scholarly journals Cell surface galactosyltransferase mediates the initiation of neurite outgrowth from PC12 cells on laminin.

1990 ◽  
Vol 110 (2) ◽  
pp. 461-470 ◽  
Author(s):  
P C Begovac ◽  
B D Shur
Keyword(s):  
Cell Surface ◽  
Neurite Outgrowth ◽  
Enzymatic Reaction ◽  
Mesenchymal Cell ◽  
Laminin Receptor ◽  
Extracellular Matrix Molecule ◽  
Neurite Initiation ◽  
Neurite Formation ◽  
And Migration

Neurite outgrowth from PC12 pheochromocytoma cells, as well as from peripheral and central nervous system neurons in vitro, is mediated by the extracellular matrix molecule, laminin. We have recently shown that mesenchymal cell spreading and migration on laminin is mediated, in part, by the cell surface enzyme, beta 1,4 galactosyltransferase (GalTase). GalTase is localized on lamellipodia of migrating cells where it functions as a laminin receptor by binding to specific N-linked oligosaccharides in laminin (Runyan et al., 1988; Eckstein and Shur, 1989). In the present study, we examined whether GalTase functions similarly during neutrite outgrowth on laminin using biochemical and immunological analyses. PC12 neurite outgrowth was inhibited by reagents that perturb cell surface GalTase activity, including anti-GalTase IgG and Fab fragments, as well as the GalTase modifier protein alpha-lactalbumin. Control reagents had no effect on neurite outgrowth. Furthermore, blocking GalTase substrates on laminin matrices by earlier galactosyltion or enzymatic removal of GalTase substrates also inhibited neurite outgrowth. Conversely, neurite outgrowth was enhanced by the addition of UDP-galactose, which completes the GalTase enzymatic reaction, while inappropriate sugar nucleotides had no effect. The effects of all these treatments were dose and/or time dependent. Surface GalTase was shown to function during both neurite initiation and elongation, although the effects of GalTase perturbation were most striking during the initiation stages of neurite formation. Consistent with this, surface GalTase was localized by indirect immunofluorescence to the growth cone and developing neurite. Collectively, these results demonstrate that GalTase mediates the initiation of neurite outgrowth on laminin, and to a lesser extent, neurite elongation. Furthermore, this study demonstrates that process extension from both mesenchymal cells and neuronal cells is partly dependent upon specific oligosaccharide residues in laminin.

Overexpressing cell surface beta 1.4-galactosyltransferase in PC12 cells increases neurite outgrowth on laminin

1995 ◽  
Vol 108 (2) ◽  
pp. 839-847
Author(s):  
Q. Huang ◽  
B.D. Shur ◽  
P.C. Begovac
Keyword(s):  
Cell Surface ◽  
Neurite Outgrowth ◽  
Pc12 Cells ◽  
Intact Cells ◽  
Surface Receptors ◽  
Laminin Receptors ◽  
Neurite Initiation ◽  
Neurite Formation ◽  
Overexpressing Cell ◽  
Rate Limiting

Neurite outgrowth on cellular and extracellular matrices is mediated by a variety of cell surface receptors. Some of these receptors recognize peptide determinants, whereas others bind oligosaccharide ligands. Previous studies have suggested that cell surface beta 1.4-galactosyltransferase functions as one of these receptors during neurite outgrowth on basal lamina by binding to N-linked oligosaccharides in the E8 domain of laminin. However, these previous investigations have been limited to the use of galactosyltransferase inhibitory reagents to block neurite formation. Therefore, in this study, we investigated whether the level of surface galactosyltransferase directly affects the efficiency of neurite outgrowth, or rather, is incidental to neurite formation. Northern blot analysis and cell surface galactosyltransferase assays were used to select two stable PC12 transfectants that overexpress surface galactosyltransferase by approximately four-fold. Radiolabeled antibody binding to intact cells and indirect immunofluorescence confirmed the higher expression of surface galactosyltransferase on transfected cells, compared to controls. Both galactosyltransferase transfected cell lines exhibited markedly enhanced neurite initiation, neurite formation, and rates of neurite elongation by two- to three-fold. These studies demonstrate that the expression of laminin receptors can be rate-limiting during neurite outgrowth, and that the level of surface galactosyltransferase can modulate the frequency and rate of neurite formation from PC12 cells on laminin.

scholarly journals Laminin fragment E8 mediates PC12 cell neurite outgrowth by binding to cell surface beta 1,4 galactosyltransferase.

