Limits...
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.

Show MeSH

Related in: MedlinePlus

cPTPRO–Fc induces collapse of retinal growth cones. E6 chick retinal neurons were plated on glass coverslips coated with PDL and 10 μg/ml LN. After 10 h of culture, cPTPRO–Fc or mIgG-1 was added to the wells in 100 μl of medium. The same volume of medium was added to control wells. After 30 min, the cells were fixed and growth cones were scored as open or collapsed. (A) Control neurites had well-spread growth cones, in contrast to neurites treated with 6 nM cPTPRO–Fc (B). (C) Quantification of the growth cone collapsing effect. The percentage of collapsed growth cones (mean ± SEM) was plotted for control neurons and neurons treated with various concentrations of cPTPRO–Fc or mIgG. Concentrations are given in nM. Neurons treated with cPTPRO–Fc showed significantly higher levels of growth cone collapse than controls. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196468&req=5

fig8: cPTPRO–Fc induces collapse of retinal growth cones. E6 chick retinal neurons were plated on glass coverslips coated with PDL and 10 μg/ml LN. After 10 h of culture, cPTPRO–Fc or mIgG-1 was added to the wells in 100 μl of medium. The same volume of medium was added to control wells. After 30 min, the cells were fixed and growth cones were scored as open or collapsed. (A) Control neurites had well-spread growth cones, in contrast to neurites treated with 6 nM cPTPRO–Fc (B). (C) Quantification of the growth cone collapsing effect. The percentage of collapsed growth cones (mean ± SEM) was plotted for control neurons and neurons treated with various concentrations of cPTPRO–Fc or mIgG. Concentrations are given in nM. Neurons treated with cPTPRO–Fc showed significantly higher levels of growth cone collapse than controls. Bar, 10 μm.

Mentions: Proteins that inhibit neurite outgrowth by interacting with specific inhibitory receptors are candidates for repulsive axonal guidance cues. Such repulsive guidance cues may be assayed in vitro using growth cone collapse assays. For example, several of the best-known families of repulsive guidance proteins, including semaphorins, ephrins, slits, and bone morphogenetic proteins, exhibit growth cone collapsing activity in vitro (Luo et al., 1993; Drescher et al., 1995). Therefore, we tested the growth cone collapsing ability of cPTPRO to determine whether cPTPRO might also act as a repulsive guidance cue. In these assays, we added various concentrations of soluble cPTPRO–Fc (or mIgG-1 as a control) to cultures of E6 retinal explants 30 min before fixation. We found that concentrations of cPTPRO as low as 60 pM had clear collapsing ability, and that this activity was already saturated at 0.6 nM (Fig. 8) . The potency of the cPTPRO ECD in collapsing growth cones, as well as the percentage of growth cones collapsed at saturating cPTPRO concentrations, were similar to the values seen previously for ephrin A5–Fc, another repulsive guidance cue for retinal neurons (Wahl et al., 2000), and for the collapse of dorsal root ganglia growth cones by semaphorin IIIA (Luo et al., 1993).


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–Fc induces collapse of retinal growth cones. E6 chick retinal neurons were plated on glass coverslips coated with PDL and 10 μg/ml LN. After 10 h of culture, cPTPRO–Fc or mIgG-1 was added to the wells in 100 μl of medium. The same volume of medium was added to control wells. After 30 min, the cells were fixed and growth cones were scored as open or collapsed. (A) Control neurites had well-spread growth cones, in contrast to neurites treated with 6 nM cPTPRO–Fc (B). (C) Quantification of the growth cone collapsing effect. The percentage of collapsed growth cones (mean ± SEM) was plotted for control neurons and neurons treated with various concentrations of cPTPRO–Fc or mIgG. Concentrations are given in nM. Neurons treated with cPTPRO–Fc showed significantly higher levels of growth cone collapse than controls. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196468&req=5

fig8: cPTPRO–Fc induces collapse of retinal growth cones. E6 chick retinal neurons were plated on glass coverslips coated with PDL and 10 μg/ml LN. After 10 h of culture, cPTPRO–Fc or mIgG-1 was added to the wells in 100 μl of medium. The same volume of medium was added to control wells. After 30 min, the cells were fixed and growth cones were scored as open or collapsed. (A) Control neurites had well-spread growth cones, in contrast to neurites treated with 6 nM cPTPRO–Fc (B). (C) Quantification of the growth cone collapsing effect. The percentage of collapsed growth cones (mean ± SEM) was plotted for control neurons and neurons treated with various concentrations of cPTPRO–Fc or mIgG. Concentrations are given in nM. Neurons treated with cPTPRO–Fc showed significantly higher levels of growth cone collapse than controls. Bar, 10 μm.
Mentions: Proteins that inhibit neurite outgrowth by interacting with specific inhibitory receptors are candidates for repulsive axonal guidance cues. Such repulsive guidance cues may be assayed in vitro using growth cone collapse assays. For example, several of the best-known families of repulsive guidance proteins, including semaphorins, ephrins, slits, and bone morphogenetic proteins, exhibit growth cone collapsing activity in vitro (Luo et al., 1993; Drescher et al., 1995). Therefore, we tested the growth cone collapsing ability of cPTPRO to determine whether cPTPRO might also act as a repulsive guidance cue. In these assays, we added various concentrations of soluble cPTPRO–Fc (or mIgG-1 as a control) to cultures of E6 retinal explants 30 min before fixation. We found that concentrations of cPTPRO as low as 60 pM had clear collapsing ability, and that this activity was already saturated at 0.6 nM (Fig. 8) . The potency of the cPTPRO ECD in collapsing growth cones, as well as the percentage of growth cones collapsed at saturating cPTPRO concentrations, were similar to the values seen previously for ephrin A5–Fc, another repulsive guidance cue for retinal neurons (Wahl et al., 2000), and for the collapse of dorsal root ganglia growth cones by semaphorin IIIA (Luo et al., 1993).

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