Cytoskeletal changes during neurogenesis in cultures of avain neural crest cells

Neural crest cells are motile and mitotic, whereas their neuronal derivatives are terminally post-mitotic and consist of stationary cell body from which processes grow. The present study documents changes in the cytoskeleton that occur during neurogenesis in cultures of avain neural crest cells. The undifferentiated neural crest cells contain dense bundles of actin filaments throughout their cytoplasm, and a splayed array of microtubules attached to the centrosome. In newly differentiating neurons, the actin bundles are disrupted and most of the remaining actin filaments are reorganized into a cortical layer underlying the plasma membrane of the cell body and processes. Microtubules are more abundant in newly-differentiating neurons than in the undifferentiated cells, and individual microtubules can be seen dissociated from the centrosome. Neuron-specific beta-III tubulin appears in some crest cells prior to cessation of motility and cell division, and expression increases with total microtubule levels during neurogenesis. To investigate how these early cytoskeletal changes might contribute to alterations in morphology during neurogenesis, we have disrupted the cytoskeleton with pharmacologic agents. Microfilament disruption by cytochalasin immediately arrests the movement of neural crest cells and causes them to round-up, but does not significantly change the morphology of the immature neurons. Microtubule depolymerization by nocodazole slows the movement of undifferentiated cells and causes retraction of processes extended by the immature neurons. These results suggest that changes in the actin and microtubule arrays within neural crest cells govern distinct aspects of their morphogenesis into neurons.     

Upregulation and redistribution of E-MAP-115 (epithelial microtubule-associated protein of 115 kDa) in terminally differentiating keratinocytes is coincident with the formation of intercellular contacts

Microtubules are involved in the positioning and movement of organelles and vesicles and therefore play fundamental roles in cell polarization and differentiation. Their organization and properties are cell-type specific and are controlled by microtubule-associated proteins (MAP). E-MAP-115 (epithelial microtubule-associated protein of 115 kDa) has been identified as a microtubule-stabilizing protein predominantly expressed in epithelial cells. We have used human skin and primary keratinocytes as a model to assess a putative function of E-MAP-115 in stabilizing and reorganizing the microtubule network during epithelial cell differentiation. Immunolabeling of skin sections indicated that E-MAP-115 is predominantly expressed in the suprabasal layers of the normal epidermis and, in agreement with this observation, is relatively abundant in squamous cell carcinomas but barely detectable in basal cell carcinomas. In primary keratinocytes whose terminal differentiation was induced by increasing the Ca2+ concentration of the medium, E-MAP-115 expression significantly increased during the first day, as observed by northern and western blot analysis. Parallel immunofluorescence studies showed an early redistribution of E-MAP-115 from microtubules with a paranuclear localization to cortical microtubules organized in spike-like bundles facing intercellular contacts. This phenomenon is transient and can be reversed by Ca2+ depletion. Treatment of cells with cytoskeleton-active drugs after raising the Ca2+ concentration indicated that E-MAP-115 is associated with a subset of stable microtubules and that the cortical localization of these microtubules is dependent on other microtubules but not on strong interactions with the actin cytoskeleton or the plasma membrane. The mechanism whereby E-MAP-115 would redistribute to and stabilize cortical microtubules used for the polarized transport of vesicles towards the plasma membrane, where important reorganizations take place upon stratification, is discussed.     

Coronin promotes the rapid assembly and cross-linking of actin filaments and may link the actin and microtubule cytoskeletons in yeast

