The drebrin/EB3 pathway drives invasive activity in prostate cancer.

Prostate cancer is the most common cancer in men and the metastatic form of the disease is incurable. We show here that the drebrin/EB3 pathway, which co-ordinates dynamic microtubule/actin filament interactions underlying cell shape changes in response to guidance cues, plays a role in prostate cancer cell invasion. Drebrin expression is restricted to basal epithelial cells in benign human prostate but is upregulated in luminal epithelial cells in foci of prostatic malignancy. Drebrin is also upregulated in human prostate cancer cell lines and co-localizes with actin filaments and dynamic microtubules in filopodia of pseudopods of invading cells under a chemotactic gradient of the chemokine CXCL12. Disruption of the drebrin/EB3 pathway using BTP2, a small molecule inhibitor of drebrin binding to actin filaments, reduced the invasion of prostate cancer cell lines in 3D in vitro assays. Furthermore, gain- or loss-of-function of drebrin or EB3 by over-expression or siRNA-mediated knockdown increases or decreases invasion of prostate cancer cell lines in 3D in vitro assays, respectively. Finally, expression of a dominant-negative construct that competes with EB3 binding to drebrin, also inhibited invasion of prostate cancer cell lines in 3D in vitro assays. Our findings show that co-ordination of dynamic microtubules and actin filaments by the drebrin/EB3 pathway drives prostate cancer cell invasion and is therefore implicated in disease progression.

Glycogen synthase kinase 3beta and the regulation of axon growth.

One of the earliest hallmarks that distinguish growing axons from dendrites is their growth rate; axons grow faster than dendrites. In vertebrates, where axons are required to grow for considerable distances, particularly in the peripheral nervous system, a fast axon growth rate is a requisite property. In neurons that respond to the neurotrophin growth factor/nerve growth factor with increased axon growth rates, two distinct intracellular signalling pathways are recruited: the MAPK (mitogen-activated protein kinase) pathway and the phosphatidylinositol-3 kinase pathway. The activation of either pathway leads to changes in microtubule dynamics within growing axons and growth cones and these underlie fast axon growth rates. Microtubule dynamics is regulated by microtubule-associated proteins and in the MAPK pathway this function is subserved by microtubule-associated protein 1B, whereas in the phosphatidylinositol-3 kinase pathway, adenomatous polyposis coli is the regulating microtubule-associated protein.

The non-immunosuppressive immunophilin ligand GPI-1046 potently stimulates regenerating axon growth from adult mouse dorsal root ganglia cultured in Matrigel.

We used explant cultures of adult mouse dorsal root ganglia with spinal nerve attached growing in Matrigel to assess the effects of the non-immunosuppressive immunophilin ligand GPI-1046 [Snyder et al. (1998) TIPS 19, 21-26] on the growth rate of regenerating sensory axons and found a potent stimulation of axon growth. In these explant cultures, naked, unfasciculated axons emerge from the cut end of the spinal nerve and continue to grow in the Matrigel for up to eight days [Tonge et al. (1996) Neuroscience 73, 541-551]. Some axons are entirely smooth whilst others show prominent varicosities. Some of the former express the phosphorylated neurofilament epitope recognised by monoclonal antibody RT97, a marker for large calibre, myelinated axons, whilst the latter express calcitonin gene-related peptide, predominantly a marker for unmyelinated, and small diameter myelinated sensory axons. Many of the axons in these cultures also express the low-affinity neurotrophin receptor p75. GPI-1046 has been shown to have striking stimulatory effects on embryonic primary sensory axons growing in vitro and it was therefore of interest to see whether it could also enhance regenerating sensory axon growth from the adult ganglia in our cultures. GPI-1046 potently stimulated axon growth in our cultures in a dose-dependent manner. The stimulatory effect was not dependent on the class of sensory axon. These observations show that GPI-1046 is a potent stimulator of regenerating axons from adult, primary sensory neurones. The cellular site of action of GPI-1046 is unknown. To distinguish between a direct effect of the drug on neurones and an indirect effect we compared the effects of GPI-1046 on explant and dissociated cultures. In confirmation of previous results, we found that GPI-1046 potently stimulated axon outgrowth from explants of embryonic chick dorsal root ganglia. However, the drug was without effect on dissociated embryonic dorsal root ganglion neurones, suggesting that non-neuronal cells are important for axon growth stimulation.

