Growth cones isolated from identified Aplysia neurons in vitro: biochemical and morphological characterization

The right upper quadrant (RUQ) cells (R3-R13) of Aplysia regenerating in dissociated cell culture form unusually large growth cones. The movement of these growth cones was observed by time-lapse phase microscopy and their ultrastructure was examined by transmission electron microscopy. Their behavior and ultrastructure have features that are typical of growth cones in vitro. Additionally, they contain neurosecretory granules similar to those found in these cells in vivo. Because RUQ growth cones are large, they can be isolated by manual dissection. RUQ cells were grown in the presence of [35S]methionine and the labeled proteins transported to the growth cones were analyzed by SDS-PAGE. These proteins were compared to those in RUQ cell bodies, RUQ neurites, and to those in the neurites and cell bodies of other identified neurons grown in vitro. Most proteins synthesized by RUQ cells in vitro are transported to their growth cones, including several glycoproteins and the precursor to the R3-R14 neuropeptide. Neuropeptides are also synthesized by a number of other Aplysia neurons growing in vitro. We examined R2, LPL1, R15, and left upper quadrant neurons and found that their precursor peptides, like those of R3-R14, are readily recognized as major cell-specific radiolabeled bands on SDS gels. The presence in regenerating growth cones of neuropeptides, neurosecretory granules, and glycoproteins known to be rapidly transported toward synapses in vivo supports the emerging view that the growth cone in vitro contains not only a motility apparatus but also a macromolecular assembly capable of forming an active synapse immediately upon or shortly after contacting targets.     

WGA binding to the surface of two autologous human melanoma cell lines: different expression of sialyl and N-acetylglucosaminyl residues

Two autologous human melanoma cell lines were studied to determine their capacities to bind wheat germ agglutinin (WGA). Both cell lines were derived from the same patient, the first, IGR 39, originated from the primary tumor, the second, IGR 37, was established from a metastatic lymph node. WGA binding sites on the surface of these cell lines were compared before and after sialidase and/or tunicamycin treatments. IGR 39 cells exhibited two classes of WGA binding sites with high and low affinities, whereas IGR 37 cells had only one class of high affinity binding sites. After tunicamycin treatment, the capacity of IGR 39 cells to bind WGA was markedly altered, since only one class of WGA binding sites with high affinity was observed under these conditions, whereas tunicamycin did not induce significant changes in the lectin binding of IGR 37 cells. The low affinity WGA binding sites, which were only found on IGR 39 cells, corresponded to sialyl residues present in N-linked glycoproteins. The high affinity binding sites present on both cell lines probably involved sialyl and N-acetyl-glucosaminyl residues associated with O-linked glycoproteins and/or glycolipids. No direct correlation could be drawn between the number of WGA binding sites and the overall sialic acid levels exposed to sialidase treatment. The 3-fold increase in the amount of cell surface glycopeptides obtained after pronase digestion and specifically binding to WGA-Sepharose was in good agreement with the overall higher number of WGA binding sites on IGR 39 compared to IGR 37 cells. Thus, subtle carbohydrate changes of cell surface glycoconjugates might account for the differences between the biological properties of human melanoma cell lines of low and high tumorigenicity.     

Growth cones stall and collapse during axon outgrowth in Caenorhabditis elegans.

During nervous system development, neurons form synaptic contacts with distant target cells. These connections are formed by the extension of axonal processes along predetermined pathways. Axon outgrowth is directed by growth cones located at the tips of these neuronal processes. Although the behavior of growth cones has been well-characterized in vitro, it is difficult to observe growth cones in vivo. We have observed motor neuron growth cones migrating in living Caenorhabditis elegans larvae using time-lapse confocal microscopy. Specifically, we observed the VD motor neurons extend axons from the ventral to dorsal nerve cord during the L2 stage. The growth cones of these neurons are round and migrate rapidly across the epidermis if they are unobstructed. When they contact axons of the lateral nerve fascicles, growth cones stall and spread out along the fascicle to form anvil-shaped structures. After pausing for a few minutes, they extend lamellipodia beyond the fascicle and resume migration toward the dorsal nerve cord. Growth cones stall again when they contact the body wall muscles. These muscles are tightly attached to the epidermis by narrowly spaced circumferential attachment structures. Stalled growth cones extend fingers dorsally between these hypodermal attachment structures. When a single finger has projected through the body wall muscle quadrant, the growth cone located on the ventral side of the muscle collapses and a new growth cone forms at the dorsal tip of the predominating finger. Thus, we observe that complete growth cone collapse occurs in vivo and not just in culture assays. In contrast to studies indicating that collapse occurs upon contact with repulsive substrata, collapse of the VD growth cones may result from an intrinsic signal that serves to maintain growth cone primacy and conserve cellular material.     

