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P R Gordon-Weeks

Publications and source records attributed to P R Gordon-Weeks.

At least 37 records · Page 2Linked to original sources
PubMed1993

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.

Animals↗
PubMed1993

The organization of F-actin and microtubules in growth cones exposed to a brain-derived collapsing factor.

In previous work we characterized a brain derived collapsing factor that induces the collapse of dorsal root ganglion growth cones in culture (Raper and Kapfhammer, 1990). To determine how the growth cone cytoskeleton is rearranged during collapse, we have compared the distributions of F-actin and microtubules in normal and partially collapsed growth cones. The relative concentration of F-actin as compared to all proteins can be measured in growth cones by rationing the intensity of rhodamine-phalloidin staining of F-actin to the intensity of a general protein stain. The relative concentration of F-actin is decreased by about one half in growth cones exposed to collapsing factor for five minutes, a time at which they are just beginning to collapse. During this period the relative concentration of F-actin in the leading edges of growth cones decreases dramatically while the concentration of F-actin in the centers decreases little. These results suggest that collapse is associated with a net loss of F-actin at the leading edge. The distributions of microtubules in normal and collapsing factor treated growth cones were examined with antibodies to tyrosinated and detyrosinated isoforms of alpha-tubulin. The tyrosinated form is found in newly polymerized microtubules while the detyrosinated form is not. The relative proximal-distal distributions of these isoforms are not altered during collapse, suggesting that rates of microtubule polymerization and depolymerization are not greatly affected by the presence of collapsing factor. An analysis of the distributions of microtubules before and after collapse suggests that microtubules are rearranged, but their polymerization state is unaffected during collapse. These results are consistent with the hypothesis that the brain derived collapsing factor has little effect on microtubule polymerization or depolymerization. Instead it appears to induce a net loss of F-actin at the leading edge of the growth cone.

Actins↗
PubMed1992

A study of the expression of laminin in the spinal cord of the frog during development and regeneration.

In the present experiments, we have used an affinity-purified polyclonal antibody to laminin to determine the time course of expression of laminin in the central nervous system (CNS) of Rana temporaria tadpoles during normal development and during restoration of the dorsal columns of the spinal cord. Immunoblotting analysis indicated that in the peripheral nervous system (PNS) of adult frogs the antibody recognized proteins of molecular weights 350-400 and 205-220 kDa, corresponding to the A and B chains respectively of mammalian laminin. Immunohistochemistry with our antibody suggested that laminin was absent from the tadpole spinal cord, and did not appear even in the basal lamina of the blood vessels within the spinal cord until after metamorphosis. Furthermore, there was no evidence of laminin expression in the dorsal columns after hemisection of the spinal cord. However, throughout development laminin was present in basal lamina outside the CNS, in particular in the pial membranes, and in the basal lamina of blood vessels and sheath cells in the dorsal and ventral roots. Electron microscopy showed that the blood vessels of CNS capillaries had basal laminae throughout development that was morphologically indistinguishable from that seen in peripheral vessels.

Animals↗
PubMed1991

Growth cones: the mechanism of neurite advance.

Growth cones are the highly motile structures found at the tips of growing axons and dendrites (neurites), which extend from neurones, during the development of the nervous system. They function both as detectors and transducers of extrinsic guidance cues and as regions where the neurite assembly, advance cannot occur. Assembly of the neurite cytoskeleton in growing neurites chiefly involves microtubule assembly at the growth cone. Some of the factors that may influence microtubule assembly in growth cones are becoming apparent and include post-translational modification of tubulin itself and microtubule associated proteins, particularly tau and MAP1B.

Actin Cytoskeleton↗
PubMed1991

Dynamic post-translational modification of tubulin in rat cerebral cortical neurons extending neurites in culture: effects of taxol.