Microtubule-associated protein 1B phosphorylation by glycogen synthase kinase 3beta is induced during PC12 cell differentiation.

In recent studies we have demonstrated that glycogen synthase kinase 3beta (GSK3beta) and its substrate microtubule-associated protein 1B (MAP1B) regulate the microtubule cytoskeleton during axon outgrowth. To further examine the role GSK3beta plays in axon outgrowth we investigated the expression of GSK3beta and its activity towards MAP1B during nerve growth factor (NGF)-stimulated PC12 cell differentiation. Levels of GSK3beta expression increase relatively little during the course of differentiation. However, the expression of a novel GSK3beta isoform characterised by a reduced mobility on SDS gels is induced by NGF. Expression of this isoform and the GSK3beta-phosphorylated isoform of MAP1B (MAP1B-P) are induced in parallel in response to NGF. This increase lags behind initial neurite formation and the expression of MAP1B in these cells by about two days and coincides with a period when the majority of cells are extending existing neurites. MAP1B and GSK3beta are expressed throughout the PC12 cell but MAP1B-P expression is restricted to the growth cones and neurites. Consistent with these observations, we find that neurite extension is more sensitive to the GSK3 inhibitor Li+ than neurite formation and that this correlates with an inhibition of MAP1B phosphorylation. Additionally, GSK3beta from PC12 cells not exposed to NGF can not phosphorylate MAP1B in vitro. However, a soluble factor in differentiated PC12 cell extracts depleted of GSK3beta can activate MAP1B phosphorylation from undifferentiated cell extracts otherwise devoid of kinase activity. These experiments provide evidence for an NGF-mediated regulation of MAP1B phosphorylation in growing neurites by the induction of a novel isoform of GSK3beta.

MAP1B expression and microtubule stability in growing and regenerating axons.

MAP1B is a microtubule-associated phosphoprotein that is particularly highly expressed in developing neurons. There is experimental evidence that it plays an important role in neuronal differentiation, especially the extension of axons and dendrites, but exactly what role is unclear. Recent experiments have shed light on the gene structure of MAP1B and identified some of the kinases that phosphorylate the protein. Implicit in these findings is the idea that MAP1B regulates the organisation of microtubules in neurites and is itself regulated in a complex way and at a number of levels.

Glycogen synthase kinase 3beta phosphorylation of microtubule-associated protein 1B regulates the stability of microtubules in growth cones.

We have recently shown that glycogen synthase kinase 3beta (GSK3beta) phosphorylates the microtubule-associated protein (MAP) 1B in an in vitro kinase assay and in cultured cerebellar granule cells. Mapping studies identified a region of MAP1B high in serine-proline motifs that is phosphorylated by GSK3beta. Here we show that COS cells, transiently transfected with both MAP1B and GSK3beta, express high levels of the phosphorylated isoform of MAP1B (MAP1B-P) generated by GSK3beta. To investigate effects of MAP1B-P on microtubule dynamics, double transfected cells were labelled with antibodies to tyrosinated and detyrosinated tubulin markers for stable and unstable microtubules. This showed that high levels of MAP1B-P expression are associated with the loss of a population of detyrosinated microtubules in these cells. Transfection with MAP1B protected microtubules in COS cells against nocodazole depolymerisation, confirming previous studies. However, this protective effect was greatly reduced in cells containing high levels of MAP1B-P following transfection with both MAP1B and GSK3beta. Since we also found that MAP1B binds to tyrosinated, but not to detyrosinated, microtubules in transfected cells, we propose that MAP1B-P prevents tubulin detyrosination and subsequent conversion of unstable to stable microtubules and that this involves binding of MAP1B-P to unstable microtubules. The highest levels of MAP1B-P are found in neuronal growth cones and therefore our findings suggest that a primary role of MAP1B-P in growing axons may be to maintain growth cone microtubules in a dynamically unstable state, a known requirement of growth cone microtubules during pathfinding. To test this prediction, we reduced the levels of MAP1B-P in neuronal growth cones of dorsal root ganglion cells in culture by inhibiting GSK3beta with lithium. In confirmation of the proposed role of MAP1B-P in maintaining microtubule dynamics we found that lithium treatment dramatically increased the numbers of stable (detyrosinated) microtubules in the growth cones of these neurons.