Identification of two binding sites for wheat-germ agglutinin on polylactosamine-type oligosaccharides.

The carbohydrate-binding properties of wheat-germ agglutinin (WGA) have been studied by using glycopeptides isolated from the cell surfaces of a cultured murine myeloid cell line (416B). The glycopeptides were passed through affinity columns of lentil lectin (LCA), concanavalin A (Con A) and WGA arranged in series so that material reaching the WGA column had failed to bind to LCA or Con A. WGA-binding glycopeptides were step-eluted with 0.01 M, 0.1 M and 0.5 M-N-acetylglucosamine (GlcNAc), to yield weak (WGA-W), intermediate (WGA-I) and strong (WGA-S) affinity fractions. WGA-W and WGA-I contained 'N'- and 'O'-linked oligosaccharides bound to separate polypeptides. WGA-S consisted almost entirely of N-linked components. Our analytical work was concentrated mainly on the N-linked fractions. In these carbohydrates WGA affinity was directly proportional to molecular size but inversely related to N-acetylneuraminic acid content. The binding of the weak-affinity fraction was dependent on N-acetylneuraminic acid, but the intermediate- and strong-binding species interacted with the lectin by N-acetylneuraminic acid-independent mechanisms. N-linked glycopeptides in each WGA-binding class were almost totally degraded to monosaccharides by the concerted action of the exoglycosidases neuraminidase, beta-galactosidase and beta-N-acetylglucosaminidase. Treatment with endo-beta-galactosidase caused partial depolymerization, yielding some disaccharides but also a heterogeneous population of partially degraded components. These findings suggest that WGA binds with high affinity to internal GlcNAc residues in large oligosaccharides containing repeat sequences of Gal beta(1----4)GlcNAc beta(1----3) (i.e. polylactosamine-type glycans). N-Acetylneuraminic acid is involved only in low-affinity interactions with WGA. WGA therefore displays an intricate pattern of saccharide specificities that can be profitably utilized for structural analysis of complex carbohydrates.     

Ca2+ dynamics in neuronal growth cones: regulation and changing patterns of Ca2+ entry

Digital ratio imaging of Fura-2 fluorescence was used to determine spatially resolved dynamics of Ca2+ changes in neuronal growth cones from the molluscs, Helisoma and Aplysia. Time resolution was approximately 1 s and spatial resolution a few mm depending upon the thickness of the cell region examined. Isolated growth cones of Helisoma were shown to recover from large Ca2+ loads over a time course of minutes, therefore demonstrating Ca2+ regulation mechanisms not dependent on the rest of the cell. Ca2+ changes monitored during action potential discharge showed sharply defined spatial gradients within the growth cones, probably arising from clustering of voltage-gated Ca-channels in the surface membrane. The regions of peak concentration change appeared to shift from central regions to the growth cone periphery as the growth cones matured. There was a marked difference in soma Ca2+ changes produced by action potentials depending on whether or not the soma had sprouted neurites. Neurite-free somata showed large Ca2+ changes, whereas in somata that had recently sprouted neurites there were almost no changes for similar electrical stimulation. Measurements on growth cones of N1E115 neuroblastoma cells showed static distributions of Ca2+ similar to those in the molluscan neurons.     

Fate of constitutive endocytic vesicles formed in the growth cone: transport of vesicles from one growth cone to another in the same neuron

A developing neuron must have multiple paths of communication coordinating events among all its parts. One of these is the transport to the cell body of endocytic vesicles formed in growth cones. In order to observe this at single cell resolution, we developed a technique in which the fluorescent dye FM1-43 was applied to a single growth cone and newly formed constitutive endocytic vesicles were labeled. Using low light, time-lapse microscopy we were able to follow the movement of these vesicles throughout the neuron. The vast majority of the transported vesicles went to the cell body. However, many were observed to enter secondary neurites and to be transported to other growth cones. These new, more direct paths of transport that link the multiple growth cones of a neuron may play a role in several important developmental events involving interactions between the multiple neurites of a single neuron.     

