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.     

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.     

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

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

The microtubule binding domain of microtubule-associated protein MAP1B contains a repeated sequence motif unrelated to that of MAP2 and tau

We report the complete sequence of the microtubule-associated protein MAP1B, deduced from a series of overlapping genomic and cDNA clones. The encoded protein has a predicted molecular mass of 255,534 D and contains two unusual sequences. The first is a highly basic region that includes multiple copies of a short motif of the form KKEE or KKEVI that are repeated, but not at exact intervals. The second is a set of 12 imperfect repeats, each of 15 amino acids and each spaced by two amino acids. Subcloned fragments spanning these two distinctive regions were expressed as labeled polypeptides by translation in a cell-free system in vitro. These polypeptides were tested for their ability to copurify with unlabeled brain microtubules through successive cycles of polymerization and depolymerization. The peptide corresponding to the region containing the KKEE and KKEVI motifs cycled with brain microtubules, whereas the peptide corresponding to the set of 12 imperfect repeats did not. To define the microtubule binding domain in vivo, full-length and deletion constructs encoding MAP1B were assembled and introduced into cultured cells by transfection. The expression of transfected polypeptides was monitored by indirect immunofluorescence using anti-MAP1B-specific antisera. These experiments showed that the basic region containing the KKEE and KKEVI motifs is responsible for the interaction between MAP1B and microtubules in vivo. This region bears no sequence relationship to the microtubule binding domains of kinesin, MAP2, or tau.     

Microtubule-associated proteins MAP65-1 and MAP65-2 positively regulate axial cell growth in etiolated Arabidopsis hypocotyls

The Arabidopsis thaliana MAP65-1 and MAP65-2 genes are members of the larger eukaryotic MAP65/ASE1/PRC gene family of microtubule-associated proteins. We created fluorescent protein fusions driven by native promoters that colocalized MAP65-1 and MAP65-2 to a subset of interphase microtubule bundles in all epidermal hypocotyl cells. MAP65-1 and MAP65-2 labeling was highly dynamic within microtubule bundles, showing episodes of linear extension and retraction coincident with microtubule growth and shortening. Dynamic colocalization of MAP65-1/2 with polymerizing microtubules provides in vivo evidence that plant cortical microtubules bundle through a microtubule-microtubule templating mechanism. Analysis of etiolated hypocotyl length in map65-1 and map65-2 mutants revealed a critical role for MAP65-2 in modulating axial cell growth. Double map65-1 map65-2 mutants showed significant growth retardation with no obvious cell swelling, twisting, or morphological defects. Surprisingly, interphase microtubules formed coaligned arrays transverse to the plant growth axis in dark-grown and GA(4)-treated light-grown map65-1 map65-2 mutant plants. We conclude that MAP65-1 and MAP65-2 play a critical role in the microtubule-dependent mechanism for specifying axial cell growth in the expanding hypocotyl, independent of any mechanical role in microtubule array organization.     

Stable expression of heterologous microtubule-associated proteins (MAPs) in Chinese hamster ovary cells: evidence for differing roles of MAPs in microtubule organization

To study the effects of microtubule-associated proteins (MAPs) on in vivo microtubule assembly, cDNAs containing the complete coding sequences of a Drosophila 205-kD heat stable MAP, human MAP 4, and human tau were stably transfected into CHO cells. Constitutive expression of the transfected genes was low in most cases and had no obvious effects on the viability of the transfected cell lines. High levels of expression, as judged by Western blots, immunofluorescence, and Northern blots, could be induced by treating cells with sodium butyrate. High levels of MAPs were maintained for at least 24-48 h after removal of the sodium butyrate. Immunofluorescence analysis indicated that all three MAPs bound to cellular microtubules, but only the transfected tau caused a rearrangement of microtubules into bundles. Despite high levels of expression of these exogenous MAPs and the bundling of microtubules in cells expressing tau, transfected cells had normal levels of assembled and unassembled tubulin. With the exception of the tau-induced bundles, microtubules in transfected cells showed the same sensitivity as control cells to microtubule depolymerization by Colcemid. Further, all three MAPs were ineffective in reversing the taxol-dependent phenotype of a CHO mutant cell line. The absence of a quantitative effect of any of these heterologous proteins on the assembly of tubulin suggests that these MAPs may have different roles in vivo from those inferred previously from in vitro experiments.     