Coronin is a highly conserved actin-associated protein that until now has had unknown biochemical activities. Using microtubule affinity chromatography, we coisolated actin and a homologue of coronin, Crn1p, from Saccharomyces cerevisiae cell extracts. Crn1p is an abundant component of the cortical actin cytoskeleton and binds to F-actin with high affinity (Kd 6 x 10(-9) M). Crn1p promotes the rapid barbed-end assembly of actin filaments and cross-links filaments into bundles and more complex networks, but does not stabilize them. Genetic analyses with a crn1Delta deletion mutation also are consistent with Crn1p regulating filament assembly rather than stability. Filament cross-linking depends on the coiled coil domain of Crn1p, suggesting a requirement for Crn1p dimerization. Assembly-promoting activity is independent of cross-linking and could be due to nucleation and/or accelerated polymerization. Crn1p also binds to microtubules in vitro, and microtubule binding is enhanced by the presence of actin filaments. Microtubule binding is mediated by a region of Crn1p that contains sequences (not found in other coronins) homologous to the microtubule binding region of MAP1B. These activities, considered with microtubule defects observed in crn1Delta cells and in cells overexpressing Crn1p, suggest that Crn1p may provide a functional link between the actin and microtubule cytoskeletons in yeast.     

Disassembly of actin filaments leads to increased rate and frequency of mitochondrial movement along microtubules

In activated sea urchin coelomocytes, cytoplasmic organelles move along distinct actin and microtubule dependent pathways, actin-based motility is driven by an unconventional myosin, and microtubule disassembly does not effect actin-dependent organelle motility [D'Andrea et al., 1994: J. Cell Sci. 107:2081-2094]. Given the growing evidence for potential interactions between components of the actin and microtubule cytoskeletons, we examined the effect of actin filament disassembly on the movement of mitochondria along microtubules in activated coelomocytes. Coelomocytes treated with cytochalasin B (CB), to disrupt actin filaments, exhibited a thinning of the cytoplasm, enhanced lateral undulation of microtubules, and ceased centripetal cortical flow of actin. Interestingly, the loss of actin filaments resulted in a approximately 1.5-fold increase in the average velocity of outward and inward moving mitochondria and increased the frequency of centripetal movement. To test if enhanced motility along microtubules was a consequence of decreased actin-myosin interaction, coelomocytes were treated with 2,3-butanedione monoxime (BDM), a potent inhibitor of myosin activity [Cramer and Mitchison, 1995: J. Cell Biol. 131:179-189]. BDM inhibited all types of actin-based motility observed in these cells including retrograde cortical flow, protrusion and retraction of the cell edge, and movement of intracellular organelles. Surprisingly, BDM treatment stopped the movement of mitochondria in CB-exposed cells, suggesting that BDM can also act as an inhibitor of organelle movement along microtubules. Collectively, these data demonstrated that microtubule-dependent mitochondrial motility and microtubule movement were sensitive to changes in the assembly state of the actin cytoskeleton.     

Herpes simplex virus type 1 tegument protein VP22 induces the stabilization and hyperacetylation of microtubules

The role of the herpes simplex virus type 1 tegument protein VP22 during infection is as yet undefined. We have previously shown that VP22 has the unusual property of efficient intercellular transport, such that the protein spreads from single expressing cells into large numbers of surrounding cells. We also noted that in cells expressing VP22 by transient transfection, the protein localizes in a distinctive cytoplasmic filamentous pattern. Here we show that this pattern represents a colocalization between VP22 and cellular microtubules. Moreover, we show that VP22 reorganizes microtubules into thick bundles which are easily distinguishable from nonbundled microtubules. These bundles are highly resistant to microtubule-depolymerizing agents such as nocodazole and incubation at 4 degreesC, suggesting that VP22 has the capacity to stabilize the microtubule network. In addition, we show that the microtubules contained in these bundles are modified by acetylation, a marker for microtubule stability. Analysis of infected cells by both immunofluorescence and measurement of microtubule acetylation further showed that colocalization between VP22 and microtubules, and induction of microtubule acetylation, also occurs during infection. Taken together, these results suggest that VP22 exhibits the properties of a classical microtubule-associated protein (MAP) during both transfection and infection. This is the first demonstration of a MAP encoded by an animal virus.     