Growth cone-growth cone interactions in cultures of rat sympathetic neurons

Growth cones of sympathetic neurons from the superior cervical ganglia of neonatal rats were studied using video-microscopy to determine events following contact between growth cones and other cell surfaces, including other growth cones and neurites. A variety of behaviors were observed to occur upon contact between growth cones. Most commonly, one growth cone would collapse and subsequently retract upon establishing filopodial contact with the growth cone of another sympathetic neuron. Contacts resulting in collapse and retraction were often accompanied by a rapid and transient burst of lamellipodial activity along the neurite 30-50 microns proximal to the retracting growth cone. In no instances did interactions between growth cones and either fibroblasts or red blood cells result in the growth cone collapsing, suggesting that a specific recognition event was involved. On several occasions, growth cones were seen to track other growth cones, although fasciculation was rare. In some cases, there was no obvious response between contacting growth cones. Growth cone-growth cone contact was almost four times more likely to result in collapse and retraction than was growth cone-neurite contact (45% vs 12%, respectively). These observations suggest that the superior cervical ganglion may be composed of neurons with different cell surface determinants and that these determinants are more concentrated on the surface of growth cones than on neurites. These results further suggest that contact-mediated inhibition of growth cone locomotion may play an important role in growth cone guidance.     

Substrate-bound factors stimulate engorgement of growth cone lamellipodia during neurite elongation.

The surfaces on which neurons grow greatly affect neurite elongation, but it is unclear how substrates influence the events within the growth cone that bring about elongation. Neurite elongation by Aplysia californica neurons in culture occurs through a series of transformations of the structures of the growth cone (Goldberg and Burmeister, J. Cell Biol., 103:1921-1931, 1986). The growth cone produces actin-rich protrusions, veils, and lamellipodia, which can then mature into the central body of the growth cone through the net advance of microtubules and membranous organelles from contiguous central regions, a process called "engorgement." Aplysia neurons form growth cones on poly-l-lysine-treated substrates, but their rate of neurite elongation is greatly enhanced on substrates additionally exposed to Aplysia hemolymph. The acute application of hemolymph to slowly growing neurites brings about a rapid acceleration of neurite elongation and engorgement. The enhancement of engorgement was effected with material eluted from hemolymph-treated substrates and was not seen when hemolymph was added to neurons cultured on hemolymph-treated substrates inactivated by exposure to UV radiation. Thus, we conclude that the rapid acceleration of engorgement caused by hemolymph is, in large part, a substrate-mediated effect. We propose that extracellular substrate molecules can modulate the rate of neurite growth through the regulation of the engorgement of lamellipodia. The microtubule disrupters colcemid and nocodazole inhibit the advance of vesicular elements into the lamellipodia following hemolymph treatment, but taxol, which promotes the polymerization and stabilization of microtubules, does not itself enhance engorgement. The microfilament disrupter cytochalasin B, however, stimulates engorgement. Our results suggest that regulating the resistance of the peripheral actin meshwork to penetration by microtubules and vesicles may be a mechanism by which substrate-attached molecules regulate neurite advance.     

Analysis of the glycopeptides derived from the extracellular matrix secreted by cultured bovine aortic smooth muscle cells

Modulation of smooth muscle cell behaviour in culture has been associated with changes in the extracellular matrix. In the present study cultures of bovine aortic smooth muscle cells were compared in the rapidly proliferating and confluent phases of growth. The extracellular matrix was similar in both phases of growth and consisted of glycoproteins ranging from molecular weight 20,000 to over 200,000. The glycopeptides derived from these components displayed several differences. N-linked heteropolysaccharides of the biantennary and complex (more than two branches) types were predominant in the matrix of the confluent phase. Larger amounts of high mannose glycopeptides were present in the preparations from proliferating cells. O-Glycosidic glycopeptides were minor components in both preparations, but a slight increase was noted in the confluent phase of growth. Some of the changes in glycopeptides were interpreted in terms of the levels of the major components of the matrix such as the interstitial procollagens and fibronectin. The results indicate that processing of oligosaccharides associated with secreted glycoproteins of the extracellular matrix correlates with the state of growth of smooth muscle cells in culture.     