τ and Microtubule-Associated Protein 2c Transfection and Neurite Outgrowth in ND 7/23 Cells

Abstract: Neuronal hybrid ND 7/23 cells, which display sensorylike properties, develop neurites when cultured in the presence of either dibutyryl cyclic AMP plus nerve growth factor (DBcAMP + NGF) or retinoic acid or a phorbol ester derivative, although they express only trace amounts of the microtubule-associated τ proteins and low levels of microtubule-associated protein 2c (MAP2c). Nondifferentiated ND cells transfected with τ cDNAs did not develop neurites, whereas very short cell processes were formed in MAP2c-transfected cells. τ and MAP2 antibodies labeled microtubule bundles displayed in a ring array underneath the surface of the transfected cells and short microtubules starting from the cell center. After differentiation in the presence of DBcAMP + NGF, the same bundle organization was observed in the transfected cells. In addition, τ and MAP2 antibodies stained a short section of the formed neurites. These data demonstrate that the expression of τ protein is not sufficient to induce neurite extension and that other proteins induced by morphogens are more important to initiate morphological differentiation of this cell line.     

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     

The functional cooperation of MAP1A heavy chain and light chain 2 in the binding of microtubules

Microtubule-associated protein 1A (MAP1A) is a high-molecular-weight protein that is comprised of a heavy chain and a light chain (LC2) and is widely distributed along the microtubules in both mature neurons and glial cells. To illustrate the interaction among the MAP1A heavy chain, light chain, and microtubule, we prepared DNA constructs with Myc-, EGFP-, or DsRed-tags for full-length MAP1A DNA expressing whole MAP1A protein, two domains of MAP1A heavy chain, and light chain. Distribution patterns of various MAP1A domains as well as their interactions with microtubules were monitored in a non-neuronal COS7 and a neuronal Neuro2A cells. Our data revealed that a complete MAP1A protein, which contains both heavy chain and LC2, could be colocalized with microtubule networks not only in Neuro2A cells but also in transfected COS7 cells. Filamentous structures failed to be visualized along microtubules in COS7 cells transfected with MAP1A heavy chain or LC2 alone. Whereas, after introducing MAP1A heavy chain with LC2 into COS7 cells, both heavy chain and LC2 could be colocalized with microtubules. From our functional analysis, both MAP1A and its LC2 could protect microtubules against the challenge of nacodazol. Data collected from yeast two-hybrid assays of various MAP1A domains confirmed that the interaction of LC2 and NH2-terminal of MAP1A heavy chain is important for microtubule binding. From our analysis of MAP1A functional domains, we suggest that interactions between MAP1A heavy chain and LC2 are critical for the binding of microtubules.     

Membrane heparan sulfate proteoglycan-supported FGF2-FGFR1 signaling: evidence in support of the "cooperative end structures" model

Fibroblast growth factor 2 (FGF2)-initiated FGF receptor (FGFR)-signaling requires the assistance of heparin/heparan sulfate. Here, we evaluated the effects of different heparan sulfate proteoglycan (HSPG)-expressing cell lines and HSPGs derived from these cells on FGF2-induced FGFR1-phosphorylation in heparan sulfate-negative BaF3 cells. HSPGs supplied in membrane-associated form, by presenting cells, were all effective promotors of FGF2-initiated FGFR1 phosphorylation, independently of their nature (syndecan/glypican) or cellular origin (human lung fibroblasts, transfected Namalwa cells, or transfected K562 cells). A treatment with heparitinase initially stimulated, but finally completely inhibited, the activity of these presenting cells. In comparison, equivalent amounts of soluble HSPGs, obtained by trypsinization of these cells or by immunopurification from cell extracts, did not promote FGF2-induced FGFR1-phosphorylation, yet removal of the less anionic species or a further treatment with heparitinase converted these soluble fractions into potent activators of FGF2/FGFR1 signaling. Extrapolating from current structural models, we suggest that FGFR dimerization and autophosphorylation is supported by cooperative "heparin-like end structures," and that cell surface association and concentration compensate for the relative scarcity of such end structures in native HSPGs. In this model, "proteolytic" shedding of heparan sulfate would act as a diluting, down-regulatory mechanism, while "heparanolytic" shedding might act as an up-regulatory mechanism, by increasing the concentration of these end structures.     

