Cooperativity of ganglioside-dependent with protein-dependent substratum adhesion and neurite extension of human neuroblastoma cells

The potential involvement of gangliosides in the adherence and neurite extension of human neuroblastoma cells (Platt and La-N1) was investigated on tissue culture substrata coated with the ganglioside GM1-binding protein, cholera toxin B (CTB) subunit, for comparison with similar processes on plasma fibronectin (pFN)-coated substrata. Cells attached with reduced efficiency on CTB substrata as compared with pFN substrata and required a much longer time to form neurite processes for a small percentage of cells on CTB. The specificity of these processes for GM1 binding was tested in a variety of ways. Supplementation of the cells with exogenous GM1, but not GD1a, identified a larger population of cells adherent on CTB (comparable to pFN-adherent cells) and dramatically increased the proportion of cells capable of forming neurites without reducing the time requirement. In ultrastructural studies using the scanning electron microscope (SEM) and immunofluorescence (IF) analyses to discriminate microtubule distributions, neurites of GM1-supplemented cells on CTB were virtually identical with pFN-adherent neurites, whereas unsupplemented cells on CTB generated processes with fine-structural differences. Treatment of cells during the GM1 supplementation period with cycloheximide completely abolished the ability of cells to generate neurites on CTB and decreased the adhesive capacity of cells as well; a similar treatment of cells had no adverse effect on adherence or neurite extension on pFN. The importance of one or more proteins in GM1-dependent processes was further confirmed by demonstrating the trypsin sensitivity of a cell surface component(s) required to achieve maximal attachment on CTB; in contrast, adherence and neurite extension on pFN were much more resistant to this treatment process. Therefore, these experiments demonstrate (a) that certain cell surface gangliosides are capable of mediating adherence and neurite outgrowth of human neuroblastoma cells on a suitable ganglioside-binding substratum; (b) this ganglioside dependence is cooperative with one or more cell surface proteins which can now be analysed. These results are discussed in light of the identification in ref. [16] (Exp cell res 169 (1987) 311) of a second ‘cell-binding’ domain on the pFN molecule competent for adherence and neurite extension of these neuroblastoma cells, as well as the potential role of pFN binding to a complex ganglioside on the surface of these neural tumor cells in these processes.     

Ganglioside-dependent adhesion events of human neuroblastoma cells regulated by the RGDS-dependent fibronectin receptor and proteoglycans

Human neuroblastoma cells (Platt and La-N1) adhere and extend neurites on a ganglioside GM1-binding substratum provided by cholera toxin B (CTB). These adhesive responses, similar to those on plasma fibronectin (pFN), require the mediation of one or more cell-surface proteins [G. Mugnai and L. A. Culp (1987) Exp. Cell Res. 169, 328]. The involvement of two pFN receptor molecules in ganglioside GM1-mediated responses on CTB have now been tested. In order to test the role of cellular FN binding to its glycoprotein receptor integrin, a soluble peptide containing the Arg-Gly-Asp-Ser (RGDS) sequence was added to the medium. It did not inhibit attachment on CTB but completely inhibited formation of neurites; in contrast, the RGDS peptide minimally inhibited attachment or neurite formation on pFN. Once formed, neurites on CTB became resistant to the peptide. In order to test the role of cell-surface heparan sulfate proteoglycan (HS-PG), two approaches were used. First, the HS-binding protein platelet factor-4 (PF4) was used to dilute CTB or pFN on the substratum or, alternatively, added to the medium. Diluting the substratum ligand with PF4 had no effects on attachment on either CTB or pFN. However, neurite formation on CTB was readily inhibited and on pFN partially inhibited; the effects of PF4 were far greater than a similar dilution with nonbinding albumin. When PF4 was added to the medium of cells, attachment on either substratum was unaffected as was neurite outgrowth on pFN, revealing differences in PF4's inhibition as the substratum-bound or medium-borne component. In contrast, PF4 in the medium at low concentrations (1 microgram/ml) was highly inhibitory for neurite formation on CTB. The second approach utilized the addition of bovine cartilage dermatan sulfate proteoglycan (DS-PG), shown to bind to pFN as well as to substratum-bound CTB by ELISA, or cartilage chondroitin sulfate/keratan sulfate proteoglycan (CS/KS-PG) to the substratum or to the medium. At low concentrations, DS-PG but not CS/KS-PG actually stimulated neurite formation on CTB while at higher concentrations DS-PG completely inhibited attachment and neurite formation. While DS-PG partially inhibited attachment on pFN, it had no effect on neurite formation of the attached cells. Neuroblastoma cells adhered to some extent to substrata coated only with DS-PG, indicating "receptors" for PGs that permit stable interaction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Substratum contacts and cytoskeletal reorganization of BALB/c 3T3 cells on a cell-binding fragment and heparin-binding fragments of plasma fibronectin

