin-dependent protein kinase (640K enzyme) from rat brain. The phosphorylation of MAP2 and τ factor separately induced the inhibition of microtubule assembly, in accordance with the degree. Tubulin phosphorylation by the 640K enzyme induced the inhibition of microtubule assembly, whereas the effect of tubulin phosphorylation by the catalytic subunit was undetectable. The effects of tubulin and MAPs phosphorylation on microtubule assembly were greater than that of either tubulin or MAPs phosphorylation. Because MAP2, τ factor, and tubulin were also phosphorylated by the catalytic subunit of type-II cyclic AMP-dependent protein kinase from rat brain, the kinetic properties and phosphorylation sites were compared. The amount of phosphate incorporated into each microtubule protein was three to five times higher by the 640K enzyme than by the catalytic subunit. The K _m values of the 640K enzyme for microtubule proteins were four to 24 times lower than those of the catalytic subunit. The peptide mapping analysis showed that the 640K enzyme and the catalytic subunit incorporated phosphate into different sites on MAP2, τ factor, and tubulin. Investigation of phosphoamino acids revealed that only the seryl residue was phosphorylated by the catalytic subunit, whereas both seryl and threonyl residues were phosphorylated by the 640K enzyme. These data suggest that the Ca²⁺, calmodulin system via phosphorylation of MAP2, τ factor, and tubulin by the 640K enzyme is more effective than the cyclic AMP system on the regulation of microtubule assembly.     

Exercise ameliorates high-fat diet-induced impairment of differentiation of adipose-derived stem cells into neuron-like cells in rats

Adipose-derived stem cells (ADSCs) can differentiate into neurons under particular conditions. It remains largely unknown whether this differentiation potential is affected by physical conditions such as obesity, which modulates the functions of adipose tissue. In this study, we determined the impact of either a 9-week high-fat diet (60% fat; HFD) or 9-week exercise training on the differentiation potential of ADSCs into neuron-like cells in male Wistar rats. Rats were randomly assigned to a normal diet-fed (ND-SED) group, HFD-fed (HFD-SED) group, or exercise-trained HFD-fed group (HFD-EX). After a 9-week intervention, ADSCs from all groups differentiated into neuron-like cells. Expression of neuronal marker proteins (nestin, βIII-tubulin, and microtubule-associated protein 2 [MAP2]) and the average length of cell neurites were lower in cells from HFD-SED rats than in other groups. Instead, protein expression of COX IV and Cyt-c, the Bax/Bcl-2 and LC3-II/I ratio, and the malondialdehyde level in culture medium were higher in cells from HFD-SED rats. No significant difference between ND-SED and HFD-EX rats was observed, except for the average length of cell neurites in MAP2. Thus, HFD impaired the differentiation potential of ADSCs into neuron-like cells, which was accompanied by increases in apoptotic activity and oxidative stress. Importantly, exercise training ameliorated the HFD-induced impairment of neurogenesis in ADSCs. The adipose tissue microenvironment could influence the differentiation potential of ADSCs, a source of autologous stem cell therapy.     

Reorganisation of the microtubular cytoskeleton by embryonic microtubule-associated protein 2 (MAP2c).

Microtubule-associated protein 2c (MAP2c) is one of a set of embryonic MAP forms that are expressed during neuronal differentiation in the developing nervous system. We have investigated its mode of action by expressing recombinant protein in non-neuronal cell lines using cell cDNA transfection techniques. At every level of expression, all the MAP2c was bound to cellular microtubules. At low MAP2c levels, the microtubules retained their normal arrangement, radiating from the centrosomal microtubule-organising centre (MTOC) but at higher levels an increasing proportion of microtubules occurred independently of the MTOC. In most cells, radially oriented microtubules still attached to the MTOC co-existed with detached microtubules, suggesting that the primary effect of MAP2 is to increase the probability that tubulin polymerisation will occur independently of the MTOC. The MTOC-independent microtubules formed bundles whose distribution depended on their length in relation to the diameter of the transfected cell. Short bundles were attached to the cell cortex at one end and followed a straight course through the cytoplasm, whereas longer bundles followed a curved path around the periphery of the cell. By comparing these patterns to those produced by two chemical agents that stabilise microtubules, taxol and dimethyl sulphoxide, we conclude that effects of MAP2c arise from two sources. It stabilises microtubules without providing assembly initiation sites and as a result produces relatively few, long microtubule bundles. These bend only when they encounter the restraining influence of the cortical cytoskeleton of the cell, indicating that MAP2c also imparts stiffness to them. By conferring these properties of stability and stiffness to neuronal microtubules MAP2c contributes to supporting the structure of developing neurites.