GSK3beta-mediated phosphorylation of the microtubule-associated protein 2C (MAP2C) prevents microtubule bundling

A major determinant of neuronal morphology is the cytoskeleton. And one of the main regulatory mechanisms of cytoskeletal proteins is the modification of their phosphorylation state via changes in the relative activities of protein kinases and phosphatases in neurons. In particular, the microtubule-associated protein 2 (MAP2) family of proteins are abundant cytoskeletal components predominantly expressed in neurons and have been found to be substrates for most of protein kinases and phosphatases present in neurons, including glycogen-synthase kinase 3 (GSK3). It has been suggested that changes in GSK3-mediated MAP phosphorylation may modify MT stability and could control neuronal development. We have previously shown that MAP2 is phosphorylated in vitro and in situ by GSK3 at Thr1620 and Thr1623, located in the proline-rich region of MAP2 and recognized by antibody 305. However, the function of the phosphorylation of this site of MAP2 is still unknown. In this study, non-neuronal COS-1 cells have been co-transfected with cDNAs encoding MAP2C and either wild type or mutated GSK3beta to analyze possible effects on microtubule stability and on the association of MAP2 with microtubules. We have found that GSK3beta phosphorylates MAP2C in co-transfected cells. Moreover, this phosphorylation is inhibited by the specific GSK3 inhibitor lithium chloride. Additionally, the formation of microtubule bundles, which is observed after transfection with MAP2C, was decreased when MAP2C was co-transfected with GSK3beta wild type. Microtubule bundles were not observed in cells expressing MAP2C phosphorylated at the site recognized by antibody 305. The absence of microtubule bundles was reverted after treatment of MAP2C/GSK3beta wild type transfected cells with lithium chloride. Highly phosphorylated MAP2C species, which were phosphorylated at the site recognized by antibody 305, appeared in cells co-transfected with MAP2C and GSK3beta wild type. Interestingly, these MAP2C species were enriched in cytoskeleton-unbound protein preparations. These data suggests that GSK3-mediated phosphorylation of MAP2 may modify its binding to microtubules and regulate microtubule stability.     

Juvenile and mature MAP2 isoforms induce distinct patterns of process outgrowth.

Microtubule-associated protein-2 (MAP2) is the most abundant MAP in neurons, where its distribution is restricted to the somatodendritic compartment. This molecule undergoes developmentally regulated alternative splicing, resulting in at least two isoforms, a juvenile isoform (termed MAP2c) and a mature isoform (MAP2), with greatly different molecular masses. Spodoptera frugiperda (Sf9) cell expression of the juvenile versus the mature MAP2 isoform generates two distinct patterns of process outgrowth. The smaller juvenile isoform induces multiple short thin processes. Mature MAP2 tends to induce single processes that are considerably thicker than those processes induced by juvenile MAP2. We found important differences in the variability of spacing between microtubules and the number of microtubules along the processes induced by MAP2c and mature MAP2. MAP2c showed variability with most microtubules spaced as closely as with tau, but some spaced as far apart as with mature MAP2. Over their length, the mature MAP2 processes demonstrate proximo-distal taper, which corresponds to a narrowing of the spacing between microtubules from 90 nm to 40 nm. Moreover, there is a decreased number of microtubules in mature MAP2-induced processes whereas in tau and MAP2-induced processes, the number of microtubules is constant along the length. Based on these observations, we conclude that MAP2 isoforms can serve as architectural elements by establishing specific morphological features of processes and specific arrangements of their microtubules.     

