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 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.

scholarly journals Early detachment of colon carcinoma cells during CD95(APO-1/Fas)-mediated apoptosis. I. De-adhesion from hyaluronate by shedding of CD44.

1996 ◽  
Vol 134 (4) ◽  
pp. 1089-1096 ◽  
Author(s):  
A R Günthert ◽  
J Sträter ◽  
U von Reyher ◽  
C Henne ◽  
S Joos ◽  
...  
Keyword(s):  
Cell Surface ◽  
Colon Carcinoma ◽  
Substantial Reduction ◽  
Carcinoma Cells ◽  
Surface Adhesion ◽  
Intact Cells ◽  
Surface Receptors ◽  
Colon Carcinoma Cells ◽  
Soluble Cd44

Ligation of CD95 (APO-1/Fas) cell surface receptors induces death in apoptosis-sensitive cells. Induction of apoptosis in adherent gamma interferon-stimulated HT-29 and COLO 205 colon carcinoma cells by cross-linking CD95 with anti-APO-1 monoclonal antibody resulted in detachment of the cells from hyaluronate starting about 1 h after antibody exposure. Loss of adhesion was paralleled by a substantial reduction of the multifunctional cell surface adhesion molecule CD44. As evidenced by cycloheximide treatment, this effect was not caused by impaired protein synthesis. Depletion of surface CD44 was also not due to membrane blebbing, since cytochalasin B failed to inhibit ascension from hyaluronate. Instead, ELISA and time kinetics showed increasing amounts of soluble CD44 in the supernatant of CD95-triggered cells. SDS-PAGE revealed that soluble CD44 had an apparent molecular mass of about 20 kD less than CD44 immunoprecipitated from intact cells. Thus, CD95-triggering induced shedding of CD44. Shedding is a novel mechanism operative in early steps of CD95-mediated apoptosis. Shedding surface molecules like CD44 might contribute to the active disintegration of dying epithelial cells in vivo.

scholarly journals Neurite Outgrowth-Promoting Activity of Compounds in PC12 Cells from Sunflower Seeds

Molecules ◽  
2020 ◽  
Vol 25 (20) ◽  
pp. 4748
Author(s):  
Takeru Koga ◽  
Takaiku Sakamoto ◽  
Eiji Sakuradani ◽  
Akihiro Tai
Keyword(s):  
Neurite Outgrowth ◽  
Pc12 Cells ◽  
Sunflower Seed ◽  
Seed Extract ◽  
Proton Nuclear Magnetic Resonance ◽  
Gas Chromatography Mass Spectrometry ◽  
Sunflower Seeds ◽  
Active Fraction ◽  
Neurite Formation ◽  
Main Component

In the current super-aging society, the establishment of methods for prevention and treatment of Alzheimer’s disease (AD) is an urgent task. One of the causes of AD is thought to be a decrease in the revel of nerve growth factor (NGF) in the brain. Compounds showing NGF-mimicking activity and NGF-enhancing activity have been examined as possible agents for improving symptoms. In the present study, sunflower seed extract was found to have neurite outgrowth-promoting activity, which is an NGF-enhancing activity, in PC12 cells. To investigate neurite outgrowth-promoting compounds from sunflower seed extract, bioassay-guided purification was carried out. The purified active fraction was obtained by liquid-liquid partition followed by some column chromatographies. Proton nuclear magnetic resonance and gas chromatography-mass spectrometry analyses of the purified active fraction indicated that the fraction was a mixture of β-sitosterol, stigmasterol and campesterol, with β-sitosterol being the main component. Neurite outgrowth-promoting activities of β-sitosterol, stigmasterol, campesterol and cholesterol were evaluated in PC12 cells. β-Sitosterol and stigmasterol showed the strongest activity of the four sterol compounds (β-sitosterol ≈ stigmasterol > campesterol > cholesterol), and cholesterol did not show any activity. The results indicated that β-sitosterol was the major component responsible for the neurite outgrowth-promoting activity of sunflower seeds. Results of immunostaining also showed that promotion by β-sitosterol of neurite formation induced by NGF was accompanied by neurofilament expression. β-Sitosterol, which showed NGF-enhancing activity, might be a candidate ingredient in food for prevention of AD.

scholarly journals Inositol 1,4,5-trisphosphorothioate, a stable analogue of inositol trisphosphate which mobilizes intracellular calcium

1989 ◽  
Vol 259 (3) ◽  
pp. 645-650 ◽  
Author(s):  
C W Taylor ◽  
M J Berridge ◽  
A M Cooke ◽  
B V L Potter
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Intracellular Calcium ◽  
Racemic Mixture ◽  
Synthetic Analogue ◽  
Intact Cells ◽  
Surface Receptors ◽  
Prolonged Stimulation ◽  
Crude Preparation ◽  
Intracellular Messenger