BALB/c 3T3 cells make both close contacts and tight-focal contacts (with associated microfilament stress fibers) on plasma fibronectin (pFN)-coated substrata. To resolve the importance of the heparan sulfate-binding or cell-binding activities of the pFN molecule in these adhesive responses, a cell-binding fragment (120K) (CBF) free of any heparan sulfate-binding activity was prepared from human pFN by chymotrypic digestion and isolated as described by Pierschbacher et al. (Cell 26 (1981) 259). These adhesive responses to CBF were also compared to those of the model heparan sulfate-binding protein, platelet factor-4 (PF4), or heparin-binding fragments (HBF) of pFN. On intact pFN, greater than 70% of the cells formed tight-focal contacts and associated stress fibers by 4 h, the latter staining with NBD-phallacidin. In contrast, cells spread differently on CBF and failed to form tight-focal contacts; staining with NBD-phallacidin was localized to spiky projections at the cell margin with no detectable stress fiber formation. On PF4 or HBF, cells failed to form tight-focal contacts but did spread well and formed long microfilament bundles in peripheral lamellae. Spreading on CBF, HBF, or PF4 was paralleled by formation of close contacts. Spreading and to some extent attachment of cells on CBF was inhibited with a small peptide containing the Arg-Gly-Asp-Ser sequence; responses on HBF were unaffected by this peptide. When mixtures of CBF and PF4 were tested, cells still failed to form tight-focal contacts and stress fibers. These results demonstrate that the binding of CBF to its probable receptor under conditions routinely used to assay spreading activity results in an incomplete adhesive response compared with intact pFN. While this partial response may result from quantitative differences in the density of active cell-binding domains on the substratum, the pattern of microfilament reorganization produced by the binding of PF4 to cell surface heparan sulfate proteoglycans suggests that the ability of pFN to promote formation of tight-focal contacts and stress fibers may reside in the coordinate interaction of two or more binding activities in the intact molecule.     

Neurite extension by neuroblastoma cells on substratum-bound fibronectin''s cell-binding fragment but not on the heparan sulfate-binding protein, platelet factor-4

Human and rat neuroblastoma cells extend neurites over plasma fibronectin (pFN)-coated substrata. For resolution of which fibronectin binding activities (the cell-binding domain (CBD), the heparan sulfate-binding domains, or a combination of the two) are responsible for neurite outgrowth, CBD was prepared free of heparan sulfate-binding activity as described by Pierschbacher et al. (Cell 26 (1981) 259-267). Neuroblastoma cells attached and extended neurites as stably and as effectively on CBD-coated substrata as on intact pFN, while cytoplasmic spreading was more extensive on pFN-coated substrata. The structures of growth cones on CBD or pFN were virtually identical. On substrata coated with the model heparan sulfate-binding protein, platelet factor 4 (PF4), cells attached and spread somewhat but never extended neurites. When cells were challenged with substrata coated with various ratios of CBD and PF4, PF4 was found to be an effective inhibitor of CBD-mediated neurite extension. Similarly, cells grown on substrata coated at different locations with CBD or PF4 in order to evaluate topographical dependence of growth cone formation extended neurites only onto the CBD-coated region or along the interface between these two proteins, but never onto the PF4 side of cells that bridged the interface. These studies indicate that (a) the CBD activity of pFN, and not its heparan sulfate-binding activity, is the critical determinant in neurite extension of these neural tumor cells from the central nervous system; (b) under some circumstances, heparan sulfate-binding activity can be antagonistic to neurite extension; (c) the chemical nature of the substratum controls the direction of neurite extension; (d) these neuroblastoma cells respond to these binding proteins very differently than fibroblasts or neurons from the peripheral nervous system.     

Multiple and alternative adhesive responses on defined substrata of an immortalized dorsal root neuron hybrid cell line