Targeting of neuromodulin (GAP-43) fusion proteins to growth cones in cultured rat embryonic neurons.

Neuromodulin (GAP-43) is a membrane protein that is transported to neuronal growth cones. Zuber and co-workers have proposed that the N-terminal 10 amino acid sequence of neuromodulin is sufficient to target proteins to growth cones. We demonstrate that a neuromodulin-beta-galactosidase fusion protein is transported to growth cones of cultured rat neurons, whereas a fusion protein containing the N-terminal 10 amino acids of neuromodulin and beta-galactosidase is not. A mutant neuromodulin lacking cysteines 3 and 4, the palmitylation sites required for membrane attachment, does not target beta-galactosidase to growth cones. We conclude that membrane attachment is required for growth cone accumulation and that structural elements, in addition to the first 10 amino acids of neuromodulin, may be required for growth cone targeting.     

Geometry of isolated sensory neurons in culture. Effects of embryonic age and culture substratum

Sensory neurons were dissociated from lumbar dorsal root ganglia of embryonic chick and put into culture, either directly or after removing non-neuronal cells by density gradient centrifugation. The cells were grown on culture substrata of various kinds in medium containing nerve growth factor (NGF). After 24 h the cultures were fixed, mounted and analysed. Lengths of neurites were measured, and the numbers of primary processes formed at the cell body and of growth cones were counted. From these values, the rates of growth cone advance and frequency of growth cone branching were calculated. Neuronal outgrowths increased strikingly in length and complexity with embryonic age; there was a 3.5-fold increase in total neurite length and a 3-fold increase in the number of growth cones when neurons from 15-day embryos (E15) were compared with those from 8-day embryos (E8) grown on the same substratum (glass). Growth was markedly greater on surfaces prepared with laminin or conditioned medium compared with plain glass or air-dried collagen. When E15 neurons grown on glass were compared with those grown on laminin, for example, a 2.5-fold increase in total neurite length and a 3-fold increase in the number of growth cones was observed. Calculations showed that a major factor in these changes was an increase in the frequency of growth cone branching. The number of initial processes emanating from the cell body changed with age, but not with the different substrata tested. Non-neuronal cells when present in low numbers and in contact with neurons did not appear to influence neuronal geometry in a systematic way. Our results document the fact that both external factors (in this case, the nature of the culture substratum) and intrinsic factors (stage of development of the neuron) can influence the geometry of neurite outgrowth.     

Binding of wheat germ agglutinin to extracellular network produced by cultured human fibroblasts

The occurrence of diverse carbohydrate moieties on the cell surface and in the extracellular matrix, makes lectins the suitable probes to study the distribution of appropriate determinants produced in cell culture. Biotin-labelled wheat germ agglutinin (WGA) was used in microscopic and photometric detection of lectin binding to monolayer of human skin fibroblasts. The incubation of confluent fibroblast monolayer with labelled WGA reveals two principal patterns of binding of this lectin: to cell surface structures and, predominantly, to extracellular fibres; the alignment and density of extracellular network are not uniform. After binding of WGA to confluent culture, light microscopic analysis revealed the ubiquitous fibrillar network between and over cells, with some regions of increased compactness and altered orientation of fibrils. Binding to cell surfaces (manifested as specks) was predominant for the fibroblasts at the logarithmic phase of growth. N-acetylglucosamine (0.2 M) and native lectin (100 microg/ml) had a partial inhibitory effect on WGA binding to the extracellular network. Treatment with neuraminidase (0.1 unit/ml) of untreated or prefixed monolayers resulted in a significant decrease in WGA binding to fibrils (and increase in PNA binding), indicating that terminal sialic acid residues are mainly involved in the network-WGA interaction. Mild trypsinization (10 microg/ml) removed the target sites, which retained the ability to bind WGA, being spotted on hydrophobic Immobilon P paper; biotinylated lectin, bound to adsorbed glycopeptides, could be eluted and quantified in solid-phase inhibition assay.     

