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.     

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     

Associations of elements of the Golgi apparatus with microtubules

The intracellular spatial relationships between elements of the Golgi apparatus (GA) and microtubules in interphase cells have been explored by double immunofluorescence microscopy. By using cultured cells infected with the temperature-sensitive Orsay-45 mutant of vesicular stomatitis virus and a temperature shift-down protocol, we visualized functional elements of the GA by immunolabeling of the G protein of the virus that was arrested in the GA during its intracellular passage to the plasma membrane 13 min after the temperature shift-down. Complete disassembly of the cytoplasmic microtubules by nocodazole at the nonpermissive temperature before the temperature shift led to the dispersal of the GA elements, from their normal compact perinuclear configuration close to the microtubule-organizing center (MTOC) into the cell periphery. Washout of the nocodazole that led to the reassembly of the microtubules from the MTOC also led to the recompaction of the GA elements to their normal configuration. During this recompaction process, GA elements were seen in close lateral apposition to microtubules. In cells treated with nocodazole followed by taxol, an MTOC developed, but most of the microtubules were free of the MTOC and were assembled into bundles in the cell periphery. Under these circumstances, the GA elements that had been dispersed into the cell periphery by the nocodazole treatment remained dispersed despite the presence of an MTOC. In cells treated directly with taxol, free microtubules were seen in the cytoplasm in widely different, bundled configurations from one cell to another, but, in each case, elements of the GA appeared to be associated with one of the two end regions of the microtubule bundles, and to be uncorrelated with the locations of the vimentin intermediate filaments in these cells. These results are interpreted to suggest two types of associations of elements of the GA with microtubules: one lateral, and the other (more stable) end-on. The end-on association is suggested to involve the minus-end regions of microtubules, and it is proposed that this accounts for the GA-MTOC association in normal cells.     

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.     

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.     

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.     

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.     

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.     

MAP2 and tau bind longitudinally along the outer ridges of microtubule protofilaments

MAP2 and tau exhibit microtubule-stabilizing activities that are implicated in the development and maintenance of neuronal axons and dendrites. The proteins share a homologous COOH-terminal domain, composed of three or four microtubule binding repeats separated by inter-repeats (IRs). To investigate how MAP2 and tau stabilize microtubules, we calculated 3D maps of microtubules fully decorated with MAP2c or tau using cryo-EM and helical image analysis. Comparing these maps with an undecorated microtubule map revealed additional densities along protofilament ridges on the microtubule exterior, indicating that MAP2c and tau form an ordered structure when they bind microtubules. Localization of undecagold attached to the second IR of MAP2c showed that IRs also lie along the ridges, not between protofilaments. The densities attributable to the microtubule-associated proteins lie in close proximity to helices 11 and 12 and the COOH terminus of tubulin. Our data further suggest that the evolutionarily maintained differences observed in the repeat domain may be important for the specific targeting of different repeats to either alpha or beta tubulin. These results provide strong evidence suggesting that MAP2c and tau stabilize microtubules by binding along individual protofilaments, possibly by bridging the tubulin interfaces.     

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.     

Developmental regulation of two microtubule-associated proteins (MAP2 and MAP5) in the embryonic avian retina

Previous studies with the mammalian brain have shown that the expression of a number of neuronal microtubule-associated proteins (MAPs) is developmentally regulated. For example, the low-molecular-weight form of MAP2 (MAP2c) is abundant in neonatal rat brains and is less abundant in adults. Similarly, MAP5 levels decrease during postnatal development. Using monoclonal antibodies, we have followed the time of first appearance, cellular distribution, and molecular form of MAP2 and MAP5 during the morphogenesis of the quail retina. MAP2 first appears in ganglion cell bodies and in the axons of the optic fibre layer (OFL) at embryonic day 4 (E4). Anti-MAP2 staining remains restricted to these sites until E10, when staining appears in the inner plexiform layer (IPL). At E14, one day before hatching, anti-MAP2 staining is found in three broad laminae in the IPL, as well as in photosensitive cells. MAP5 is present in ganglion cell axons from the onset of neurite elongation at E3 and is limited to the OFL until E10. The intensity of anti-MAP5 staining in the OFL and optic nerve decreases after E7, which corresponds with a decrease in the number of actively growing ganglion cell axons. By E14, anti-MAP5 stains five layers in the IPL that correspond with layers of amacrine cell process arborizations. Western blots of E10 brain microtubule proteins show that MAP2 is represented by both a 260 x 10(3) Mr protein and a 60-65 x 10(3) Mr protein; the latter is much more abundant. Anti-MAP5 recognizes a 320 x 10(3) Mr brain microtubule protein in both the quail and the rat. We conclude that the cellular distribution, developmental regulation and molecular forms of MAP2 and MAP5 are similar in the rat and quail, suggesting that these molecules have conserved and hence fundamental roles in the growth and differentiation of neuronal processes.     