Domains of neuronal microtubule-associated proteins and flexural rigidity of microtubules

Microtubules are flexible polymers whose mechanical properties are an important factor in the determination of cell architecture and function. It has been proposed that the two most prominent neuronal microtubule-associated proteins (MAPs), tau and MAP2, whose microtubule binding regions are largely homologous, make an important contribution to the formation and maintenance of neuronal processes, putatively by increasing the rigidity of microtubules. Using optical tweezers to manipulate single microtubules, we have measured their flexural rigidity in the presence of various constructs of tau and MAP2c. The results show a three- or fourfold increase of microtubule rigidity in the presence of wild-type tau or MAP2c, respectively. Unexpectedly, even low concentrations of MAPs promote a substantial increase in microtubule rigidity. Thus at approximately 20% saturation with full-length tau, a microtubule exhibits >80% of the rigidity observed at near saturating concentrations. Several different constructs of tau or MAP2 were used to determine the relative contribution of certain subdomains in the microtubule-binding region. All constructs tested increase microtubule rigidity, albeit to different extents. Thus, the repeat domains alone increase microtubule rigidity only marginally, whereas the domains flanking the repeats make a significant contribution. Overall, there is an excellent correlation between the strength of binding of a MAP construct to microtubules (as represented by its dissociation constant Kd) and the increase in microtubule rigidity. These findings demonstrate that neuronal MAPs as well as constructs derived from them increase microtubule rigidity, and that the changes in rigidity observed with different constructs correlate well with other biochemical and physiological parameters.     

Single-channel characterization of the pharmacological properties of the K(Ca2+) channel of intermediate conductance in bovine aortic endothelial cells

The effects of scatter factor, HGF/SF, on multinuclear MDCK epitheliocytes were examined. Multinuclear cells were obtained by blocking cytokinesis by low concentration of cytochalasin D; these large cells had discoid shape and did not move much on the substrate. Incubation of these cells with HGF/SF induced their profound reorganization: their cytoplasm was reversibly segregated into several individually moving motile flattened domains, termed lamelloplasts and connected with one another by cylindrical domains termed cables. One or several nuclei were present in many lamelloplasts, but some lamelloplasts were anuclear. Nuclei were absent from the cables. Lamelloplasts continuously formed actin-rich ruffles at their edges; their cytoplasm contained small actin bundles and numerous focal adhesions. In contrast, cable, had no ruffles or focal adhesions. Dense networks of vimentin and keratin intermediate filaments were present in lamelloplasts; bundles of filaments of both types were seen in the cables. Segregation was accompanied by redistribution of centrosomes from perinuclear zone into lamelloplasts. As a result each lamelloplast in segregated cell acquired individual complex of centrosome and radiating microtubules. The cables contained numerous parallel microtubules but never had centrosomes. This reorganization of microtubular system was essential for segregation as alterations of shape and actin cytoskeleton were prevented by microtubule specific drugs: colcemid and Taxol (paclitaxel). It is suggested that mechanism of segregation is based on activation of two types of opposite actin reorganization: formation of actin networks in lamelloplasts and their dismantlement in the cables. Spatial distribution of the domains in which these opposite types of reorganizations occur may be regulated by microtubular system. It is also suggested that mechanisms of HGF/SF-induced segregation may be closely related to the mechanisms of important physiological reorganizations of cells, such as polarization of pseudopodial activities in motile cells and cytokinesis.     

The role of Saccharomyces cerevisiae coronin in the actin and microtubule cytoskeletons

Coronin was originally identified as a cortical protein associated with the actin cytoskeleton in Dictyostelium [1]. More recent studies have revealed that coronin is involved in actin-based motility, cytokinesis and phagocytosis [2,3]. Here, we describe the identification of a single homolog of coronin in Saccharomyces cerevisiae, which we show localizes to cortical actin patches in an actin-dependent manner. Unlike Dictyostelium mutants that lack coronin, yeast strains lacking coronin had no detectable defects in actin-based processes. This may reflect differences in the functions of the actin cytoskeleton in these two organisms. Previous studies have shown that cortical actin may mediate astral microtubule-based movements of the mitotic spindle in S. cerevisiae [4,5] and that, during mitosis in Dictyostelium, the regions of the cell cortex that overlap with astral microtubules become enriched in actin and coronin [6]. We therefore examined whether yeast lacking coronin had defects in the microtubule cytoskeleton. The mutant strains had increased sensitivity to the microtubule-destabilizing drug benomyl and an increased number of large-budded cells with short spindles. Further examination of microtubule-related processes, including spindle formation, migration of the mitotic spindle to the bud neck, spindle elongation, and translocation of the elongating spindle through the bud neck, failed to reveal any defects in the coronin mutant. Taken together, these results suggest that S. cerevisiae coronin is a component of the actin cytoskeleton that may interact with the microtubule cytoskeleton.     