Attachment and neurite extension processes have been evaluated for an immortalized derivative cell of a rat dorsal root neuron after fusion with a mouse neuroblastoma cell (the clonal F11 hybrid cell line) and these processes compared with previous studies of neuroblastoma cells, since both cell types may be derived from the neural crest of the developing embryo. Biochemically defined substrata were provided by human plasma fibronectin (pFN), the heparan sulfate-binding protein platelet factor-4 (PF4), and the ganglioside GM1-binding protein cholera toxin B subunit (CTB). While some attachment of unsupplemented cells was noted on CTB substrata, GM1 supplementation permitted F11 cells to attach as well on CTB as on pFN or PF4. On PF4, very few neurite processes were observed while on pFN two morphologically distinct types of neurites could be identified: short, linear processes in a low percentage of cells resembling those of neuroblastoma cells and long, irregular and narrow processes in a higher percentage of cells resembling those of dorsal root neurons. On CTB, neurites of the latter class were even more prominent; however, cell bodies on CTB failed to spread by cytoplasmic extension as commonly observed in F11 cells on pFN and, to some extent, on PF4. The formation of both neurite classes on either pFN or CTB was completely inhibited by low concentrations of an RGDS (Arg-Gly-Asp-Ser) peptide in the medium of cultures, indicating the significance of pFN's binding to cell surface integrin or ganglioside GM1's possible interaction with integrin for mediating the differentiative process. In contrast, neurite formation of neuroblastoma cells is refractile to the soluble peptide as reported previously. Neurite extensions of F11 cells on either pFN or CTB were comparably sensitive to low concentrations of cytochalasin D, revealing the mediation of microfilament reorganization in these processes. Treatment of F11 cells with cycloheximide failed to inhibit neurite extension on pFN but did partially inhibit extension on CTB; this contrasts with the very high sensitivity of neurite formation by neuroblastoma cells on CTB substrata reported previously.(ABSTRACT TRUNCATED AT 400 WORDS)     

Clonal segregation of multiple and overlapping matrix adhesive responses in dorsal root neuronal derivative cells

Clones of F11 hybrid (neuroblastoma X dorsal root neuron) cells have been tested for adherence and neurite outgrowth on three different substrata on which the parental cells display some competence--plasma fibronectin (pFN) with its multiple receptors, cholera toxin subunit B(CTB) as a model ganglioside GM1-binding substratum, and platelet factor-4 (PF4) as a model proteoglycan-binding substratum. This paradigm tests for independently segregating and overlapping mechanisms of neuritogenesis via transmembrane processes in pluripotent hybrid cells based on random loss of chromosomes contributed by the parent neural cells. For the nine clones tested, attachment was significantly lower on CTB but much higher on PF4 for all clones when compared to their attachment on pFN. Supplementation of cells with GM1 stimulated attachment of only two clones (on all three substrata). Neurite outgrowth was observed in a substratum-specific pattern and varied from 0 to greater than 60% on pFN; on CTB and PF4 substrata, the patterns were similar to each other but differed markedly from the pattern on pFN. In contrast, PF4- and CTB-directed neurites differed morphologically from each other while sharing some characteristics with neurites on pFN. Supplementation with GM1 or GT1b, but not GD1a, was inhibitory for neurite outgrowth in certain clones. Cycloheximide pretreatment distinguished several classes of clones based on inhibition of neuritogenesis. While most clones on pFN were unaffected, all clones on CTB and PF4 displayed significant and comparable degrees of inhibition, suggesting the sharing of some protein intermediate(s) on these substrata. Exposure to cycloheximide only during the active period of neuritogenesis generated higher percentages and longer neurites for all clones, indicating a widely-used negative regulation mechanism. Based on substratum type and cycloheximide protocols, these data have resolved six or more different transmembrane signalling processes for generating different classes of neurites. Some mechanisms have been segregated into individual clones while others overlap in other clones, providing cell systems for biochemical and molecular biological dissection of these processes.     

Fibronectin-mediated adhesion of fibroblasts: inhibition by dermatan sulfate proteoglycan and evidence for a cryptic glycosaminoglycan-binding domain

Dermatan sulfate proteoglycans (DS-PGs) isolated from bovine articular cartilage have been examined for their effects on the adhesive responses of BALB/c 3T3 cells and bovine dermal fibroblasts on plasma fibronectin (pFN) and/or type I collagen matrices, and compared to the effects of the chondroitin sulfate/keratan sulfate proteoglycan monomers (CS/KS-PGs) from cartilage. DS-PGs inhibited the attachment and spreading of 3T3 cells on pFN-coated tissue culture substrata much more effectively than the cartilage CS/KS-PGs reported previously; in contrast, dermal fibroblasts were much less sensitive to either proteoglycan class unless they were pretreated with cycloheximide. Both cell types failed to adhere to substrata coated only with the proteoglycans; binding of the proteoglycans to various substrata has also been quantitated. While a strong inhibitory effect was obtained with the native intact DS-PGs, little inhibitory effect was obtained with isolated DS chains (liberated by alkaline-borohydride cleavage) or with core protein preparations (liberated by chondroitinase ABC digestion). In marked contrast, DS-PGs did not inhibit attachment or spreading responses of either 3T3 or dermal fibroblasts on type I collagen-coated substrata when the collagen was absorbed with pFN alone, DS-PGs alone, or the two in combination. These results support evidence for (a) collagen-dependent, fibronectin-independent mechanisms of adhesion of fibroblasts, and (b) different sites on the collagen fibrils where DS-PGs bind and where cell surface "receptors" for collagen bind. Experiments were developed to determine the mechanism(s) of inhibition. All evidence indicated that the mechanism using the intact pFN molecule involved the binding of the DS-PGs to the glycosaminoglycan (GAG)-binding sites of substratum-bound pFN, thereby inhibiting the interaction of the fibronectin with receptors on the cell surface. This was supported by affinity chromatography studies demonstrating that DS-PGs bind completely and effectively to pFN-Sepharose columns whereas only a subset of the cartilage CS/KS-PG binds weakly to these columns. In contrast, when a 120-kD chymotrypsin-generated cell-binding fragment of pFN (CBF which has no detectable GAG-binding activity as a soluble ligand) was tested in adhesion assays, DS-PGs inhibited 3T3 adherence on CBF more effectively than on intact pFN. A variety of experiments indicated that the mechanism of this inhibition also involved the binding of DS-PGs to only substratum-bound CBF due to the presence of a cryptic GAG-binding domain not observed in the soluble CBF.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of matrix adhesive responses of human neuroblastoma cells by neighboring sequences in the fibronectins