Colocalization of microtubule-associated protein 1A and microtubule- associated protein 2 on neuronal microtubules in situ revealed with double-label immunoelectron microscopy

Microtubule-associated protein 1A (MAP1A) and microtubule-associated protein 2 (MAP2) were shown to be colocalized on the same microtubules (MTs) within neuronal cytoskeletons by double-label immunoelectron microscopy. To investigate the electron microscopic disposition of MAP1A and MAP2 and their relationship to MTs in vivo, and to determine whether there are different subsets of MTs which specifically bind either MAP1 or MAP2, we employed a double-label immunogold procedure on rat cerebella using mouse monoclonal antibody against rat brain MAP1A and affinity-purified rabbit polyclonal antibody against rat brain MAP2. MAP1A and MAP2 were identified with secondary antibodies coupled to 10- and 5-nm gold particles, respectively. In Purkinje cell dendrites, both 10- and 5-nm gold particles were observed to be studded on the fuzzy structures attached to the same MTs. Many such structures connected MTs to each other. There was no particular MT which bound either MAP1A or MAP2 alone. Furthermore, there seemed to be no specific regions on MTs where either MAP1A or MAP2 was specifically attached. Hence, we conclude that MAP1A and MAP2 are colocalized on MTs in dendrites and assume that MAP1A and MAP2 have some interrelationship in vivo and that their interactions are responsible for forming the network of cross-bridges between MTs and MTs in neuronal cytoskeletons.     

Microtubule-associated protein 1B: molecular structure, localization, and phosphorylation-dependent expression in developing neurons

Two monoclonal antibodies, 5E6 and 1B6, were raised against microtubule-associated protein 1B (MAP1B), a major component of the neuronal cytoskeleton. 5E6 recognized the entire MAP1B population, while 1B6 detected only phosphorylated forms. Affinity-purified MAP1B appeared as a long, filamentous molecule (186 +/- 38 nm) with a small spherical portion at one end, forming long cross-bridges between microtubules in vitro. These results, together with in vivo data from immunogold methods, demonstrate that MAP1B is a component of cross-bridges between microtubules in neurons. By immunohistochemical analysis, phosphorylated forms were shown to exist mainly in axons, whereas unphosphorylated forms were limited to cell bodies and dendrites. Phosphorylated MAP1B was quite abundant in developing axons, suggesting its essential role in axonal elongation.     

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 ontogeny and phylogenetic conservation of MAP 2 forms

Summary In the adult rat brain, MAP 2 is a high-molecular weight protein that is highly concentrated in dendrites. Immunoblots of homogenates of developing rat brain have indicated that a low-molecular weight form of MAP 2, MAP 2 c, is transiently expressed as the brain is undergoing morphogenesis. Using MAP 2-specific monoclonal antibodies, we have demonstrated that the compartmentalization of high-molecular weight MAP 2 and the developmental regulation of MAP 2 are conserved in mammalian, avian, and amphibian brain. We have also determined the distribution of MAP 2 c in developing neuronal tissue. MAP 2 c appears before high-molecular weight MAP 2 in developing neurons, and in contrast to the dendrite-specific high-molecular weight forms of MAP 2, MAP 2 c is present in axons and glia. We have also shown that MAP 2 c is present in the adult rat retina, where it is concentrated in regenerative photosensitive cells. The transient expression of MAP 2 c in the developing brain of three species as well as in adult photosensitive cells suggests a role for this protein in neurite growth and plasticity.Abbreviations MAP microtubule-associated protein - E embryonic day - P postnatal day     

The sperm of Matsucoccus feytaudi (Insecta,Coccoidea): Can the microtubular bundle be considered as a true flagellum?