Dystonin is essential for maintaining neuronal cytoskeleton organization

The mouse neurological mutant dystonia musculorum (dt) suffers from a hereditary sensory neuropathy. We have previously described the cloning and characterization of the dt gene, which we named dystonin (Dst). We had shown that dystonin is a neural isoform of bullous pemphigoid antigen 1 (Bpag1) with an N-terminal actin-binding domain. It has been shown previously that dystonin is a cytoskeletal linker protein, forming a bridge between F-actin and intermediate filaments. Here, we have used two different antibody preparations against dystonin and detected a high-molecular-weight protein in immunoblot analysis of spinal cord extracts. We also show that this high-molecular-weight protein was not detectable in the nervous system of all dt alleles tested. Immunohistochemical analysis revealed that dystonin was present in different compartments of neurons--cell bodies, dendrites, and axons, regions which are rich in the three elements of the cytoskeleton (F-actin, neurofilaments, and microtubules). Ultrastructural analysis of dt dorsal root axons revealed disorganization of the neurofilament network and surprisingly also of the microtubule network. In this context it is of interest that we observed altered levels of the microtubule-associated proteins MAP2 and tau in spinal cord neurons of different dt alleles. Finally, dt dorsal root ganglion neurons formed neurites in culture, but the cytoskeleton was disorganized within these neurites. Our results demonstrate that dystonin is essential for maintaining neuronal cytoskeleton integrity but is not required for establishing neuronal morphology.     

The let-99 gene is required for proper spindle orientation during cleavage of the C. elegans embryo

The orientation of cell division is a critical aspect of development. In 2-cell C. elegans embryos, the spindle in the posterior cell is aligned along the long axis of the embryo and contributes to the unequal partitioning of cytoplasm, while the spindle in the anterior cell is oriented transverse to the long axis. Differing spindle alignments arise from blastomere-specific rotations of the nuclear-centrosome complex at prophase. We have found that mutations in the maternally expressed gene let-99 affect spindle orientation in all cells during the first three cleavages. During these divisions, the nuclear-centrosome complex appears unstable in position. In addition, in almost half of the mutant embryos, there are reversals of the normal pattern of spindle orientations at second cleavage: the spindle of the anterior cell is aligned with the long axis of the embryo and nuclear rotation fails in the posterior cell causing the spindle to form transverse to the long axis. In most of the remaining embryos, spindles in both cells are transverse at second cleavage. The distributions of several asymmetrically localized proteins, including P granules and PAR-3, are normal in early let-99 embryos, but are perturbed by the abnormal cell division orientations at second cleavage. The accumulation of actin and actin capping protein, which marks the site involved in nuclear rotation in 2-cell wild-type embryos, is abnormal but is not reversed in let-99 mutant embryos. Based on these data, we conclude that let-99(+) is required for the proper orientation of spindles after the establishment of polarity, and we postulate that let-99(+) plays a role in interactions between the astral microtubules and the cortical cytoskeleton.     