The protein MAP-1B links GABA(C) receptors to the cytoskeleton at retinal synapses.

The ionotropic type-A and type-C receptors for the neurotransmitter gamma-aminobutyric acid (GABA(A) and GABA(C) receptors) are the principal sites of fast synaptic inhibition in the central nervous system, but it is not known how these receptors are localized at GABA-dependent synapses. GABA(C) receptors, which are composed of rho-subunits, are expressed almost exclusively in the retina of adult vertebrates, where they are enriched on bipolar cell axon terminals. Here we show that the microtubule-associated protein 1B (MAP-1B) specifically interacts with the GABA(C) rho1 subunit but not with GABA(A) receptor subunits. Furthermore, GABA(C) receptors and MAP-1B co-localize at postsynaptic sites on bipolar cell axon terminals. Co-expression of MAP-1B and the rho1 subunit in COS cells results in a dramatic redistribution of the rho1 subunit. Our observations suggest a novel mechanism for localizing ionotropic GABA receptors to synaptic sites. This mechanism, which is specific for GABA(C) but not GABA(A) receptors, may allow these receptor subtypes, which have distinct physiological and pharmacological properties, to be differentially localized at inhibitory synapses.

Monoclonal antibody 2G13, a new axonal growth cone marker.

Growth cones are specialized sensorimotor structures at the tips of neurites implicated in pathfinding decisions and axonal outgrowth during neuronal development. We generated a mouse monoclonal antibody (mAb 2G13) against chick tectum and found that the antibody exclusively labelled axonal growth cones, particularly their filopodia and lamellipodia, in developing rat CNS and in embryonic neurons in culture. The high fidelity of the staining of growth cones by mAb 2G13 means that the antibody will be a useful marker for identifying growth cones. In growth cones of cultured neurons, mAb 2G13 labelling is intracellular and mainly associated with the filamentous actin cytoskeleton. Experiments with cytochalasins, which depolymerise filamentous actin, showed that 2G13p (the protein recognised by mAb 2G13) is physically associated with filamentous actin in growth cones. These properties of 2G13p suggest a role in growth cone motility.

Inhibition of GSK-3beta leading to the loss of phosphorylated MAP-1B is an early event in axonal remodelling induced by WNT-7a or lithium.

WNT-7a induces axonal spreading and branching in developing cerebellar granule neurons. This effect is mediated through the inhibition of GSK-3beta, a serine/threonine kinase and a component of the WNT pathway. Lithium, an inhibitor of GSK-3beta, mimics WNT-7a in granule cells. Here we examined further the effect of GSK-3beta inhibition on cytoskeletal re-organisation. Lithium induces axonal spreading and increases growth cone area and perimeter. This effect is associated with the absence or reduction of stable microtubules in spread areas. Lithium induces the loss of a phosphorylated form of MAP-1B, a microtubule associated protein involved in axonal outgrowth. Down-regulation of the phosphorylated MAP-1B, MAP-1B-P, from axonal processes occurs before axonal remodelling is evident. In vitro phosphorylation assays show that MAP-1B-P is generated by direct phosphorylation of MAP-1B by GSK-3beta. WNT-7a, like lithium, also leads to loss of MAP-1B-P from spread axons and growth cones. Our data suggest that WNT-7a and lithium induce changes in microtubule dynamics by inhibiting GSK-3beta which in turn lead to changes in the phosphorylation of MAP-1B. These findings suggest a novel role for GSK-3beta and WNTs in axonal remodelling and identify MAP-1B as a new target for GSK-3beta and WNT.