Identification of a neural cell specific variant of microtubule-associated protein 4

The microtubule-binding domain of MAP4, a ubiquitous microtubule-associated protein, contains a region rich in proline and basic residues (proline-rich region). We searched the bovine adrenal gland for MAP4 isoforms, and identified a novel variant lacking 72 consecutive amino acid residues within the proline-rich region, as compared with the full-length MAP4. The amino acid sequence of the missing region was highly conserved (about 85% identity/similarity) among the corresponding regions of bovine, human, mouse, and rat MAP4, which suggested the functional significance of this region. A comparison of the genomic sequence with the cDNA sequence revealed that the missing region is encoded by a single exon. A MAP4 variant cDNA homologous to the bovine form was also detected in rat cells, suggesting that the new variant can be generated by alternative splicing, not only in bovine but also in other mammalian species. The mRNA expression of the novel isoform was restricted to the brain and the adrenal medulla, suggesting that this isoform is specific to a certain cell type. Using a bacterially expressed fragment corresponding to the microtubule-binding domain of the novel isoform, we analyzed its in vitro characteristics. The fragment induced microtubule assembly and bound to preformed microtubules, but the activities were slightly lower than those of the conventional MAP4 fragment, which carries the full-length proline-rich region. The microtubules assembled in the presence of the fragment failed to be bundled. Instead, a constant spacing between neighboring microtubules was observed.     

Analysis of MAP 4 function in living cells using green fluorescent protein (GFP) chimeras

MAP 4 is a ubiquitous microtubule-associated protein thought to play a role in the polymerization and stability of microtubules in interphase and mitotic cells. We have analyzed the behavior of protein domains of MAP 4 in vivo using chimeras constructed from these polypeptides and the green fluorescent protein (GFP). GFP-MAP 4 localizes to microtubules; this is confirmed by colocalization of GFP-MAP 4 with microtubules that have incorporated microinjected rhodamine-tubulin, and by loss of localized fluorescence after treatment of cells with anti-microtubule agents. Different subdomains of MAP 4 have distinct effects on microtubule organization and dynamics. The entire basic domain of MAP 4 reorganizes microtubules into bundles and stabilizes these arrays against depolymerization with nocodazole. Within the basic domain, the PGGG repeats, which are conserved with MAP 2 and tau, have a weak affinity for microtubules and are dispensable for microtubule binding, whereas the MAP 4-unique PSP region can function independently in binding. The projection domain shows no microtubule localization, but does modulate the association of various binding subdomains with microtubules. The acidic carboxy terminus of MAP 4 strongly affects the microtubule binding characteristics of the other domains, despite constituting less than 6% of the protein. These data show that MAP 4 association with microtubules is modulated by sequences both within and outside the basic domain. Further, our work demonstrates that GFP chimeras will allow an in vivo analysis of the effects of MAPs and their variants on microtubule dynamics in real time.     

Characterization and Intracellular Distribution of Microtubule-Associated Protein 2 in Differentiating Human Neuroblastoma Cells

The use of a panel of monoclonal antibodies (mAbs) directed against different determinants of microtubule-associated protein 2 (MAP2) enabled us to identify two distinct high-molecular-mass MAP2 species (270 and 250 kDa) and a substantial amount of MAP2c (70 kDa) in human neuroblastoma cells. The 250-kDa MAP2 species appears to be confined to the human neuroblastoma cells and was not observed in microtubules (MTs) from bovine and rat brain, mouse neuroblastoma, or MTs from human cerebellum. A new overlay method was developed, which demonstrates binding of tubulin to human neuroblastoma high-molecular-mass MAP2 by exposing nitrocellulose-bound MT proteins under polymerization conditions to tubulin. Bound tubulin was detected with a mAb directed against beta-tubulin. The binding of tubulin to MAP2 could be abolished by a peptide homologous to positions 426-445 of the C-terminal region of beta-tubulin. Immunological cross-reactivity with several mAbs directed against bovine brain MAP2, taxol-promoted coassembly into MTs, and immunocytochemical visualization within cells were further criteria utilized to characterize these proteins as true MAPs. Indirect immunofluorescence with anti-MAP2 and anti-beta-tubulin mAbs demonstrated that there is a change in the spatial organization of MTs during induced cell differentiation, as indicated by the appearance of MT bundles and the redistribution of MAP2.     