Microtubule nucleation by gamma-tubulin-containing rings in the centrosome

The microtubule cytoskeleton of animal cells does not assemble spontaneously, but instead requires the centrosome. This organelle consists of a pair of centrioles surrounded by a complex collection of proteins known as the pericentriolar material (PCM). The PCM is required for microtubule nucleation. The minus, or slow-growing, ends of microtubules are embedded in the PCM and the plus, or fast-growing, ends project outwards into the cytoplasm during interphase, or into the spindle apparatus during mitosis. gamma-Tubulin is the only component of the PCM that is so far implicated in microtubule nucleation. Here we use immuno-electron microscopic tomography to show that gamma-tubulin is localized in ring structures in the PCM of purified centrosomes without microtubules. When these centrosomes are used to nucleate microtubule growth, gamma-tubulin is localized at the minus ends of the microtubules. We conclude that microtubule-nucleating sites within the PCM are ring-shaped templates that contain multiple copies of gamma-tubulin.     

Regulation of the cortical actin cytoskeleton in budding yeast by twinfilin, a ubiquitous actin monomer-sequestering protein

Here we describe the identification of a novel 37-kD actin monomer binding protein in budding yeast. This protein, which we named twinfilin, is composed of two cofilin-like regions. In our sequence database searches we also identified human, mouse, and Caenorhabditis elegans homologues of yeast twinfilin, suggesting that twinfilins form an evolutionarily conserved family of actin-binding proteins. Purified recombinant twinfilin prevents actin filament assembly by forming a 1:1 complex with actin monomers, and inhibits the nucleotide exchange reaction of actin monomers. Despite the sequence homology with the actin filament depolymerizing cofilin/actin-depolymerizing factor (ADF) proteins, our data suggests that twinfilin does not induce actin filament depolymerization. In yeast cells, a green fluorescent protein (GFP)-twinfilin fusion protein localizes primarily to cytoplasm, but also to cortical actin patches. Overexpression of the twinfilin gene (TWF1) results in depolarization of the cortical actin patches. A twf1 null mutation appears to result in increased assembly of cortical actin structures and is synthetically lethal with the yeast cofilin mutant cof1-22, shown previously to cause pronounced reduction in turnover of cortical actin filaments. Taken together, these results demonstrate that twinfilin is a novel, highly conserved actin monomer-sequestering protein involved in regulation of the cortical actin cytoskeleton.     

In vivo analyses of cytoplasmic transport and cytoskeletal organization during Drosophila oogenesis: characterization of a multi-step anterior localization pathway

Anterior patterning of the Drosophila embryo depends on localization of bicoid (bcd) mRNA to the anterior pole of the developing oocyte, and bcd mRNA localization requires both the exuperantia (exu) gene and an intact microtubule cytoskeleton. To gain insight into the mechanism of anterior patterning, we have used time lapse laser scanning confocal microscopy to analyze transport of particles containing a Green Fluorescent Protein-Exu fusion (GFP-Exu), and to directly image microtubule organization in vivo. Our observations indicate that microtubules are required for three forms of particle movement within the nurse cells, while transport through the ring canals linking the nurse cells and oocyte appears to be independent of both microtubules and actin filaments. As particles enter the oocyte, a final microtubule-dependent step directs movement to the oocyte cortex. However, our observations and previous studies suggest that the polarity of the oocyte microtubule network is not in itself sufficient to generate anterior asymmetry, and that additional factors are required to restrict morphogens to the anterior pole. Based on these observations, we propose a multi-step anterior localization pathway.     

Female genital mutilation: an overview

A method for biochemically isolating microtubule-associated proteins (MAPs) from the detergent-extracted cytoskeletons of carrot suspension cells has been devised. The advantage of cytoskeletons is that filamentous proteins are enriched and separated from vacuolar contents. Depolymerization of cytoskeletal microtubules with calcium at 4 degrees C releases MAPs which are then isolated by association with taxol stabilized neurotubules. Stripped from microtubules (MTs) by salt, then dialysed, the resulting fraction contains a limited number of high molecular weight proteins. Turbidimetric assays demonstrate that this MAP fraction stimulates polymerization of tubulin at concentrations at which it does not self-assemble. By adding it to rhodamine-conjugated tubulin, the fraction can be seen to form radiating arrays of long filaments, unlike MTs induced by taxol. In the electron microscope, these arrays are seen to be composed of mainly single microtubules. Blot-affinity purified antibodies confirm that two of the proteins decorate cellular microtubules and fulfil the criteria for MAPs. Antibodies to an antigenically related triplet of proteins about 60-68 kDa (MAP 65) stain interphase, preprophase band, spindle and phragmoplast microtubules. Antibodies to the 120 kDa MAP also stain all of the MT arrays but labelling of the cortical MTs is more punctate and, unlike anti-MAP 65, the nuclear periphery is also stained. Both the anti-65 kDa and the anti-120 kDa antibodies stain cortical MTs in detergent-extracted, substrate-attached plasma membrane disks ('footprints'). Since the 120 kDa protein is detected at two surfaces (nucleus and plasma membrane) known to support MT growth in plants, it is hypothesized that it may function there in the attachment or nucleation of MTs.     