Localisation of microtubule-associated protein 1B phosphorylation sites recognised by monoclonal antibody SMI-31.

MAP 1B is a microtubule-associated phosphoprotein that is expressed early in neurons and plays a role in axon growth. MAP 1B has two types of phospho-isoforms, one of which is developmentally down-regulated after neuronal maturation and one of which persists into adulthood. Because phosphorylation regulates MAP 1B binding activity, characterisation of the phosphorylation sites and identification of the corresponding kinases/phosphatases are important goals. We have characterised the developmentally down-regulated phosphorylation sites recognised by monoclonal antibody (mAb) SMI-31. We purified MAP 1B from neonatal rat brain and mapped the mAb SMI-31 sites to specific MAP 1B fragments after chemical cleavage. We then developed an in vitro kinase assay by using a high-speed spin supernatant from neonatal rat brain in the presence of ATP and recombinant proteins encoding selective regions of the MAP 1B molecule. Phosphorylation of the recombinant protein was detected on western blots using mAb SMI-31. This analysis showed that mAb SMI-31 recognises two recombinant proteins corresponding to residues 1,109-1,360 and 1,836-2,076 of rat MAP 1B after in vitro phosphorylation. The former phosphorylation site was further defined in the in vitro kinase assay by inhibition with peptides and antibodies from candidate regions of the MAP 1B sequence. This approach identified a region of 20 amino acids, from 1,244 to 1,264, characterised by a high concentration of serines immediately upstream of prolines, indicating that the kinase responsible is a proline-directed serine kinase.

The neurofilament antibody RT97 recognises a developmentally regulated phosphorylation epitope on microtubule-associated protein 1B.

Microtubules are important for the growth and maintenance of stable neuronal processes and their organisation is controlled partly by microtubule-associated proteins (MAPs). MAP 1B is the first MAP to be expressed in neurons and plays an important role in neurite outgrowth. MAP 1B is phosphorylated at multiple sites and it is believed that the function of the protein is regulated by its phosphorylation state. We have shown that the monoclonal antibody (mAb) RT97, which recognises phosphorylated epitopes on neurofilament proteins, fetal tau, and on Alzheimer's paired helical filament-tau, also recognises a developmentally regulated phosphorylation epitope on MAP 1B. In the rat cerebellum, Western blot analysis shows that mAb RT97 recognises the upper band of the MAP 1B doublet and that the amount of this epitope peaks very early postnatally and decreases with increasing age so that it is absent in the adult, despite the continued expression of MAP 1B in the adult. We confirmed that mAb RT97 binds to MAP 1B by showing that it recognises MAP 1B immunoprecipitated from postnatal rat cerebellum using polyclonal antibodies to recombinant MAP 1B proteins. We established that the RT97 epitope on MAP 1B is phosphorylated by showing that antibody binding was abolished by alkaline phosphatase treatment of immunoblots. Epitope mapping experiments suggest that the mAb RT97 site on MAP 1B is near the N-terminus of the molecule. Despite our immunoblotting data, immunostaining of sections of postnatal rat cerebellum with mAb RT97 shows a staining pattern typical of neurofilaments with no apparent staining of MAP 1B. For instance, basket cell axons and axons in the granule cell layer and white matter stained, whereas parallel fibres did not. These results suggest that the MAP 1B epitope is masked or lost under the immunocytochemical conditions in which the cerebellar sections are prepared. The upper band of the MAP 1B doublet is believed to be predominantly phosphorylated by proline-directed protein kinases (PDPKs). PDPKs are also good candidates for phosphorylating neurofilament proteins and tau and therefore we postulate that the sites recognised by RT97 on these neuronal cytoskeletal proteins may be phosphorylated by similar kinases. Important goals are to determine the precise location of the RT97 epitope on MAP 1B and the kinase responsible.