In the present work the spermiogenesis and sperm structure of Matsucoccus feytaudi, a primary pest of the maritime pine in southern eastern Europe, is studied. In addition to the already known characteristics of coccid sperm, such as the absence of the acrosome and mitochondria, and the presence of a bundle of microtubules responsible for sperm motility, a peculiar structure from which the microtubule bundle takes origin is described. Such a structure – a short cylinder provided with a central hub surrounded by several microtubules with a dense wall – is regarded as a Microtubule Organizing Centre (MTOC). During spermiogenesis, quartets of fused spermatids are formed; from each spermatid, a bundle of microtubules, generated by the MTOC, projects from the cell surface. Each cell has two centrioles, suggesting the lack of a meiotic process and the occurrence of parthenogenesis. At the end of the spermiogenesis, when the cysts containing bundles of sperm are formed, part of the nuclear material together with the MTOC structure is eliminated. Based on the origin of the microtubular bundle from the MTOC, the nature of the bundle as a flagellum is discussed.     

Influence of Monoclonal Antibodies on Microtubule Assembly

The influence on microtubule assembly in vitro of monoclonal antibodies against microtubule-associated proteins (MAPs) was studied. Light scattering was used for measuring net polymer formation and electron microscopy for determining the influence of antibodies on microtubule morphology. Control experiments showed that nonimmune mouse IgG had no effect on either the assembly or appearance of microtubules. The same was true for monoclonal antibodies against MAP1. At low levels, antibodies against MAP2 caused the aggregation of microtubules into bundles, an effect that did not occur with antibodies against any other MAP type studied. At increasing concentrations, anti-MAP2 progressively inhibited tubulin polymerization, producing irregular, shortened filaments. Anti-MAP5 produced a striking fragmentation of microtubules into very short pieces that were otherwise morphologically identical to control microtubules. The different effects of these antibodies show the potential of monoclonal antibodies for investigating MAP function and form an important adjunct to cellular microinjection experiments.     

A 70-Kilodalton Microtubule-Associated Protein (MAP2c), Related to MAP2

Microtubule-associated protein 2 (MAP2) from adult brain consists of a pair of high molecular mass (280 kilodaltons) polypeptides, MAP2a and MAP2b. Juvenile brain microtubules also contain a 70-kilodalton protein that cross-reacts with monoclonal antibodies against these high molecular weight MAP2s. We have analyzed the relationship between this 70-kilodalton protein and MAP2 by peptide mapping. Our results show that the 70-kilodalton species bears strong homology to the MAP2 molecules and that it is distinct from the tau MAPs. We propose the name MAP2c for this low molecular weight MAP2 species. MAP2c is developmentally regulated in brain, being more abundant in neonatal tissue than in the adult. In several cell lines, MAP2c is the sole MAP2 species expressed. We examined homogenates from both juvenile brain and MAP2c-containing cell lines for evidence of a protease activity that might be responsible for generating MAP2c from either MAP2a or MAP2b. No such activity was found, suggesting that MAP2c is an independently synthesized MAP2 species some 200 kilodaltons smaller than the previously recognized forms.     

Self-organization of microtubule bundles in anucleate fission yeast cells

Self-organization of cellular structures is an emerging principle underlying cellular architecture. Properties of dynamic microtubules and microtubule-binding proteins contribute to the self-assembly of structures such as microtubule asters. In the fission yeast Schizosaccharomyces pombe, longitudinal arrays of cytoplasmic microtubule bundles regulate cell polarity and nuclear positioning. These bundles are thought to be organized from the nucleus at multiple interphase microtubule organizing centres (iMTOCs). Here, we find that microtubule bundles assemble even in cells that lack a nucleus. These bundles have normal organization, dynamics and orientation, and exhibit anti-parallel overlaps in the middle of the cell. The mechanisms that are responsible for formation of these microtubule bundles include cytoplasmic microtubule nucleation, microtubule release from the equatorial MTOC (eMTOC), and the dynamic fusion and splitting of microtubule bundles. Bundle formation and organization are dependent on mto1p (gamma-TUC associated protein), ase1p (PRC1), klp2p (kinesin-14) and tip1p (CLIP-170). Positioning of nuclear fragments and polarity factors by these microtubules illustrates how self-organization of these bundles contributes to establishing global spatial order.     

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.