Attachment and neurite extension have been measured when Platt or La-N1 human neuroblastoma cells respond to tissue culture substrata coated with a panel of complementary fragments from the individual chains of human plasma (pFN) or cellular fibronectins (cFN) purified from thermolysin digests. A 110-kD fragment (f110), which contains the Arg-Gly-Asp-Ser sequence (RGDS)-dependent cell-binding domain but no heparin-binding domains and whose sequences are shared in common by both the alpha- and beta-subunits of pFN, facilitated attachment of cells that approached the level observed with either intact pFN or the heparan sulfate-binding platelet factor-4 (PF4). This attachment on f110 was resistant to RGDS-containing peptide in the medium. Neurite outgrowth was also maximal on f110, and half of these neurites were also resistant to soluble RGDS peptide. Treatment of cells with glycosaminoglycan lyases failed to alter these responses on f110. Therefore, there is a second "cell-binding" domain in the sequences represented by f110 that is not RGDS- or heparan sulfate-dependent and that facilitates stable attachment and some neurite outgrowth; this domain appears to be conformation-dependent. Comparisons were also made between two larger fragments generated from the two subunits of pFN-f145 from the alpha-subunit and f155 from the beta-subunit--both of which contain the RGDS-dependent cell-binding domain and the COOH-terminal heparin-binding domain but which differ in the former's containing some IIICS sequence at its COOH terminus and the latter's having an additional type III homology unit. Heparin-binding fragments (with no RGDS activity) of f29 and f38, derived from f145 or f155 of pFN, respectively, and having the same differences in sequence, were also compared with f44 + 47 having the "extra domain" characteristic of cFN. Attachment on f145 was slightly sensitive to soluble RGDS peptide; attachment on f155 was much more sensitive. There were also differences in the percentage of cells with neurites on f145 vs. f155 but neurites on either fragment were completely sensitive to RGDS peptide. Mixing of f29, f38, or PF4 with f110 could not reconstitute the activities demonstrated in f145 or f155, demonstrating that covalently linked sequences are critical in modulating these responses. However, mixing of f44 + 47 from cFN with f110 from pFN increased the sensitivity to RGDS peptide.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of fibronectin adhesive functions for fibroblasts and neural cells by chemically derivatized substrata

Adhesion responses of fibroblasts (Balb/c 3T3 cells) and human neuron-derived (Platt neuroblastoma) cells have been examined with plasma fibronectin (pFN) adsorbed to glass surfaces derivatized with an alkyl chain and six chemical end groups interfacing with the bound pFN to test regulation of pFN function. Using new derivatization protocols, the following surfaces have been tested in order of increasing polarity: [CH3], [C = C], [Br], [CN], [Diol], [COOH], and underivatized glass [( SiOH]). For all substrata, pFN bound equivalently using either a supersaturating amount of pFN or a subsaturating amount in competition with bovine albumin. Attachment of both cell types was also equivalent on all substrata. However, spreading/differentiation responses varied considerably. F-actin reorganization was tested in 3T3 cells with rhodamine-phalloidin staining. While stress fibers formed effectively on pFN-coated [SiOH] and [Br] substrata, only small linear bundles of F-actin and a few thin stress fibers were observed on the [COOH], [Diol], and [CN] substrata; the hydrophobic substrata [( CH3] and [C = C]) gave an intermediate response. When a synthetic peptide containing the Arg-Gly-Asp-Ser sequence required for integrin binding to FNs was included in the medium as an inhibitor, additional differences were noted: Stress fiber formation was completely inhibited on [SiOH] but not on [Br] and stress fiber formation was very sensitive to inhibition on the hydrophobic substrata while the F-actin patterns on the [CN] and [COOH] substrata were unaffected. Evaluation of neurite outgrowth by neuroblastoma cells on these substrata revealed both qualitative and quantitative differences as follows: [Diol] = [COOH] greater than [SiOH] much greater than [CN] = [Br] greater than [CH3] = [C = C]. While there was poor cytoplasmic spreading and virtually no neurites formed on the hydrophobic surfaces when pFN alone was adsorbed, neurite formation could be "rescued" if a mixture of pFN with an excess of bovine albumin was adsorbed, demonstrating complex conformational interactions between substratum-bound pFN and adhesion-inert neighboring molecules. In summary, these studies demonstrate that different chemical end groups on the substratum modulate pFN functions for cell adhesion, principally by affecting the conformation of these molecules rather than the amounts bound. Furthermore, these studies confirm multiple-receptor interactions with the FN molecules in cell type-specific adhesion patterns.(ABSTRACT TRUNCATED AT 400 WORDS)     