Microtubule reorganization is obligatory for growth cone turning.

To examine the role of microtubules in growth cone turning, we have compared the microtubule organization in growth cones advancing on uniform laminin substrates with their organization in growth cones turning at a laminin-tenascin border. The majority (82%) of growth cones on laminin had a symmetrical microtubule organization, in which the microtubules entering the growth cone splay out toward the periphery of the growth cone. Growth cones at tenascin borders had symmetrically arranged microtubules in only 34% of cases, whereas in the majority of cases the microtubules were displaced toward one-half of the growth cone, presumably stabilizing in the direction of the turn along the tenascin border. These results suggest that reorganization of microtubules could underlie growth cone turning. Further evidence for the involvement of microtubule rearrangement in growth cone turning was provided by experiments in which growth cones approached tenascin borders in the presence of nanomolar concentrations of the microtubule stabilizing compound, Taxol. Taxol altered the organization of microtubules in growth cones growing on laminin by restricting their distribution to the proximal regions of the growth cone and increasing their bundling. Taxol did not stop growth cone advance on laminin. When growing in the presence of Taxol, growth cones at tenascin borders were not able to turn and grow along the laminin-tenascin border, and consequently stopped at the border. Growth cones were arrested at borders for as long as Taxol was present (up to 6 h) without showing any signs of drug toxicity. These effects of Taxol were reversible. Together, these results suggest that microtubule reorganization in growth cones is a necessary event in growth cone turning.

Expression of a developmentally regulated, phosphorylated isoform of microtubule-associated protein 1B in sprouting and regenerating axons in vitro.

We have developed a novel culture system for studying axonal regeneration. Short lengths of spinal nerves with their attached dorsal root ganglia were removed from adult mice, explanted into Matrigel and maintained in serum-free medium for up to eight days. Profuse outgrowth of unfasciculated, naked axons occurred within 6 h from the cut ends of the peripheral nerve, dorsal roots and eventually from the ganglion itself, and continued to grow throughout the observation period. Some axons were entirely smooth, whilst others showed prominent varicosities. The former stained with antibody RT97, a marker for large-calibre, myelinated axons, whilst the latter stained with antibodies to calcitonin gene-related peptide, predominantly a marker for unmyelinated and small-diameter myelinated sensory axons. All axons stained with a monoclonal antibody (150) that recognizes a developmentally regulated phosphorylated isoform of the microtubule-associated protein 1B [Gordon-Weeks P. R. et al. (1993) Eur. J. Neurosci. 5, 1302-1311]. Monoclonal antibody 150 staining was observed along the entire length of all axons growing out of the explant; the proximal regions of these axons within the explant itself did not stain. The staining extended to the growth cones, which had elaborate morphologies. Other antibodies (e.g. to growth-associated protein 43) labelled axons within the nerve, as well as those growing in Matrigel. In preparations where the peripheral nerve had been crushed half-way along its length at the time of explantation, monoclonal antibody 150 staining was absent from axons in the nerve proximal to the crush, but present in axons which had regenerated within the nerve distal to the crush. The results indicate that re-expression during axonal regeneration of the phosphorylated isoform of microtubule-associated protein 1B recognized by monoclonal antibody 150 is restricted to the newly formed lengths of regenerated axons. The correlation between its expression and axonal growth during development and regeneration suggests that it may play a role in axonal extension. Our observations also demonstrate the usefulness of these explant cultures for studying axonal regeneration.

Expression of a developmentally regulated, phosphorylated isoform of microtubule-associated protein 1B in regenerating axons of the sciatic nerve.