Association of ebola virus matrix protein VP40 with microtubules

Viruses exploit a variety of cellular components to complete their life cycles, and it has become increasingly clear that use of host cell microtubules is a vital part of the infection process for many viruses. A variety of viral proteins have been identified that interact with microtubules, either directly or via a microtubule-associated motor protein. Here, we report that Ebola virus associates with microtubules via the matrix protein VP40. When transfected into mammalian cells, a fraction of VP40 colocalized with microtubule bundles and VP40 coimmunoprecipitated with tubulin. The degree of colocalization and microtubule bundling in cells was markedly intensified by truncation of the C terminus to a length of 317 amino acids. Further truncation to 308 or fewer amino acids abolished the association with microtubules. Both the full-length and the 317-amino-acid truncation mutant stabilized microtubules against depolymerization with nocodazole. Direct physical interaction between purified VP40 and tubulin proteins was demonstrated in vitro. A region of moderate homology to the tubulin binding motif of the microtubule-associated protein MAP2 was identified in VP40. Deleting this region resulted in loss of microtubule stabilization against drug-induced depolymerization. The presence of VP40-associated microtubules in cells continuously treated with nocodazole suggested that VP40 promotes tubulin polymerization. Using an in vitro polymerization assay, we demonstrated that VP40 directly enhances tubulin polymerization without any cellular mediators. These results suggest that microtubules may play an important role in the Ebola virus life cycle and potentially provide a novel target for therapeutic intervention against this highly pathogenic virus.     

Interaction of microtubule-associated protein-2 and p63: a new link between microtubules and rough endoplasmic reticulum membranes in neurons

Neurons are polarized cells presenting two distinct compartments, dendrites and an axon. Dendrites can be distinguished from the axon by the presence of rough endoplasmic reticulum (RER). The mechanism by which the structure and distribution of the RER is maintained in these cells is poorly understood. In the present study, we investigated the role of the dendritic microtubule-associated protein-2 (MAP2) in the RER membrane positioning by comparing their distribution in brain subcellular fractions and in primary hippocampal cells and by examining the MAP2-microtubule interaction with RER membranes in vitro. Subcellular fractionation of rat brain revealed a high MAP2 content in a subfraction enriched with the endoplasmic reticulum markers ribophorin and p63. Electron microscope morphometry confirmed the enrichment of this subfraction with RER membranes. In cultured hippocampal neurons, MAP2 and p63 were found to concomitantly compartmentalize to the dendritic processes during neuronal differentiation. Protein blot overlays using purified MAP2c protein revealed its interaction with p63, and immunoprecipitation experiments performed in HeLa cells showed that this interaction involves the projection domain of MAP2. In an in vitro reconstitution assay, MAP2-containing microtubules were observed to bind to RER membranes in contrast to microtubules containing tau, the axonal MAP. This binding of MAP2c microtubules was reduced when an anti-p63 antibody was added to the assay. The present results suggest that MAP2 is involved in the association of RER membranes with microtubules and thereby could participate in the differential distribution of RER membranes within a neuron.     