Dissociated embryonic (E18-E20) rat cortical neurons were grown in culture and double-labelled by immunofluorescence with antibodies directed against tyrosinated (YL 1/2), detyrosinated (SUP GLU), and acetylated (6-11B-1) alpha-tubulin. Within 90 min of plating, neurons extended growth cones that were YL 1/2+ but SUP GLU- and 6-11B-1-. The neurite that forms behind the advancing growth cone is also, initially, YL 1/2+ and SUP GLU-/6-11B-1-. However, when it has attained a length of about half the cell body diameter, it becomes SUP GLU+ and 6-11B-1+. The effects of the microtubule polymerizing agent taxol (15 microM) on growth cone and neurite alpha-tubulins was investigated. Taxol, as reported previously, caused the formation of microtubule loops in the central domain of the growth cone, a loss of filopodia, and the collapse of the growth cone onto the loops. The taxol effects peaked at 60 min, when over 85% of neurites showed microtubule loops, and declined thereafter, so that at 420 min in taxol, only about 23% of neurites had microtubule loops. Over this period there was an inverse correlation between the presence of microtubule loops and growth cones. Taxol had striking effects on the intensity of SUP GLU and 6-11B-1 staining in neurons. In 48 h cultures, a 30 min exposure to taxol enhanced the SUP GLU and 6-11B-1 staining of dendrites and axons and produced a loss of YL 1/2 staining in axons. Immunoblotting experiments confirmed that there was an overall reduction in YL 1/2 immunoreactivity and an increase in SUP GLU immunoreactivity. These observations support previous suggestions that the neurite microtubules are assembled in the growth cone and post-translationally modified in the neurite and, in addition, imply that growth cones can overcome the effects of taxol in the continued presence of the compound.

Alkaloids↗
PubMed1991

The distribution and phosphorylation of the microtubule-associated protein MAP 1B in growth cones.

Primary cultures of dissociated embryonic day 18 rat cerebral cortices were labelled by immunofluorescence with antibodies directed either against phosphorylated and non-phosphorylated MAP 1B (antibody 81) or against phosphorylated MAP 1B (antibody 150). Both antibodies stain cortical neurons, including their neurites and growth cones, in early (18 h) cultures, whereas only antibody 81 stained glial cells. By 4 days in culture, phosphorylated MAP 1B is largely restricted to axonal processes and growth cones, where it is often distributed in a gradient that is highest distally. In axonal processes and growth cones after 18 h and 4 days in culture, the phosphorylated form of MAP 1B is present both in a soluble form and bound to microtubules. Growth cones isolated from postnatal day 5 rat forebrain were labelled in vitro with 32P-orthophosphate and detergent soluble and insoluble (cytoskeleton) fractions prepared. SDS-PAGE analysis revealed several major phosphoproteins in isolated growth cone cytoskeletons, including MAP 1B. Phosphorylated MAP 1B was also present in the detergent soluble fraction of growth cones. Immunoblotting and immunoprecipitation with MAP 1B antibodies confirmed the identification of MAP 1B and that the protein is phosphorylated in growth cones. These data show that MAP 1B, in particular the phosphorylated isoform, is present in growth cones and suggest that phosphorylation of MAP 1B may play an important role in neurite elongation.

Animals↗
PubMed1991

PAC 1: an epitope associated with two novel glycoprotein components of isolated postsynaptic densities and a novel cytoskeleton-associated polypeptide.

A monoclonal antibody has been raised which recognizes an epitope, PAC 1 (postsynaptic density and cytoskeleton enriched), which is specifically associated with two novel glycoprotein components of forebrain postsynaptic density preparations and a novel neuronal cytoskeletal-associated polypeptide. The monoclonal antibody has been used to study the cellular and subcellular localization of these molecules and for the partial characterization of all three PAC 1 antigens in the rat. The PAC 1 epitope is present on two concanavalin A binding glycoproteins of apparent molecular weights 130,000 (pgp130) and 117,000 (pgp117). Both species are enriched in preparations of rat forebrain postsynaptic densities and to a lesser extent in synaptic membranes. The epitope is also expressed by a polypeptide of 155,000 mol. wt, cp155. This molecule is highly enriched in cytoskeleton rather than membrane preparations. Enzymic removal of N-linked carbohydrate lowers the molecular weights of the PAC 1 glycoproteins pgp130 and pgp117 by 11,000 and 14,000 respectively, and suggests that cp155 is not glycosylated. Detergent, alkaline and salt extractions of postsynaptic densities and synaptic membranes indicate that pgp130 and pgp117 are integral membrane glycoproteins and are tightly bound components of postsynaptic density preparations. Immunocytochemical studies of adult rat forebrain show prominent staining of pyramidal cell dendrites and perikarya. There is no evidence of glial staining. Electron microscope studies show staining of microtubules together with punctate deposits of plasma membrane-associated reaction product. Several criteria have been used to show that pgp130 and pgp117 do not correspond to other known neuronal glycoproteins of similar molecular weight. We conclude that the PAC 1 epitope is expressed by two novel synaptic glycoproteins which are very probably integral components of the postsynaptic density and by a novel neuronal cytoskeleton-associated protein.