D-Ins(1,4,5)P3 is now recognized as an intracellular messenger that mediates the actions of many cell-surface receptors on intracellular Ca2+ pools, but its complex and rapid metabolism in intact cells has confused interpretation of its possible roles in oscillatory changes in intracellular [Ca2+] and in controlling Ca2+ entry at the plasma membrane. We now report the actions and metabolic stability of a synthetic analogue of Ins(1,4,5)P3, DL-inositol 1,4,5-trisphosphorothioate [DL-Ins(1,4,5)P3[S]3]. In permeabilized hepatocytes, DL-Ins(1,4,5)P3[S]3 and synthetic DL-Ins(1,4,5)P3 stimulated Ca2+ release from the same intracellular stores, though the concentration required for half-maximal release was 3-fold higher for DL-Ins(1,4,5)P3[S]3. Since L-Ins(1,4,5)P3 neither antagonized the effects of D-Ins(1,4,5)P3 nor itself stimulated appreciable Ca2+ release, the activity of the racemic mixture of Ins(1,4,5)P3, and presumably also of Ins(1,4,5)P3[S]3, is attributable to the D-isomer. Under conditions where there was negligible metabolism of D-[3H]Ins(1,4,5)P3, both DL-Ins(1,4,5)P3 and DL-Ins(1,4,5)P3[S]3 elicited rapid Ca2+ release from intracellular stores, and the stores remained empty during prolonged stimulation. When cells were incubated at high density, both compounds stimulated rapid Ca2+ release, but while the stores soon refilled as Ins(1,4,5)P3 was degraded to Ins(1,4)P2, there was no refilling of the pools after stimulation with DL-Ins(1,4,5)P3[S]3. When DL-Ins(1,4,5)P3 or DL-Ins(1,4,5)P3[S]3 was treated with a crude preparation of Ins(1,4,5)P3 3-kinase and ATP, and the Ca2+-releasing activity of the products subsequently assayed, DL-Ins(1,4,5)P3 was completely inactivated by phosphorylation, but there was no loss of activity of the phosphorothioate analogue. In additional experiments, DL-Ins(1,4,5)P3[S]3 (10 microM) did not affect the rate of phosphorylation of D-[3H]Ins(1,4,5)P3 (1 microM). We conclude that Ins(1,4,5)P3[S]3 is a full agonist and only 3-fold less potent than Ins(1,4,5)P3 in mobilizing intracellular Ca2+ stores, but unlike the natural messenger it is resistant to both phosphorylation and dephosphorylation. We propose that this stable analogue will allow the direct actions of Ins(1,4,5)P3 to be resolved from those that require its metabolism.

Measurements of associations of cell-surface receptors by single-particle fluorescence imaging

2003 ◽  
Vol 31 (5) ◽  
pp. 1028-1031 ◽  
Author(s):  
R.J. Cherry ◽  
I.E.G. Morrison ◽  
I. Karakikes ◽  
R.E. Barber ◽  
G. Silkstone ◽  
...  
Keyword(s):  
Cell Surface ◽  
Fluorescence Imaging ◽  
Mhc Class Ii ◽  
Single Particle ◽  
Gaussian Function ◽  
Human Leukocyte ◽  
Cell Surface Receptors ◽  
Intact Cells ◽  
Surface Receptors ◽  
Hla Dr

SPFI (single-particle fluorescence imaging) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labelled with small fluorescent particles. The images of particles are diffraction-limited spots that are analysed by fitting with a two-dimensional Gaussian function. The spot intensities depend on whether they arise from one or more particles; this provides the basis for determining self-association of cell-surface receptors. We have used this approach to determine dimerization of MHC class II molecules and its disruption by interface peptides. We have also exploited the positional information obtained from SPFI to detect co-localization of cell-surface molecules. This involves labelling two different molecules with different coloured fluorophores and determining their positions separately by dual wavelength imaging. The images are analysed to quantify the overlap of the particle images and hence determine the extent of co-localization of the labelled molecules. The technique provides quantification of the extent of co-localization and can detect whether co-localized molecules occur singly or in clusters. We have obtained preliminary data for co-localization of lipopolysaccharide and CD14 on intact cells. We also show that HLA-DR (human leukocyte antigen-DR) and CD74 are partially co-localized and that interaction between these molecules involves the peptide-binding groove of HLA-DR.

scholarly journals Yeast β-1,6-Glucan Is a Primary Target for the Saccharomyces cerevisiae K2 Toxin

2015 ◽  
Vol 14 (4) ◽  
pp. 406-414 ◽  
Author(s):  
Juliana Lukša ◽  
Monika Podoliankaitė ◽  
Iglė Vepštaitė ◽  
Živilė Strazdaitė-Žielienė ◽  
Jaunius Urbonavičius ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Yeast Cell ◽  
Membrane Integrity ◽  
Ion Leakage ◽  
Primary Target ◽  
Intact Cells ◽  
Content Type ◽  
Surface Receptors