MAP2c, but not tau, binds and bundles F-actin via its microtubule binding domain

BACKGROUND: MAP2 and tau are abundant microtubule-associated proteins (MAPs) in neurons. The development of neuronal dendrites and axons requires a dynamic interaction between microtubules and actin filaments. MAPs represent good candidates to mediate such interactions. Although MAP2c and tau have similar, well-characterized microtubule binding activities, their actin interaction is poorly understood. RESULTS: Here, we show by using a cosedimentation assay that MAP2c binds F-actin. Upon actin binding, MAP2c organizes F-actin into closely packed actin bundles. Moreover, we show by using a deletion approach that MAP2c's microtubule binding domain (MTBD) is both necessary and sufficient for both F-actin binding and bundling activities. Surprisingly, even though the MAP2 and tau MTBDs share high sequence homology and possess similar microtubule binding activities, tau is unable to bind or bundle F-actin. Furthermore, experiments with chimeric proteins demonstrate that the actin binding activity fully correlates with the ability to promote neurite initiation in neuroblastoma cells. CONCLUSIONS: These results provide the first demonstration that the MAP2c and tau MTBD domains exhibit distinct properties, diverging in actin binding and neurite initiation activities. These results implicate a novel actin function for MAP2c in neuronal morphogenesis and furthermore suggest that actin interactions could contribute to functional differences between MAP2 and tau in neurons.     

The control of microtubule stability in vitro and in transfected cells by MAP1B and SCG10

In neurons, the regulation of microtubules plays an important role for neurite outgrowth, axonal elongation, and growth cone steering. SCG10 family proteins are the only known neuronal proteins that have a strong destabilizing effect, are highly enriched in growth cones and are thought to play an important role during axonal elongation. MAP1B, a microtubule-stabilizing protein, is found in growth cones as well, therefore it was important to test their effect on microtubules in the presence of both proteins. We used recombinant proteins in microtubule assembly assays and in transfected COS-7 cells to analyze their combined effects in vitro and in living cells, respectively. Individually, both proteins showed their expected activities in microtubule stabilization and destruction respectively. In MAP1B/SCG10 double-transfected cells, MAP1B could not protect microtubules from SCG10-induced disassembly in most cells, in particular not in cells that contained high levels of SCG10. This suggests that SCG10 is more potent to destabilize microtubules than MAP1B to rescue them. In microtubule assembly assays, MAP1B promoted microtubule formation at a ratio of 1 MAP1B per 70 tubulin dimers while a ratio of 1 SCG10 per two tubulin dimers was needed to destroy microtubules. In addition to its known binding to tubulin dimers, SCG10 binds also to purified microtubules in growth cones of dorsal root ganglion neurons in culture. In conclusion, neuronal microtubules are regulated by antagonistic effects of MAP1B and SCG10 and a fine tuning of the balance of these proteins may be critical for the regulation of microtubule dynamics in growth cones.     

Microtubule-associated Protein 2c Reorganizes Both Microtubules and Microfilaments into Distinct Cytological Structures in an Actin-binding Protein-280–deficient Melanoma Cell Line

The emergence of processes from cells often involves interactions between microtubules and microfilaments. Interactions between these two cytoskeletal systems are particularly apparent in neuronal growth cones. The juvenile isoform of the neuronal microtubule-associated protein 2 (MAP2c) is present in growth cones, where we hypothesize it mediates interactions between microfilaments and microtubules. To approach this problem in vivo, we used the human melanoma cell, M2, which lacks actin-binding protein-280 (ABP-280) and forms membrane blebs, which are not seen in wild-type or ABP-transfected cells. The microinjection of tau or mature MAP2 rescued the blebbing phenotype; MAP2c not only caused cessation of blebbing but also induced the formation of two distinct cellular structures. These were actin-rich lamellae, which often included membrane ruffles, and microtubule-bearing processes. The lamellae collapsed after treatment with cytochalasin D, and the processes retracted after treatment with colchicine. MAP2c was immunocytochemically visualized in zones of the cell that were devoid of tubulin, such as regions within the lamellae and in association with membrane ruffles. In vitro rheometry confirmed that MAP2c is an efficient actin gelation protein capable of organizing actin filaments into an isotropic array at very low concentrations; tau and mature MAP2 do not share this rheologic property. These results suggest that MAP2c engages in functionally specific interactions not only with microtubules but also with microfilaments.