Animals↗
PubMed1991

Evidence for microtubule capture by filopodial actin filaments in growth cones.

We have studied, by immunofluorescence, the organization of microtubules in growth cones in dissociated neuronal cell cultures. Growth cones have a large pool of soluble tubulin and in conventionally fixed cultures the entire growth cone, including the filopodia, is labelled with tubulin antibodies, thus obscuring the microtubules. In contrast, in cultures fixed with fixatives containing detergent, the soluble pool of tubulin is removed allowing clear views of the microtubules. On entering the growth cone from the neurite, microtubules splay out and may extend as far as the base of filopodia. In most growth cones, one or more individual microtubules extend into filopodia, aligning with the parallel bundle of microfilaments. This observation suggests one way growth cone motility and neurite advance may be coupled.

Actins↗
PubMed1991

Calcium-independent gamma-aminobutyric acid release from growth cones: role of gamma-aminobutyric acid transport.

Neuronal growth cones isolated in bulk from neonatal rat forebrain have uptake and K(+)-stimulated release mechanisms for gamma-aminobutyric acid (GABA). Up to and including postnatal day 5, the K(+)-stimulated release of [3H]GABA and endogenous GABA is Ca2+ independent. At these ages, isolated growth cones neither contain synaptic vesicles nor stain for synaptic vesicle antigens. Here we examined the possibility that the release mechanism underlying Ca2(+)-independent GABA release from isolated growth cones is by reversal of the plasma membrane GABA transporter. The effects of two GABA transporter inhibitors, nipecotic acid and an analogue of nipecotic acid, SKF 89976-A, on K(+)-stimulated release of [3H]GABA from superfused growth cones were examined. Nipecotic acid both stimulated basal [3H]GABA release and enhanced K(+)-stimulated release of [3H]GABA, which indicates that this agent can stimulate GABA release and is, therefore, not a useful inhibitor with which to test the role of the GABA transporter in K(+)-stimulated GABA release from growth cones. In contrast, SKF 89976-A profoundly depressed both basal and K(+)-stimulated [3H]GABA release. This occurred at similar concentrations at which uptake was blocked. These observations provide evidence for a major role of the GABA transporter in GABA release from neuronal growth cones.

Animals↗
PubMed1991

Control of microtubule assembly in growth cones.

Growth cones are transient structures present at the tips of growing axons and dendrites (neurites). They are crucial to neuronal development because of their pathfinding ability and their role in synaptogenesis (reviewed by Dodd and Jessell, 1988). In the last few years it has become apparent that growth cones are also involved in the assembly of the cytoskeleton of the elongating neurite (reviewed by Gordon-Weeks, 1989, 1991). We are particularly interested in the assembly of microtubules in the growth cone and its importance for neurite advance. The microtubules in the neurite are bundled into fascicles, presumably by the cross-linking action of microtubule-associated proteins (MAPs), and on entering the growth cone they splay out like the ribs of a fan with their 'plus' ends, the ends at which assembly takes place preferentially, oriented distally. Within the growth cone there is a large pool of assembly competent tubulin that provides subunits for microtubule elongation. Several observations point to the existence of precise mechanisms controlling assembly of this soluble tubulin pool. If the control mechanisms are disrupted the ability of the neurite to advance is severely compromised. We have examined the possibility that MAPs are important components in this control mechanism. Many of the known MAPs are present within growth cones, including MAP 1B, MAP 2 and tau. Experiments with neuronal cultures and growth cones isolated as a subcellular fraction from developing rat brain point particularly toward the phosphorylated form of MAP 1B as an essential component in the concerted assembly of microtubules at the growth cone and in particular in the bundling of microtubules in the neurite.

Animals↗
PubMed1990

GABAergic growth cones: release of endogenous gamma-aminobutyric acid precedes the expression of synaptic vesicle antigens.