Pincher-generated Nogo-A endosomes mediate growth cone collapse and retrograde signaling

Nogo-A is one of the most potent myelin-associated inhibitors for axonal growth, regeneration, and plasticity in the adult central nervous system. The Nogo-A–specific fragment NogoΔ20 induces growth cone collapse, and inhibits neurite outgrowth and cell spreading by activating RhoA. Here, we show that NogoΔ20 is internalized into neuronal cells by a Pincher- and rac-dependent, but clathrin- and dynamin-independent, mechanism. Pincher-mediated macroendocytosis results in the formation of NogoΔ20-containing signalosomes that direct RhoA activation and growth cone collapse. In compartmentalized chamber cultures, NogoΔ20 is endocytosed into neurites and retrogradely transported to the cell bodies of dorsal root ganglion neurons, triggering RhoA activation en route and decreasing phosphorylated cAMP response element binding levels in cell bodies. Thus, Pincher-dependent macroendocytosis leads to the formation of Nogo-A signaling endosomes, which act both within growth cones and after retrograde transport in the cell body to negatively regulate the neuronal growth program.     

Neuron-specific membrane glycoproteins promoting neurite fasciculation in Aplysia californica

We have generated a library of mouse monoclonal antibodies against membrane proteins of the nervous system of the marine snail Aplysia californica. Two of these antibodies, 4E8 and 3D9, recognize a group of membrane glycoproteins with molecular masses of 100-150 kD. We have called these proteins ap100, from the molecular mass of the most abundant species. Based on Western blots, these proteins appear to be specific for the nervous system. They are enriched in the neuropil of central nervous system ganglia, and are present on the surface of neurites and growth cones of neurons in culture. They are not expressed on the surface of nonneuronal cells. Staining of living cells with fluorescently labeled mAb demonstrates that the epitope(s) are on the outside of the cell. The antibodies against the proteins defasciculate growing axons and alter the morphology of growth cones, but affect much less adhesion between neuritic shafts. In addition, the level of expression of these molecules appears to correlate with the degree of fasciculation of neurites. These observations suggest that the ap100 proteins are cell adhesion molecules that play a role in axon growth in the nervous system of Aplysia. The fact that they are enriched in the neuropil and possibly in varicosities suggest that they may also be relevant for the structure of mature synapses.     

Three types of transmitter release from embryonic neurons

Prior to the contact with their target muscle cells in culture, growth cones of many isolated Xenopus embryonic neurons release acetylcholine (ACh) spontaneously. Using patch clamp techniques, this release can be detected by an outside-out patch of muscle membrane placed near the growth cone. Intracellular recording from innervated muscle cells showed spontaneous miniature endplate potentials (MEPPs) of varying amplitudes. Amplitude histograms showed a skewed distribution with multiple peaks, suggesting the existence of subunits in either the quantal packages of ACh released by the nerve terminal or in the postsynaptic muscle response. In addition to the quantal ACh release reflected by MEPPs, nerve terminal also release a large amount of ACh in a non-quantal fashion. This non-quantal ACh release is revealed by the hyperpolarization of the muscle membrane following extracellular application of curare or alpha-bungarotoxin, as well as by denervation of the muscle cell.     

Lectin binding and uptake and glycoprotein characterization of isolated porcine aortic endothelial and smooth muscle cells

Endothelial and smooth muscle cells were isolated from porcine aorta and kept in short-term culture. To determine the terminal carbohydate composition of the plasma membranes from both cell populations, the cells were incubated with a panel of fluorescein-labelled lectins. Both cell populations shared a number of terminal carbohydrates, but the N-galactosamine specific lectin Wistaria floribunda agglutinin labelled only the endothelial cells. A lectin which selectively labelled smooth muscle cells was not found. Western blot analysis of isolated endothelial cell membrane glycoproteins indicated that most membrane glycoproteins are labelled by Wistaria floribunda agglutinin.     

Developmentally-related changes in surface membrane glycopeptides of murine haemopoietic cells.