1991 ◽  
Vol 113 (3) ◽  
pp. 637-644 ◽  
Author(s):  
P C Begovac ◽  
D E Hall ◽  
B D Shur
Keyword(s):  
Cell Surface ◽  
Neurite Outgrowth ◽  
Pc12 Cell ◽  
Biological Activities ◽  
Mesenchymal Cell ◽  
Binding Activity ◽  
Surface Receptors ◽  
Laminin Receptors ◽  
Neurite Initiation ◽  
Membrane Bound

A number of cell surface receptors bind to distinct laminin domains, thereby mediating laminin's diverse biological activities. Cell surface beta 1,4-galactosyltransferase (GalTase) functions as one of these laminin receptors, facilitating mesenchymal cell migration and PC12 cell neurite outgrowth on laminin. In this study, the GalTase binding site within laminin was identified as the E8 fragment by assaying purified fragments and by immunoprecipitating and immunoblotting galactosylated laminin using E8-reactive antibodies. Compared with intact laminin and other laminin fragments, E8 possessed the highest GalTase binding activity, using both membrane-bound and solubilized GalTase. More significantly, the neurite-promoting activity of fragment E8 was shown to be dependent upon its interaction with GalTase. Pregalactosylating purified E8 eliminated subsequent GalTase binding and consequently inhibited neurite initiation; parallel studies on laminin fragments E1-4 or E1 failed to affect neurite outgrowth. Furthermore, anti-GalTase IgG inhibited neurite initiation on purified E8 substrates; control IgG had no effect. These results localize the predominant GalTase binding domain in laminin to fragment E8 and demonstrate that the neurite-promoting activity of E8 is dependent upon its interaction with GalTase.

scholarly journals Neurite outgrowth in peripherin-depleted PC12 cells

1992 ◽  
Vol 117 (5) ◽  
pp. 1085-1092 ◽  
Author(s):  
CM Troy ◽  
LA Greene ◽  
ML Shelanski
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Neurite Outgrowth ◽  
Pc12 Cells ◽  
Antisense Oligonucleotide ◽  
Antisense Oligonucleotides ◽  
Biological Function ◽  
Process Stability ◽  
Neurite Initiation ◽  
Neurite Formation

Peripherin is the major neuronal intermediate filament (IF) protein in PC12 cells and both its synthesis and amount increase during nerve growth factor (NGF) promoted neuronal differentiation. To address the question of the biological function of peripherin in neurite initiation we have used an antisense oligonucleotide complementary to the 5' region of peripherin mRNA to specifically inhibit its transcription. The oligonucleotide blocks both the synthesis of peripherin and its increase in response to NGF. Peripherin was found to be a stable protein with a cellular half-life of approximately 7 d. 6 wk of incubation with the oligonucleotide decreases peripherin to 11% of the level in naive control cells and to 3% of that in NGF-treated control cells. Despite the depletion, NGF elicits apparently normal neurite outgrowth from the oligonucleotide-treated cells. As evaluated by EM, there are few IFs in these cells, either in the cell bodies or neurites. There is no compensatory increase in NF-M, NF-L, or vimentin levels as a result of the inhibition of peripherin synthesis. These findings suggest that peripherin is not required for neurite formation, but is necessary for the formation of a cellular IF network which could be involved in process stability. They also demonstrate the utility of antisense oligonucleotides for the study of proteins with long half-lives.

scholarly journals Thrombospondin-4 Is a Soluble Dermal Inflammatory Signal That Selectively Promotes Fibroblast Migration and Keratinocyte Proliferation for Skin Regeneration and Wound Healing

2021 ◽  
Vol 9 ◽  
Author(s):  
Mariliis Klaas ◽  
Kristina Mäemets-Allas ◽  
Elizabeth Heinmäe ◽  
Heli Lagus ◽  
Claudia Griselda Cárdenas-León ◽  
...  
Keyword(s):  
Wound Healing ◽  
Skin Regeneration ◽  
Keratinocyte Proliferation ◽  
Fibroblast Migration ◽  
Extracellular Matrix Molecule ◽  
Primary Fibroblasts ◽  
And Migration ◽  
Thrombospondin 4

Thrombospondin-4 (THBS4) is a non-structural extracellular matrix molecule associated with tissue regeneration and a variety of pathological processes characterized by increased cell proliferation and migration. However, the mechanisms of how THBS4 regulates cell behavior as well as the pathways contributing to its effects have remained largely unexplored. In the present study we investigated the role of THBS4 in skin regeneration both in vitro and in vivo. We found that THBS4 expression was upregulated in the dermal compartment of healing skin wounds in humans as well as in mice. Application of recombinant THBS4 protein promoted cutaneous wound healing in mice and selectively stimulated migration of primary fibroblasts as well as proliferation of keratinocytes in vitro. By using a combined proteotranscriptomic pathway analysis approach we discovered that β-catenin acted as a hub for THBS4-dependent cell signaling and likely plays a key role in promoting its downstream effects. Our results suggest that THBS4 is an important contributor to wound healing and its incorporation into novel wound healing therapies may be a promising strategy for treatment of cutaneous wounds.