Promotion of retinal neurite outgrowth by substratum-bound fibronectin

We have examined conditions under which aggregates of embryonic chick neural retina will extend neurities in vitro. Trypsin-dispersed cells from 7-day embryonic chick neural retina were aggregated in rotation culture for 8 hr and maintained in serum-free medium on a variety of standard culture substrate. Aggregates extend few neurites on untreated plastic, glass, or collagen substrata. However, pretreatment of these substrata with human plasma fibronectin enhances their capacity to support retinal neurite outgrowth. Aggregates cultured on fibronectin-treated substrata extend long, radially oriented neurites within 36 hr in vitro. The morphology of these neurites is distinct from that seen when aggregates are cultured on polylysine-treated substrata. In the latter case, neurites are highly branched and grow concentrically around the aggregate perimeter. Addition of fibronectin to polylysine-treated substrata stimulates radial neurite outgrowth. Promotion of neurite outgrowth is dependent on the amount of fibronectin bound to the culture substratum and on the pH at which binding occurs. The requirements for fibronectin-mediated neurite outgrowth are more stringent than those previously reported for fibroblast attachment and spreading.     

Effects of plant lectins on the adhesive properties of baby hamster kidney cells

We studied the effects of different lectins on the adhesive properties of baby hamster kidney (BHK) cells. The purpose of these studies was to learn more about the cell surface receptors involved in cell adhesion. Three adhesive phenomena were analyzed: 1) the adhesion of BHK cells to lectin-coated substrata; 2) the effects of lectins on the adhesion of cells to substrata coated by plasma fibronectin (pFN); and 3) the effects of lectins on the binding of pFN-coated beads to cells. Initial experiments with fluorescein-conjugated lectins indicated that concanavalin A (Con A), ricinus communis agglutinin I (RCA I), and wheat germ agglutinin (WGA) bound to BHK cells but peanut agglutinin (PNA), soybean agglutinin (SBA), and ulex europaeus agglutinin I (UEA I) dod not bind. All three of the lectins which bound to the cells promoted cell spreading on lectin substrata, and the morphology of the spread cells was similar to that observed with cells spread on pFN substrata. Protease treatment of the cells, however, was found to inhibit cell spreading on pFN substrata or WGA substrata more than on Con A substrata or RCA I substrata. In the experiment of cells with Con A or WGA inhibited cell spreading on pFN substrata, but RCA I treatment had no effect. Finally, treatment of cells with WGA inhibited binding to cells of pFN beads, but neither Con A nor RCA I affected this interaction. These results indicate that the lectins modify cellular adhesion in different ways, probably by interacting with different surface receptors. The possibility that the pFN receptor is a WGA receptor is discussed.     

The role of endogenous heparan sulfate proteoglycan in adhesion and neurite outgrowth from dorsal root ganglia

Some phases of dorsal root ganglion (DRG) substratum attachment and growth cone morphology are mediated through endogenous cell surface heparan sulfate proteoglycan. The adhesive behavior of intact embryonic chicken DRG (spinal sensory ganglia) is examined on substrata coated with fibronectin, fibronectin treated with antibody to the cell-binding site (anti-CBS), and the heparan sulfate-binding protein platelet factor four. DRG attach to fibronectin, anti-CBS-treated fibronectin, and platelet factor four. The ganglia extend an extensive halo of unfasciculated neurites on fibronectin and produce fasciculated neurite outgrowth on platelet factor four and anti-CBS antibody-treated FN. Treatment with heparinase, but not chondroitinase, abolishes adhesion to fibronectin and platelet factor four. Growth cones of DRG on fibronectin have well-spread lamellae and microspikes. On platelet factor four, and anti-CBS-treated FN, growth cones exhibit microspikes only. Isolated Schwann cells adhere equally well to fibronectin and platelet factor four, spreading more rapidly on fibronectin. Isolated DRG neurons adhere equally well on both substrata, but only 10% of the neurons extend long neurites on platelet factor four. The majority of the isolated neurons on platelet factor four exhibit persistent microspike production resembling that of the early stages of normal neurite extension. Endogenous heparan sulfate proteoglycan supports the adhesion of whole DRG, isolated DRG neurons, and Schwann cells, as well as extensive microspike activity by DRG neurons, one important part of growth cone activity.     