Monoclonal antibodies SMI-31 and 150 recognize phosphorylation epitopes on microtubule-associated protein 1B that have been shown to be developmentally down-regulated in the nervous system. We have used these antibodies to establish changes in the pattern of expression of their epitopes on microtubule-associated protein 1B in regenerating axons of the sciatic nerves in the adult mouse and rat. Immunohistochemical studies showed that, in the sciatic nerve, regenerating axons in both adult mice and rats were labelled with monoclonal antibody 150 in a proximodistal gradient which was highest at the growth cone. This is the first report of expression of a developmentally regulated, phosphorylated isoform of microtubule-associated protein 1B in regenerating axons. Immunoblotting showed that the expression of the isoform recognized by monoclonal antibody 150 is present in normal adult mouse sciatic nerve and in regenerating axons following crush or cut lesions, but was not detectable in the normal or regenerating adult rat peripheral nervous system. Regenerating axons were also labelled by monoclonal antibody SMI-31, but the labelling, unlike antibody 150 labelling, was uniform along the entire length of the axon and immunoblotting showed that it was due to recognition of neurofilament protein. We conclude that the phosphorylated isoforms of microtubule-associated protein 1B recognized by monoclonal antibody 150 that are developmentally down-regulated in the adult rat central and peripheral nervous systems and adult mouse cerebellum are maintained in the normal peripheral nervous system of the adult mouse. When peripheral axons regenerate in the adult mouse, the regenerating axons also contain these isoforms. Adult rat regenerating axons are stained by antibody 150 only in tissue sections, not in immunoblots. The maintenance of immature isoforms of microtubule-associated protein 1B in mouse peripheral axons may relate to a continual capacity for growth and remodelling. The immunohistochemical localization of the antibody 150 epitope in growth cone-like structures and sprouts in injured nerves shows that phosphorylation of microtubule-associated protein 1B is likely to be an integral part of the regenerative response. These results also show that the phosphorylation epitopes on microtubule-associated protein 1B recognized by monoclonal antibodies 150 and SMI-31 are different and that only expression of the former correlates with axonal regeneration.

An analysis of an axonal gradient of phosphorylated MAP 1B in cultured rat sensory neurons.

The present study investigated the cellular distribution of a developmentally regulated phosphorylated form of MAP 1B recognized by monoclonal antibody (mAb) 150 in cultures of dorsal root ganglia. The cell soma and the whole axon, when it first appears, are labelled, but longer axons label with a proximodistal gradient, such that the cell soma and proximal axon become unlabelled, whilst the distal axon and growth cone label strongly. Double-labelling experiments with mAb 150 and a polyclonal antibody (N1-15) that recognizes all forms of MAP 1B demonstrated that MAP 1B is distributed along the entire length of axons with gradients, so the gradient of phosphorylated MAP 1B is not due to a loss or absence of MAP 1B from the proximal axon. The proportion of axons from 20 h cultures that were labelled with a mAb 150 gradient was at least 80% and this proportion was independent of the nerve growth factor concentration of the culture medium. Analysis of axons ranging in length from 100 to 700 microm and labelled with a gradient showed that the unlabelled proximal portions of axons increased in length more slowly than the labelled distal axon. Axons labelled along their entire length accounted for no more than 19% of th axonal population and analysis of these showed them to be frequently < 400 microm long. After simultaneously fixing and detergent-extracting cultures this proportion rose significantly to 93%, suggesting that in the proximal axon the mAb 150 epitope is masked by some factor(s) that is removed by detergent extraction. The possibility that mAb 150 could not access the epitope in the proximal axon was discounted because another IgM, mAb 125, which recognizes a different phosphorylation epitope on MAP 1B, labelled the proximal axon of conventionally fixed cultures. In growth cones of fixed and extracted neurons examined by immunofluorescence, the mAb 150 labelling strongly colocalized to bundled microtubules in the distal axon shaft and the C-domain. In the P-domain, mAb 150 staining was weaker and more widely distributed than the microtubules. Immunogold electron microscopy confirmed that antibody N1-15 and mAb 150 strongly labelled the bundled microtubules in the C-domain and also showed that individual microtubules in the P-domain, some of which lie alongside actin filament bundles of filopodia, were labelled lightly and discontinuously with both antibodies. This suggests that the phosphorylated isoform of MAP 1B recognized by mAb 150 may be microtubules and actin filaments in the P-domain.