scholarly journals Functional reciprocity between Na+ channel Nav1.6 and β1 subunits in the coordinated regulation of excitability and neurite outgrowth

2010 ◽  
Vol 107 (5) ◽  
pp. 2283-2288 ◽  
Author(s):  
William J. Brackenbury ◽  
Jeffrey D. Calhoun ◽  
Chunling Chen ◽  
Haruko Miyazaki ◽  
Nobuyuki Nukina ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Neurite Outgrowth ◽  
High Frequency ◽  
Neuronal Excitability ◽  
Na Channel ◽  
Action Potential Firing ◽  
Cerebellar Neurons ◽  
Potential Firing ◽  
And Migration

Voltage-gated Na+ channel (VGSC) β1 subunits regulate cell–cell adhesion and channel activity in vitro. We previously showed that β1 promotes neurite outgrowth in cerebellar granule neurons (CGNs) via homophilic cell adhesion, fyn kinase, and contactin. Here we demonstrate that β1-mediated neurite outgrowth requires Na+ current (INa) mediated by Nav1.6. In addition, β1 is required for high-frequency action potential firing. Transient INa is unchanged in Scn1b (β1) null CGNs; however, the resurgent INa, thought to underlie high-frequency firing in Nav1.6-expressing cerebellar neurons, is reduced. The proportion of axon initial segments (AIS) expressing Nav1.6 is reduced in Scn1b null cerebellar neurons. In place of Nav1.6 at the AIS, we observed an increase in Nav1.1, whereas Nav1.2 was unchanged. This indicates that β1 is required for normal localization of Nav1.6 at the AIS during the postnatal developmental switch to Nav1.6-mediated high-frequency firing. In agreement with this, β1 is normally expressed with α subunits at the AIS of P14 CGNs. We propose reciprocity of function between β1 and Nav1.6 such that β1-mediated neurite outgrowth requires Nav1.6-mediated INa, and Nav1.6 localization and consequent high-frequency firing require β1. We conclude that VGSC subunits function in macromolecular signaling complexes regulating both neuronal excitability and migration during cerebellar development.

scholarly journals Cell Surface Sialylation and Fucosylation Are Regulated by L1 via Phospholipase Cγ and Cooperate to Modulate Neurite Outgrowth, Cell Survival and Migration

PLoS ONE ◽  
2008 ◽  
Vol 3 (12) ◽  
pp. e3841 ◽  
Author(s):  
Ya-li Li ◽  
Guang-zhi Wu ◽  
Gavin S. Dawe ◽  
Li Zeng ◽  
Shu-sen Cui ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Survival ◽  
Neurite Outgrowth ◽  
And Migration

scholarly journals Dissection of Complex Molecular Interactions of Neurofascin with Axonin-1, F11, and Tenascin-R, Which Promote Attachment and Neurite Formation of Tectal Cells

1998 ◽  
Vol 142 (4) ◽  
pp. 1083-1093 ◽  
Author(s):  
Hansjürgen Volkmer ◽  
Ute Zacharias ◽  
Ursel Nörenberg ◽  
Fritz G. Rathjen
Keyword(s):  
Alternative Splicing ◽  
Neurite Outgrowth ◽  
Molecular Interactions ◽  
Cell Attachment ◽  
Binding Assays ◽  
Ig Superfamily ◽  
Competition Binding ◽  
Neurite Formation ◽  
Additional Protein

Neurofascin is a member of the L1 subgroup of the Ig superfamily that promotes axon outgrowth by interactions with neuronal NgCAM-related cell adhesion molecule (NrCAM). We used a combination of cellular binding assays and neurite outgrowth experiments to investigate mechanisms that might modulate the interactions of neurofascin. In addition to NrCAM, we here demonstrate that neurofascin also binds to the extracellular matrix glycoprotein tenascin-R (TN-R) and to the Ig superfamily members axonin-1 and F11. Isoforms of neurofascin that are generated by alternative splicing show different preferences in ligand binding. While interactions of neurofascin with F11 are only slightly modulated, binding to axonin-1 and TN-R is strongly regulated by alternatively spliced stretches located in the NH2-terminal half, and by the proline-alanine-threonine-rich segment. In vitro neurite outgrowth and cell attachment assays on a neurofascin-Fc substrate reveal a shift of cellular receptor usage from NrCAM to axonin-1, F11, and at least one additional protein in the presence of TN-R, presumably due to competition of the neurofascin– NrCAM interaction. Thereby, F11 binds to TN-R of the neurofascin/TN-R complex, but not to neurofascin, whereas axonin-1 is not able to bind directly to the neurofascin/TN-R complex as shown by competition binding assays. In conclusion, these investigations indicate that the molecular interactions of neurofascin are regulated at different levels, including alternative splicing and by the presence of interacting proteins.

scholarly journals Ligand affinity of the 67-kD elastin/laminin binding protein is modulated by the protein's lectin domain: visualization of elastin/laminin-receptor complexes with gold-tagged ligands.

