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CRYP-2/cPTPRO is a neurite inhibitory repulsive guidance cue for retinal neurons in vitro.

Stepanek L, Sun QL, Wang J, Wang C, Bixby JL - J. Cell Biol. (2001)

Bottom Line: We found that the extracellular domain of cPTPRO is an antiadhesive, neurite inhibitory molecule for retinal neurons.This chemorepulsive effect could be regulated by the level of cGMP in the growth cone.Immunohistochemical examination of the retina indicated that cPTPRO has at least one heterophilic binding partner in the retina.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Program, University of Miami School of Medicine, Miami, FL 33136, USA.

ABSTRACT
Receptor protein tyrosine phosphatases (RPTPs) are implicated as regulators of axon growth and guidance. Genetic deletions in the fly have shown that type III RPTPs are important in axon pathfinding, but nothing is known about their function on a cellular level. Previous experiments in our lab have identified a type III RPTP, CRYP-2/cPTPRO, specifically expressed during the period of axon outgrowth in the chick brain; cPTPRO is expressed in the axons and growth cones of retinal and tectal projection neurons. We constructed a fusion protein containing the extracellular domain of cPTPRO fused to the Fc portion of mouse immunoglobulin G-1, and used it to perform in vitro functional assays. We found that the extracellular domain of cPTPRO is an antiadhesive, neurite inhibitory molecule for retinal neurons. In addition, cPTPRO had potent growth cone collapsing activity in vitro, and locally applied gradients of cPTPRO repelled growing retinal axons. This chemorepulsive effect could be regulated by the level of cGMP in the growth cone. Immunohistochemical examination of the retina indicated that cPTPRO has at least one heterophilic binding partner in the retina. Taken together, our results indicate that cPTPRO may act as a guidance cue for retinal ganglion cells during vertebrate development.

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cPTPRO is antiadhesive for retinal neurons. Dissociated E6 retinal neurons were allowed to adhere for 2 h before fixation. (A) Neurons adhered equally well to the inner spot of LN/mIgG-1 and to the outer ring of LN alone (left). However, neurons did not adhere to an inner spot of LN/cPTPRO–Fc (right). (B) Quantitative data from adhesion assays in which cPTPRO–Fc or mIgG were mixed with LN and spotted onto nitrocellulose-coated dishes. Data are expressed as a percentage of neurons adhering to a spot of LN alone (CON) and plotted as mean ± SEM for three separate experiments. A dose-dependent decrease in adhesion was apparent with increasing concentrations of cPTPRO, from 12 to 100 μg/ml. No such decrease was caused by mIgG-1. (C) Similar results were obtained from three experiments in which cPTPRO–Fc or mIgG-1 were mixed with N-cadherin. Data were plotted as the percentage of neurons adhering to a spot of N-cadherin alone (CON). Again a dose-dependent decrease in adhesion was observed with increasing concentrations of cPTPRO–Fc.
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fig4: cPTPRO is antiadhesive for retinal neurons. Dissociated E6 retinal neurons were allowed to adhere for 2 h before fixation. (A) Neurons adhered equally well to the inner spot of LN/mIgG-1 and to the outer ring of LN alone (left). However, neurons did not adhere to an inner spot of LN/cPTPRO–Fc (right). (B) Quantitative data from adhesion assays in which cPTPRO–Fc or mIgG were mixed with LN and spotted onto nitrocellulose-coated dishes. Data are expressed as a percentage of neurons adhering to a spot of LN alone (CON) and plotted as mean ± SEM for three separate experiments. A dose-dependent decrease in adhesion was apparent with increasing concentrations of cPTPRO, from 12 to 100 μg/ml. No such decrease was caused by mIgG-1. (C) Similar results were obtained from three experiments in which cPTPRO–Fc or mIgG-1 were mixed with N-cadherin. Data were plotted as the percentage of neurons adhering to a spot of N-cadherin alone (CON). Again a dose-dependent decrease in adhesion was observed with increasing concentrations of cPTPRO–Fc.

Mentions: The ECD of cPTPRO consists entirely of fibronectin type III repeats (Bodden and Bixby, 1996). Because these motifs are commonly found in the ECDs of CAMs (Baldwin et al., 1996), and in some cases appear to mediate cell adhesion (Frei et al., 1992; Appel et al., 1993), we predicted that the ECD of cPTPRO could be an adhesive substrate for neurons. To test this idea, we performed several types of cell adhesion assays. Initial adhesion experiments employed retinal neurons, as these appear to express a binding partner for the cPTPRO ECD. Surprisingly, few retinal neurons adhered to spots containing cPTPRO–Fc as a substrate, even though controls demonstrated that the neurons adhered to spots of known adhesive molecules (poly-d-lysine [PDL]) on the same dish. Similar results were obtained with forebrain, cerebellar, and ciliary ganglion neurons (unpublished data). These results suggest that the cPTPRO ECD does not support neuronal adhesion. However, because our ligand binding experiments suggest an interaction of the cPTPRO ECD with retinal neurons, we examined the possibility that cPTPRO–Fc could be an antiadhesive substrate. In these experiments, cPTPRO or mIgG as a control was spotted onto nitrocellulose substrates, and a larger spot of laminin (LN) was then placed over the first spot. This created a central spot of cPTPRO–Fc (or mIgG) mixed with LN, surrounded by a ring of LN alone. In our short-term assay, the center area containing LN plus cPTPRO–Fc did not support neuronal adhesion, though retinal neurons adhered well to the LN surround, or to the LN–mIgG mixture (Fig. 4 A). Indeed, neurons failed to adhere to or spread on the cPTPRO–LN mixture even after 24 h of culture (unpublished data). These results suggest that the cPTPRO ECD is antiadhesive for retinal neurons.