Growth cone fractions isolated from neonatal [postnatal day 3 (P3)] rat forebrain contain GABAergic growth cones as demonstrated by immunofluorescence staining with monospecific antibodies to gamma-aminobutyric acid (GABA). HPLC analysis shows that GABAergic growth cones release this endogenous GABA when stimulated with high K+. Endogenous GABA release is Ca2(+)-independent and, in this respect, similar to that seen previously with [3H]GABA. Isolated growth cone fractions also exhibit a K(+)-stimulated, Ca2(+)-independent release of endogenous taurine. None of the other amino acids shown to be present in isolated growth cone fractions were released, including glutamate, aspartate, and glycine. A population of dissociated cerebral cortical neurones prepared from P1 rat forebrain were GABA-immunoreactive after 1 day in culture. The cell body, neurites, and growth cones of these neurones were all stained with GABA antibodies. At this time in culture, neurones did not stain with either of two antibodies to synaptic vesicle antigens, i.e., p65 and synaptophysin. Growth cones isolated from P3 rat forebrain were also not immunoreactive with these antibodies. After about 8 days in culture, when neurones had established extensive networks of long, varicose axons and elaborately branched dendrites, many neurones and their neurites were immunoreactive for GABA antibodies. At this time in culture, p65 and synaptophysin antibodies did stain neuronal cell bodies and particularly their varicose axons. Dendrites were not stained with synaptic vesicle antibodies. These results suggest that GABAergic neurones synthesize GABA during neurite outgrowth and that GABA is present in, and can be released from, the growth cones of these neurones. The presence of GABA in GABAergic growth cones is not associated with synaptic vesicles, which explains the Ca2+ independency of both endogenous and [3H]GABA release from these growth cones.

Animals↗
PubMed1990

GAP-43 in growth cones is associated with areas of membrane that are tightly bound to substrate and is a component of a membrane skeleton subcellular fraction.

To ascertain the subcellular localization of the growth-associated protein GAP-43 in growth cones, we isolated growth cones from the forebrains of neonatal rats. Anti-GAP-43 immunoreactivity in these isolated growth cones (IGCs) resembled that seen in cultured growth cones in 2 respects: First, in substrate-attached IGCs, as in cultured growth cones, immunoreactivity was intracellular, punctate, and extended throughout the IGCs and their processes. Second, when IGCs were dislodged from this substrate, patches of membrane that were highly immunoreactive for GAP-43 were left behind. Extracting IGCs in nonionic detergents revealed that almost all the particulate GAP-43 colocalized with a membrane skeleton fraction: It was not present in the cytoskeleton. The association of GAP-43 with the membrane skeleton was not due to nonspecific aggregation, nor was it calcium dependent. Examination of this fraction under the electron microscope showed it to consist of membrane fragments associated with amorphous material that could be visualized with tannic acid. Immunoelectron microscopy showed that anti-GAP-43 immunoreactivity was localized to this amorphous material. Fodrin, talin, and a-actinin immunoreactivity could be detected in the membrane skeleton fraction, and actin and tubulin were also identifiable from SDS gels. The association of GAP-43 with the membrane skeleton, which by analogy with other cell types is involved in the dynamic regulation of cell shape, implies that GAP-43 in growth cones may be involved in this function.

Animals↗
PubMed1989

Direct visualisation of the soluble pool of tubulin in the neuronal growth cone: immunofluorescence studies following taxol polymerisation.

Previously we have shown that the predominant form of soluble alpha-tubulin in the growth cone is C-terminally tyrosinated (Gordon-Weeks and Lang. Dev. Brain Res., 42 (1988) 156-160). Here we show that when growth cones are incubated in taxol, this soluble pool of alpha-tubulin is polymerised onto the ends of the neurite microtubules that enter the proximal part of the growth cone indicating that it is assembly-competent.

Alkaloids↗
PubMed1989

Transient expression of laminin immunoreactivity in the developing rat hippocampus.

We have isolated and characterized a polyclonal antibody that recognizes the extracellular matrix glycoprotein laminin. In the developing rat hippocampus, in addition to staining the basal laminae of blood vessels and the glia limitans, this antibody detects a punctate deposit of non-basal lamina laminin that appears transiently, disappearing on E18. The punctate laminin is distributed throughout the hippocampus from the border of the ependyma and the neuropil to the pial surface, but is particularly concentrated in the presumptive molecular layer (marginal zone). Electron microscopy showed that the punctate laminin was in the extracellular space on the surfaces of cell bodies and, more commonly, their processes. In double-labelling experiments in which neurites were labelled with tau or MAP2 antibodies, there were occasional cases where neurites came into direct contact with punctate laminin. These observations suggest a role for laminin in hippocampal development and possibly in neurite outgrowth.

Animals↗