Selective interaction of peripheral and central nervous system cells with two distinct cell-binding domains of fibronectin

Mechanisms of cell interaction with fibronectin have been studied with proteolytic fibronectin fragments that have well-defined ligand binding properties. Results of a previous study (Rogers, S. L., J. B. McCarthy, S. L. Palm, L. T. Furcht, and P. C. Letourneau, 1985, J. Neurosci., 5:369-378) demonstrated that (a) central (CNS) and peripheral (PNS) nervous system neurons adhere to, and extend neurites on a 33-kD carboxyl terminal fibronectin fragment that also binds heparin, and (b) neurons from the PNS, but not the CNS, have stable interactions with a 75-kD cell-binding fragment and with intact fibronectin. In the present study domain-specific reagents were used in inhibition assays to further differentiate cell surface interactions with the two fibronectin domains, and to define the significance of these domains to cell interactions with the intact fibronectin molecule. These reagents are (a) a soluble synthetic tetrapeptide Arg-Gly-Asp-Ser (RGDS; Pierschbacher, M. D., and E. Ruoslahti, 1984, Nature (Lond.), 309:30-33) representing a cell-binding determinant in the 75-kD fragment, and (b) an antibody raised against the 33-kD fragment that binds specifically to that fragment. Initial cell attachment to, and neurite extension upon, fibronectin and the two different fragments was evaluated in the presence and absence of the two reagents. Attachment of both PNS and CNS cells to intact fibronectin was reduced in the presence of RGDS, the former more so than the latter. In contrast, the antibody to the 33-kD fragment did not affect attachment of PNS cells to fibronectin, but significantly decreased attachment of CNS cells to the molecule. RGDS inhibited attachment of CNS cells to the molecule. RGDS inhibited attachment of both cell types to the 75-kD fragment to a greater degree than it did attachment to the intact molecule. Cell interaction with the 33-kD fragment was not affected by RGDS. Reduction of neurite lengths (determined after 24 h of culture) by the domain-specific reagents paralleled the reduction in initial adhesion to each substratum. Therefore, it appears that (a) both PNS and CNS cells have receptors for each cell-binding domain of fibronectin, (b) the receptor(s) for the two domains are distinct, with attachment to the 33-kD fragment being independent of RGDS, and (c) the relative importance of each domain to cell interaction with intact fibronectin is different for CNS and PNS cells.     

Neurite outgrowth in dorsal root neuronal hybrid clones modulated by ganglioside GM1 and disintegrins.

Subclones of F11 neuronal hybrid cells (neuroblastoma x dorsal root ganglion neurons) have segregated differing and/or overlapping neuritogenic mechanisms on three substrata--plasma fibronectin (pFN) with its multiple receptor activities, cholera toxin B subunit (CTB) for binding to ganglioside GM1, and platelet factor-4 (PF4) for binding to heparan sulfate proteoglycans. In this study, specific cell surface receptor activities for the three substrata were tested for their modulation during neuritogenesis by several experimental paradigms, using F11 subclones representative of three differentiation classes (neuritogenic on pFN only, on CTB only, or on all three substrata). When cycloheximide was included in the medium to inhibit protein synthesis during the active period, neurite formation increased significantly for all subclones on all three substrata, virtually eliminating substratum selectivity for differentiation mediated by cell surface integrin, ganglioside GM1, or heparan sulfate proteoglycans. Therefore, one or more labile proteins (referred to as disintegrins) must modulate functions of matrix receptors (e.g., integrins) mediating neurite formation. To verify whether cycloheximide-induced neuritogenesis was also regulated by integrin interaction with cell surface GM1, two approaches were used. When (Arg-Gly-Asp-Ser)-containing peptide A was added to the medium, it completely inhibited cycloheximide-induced neuritogenesis on all three substrata of all subclones, indicating stringent requirement for cell surface integrin function in these mechanisms. In contrast, when CTB or a monoclonal anti-GM1 antibody was also added to the medium, cycloheximide-induced neuritogenesis was amplified further on pFN and sensitivity to peptide A inhibition was abolished. Therefore, in some contexts ganglioside GM1 must complex with integrin receptors at the cell surface to modulate their function. These results also indicate that (a) cycloheximide treatment leads to loss of substratum selectivity in neuritogenesis, (b) this negative regulation of neurite outgrowth is affected by integrin receptor association with labile regulatory proteins (disintegrins) as well as with GM1, and (c) complexing of GM1 by multivalent GM1-binding proteins shifts neuritogenesis from an RGDS-dependent integrin mechanism to an RGDS-independent receptor mechanism.     