Distribution and expression of developmentally regulated phosphorylation epitopes on MAP 1B and neurofilament proteins in the developing rat spinal cord.

The distribution and expression of developmentally regulated phosphorylation epitopes on the microtubule-associated protein 1B and on neurofilament proteins recognized by monoclonal antibody (mAb) 150 and mAb SMI-31 was investigated in the developing rat spinal cord. In the embryonic day 11 spinal cord, mAb 150 stained the first axons to appear, whereas mAb SMI-31 staining did not appear until embryonic day 12. At the start of axonogenesis, mAb 150 stained neuronal cell bodies and axons whereas at later times only the distal axon was stained, this is the first demonstration in vivo of a mAb 150 axonal gradient similar to that seen previously in vitro (Mansfield et al., 1991). During the postnatal period, axonal staining by mAb 150 dramatically declined so that by the third postnatal week, only the corticospinal tract, which contains axons that are still growing, was labelled. There was no evidence of dendritic staining except of adult primary motoneurons. In contrast, mAb SMI-31 staining of axons was not present as a gradient. Instead, mAb SMI-31 staining increased progressively throughout this period, persisted into adulthood and was shown by immunoblotting to be related to the increased phosphorylation of the medium and heavy neurofilament proteins. Axonal staining by mAb 150 re-appears in a sub-population of the SMI-31-labelled myelinated axons in the adult spinal cord and PNS and in the perikarya and dendrites of primary motoneurons, where it probably recognizes a phosphorylation epitope on heavy neurofilament proteins. This late appearing epitope has some similarities to that recognized by mAb SMI-31 on neurofilaments, but it is not identical. These cross-reactivities of mAbs that recognize phosphorylation epitopes on otherwise unrelated proteins dictate caution in interpreting immunohistochemical data. It may now be necessary in some cases to re-appraise published studies using these two antibodies.

Expression of PAC 1, an epitope associated with two synapse-enriched glycoproteins and a neuronal cytoskeleton-associated polypeptide in developing forebrain neurons.

The monoclonal antibody PAC 1 (postsynaptic density and cytoskeleton enriched) recognizes an epitope present on two postsynaptic density-enriched glycoproteins of 130,000 (postsynaptic density-enriched glycoprotein 130) and 117,000 mol. wt (postsynaptic density-enriched glycoprotein 117), and a cytoskeleton-enriched polypeptide of 155,000 mol. wt (cp155). The PAC 1 antibody has been used to study the development of the PAC 1 antigens in the developing rat forebrain in vivo and in tissue culture. cp155 is detected by embryonic day 14 and its level continues to rise until the sixth postnatal week. Postsynaptic density-enriched glycoproteins 130 and 117 are also expressed in embryonic brain although the level of postsynaptic density-enriched glycoprotein 130 initially increases more rapidly than that of postsynaptic density-enriched glycoprotein 117. Peak values are observed at postnatal days 4 (postsynaptic density-enriched glycoprotein 117) and 9 (postsynaptic density-enriched glycoprotein 130). The level of post synaptic density-enriched glycoprotein 117 subsequently decreases to some 50% of the peak value by postnatal day 42. Immunocytochemical studies show that PAC 1 immunoreactivity in developing cerebral cortex, detectable by postnatal day 0, is primarily associated with the perikarya and dendrites of pyramidal cells. The immunoreactivity develops as patches of PAC 1-positive neurons, uniform staining of the cortex only being fully established after postnatal day 9. Double-immunofluorescence labelling studies of forebrain cultures prepared from embryonic day 18 animals shows that many, but not all, growth-associated protein 43-positive neurons exhibit PAC 1 immunoreactivity. Some non-neuronal cells also stain with the PAC 1 monoclonal antibody. The growth cones of cultured neurons exhibit PAC 1 immunoreactivity and the PAC 1 antigens are detected on immunodeveloped western blots of isolated growth cones. The PAC 1 epitope is intracellular, but immunoreactivity does not co-localize with F-actin as detected by rhod-amine-phalloidin or with tubulin immunoreactivity. Postsynaptic density-enriched glycoprotein 130 is readily detected on PAC 1 immunodeveloped western blots of forebrain cultures maintained for up to 14 days in vitro. Postsynaptic density-enriched glycoprotein 117 is only poorly expressed by these cultures. The PAC 1 glycoproteins are present in forebrain synaptic membranes and postsynaptic densities at an early stage of development. The synaptic membrane level of postsynaptic density-enriched glycoprotein 130 and postsynaptic density-enriched glycoprotein 117 increases markedly between postnatal days 3 and 8. The level of both glycoproteins detected in postsynaptic densities remain virtually constant from postnatal days 9-90. These results are consistent with functional roles for these molecules in neuronal and synapse development.