1991 ◽  
Vol 113 (1) ◽  
pp. 187-194 ◽  
Author(s):  
R P Mecham ◽  
L Whitehouse ◽  
M Hay ◽  
A Hinek ◽  
M P Sheetz
Keyword(s):  
Cell Surface ◽  
Laminin Receptor ◽  
Live Cells ◽  
Receptor Interaction ◽  
Ligand Complex ◽  
Lectin Domain ◽  
Receptor Complexes ◽  
Laminin Binding Protein

Video-enhanced microscopy was used to examine the interaction of elastin- or laminin-coated gold particles with elastin binding proteins on the surface of live cells. By visualizing the binding events in real time, it was possible to determine the specificity and avidity of ligand binding as well as to analyze the motion of the receptor-ligand complex in the plane of the plasma membrane. Although it was difficult to interpret the rates of binding and release rigorously because of the possibility for multiple interactions between particles and the cell surface, relative changes in binding have revealed important aspects of the regulation of affinity of ligand-receptor interaction in situ. Both elastin and laminin were found to compete for binding to the cell surface and lactose dramatically decreased the affinity of the receptor(s) for both elastin and laminin. These findings were supported by in vitro studies of the detergent-solubilized receptor. Further, immobilization of the ligand-receptor complexes through binding to the cytoskeleton dramatically decreased the ability of bound particles to leave the receptor. The changes in the kinetics of ligand-coated gold binding to living cells suggest that both laminin and elastin binding is inhibited by lactose and that attachment of receptor to the cytoskeleton increases its affinity for the ligand.

Adhesion and migration of avian neural crest cells on fibronectin require the cooperating activities of multiple integrins of the (beta)1 and (beta)3 families

1999 ◽  
Vol 112 (24) ◽  
pp. 4715-4728 ◽  
Author(s):  
S. Testaz ◽  
M. Delannet ◽  
J. Duband
Keyword(s):  
Cell Adhesion ◽  
Neural Crest ◽  
Cell Population ◽  
Neural Crest Cells ◽  
Cellular Level ◽  
Extracellular Matrix Molecule ◽  
Cell Adhesion And Migration ◽  
And Migration ◽  
Alpha V Beta 3

Based on genetic, functional and histological studies, the extracellular matrix molecule fibronectin has been proposed to play a key role in the migration of neural crest cells in the vertebrate embryo. In the present study, we have analyzed in vitro the repertoire and function of integrin receptors involved in the adhesive and locomotory responses of avian truncal neural crest cells to fibronectin. Immunoprecipitation experiments showed that neural crest cells express multiple integrins, namely (alpha)3(beta)1, (alpha)4(beta)1, (alpha)5(beta)1, (alpha)8(beta)1, (alpha)v(beta)1, (alpha)v(beta)3 and a (beta)8 integrin, as potential fibronectin receptors, and flow cytometry analyses revealed no major heterogeneity among the cell population for expression of integrin subunits. In addition, the integrin repertoire expressed by neural crest cells was found not to change dramatically during migration. At the cellular level, only (alpha)v(beta)1 and (alpha)v(beta)3 were concentrated in focal adhesion sites in connection with the actin microfilaments, whereas the other integrins were predominantly diffuse over the cell surface. In inhibition assays with function-perturbing antibodies, it appeared that complete abolition of cell spreading and migration could be achieved only by blocking multiple integrins of the (beta)1 and (beta)3 families, suggesting possible functional compensations between different integrins. In addition, these studies provided evidence for functional partitioning of integrins in cell adhesion and migration. While spreading was essentially mediated by (alpha)v(beta)1 and (alpha)8(beta)1, migration involved primarily (alpha)4(beta)1, (alpha)v(beta)3 and (alpha)8(beta)1 and, more indirectly, (alpha)3(beta)1. (alpha)5(beta)1 and the (beta)8 integrin were not found to play any major role in either adhesion or migration. Finally, consistent with the results of inhibition experiments, recruitment of (alpha)4(beta)1 and (alpha)v(beta)3, individually or in combination using antibodies or recombinant VCAM-1 and PECAM-1 molecules as a substratum, was required for migration but was not sufficient to produce migration of the cell population as efficiently as with fibronectin. In conclusion, our study indicates that neural crest cells express a multiplicity of fibronectin-binding integrins and suggests that dispersion of the cell population requires cooperation between distinct integrins regulating different events of cell adhesion, locomotion and, possibly, proliferation and survival.

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