CRYP-2/cPTPRO is a neurite inhibitory repulsive guidance cue for retinal neurons in vitro.

Stepanek L, Sun QL, Wang J, Wang C, Bixby JL - J. Cell Biol. (2001)

cPTPRO is antiadhesive for retinal neurons. Dissociated E6 retinal neurons were allowed to adhere for 2 h before fixation. (A) Neurons adhered equally well to the inner spot of LN/mIgG-1 and to the outer ring of LN alone (left). However, neurons did not adhere to an inner spot of LN/cPTPRO–Fc (right). (B) Quantitative data from adhesion assays in which cPTPRO–Fc or mIgG were mixed with LN and spotted onto nitrocellulose-coated dishes. Data are expressed as a percentage of neurons adhering to a spot of LN alone (CON) and plotted as mean ± SEM for three separate experiments. A dose-dependent decrease in adhesion was apparent with increasing concentrations of cPTPRO, from 12 to 100 μg/ml. No such decrease was caused by mIgG-1. (C) Similar results were obtained from three experiments in which cPTPRO–Fc or mIgG-1 were mixed with N-cadherin. Data were plotted as the percentage of neurons adhering to a spot of N-cadherin alone (CON). Again a dose-dependent decrease in adhesion was observed with increasing concentrations of cPTPRO–Fc.
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Related In: Results  -  Collection

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fig4: cPTPRO is antiadhesive for retinal neurons. Dissociated E6 retinal neurons were allowed to adhere for 2 h before fixation. (A) Neurons adhered equally well to the inner spot of LN/mIgG-1 and to the outer ring of LN alone (left). However, neurons did not adhere to an inner spot of LN/cPTPRO–Fc (right). (B) Quantitative data from adhesion assays in which cPTPRO–Fc or mIgG were mixed with LN and spotted onto nitrocellulose-coated dishes. Data are expressed as a percentage of neurons adhering to a spot of LN alone (CON) and plotted as mean ± SEM for three separate experiments. A dose-dependent decrease in adhesion was apparent with increasing concentrations of cPTPRO, from 12 to 100 μg/ml. No such decrease was caused by mIgG-1. (C) Similar results were obtained from three experiments in which cPTPRO–Fc or mIgG-1 were mixed with N-cadherin. Data were plotted as the percentage of neurons adhering to a spot of N-cadherin alone (CON). Again a dose-dependent decrease in adhesion was observed with increasing concentrations of cPTPRO–Fc.
Mentions: The ECD of cPTPRO consists entirely of fibronectin type III repeats (Bodden and Bixby, 1996). Because these motifs are commonly found in the ECDs of CAMs (Baldwin et al., 1996), and in some cases appear to mediate cell adhesion (Frei et al., 1992; Appel et al., 1993), we predicted that the ECD of cPTPRO could be an adhesive substrate for neurons. To test this idea, we performed several types of cell adhesion assays. Initial adhesion experiments employed retinal neurons, as these appear to express a binding partner for the cPTPRO ECD. Surprisingly, few retinal neurons adhered to spots containing cPTPRO–Fc as a substrate, even though controls demonstrated that the neurons adhered to spots of known adhesive molecules (poly-d-lysine [PDL]) on the same dish. Similar results were obtained with forebrain, cerebellar, and ciliary ganglion neurons (unpublished data). These results suggest that the cPTPRO ECD does not support neuronal adhesion. However, because our ligand binding experiments suggest an interaction of the cPTPRO ECD with retinal neurons, we examined the possibility that cPTPRO–Fc could be an antiadhesive substrate. In these experiments, cPTPRO or mIgG as a control was spotted onto nitrocellulose substrates, and a larger spot of laminin (LN) was then placed over the first spot. This created a central spot of cPTPRO–Fc (or mIgG) mixed with LN, surrounded by a ring of LN alone. In our short-term assay, the center area containing LN plus cPTPRO–Fc did not support neuronal adhesion, though retinal neurons adhered well to the LN surround, or to the LN–mIgG mixture (Fig. 4 A). Indeed, neurons failed to adhere to or spread on the cPTPRO–LN mixture even after 24 h of culture (unpublished data). These results suggest that the cPTPRO ECD is antiadhesive for retinal neurons.

Bottom Line: We found that the extracellular domain of cPTPRO is an antiadhesive, neurite inhibitory molecule for retinal neurons.This chemorepulsive effect could be regulated by the level of cGMP in the growth cone.Immunohistochemical examination of the retina indicated that cPTPRO has at least one heterophilic binding partner in the retina.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Program, University of Miami School of Medicine, Miami, FL 33136, USA.

ABSTRACT
Receptor protein tyrosine phosphatases (RPTPs) are implicated as regulators of axon growth and guidance. Genetic deletions in the fly have shown that type III RPTPs are important in axon pathfinding, but nothing is known about their function on a cellular level. Previous experiments in our lab have identified a type III RPTP, CRYP-2/cPTPRO, specifically expressed during the period of axon outgrowth in the chick brain; cPTPRO is expressed in the axons and growth cones of retinal and tectal projection neurons. We constructed a fusion protein containing the extracellular domain of cPTPRO fused to the Fc portion of mouse immunoglobulin G-1, and used it to perform in vitro functional assays. We found that the extracellular domain of cPTPRO is an antiadhesive, neurite inhibitory molecule for retinal neurons. In addition, cPTPRO had potent growth cone collapsing activity in vitro, and locally applied gradients of cPTPRO repelled growing retinal axons. This chemorepulsive effect could be regulated by the level of cGMP in the growth cone. Immunohistochemical examination of the retina indicated that cPTPRO has at least one heterophilic binding partner in the retina. Taken together, our results indicate that cPTPRO may act as a guidance cue for retinal ganglion cells during vertebrate development.

Show MeSH
Related in: MedlinePlus