We have carried out a comparative study of mature murine granulocytes with two immature haemopoietic cell lines (multipotential cells, FDCP-Mix, and granulocyte progenitor cells, FDCP-2) with respect to the structure and composition of their surface membrane glycopeptides. The glycopeptides were labelled biosynthetically by incubation of the cells for 1-3 days with [3H]glucosamine. Cell-associated glycopeptides were released by treatment with trypsin and the trypsin extract was exhaustively digested with Pronase to remove most residual peptide. Radiolabelled materials were fractionated by chromatography on lectin affinity columns connected in the series: lentil lectin (LCA), concanavalin A (Con A) and wheat germ agglutinin (WGA). Lectin-binding glycopeptides were eluted with appropriate competing sugars and further analysed by gel filtration, base/borohydride elimination and susceptibility to degradation by glycosidases including endo-beta-galactosidase. Abundant quantities of N-linked polylactosamine-type glycopeptides, which bound only to the WGA columns, were identified on mature granulocytes but the molecules were highly-branched (i.e. resistant to endo-beta-galactosidase). In contrast, there seemed to be very little branching in the polylactosamine chains from FDCP-2 cells, whilst corresponding carbohydrates from multipotential FDCP-Mix cells gave evidence for both linear and branched domains in the same, large complex glycans. O-Linked tetrasaccharides of general structure: NeuAc-Gal-(NeuAc)-GalNAc were found in clusters on WGA-binding glycopeptides from all cell types, these components being especially prominent on mature granulocytes. FDCP-2 cells were distinguished by the presence of monosialylated and non-sialylated counterparts of the foregoing tetrasaccharides. The relative amount of LCA-binding glycopeptides was low on FDCP-Mix cells by comparison with FDCP-2 cells and mature granulocytes. Our findings therefore demonstrate that notable differences in gross composition and molecular fine structure of surface membrane glycopeptides are detectable in haemopoietic cells at different stages of development. The relationship of these differences to the biological properties of cell surfaces remains to be established.     

Axonal growth during regeneration: a quantitative autoradiographic study

The intraaxonal distribution of labeled glycoproteins in the regenerating hypoglossal nerve of the rabbit was studied by use of quantitative electron microscope autoradiography. 9 d after nerve crush, glycoproteins were labeled by the administration of [3H]fucose to the medulla. The distribution of transported 3H-labeled glycoproteins was determined 18 h later in segments of the regenerating nerve and in the contralateral, intact nerve. At the regenerating tip, the distribution was determined both in growth cones and in non-growth cone axons, 6 and 18 h after labeling. The distribution within the non-growth cone axons of the tips was quite different at 6 and 18 h. At 6 h, the axolemma region contained < 10% of the radioactivity; at 18 h, it contained virtually all the radioactivity. In contrast, the distribution within the growth cones was similar at both time intervals, with 30% of the radioactivity over the axolemmal region. Additional segments of the regenerating nerve also showed a preferential labeling of the axolemmal region. In the intact nerve, 3H-labeled glycoproteins were uniformly distributed. These results suggest that: (a) in this system the labeled glycoproteins reaching the tip of the regenerating axons are inserted into the axolemma between 6 and 18 h after leaving the neuronal perikaryon; (b) at the times studied, there is a fairly constant ratio between glycoproteins reaching the growth cone through axoplasmic transport and glycoproteins inserted into the growth cone axolemma; (c) the axolemma elongates by continuous insertion of membrane precursors at the growth cone; the growth cone then advances, leaving behind an immature axon with a newly formed axolemma; and (d) glycoproteins are preferentially inserted into the axolemma along the entire regenerating axon.     

Combining various strategies to increase the coverage of the plant cell wall glycoproteome

Glycoproteomics recently became a very active field, mostly in mammals. The first part of this paper consists of a mini-review on the strategies used in glycoproteomics, namely methods for enrichment in glycoproteins and mass spectrometry (MS) techniques currently used. In a second part, these strategies are applied to the cell wall glycoproteome of etiolated hypocotyls of Arabidopsis thaliana, showing their complementarity. Several sub-glycoproteomes were obtained by: (i) affinity chromatography on concanavaline A (ConA) and analysis of glycoproteins by MALDI-TOF MS; (ii) multidimensional lectin chromatography (using AIL, PNA, ConA and WGA lectins) and subsequent identification of glycoproteins by MALDI-TOF MS and LC-MS/MS; (iii) boronic acid chromatography followed by identification of glycoproteins by MALDI-TOF MS. Altogether, 127 glycoproteins were identified. Most glycoproteins were found to be putative N-glycoproteins and N-glycopeptides were predicted from MS data using the ProTerNyc bioinformatics software.