Structural proteins in the growth cone of cultured spinal cord neurons

The distribution of several structural proteins in the extending neurites and growth cones of cultured embryonic mouse spinal cord neurons was studied by indirect immunofluorescence, using affinity chromatography-purified antibodies. Fibroblastic cells in the same cultures served as internal standards for the evaluation of staining intensities. Anti-tubulin, anti-actin, and anti-clathrin stained neurons and their processes intensely, while staining with anti-α-actinin was only moderate compared with fibroblasts. Microtubules were resolved by anti-tubulin in the ‘palm’ of the growth cone but not in the neurite. Anti-actin stained even the finest lamellae and filopodia of the growth cone, and the neurite. Anti-α-actinin revealed an irregularly speckled pattern of cross-reactive material in the neurite and in the palm of the growth cone and was absent from the filopodia. Anti-clathrin stained the neurite intensely and homogeneously, and to a lesser extent the palm of the growth cone. The staining with antibodies against tubulin and clathrin differed grossly between neurons and fibroblastic cells. Within the neuron there were only gradual differences in staining intensities. The growth cone was not qualitatively different from the rest of the neurite, except for the filopodia which lacked tubulin and α-actinin, similar to the microvilli of epithelial cells.     

Influence of ganglion age,nonneuronal cells and substratum on neurite outgrowth in culture

This study characterizes the outgrowth patterns of superior cervical ganglia (SCG) obtained from embryonic (E15), perinatal (E20–21), and adult (P35) rats when placed in culture on various substrata. Outgrowth morphology, degree of fasciculation, and outgrowth length were examined on collagen (COL), polyornithine (PO), polylysine (PL), fibronectin (FN), and nonneuronal cells (NNCs) from the ganglion. COL and FN supported extensive neuritic outgrowth; PO and PL provided poor support. Outgrowth pattern, degree of fasciculation, neurite growth rate, and the number of NNCs in the outgrowth varied considerably depending upon the COL configuration. When undiluted COL (～5 mg/ml) was air dried, a three-dimensional loose fibrillar network was formed. Upon COL dilution or gelling undiluted COL by ammoniation, an essentially two-dimensional layer was formed. On two-dimensional COL, NNCs were able to proliferate and migrate extensively from ganglia of all ages; their presence influenced the form and extent of neurite growth. E15, E20, and P35 neurites responded differently to their endogenous NNCs. E15 neurites extended in relation to NNC surfaces and were predominantly nonfasciculated. E20 neurites became more fasciculated in the presence of NNCs that exhibited morphological and behavioral differences from those migrating from E15 ganglia. E20 neurite bundles became defasciculated when they extended into E15 outgrowth. Far fewer neurites grew from P35 explants in the presence of their NNCs. Three-dimensional COL greatly slowed NNC migration and thus allowed investigation of neurite outgrowth from ganglia of differing age in the absence of NNCs. We conclude that neuritic outgrowth patterns on varying substrata reflect not only neurite differences depending upon ganglion age but also variation in the behavior of accompanying NNCs.     

Role of laminin in axonal extension from olfactory receptor cells

The role of laminin, an extracellular matrix molecule believed to be involved in axon extension, was explored in the outgrowth of olfactory receptor cells and therefore in the maintenance of organization in the olfactory pathway. First, immunocytochemistry was used to examine laminin expression in the olfactory nerve and bulb during development. Laminin immunoreactivity was high in the olfactory nerve and glomerular layers. Although it declined in intensity, laminin expression continued in the nerve and in single glomeruli of adults. Second, the influence of laminin on neurite outgrowth was examined in vitro using olfactory receptor cells harvested from E14 rat embryos. We developed an in vitro assay to quantify the substrate preference of outgrowing neurites. Cells were cultured for 48 h on coverslips coated with either poly-L-lysine alone, or poly-L-lysine overlaid with laminin. On laminin-coated regions of coverslips, the primary neurites of olfactory receptor cells were 52% longer than on the poly-L-lysine control substrates. In addition, the direction of the neurite outgrowth was influenced by laminin. Fifty-six percent of all receptor cells located in a defined area surrounding a laminin zone extended neurites onto laminin. In contrast, only 7% of all receptor cells located in the corresponding laminin zone extended a neurite onto poly-L-lysine. In summary, these data suggest that laminin provides a favorable substrate for the extension of the primary neurite from olfactory receptor cells and the direction of their extension. Therefore, laminin may be a factor underlying continuous olfactory receptor cell axon outgrowth and its pathfinding in the olfactory system. © 1997 John Wiley & Sons, Inc. J Neurobiol 00: 32: 298–310, 1997     