Participation of serotonin in capsaicin-induced mouse ear edema

Microfilaments were localised by immunofluorescence and immunogold cytochemistry to examine their distribution in granular cells of the isolated frog skin epithelium. Strongly fluorescent bundles of actin were observed beneath the plasma membrane with little evidence for actin in the central regions. Higher resolution offered by cytochemistry revealed that bundles of actin filaments comprised a substantial portion of the cortical cytoskeleton. Quantitative analysis of the frequency of gold label revealed an extremely rich array of filaments beneath the apical membrane of granular cells, with markedly less label along the basolateral membrane and in the central cytoplasm. Treating cells with cytochalasin B or arginine vasopressin caused an apparent disruption of the apical actin fibres, concurrent with a decrease in gold label density. Assumably these signs are indicative of depolymerization of the filaments. Although the significance of this distribution is unknown, the apical polarisation of actin is consistent with a role in regulating the Na+ permeability of the apical membrane. The data are discussed in relation to possible roles of the cytoskeleton in the regulation of transepithelial sodium transport by vasopressin.     

Differences in the regulation of microtubule dynamics by microtubule-associated proteins MAP1B and MAP2

The regulation of microtubule dynamics in vitro by microtubule-associated proteins (MAPs) was examined, using purified porcine MAP1B and MAP2. MAP1B has a significantly smaller effect on the observed critical concentration for microtubule assembly than MAP2. Assembly is faster in the presence of either MAP, and the resulting microtubules are shorter, indicating that nucleation is substantially promoted by the MAPs. Both MAPs stabilise the microtubule lattice as observed from podophyllotoxin-induced disassembly, but the effect of MAP1B is weaker than the effect of MAP2. At steady-state of assembly MAP1B still allows microtubule dynamic instability to occur as inferred from microtubule length changes. The comparison of the effects of MAP1B and MAP2 indicates that the reduction of the observed critical concentration is attributable to the reduction of the depolymerisation rate and correlates with the extent of suppression of dynamic instability. Numerical simulations illustrate that microtubule dynamics are strongly influenced by relatively small changes in the strength of a limited subset of subunit interactions in the lattice. The observed characteristic differences between the MAPs may be important for the regulation of distinct populations of microtubules which coexist in the same cell, where differences in stability and dynamics may be essential for their different spatial roles as, for example, in developing neurons.     

Characterization of the microtubule-binding domain of microtubule-associated protein 1A and its effects on microtubule dynamics

To determine how MAP1a interacts with microtubules we expressed several 6myc-tagged MAP1a fragments in P19 EC and HeLa cells. Confocal immunofluorescence microscopy showed that the fragment consisting of amino acids (aa) 1-281 of MAP1a did not bind while the fragment consisting of aa 1-630 did, indicating that the region of MAP1a between aa 281 and 630 contains a microtubule-binding domain. Deletion of the basic repeats from aa 336-540 did not result in loss of microtubule binding, suggesting that the regions flanking the basic repeats can bind MAP1a to microtubules. These observations were confirmed using an in vitro microtubule binding assay. The levels of acetylation and detyrosination of polymerized microtubules were assessed by quantitative dot blotting in cells expressing MAP1a fragments or MAP2c. Compared with untransfected cells, the polymerized tubulin in cells expressing full-length MAP1a was more acetylated and detyrosinated, but these increases were smaller than those seen in cells expressing MAP2c. Consistent with this, the microtubules in MAP2c expressing cells were more resistant to colchicine than those in cells overexpressing MAP1a. These data implicate aa 281-336 and/or 540-630 of MAP1a in microtubule binding and suggest that MAP1a is less able to stabilize microtubules than MAP2c.     