A phosphorylation epitope on MAP 1B that is transiently expressed in growing axons in the developing rat nervous system.

We have isolated a monoclonal antibody (150) that recognizes a phosphorylation epitope on the microtubule-associated protein (MAP) 1B. Immunoblot analysis of the developing rat central nervous system shows that monoclonal antibody 150 is directed against a protein of approximately 325 kDa (MAP 1B) that copolymerizes with microtubules through successive cycles of temperature-dependent assembly and disassembly. Furthermore, immunoprecipitated MAP 1B contains the epitope recognized by monoclonal antibody 150. Removal of phosphate from blotted proteins using alkaline phosphatase abolishes the binding of monoclonal antibody 150 to MAP 1B, indicating that the epitope is phosphorylated. In the developing rat nervous system, immunohistochemistry with monoclonal antibody 150 shows that the phosphorylation epitope on MAP 1B is transiently expressed in growing axons but not in dendrites. For instance, in the neonatal rat cerebellum, the parallel fibres of granule cells are stained only during elongation and not after synaptogenesis. The monoclonal antibody 150 epitope is also transiently expressed in radial glial fibres and in certain cell nuclei. All immunostaining of sections with monoclonal antibody 150 was completely abolished by alkaline phosphatase treatment. These observations and previous ones made by us in cell culture (Mansfield et al., J. Neurocytol., 20, 654-666, 1991) suggest that the phosphorylation epitope on MAP 1B recognized by monoclonal antibody 150, which has not been previously detected in vivo, may be important in axonogenesis.

Organization of microtubules in axonal growth cones: a role for microtubule-associated protein MAP 1B.

Neuronal growth cones guide growing axons and dendrites (neurites) through developing embryos by detecting extrinsic guidance cues and transducing the signal into changes in motile behaviour. In this brief review, the role of the growth cone cytoskeleton in these events, in particular the microtubules, is discussed. Microtubules in the neurite are mainly bundled into fascicles whereas on entering the growth cone they diverge from each other and traverse the central (C)-domain of the growth cone. Occasionally, individual microtubules extend as far as the peripheral (P)-domain and may even enter filopodia. Microtubules in the growth cone are probably dynamically unstable, exchanging dimer with a large pool of soluble tubulin. It is proposed that the 'capture' of dynamically unstable microtubules by filopodial actin filament bundles is a crucial step underlying directed growth. Localised assembly of microtubules at the growth cone, rather than at the cell body followed by transport of polymer to the growth cone, may facilitate the delivery of material to specific regions of the growth cone and hence allow vectorial growth. Bundling of microtubules and capture of microtubules by filopodia both imply roles for microtubule-associated proteins (MAPs). Several microtubule-associated proteins are present within growth cones, including MAP 1B, MAP2 and tau. Recent experiments point toward a phosphorylated form of MAP 1B as an important component in neurite elongation and in particular in the bundling of microtubules in the growth cone.