Enhanced neurite outgrowth of rat neural cortical cells on surface-modified films of poly(lactic-<Emphasis Type="Italic">co</Emphasis>-glycolic acid)

Neural cortical cells, isolated from prenatal rat cerebra, were grown on surface-modified poly(lactic-co-glycolic acid, 65:35) (PLGA) films coated with poly-D-lysine (PDL) with either laminin (LN), fibronectin (FN) or collagen (CN). Immunocytochemistry showed that the isolated cells were highly immunopositive for both neurofilament and MAP-2 with well-organized neurites and somatodendritic localization. The presence of PDL with LN or FN on the PLGA films was essential for increased neural cell growth. Also, PLGA films coated with either PDL/LN or PDL/FN mixtures had higher neurite outgrowth and regular differentiation.Revisions requested 30 September 2004; Revisions received 10 November 2004     

Magnesium-dependent attachment and neurite outgrowth by PC12 cells on collagen and laminin substrata

We report a study of the substratum and medium requirements for attachment and neurite outgrowth by cells of the pheochromocytoma-derived PC12 line. In attachment medium containing both Ca2+ and Mg2+, more than 50% of cells attached within 1 hr to petri dishes coated with native collagen Types I/III or II, native or denatured collagen Type IV, laminin, wheat germ agglutinin (WGA), or poly-L-lysine; attachment to dishes coated with nerve growth factor (NGF) was only about 20% and attachment to uncoated dishes or to dishes coated with fibronectin or gelatin was almost nil. Neither prior culturing in the presence of NGF nor addition of NGF to the attachment medium significantly affected the extent of attachment to collagen or laminin. With Ca2+ (1 mM) as the sole divalent cation, cells attached normally to WGA, polylysine, and NGF, but failed to attach to collagen or laminin. With Mg2+ (1 mM) as the only divalent cation, attachment to all substrata was about the same as in medium with both Ca2+ and Mg2+. Like the ionic requirements, the kinetics of attachment, insensitivity to protease treatment of the cells, and inhibition by low temperature and sodium azide were similar for PC12 attachment to collagen and laminin, suggesting that a common molecular mechanism may underlie attachment to these substrata. The only significant difference observed was that addition of WGA (30 micrograms/ml) to the attachment medium inhibited attachment to collagen but promoted attachment to laminin. Finally, PC12 cells extended neurites on laminin, on native collagens I/III, II, and IV, and on denatured collagen IV; they did not extend neurites on denatured collagens I/III or II, NGF, or WGA. Neurite outgrowth on collagen and laminin occurred with Mg2+ as the sole divalent cation. These results suggest that the same Mg2+-dependent adhesion mechanism operates at the cell body and at the growth cone.     

Complementary adhesive responses of human skin fibroblasts to the cell-binding domain of fibronectin and the heparan sulfate-binding protein, platelet factor-4

Plasma fibronectin (pFN) contains binding domains for an unidentified receptor on the surface of fibroblasts and for heparan sulfate chains of proteoglycans on these same cells. A series of experiments were designed to assess the relative importance of these activities in mediating substratum adhesion of human skin fibroblasts (strain 4449) grown in the absence of ascorbate (asc-) or in its presence (asc+) to minimize or maximize collagen production-maturation, respectively. The cell-binding fragment (CBF) of pFN was purified from chymotryptic digests free of any heparan sulfate-binding activity. The responses of cells to CBF were then compared with those mediated by the heparan sulfate-binding protein, platelet factor-4 (PF4). At early time points when cells had spread effectively on pFN, both asc- or asc+ cells extended spiky projections on PF4 and long projections on CBF with actively ruffling membranes at their tips. By 4 h, asc+ cells had spread much more effectively on CBF than asc+ cells on PF4 or asc- cells on either binding activity. Mixtures (w/w) of CBF:PF4 between 1:1 and 9:1 generated a more physiologically normal response than to either of the binding proteins alone, particularly for asc+ cells. Examination of cytoskeletal reorganization by fluorescence analysis with an antibody to 7S tubulin (for microtubules) and NBD-phallacidin (for F-actin) revealed condensations of microfilaments at the ruffling edges of asc- cells on CBF or on PF4 and for asc+ cells on PF4; in contrast, asc+ cells on CBF generated long bundles of microfilaments in their spreading lamellae within 4 h. Microtubule networks reorganized very well on CBF but only partially on PF4 with either cell type. Microfilament reorganization was comparable to that on intact pFN with CBF:PF4 mixtures of 1:1 and 9:1 for asc+ cells, whereas asc- cells generated condensations of microfilaments but little bundling. These studies reveal that the adhesive responses to mixtures of these two binding activities are significantly greater than to the individual activities and that the responses of asc+ cells approach the properties of cells on intact pFN, whereas asc- cells remain incapable of forming stress fiber-like bundles of microfilaments under all conditions.