ABSTRACTCertainSaccharomyces cerevisiaestrains secrete different killer proteins of double-stranded-RNA origin. These proteins confer a growth advantage to their host by increasing its survival. K2 toxin affects the target cell by binding to the cell surface, disrupting the plasma membrane integrity, and inducing ion leakage. In this study, we determined that K2 toxin saturates the yeast cell surface receptors in 10 min. The apparent amount of K2 toxin, bound to a single cell of wild type yeast under saturating conditions, was estimated to be 435 to 460 molecules. It was found that an increased level of β-1,6-glucan directly correlates with the number of toxin molecules bound, thereby impacting the morphology and determining the fate of the yeast cell. We observed that the binding of K2 toxin to the yeast surface receptors proceeds in a similar manner as in case of the related K1 killer protein. It was demonstrated that the externally supplied pustulan, a poly-β-1,6-glucan, but not the glucans bearing other linkage types (such as laminarin, chitin, and pullulan) efficiently inhibits the K2 toxin killing activity. In addition, the analysis of toxin binding to the intact cells and spheroplasts confirmed that majority of K2 protein molecules attach to the β-1,6-glucan, rather than the plasma membrane-localized receptors. Taken together, our results reveal that β-1,6-glucan is a primary target of K2 toxin and is important for the execution of its killing property.

Mechanism of diminished contractile response to catecholamines during acidosis

1988 ◽  
Vol 254 (1) ◽  
pp. H20-H27 ◽  
Author(s):  
J. D. Marsh ◽  
T. I. Margolis ◽  
D. Kim
Keyword(s):  
Cell Surface ◽  
Intracellular Ph ◽  
Adrenergic Receptor ◽  
Time Course ◽  
Contractile Response ◽  
Beta Adrenergic Receptor ◽  
Similar Time ◽  
Intact Cells ◽  
Beta Adrenergic ◽  
Surface Receptors

To examine mechanisms of diminished contractile response to catecholamines during acidosis, we studied contractile properties, beta-adrenergic receptor properties, and intracellular pH of intact, cultured myocardial cells from chick embryo ventricle at pH 7.4 and 6.8. Contractile response was measured with an optical-video system. On changing the superfusing buffer from pH 7.4 to 6.8 there was a decline in contractile amplitude to 80% of control by 20 min. Fluorimetrically determined intracellular pH declined over a similar time course from 7.11 +/- 0.05 to 6.96 +/- 0.07 (P less than 0.05). After 45 min at pH 6.8 the contractile response to 1 microM isoproterenol was less than half of the response at pH 7.4. Antagonist and agonist ligand-binding properties of the beta-adrenergic receptor were determined in the intact cells under conditions identical to those for the contractility studies. With the use of the hydrophilic antagonist [3H]CGP-12177 that selectively labels cell-surface receptors, agonist competition studies demonstrated that acidosis had no significant effect on antagonist or agonist affinity but decreased beta-receptor number from 21 +/- 3 to 11 +/- 3 fmol/mg protein (P less than 0.02). It is probable that a decline in the number of beta-receptors on the cell surface contributes to contractile hyporesponsiveness to catecholamines during acidosis.

scholarly journals Reproducible Discovery of Cell-Binding Peptides "Lost" in Bulk Amplification via Emulsion Amplification in Phage Display Panning

2021 ◽  
Author(s):  
Wadim L Matochko ◽  
Frederique Deiss ◽  
Yang Yang ◽  
Ratmir Derda
Keyword(s):  
Cell Surface ◽  
Phage Display ◽  
Parasite Population ◽  
Phage Library ◽  
Cell Surface Receptors ◽  
Intact Cells ◽  
Surface Receptors ◽  
Phage Display Libraries ◽  
Phage Libraries ◽  
Oil Emulsions

Many pharmaceutically-relevant cell surface receptors are functional only in the context of intact cells. Phage display, while being a powerful method for the discovery of ligands for purified proteins often fails to identify a diverse set of ligands to receptors on a cell membrane mosaic. To understand this deficiency, we examined growth bias in naive phage display libraries and observed that it fundamentally changes selection outcomes: The presence of growth-biased (parasite) phage clones in a phage library is detrimental to selection and cell-based panning of such biased libraries is poised to yield ligands from within a small parasite population. Importantly, amplification of phage libraries in water-oil emulsions suppressed the amplification of parasites and steered the selection of biased phage libraries away from parasite population. Attenuation of the growth bias through the use of emulsion amplification reproducibly discovers the ligands for cell-surface receptors that cannot be identified in screen that use conventional "bulk" amplification.

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