Spinal locomotor generators in humans: problems in assessing effectiveness of stimulations]

The state of filament actin in Mauthner neuron (MN) of gold-fish adapted to long-term natural stimulation was investigated after introduction of actin-depolymerizing cytochalasin D. Cytochalasin was demonstrated to cause almost complete disappearance of long actin bundles that were characteristic for the cytoplasm of adapted fish and were absent from MN of intact fish. Polymeric actin supporting the cytoskeleton integrity was suggested to be involved in mechanisms underlying the increase of MN resistance extreme loads.     

The kakapo mutation affects terminal arborization and central dendritic sprouting of Drosophila motorneurons

The lethal mutation l(2)CA4 causes specific defects in local growth of neuronal processes. We uncovered four alleles of l(2)CA4 and mapped it to bands 50A-C on the polytene chromosomes and found it to be allelic to kakapo (. Genetics. 146:275- 285). In embryos carrying our kakapo mutant alleles, motorneurons form correct nerve branches, showing that long distance growth of neuronal processes is unaffected. However, neuromuscular junctions (NMJs) fail to form normal local arbors on their target muscles and are significantly reduced in size. In agreement with this finding, antibodies against kakapo (Gregory and Brown. 1998. J. Cell Biol. 143:1271-1282) detect a specific epitope at all or most Drosophila NMJs. Within the central nervous system of kakapo mutant embryos, neuronal dendrites of the RP3 motorneuron form at correct positions, but are significantly reduced in size. At the subcellular level we demonstrate two phenotypes potentially responsible for the defects in neuronal branching: first, transmembrane proteins, which can play important roles in neuronal growth regulation, are incorrectly localized along neuronal processes. Second, microtubules play an important role in neuronal growth, and kakapo appears to be required for their organization in certain ectodermal cells: On the one hand, kakapo mutant embryos exhibit impaired microtubule organization within epidermal cells leading to detachment of muscles from the cuticle. On the other, a specific type of sensory neuron (scolopidial neurons) shows defects in microtubule organization and detaches from its support cells.     

EMAP, an echinoderm microtubule-associated protein found in microtubule-ribosome complexes

The major non-tubulin polypeptide found associated with microtubules purified from unfertilized sea urchin eggs by cycles of pH-dependent assembly has a Mr of 77,000. The 77,000 Mr polypeptide is heat- and acid-labile, and is antigenically distinct from the mammalian brain MAPs, MAP-2 and tau. Affinity-purified antiserum against the 77,000 Mr polypeptide was used to survey a variety of cells and tissues for the presence of antigenically related polypeptides. A cross-reacting polypeptide, ranging in Mr from 72,000 to 80,000, was found in microtubule preparations from a wide variety of echinoderms, including sea urchins, starfish and sand dollars. Indirect immunofluorescence showed that the polypetide was found in interphase as well as mitotic microtubule arrays. No cross-reacting material was detected in microtubules isolated from marine molluscs, mammalian brain or mouse B16 cultured cells. Because the 77,000 Mr MAP is abundant in echinoderms, we have called it EMAP for echinoderm microtubule-associated protein. Although the precise function of the EMAP is not known, our data suggest that the EMAP is involved in the attachment of ribosomes to microtubules. Large numbers of ribosomes are attached to the walls of EMAP-containing microtubules, but not EMAP-deficient microtubules. Removal of the EMAP from the microtubule by salt-extraction results in the release of ribosomes from the microtubule, indicating that the EMAP may form part or all of the long tapered stalk that connects these two organelles.