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© The Rockefeller University Press, 0021-9525/1999//1323 $5.00
The Journal of Cell Biology, Volume 144, Number 6, , 1999 1323-1336

PTPµ Regulates N-Cadherin–dependent Neurite Outgrowth

Department of Molecular Biology and Microbiology, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106-4960

Cell adhesion is critical to the establishment of proper connections in the nervous system. Some receptor-type protein tyrosine phosphatases (RPTPs) have adhesion molecule–like extracellular segments with intracellular tyrosine phosphatase domains that may transduce signals in response to adhesion. PTPµ is a RPTP that mediates cell aggregation and is expressed at high levels in the nervous system. In this study, we demonstrate that PTPµ promotes neurite outgrowth of retinal ganglion cells when used as a culture substrate. In addition, PTPµ was found in a complex with N-cadherin in retinal cells. To determine the physiological significance of the association between PTPµ and N-cadherin, the expression level and enzymatic activity of PTPµ were perturbed in retinal explant cultures. Downregulation of PTPµ expression through antisense techniques resulted in a significant decrease in neurite outgrowth on an N-cadherin substrate, whereas there was no effect on laminin or L1-dependent neurite outgrowth. The overexpression of a catalytically inactive form of PTPµ significantly decreased neurite outgrowth on N-cadherin. These data indicate that PTPµ specifically regulates signals required for neurites to extend on an N-cadherin substrate, implicating reversible tyrosine phosphorylation in the control of N-cadherin function. Together, these results suggest that PTPµ plays a dual role in the regulation of neurite outgrowth.

Key Words: neurite outgrowth • protein tyrosine phosphatase • cadherin • cell adhesion • retina

Abbreviations used in this paper: CAM, cell adhesion molecule; GFP, green fluorescent protein; RGC, retinal ganglion cell; RPTP, receptor-type protein tyrosine phosphatase.

THE development of the nervous system is a complex process requiring the formation of a large number of specific connections by a variety of neuronal cell types and target tissues. Some of the factors that regulate axonal pathfinding include contact-mediated growth promoting or repulsive molecules, as well as diffusible attractant and repulsive factors (reviewed by Tessier-Lavigne and Goodman, 1996). Contact-dependent guidance is thought to be mediated by a series of adhesive events between the growth cone, the specialized region at the distal tip of a growing axon, and molecules expressed on other cells or in matrices that form the "substrate" for migration. Presumably, the growth cone responds to these cues by initiation of a local signal that ultimately regulates the direction of axonal extension. The role of adhesion molecules in contact-dependent guidance has been extensively studied, yet the signaling events involved in this process are not well understood. The present study investigates the role of a unique cell adhesion molecule, the receptor-type protein tyrosine phosphatase mu (PTPµ),¹ in contact-dependent signaling and neurite outgrowth. PTPµ is expressed in several regions of the central nervous system during development and is capable of mediating adhesion, which suggests it could be one of the adhesion molecules that plays a role in neurite outgrowth.

Due to the complexity of the developing nervous system, many studies, including the one described here, have used in vitro model systems to gain a better understanding of the mechanisms that regulate axonal growth. A number of nervous system–derived cell surface proteins have been isolated, but only a subset of these have been shown to promote neurite outgrowth when used as a substrate in vitro. These proteins are subdivided into three major classes: the integrins and their ligands the extracellular matrix molecules (ECM), cadherins, and the immunoglobulin superfamily of cell adhesion molecules (CAMs). Neuronal cells express several integrin receptors and will extend neurites on a subset of ECM molecules including laminin, vitronectin, thrombospondin, janusin, and tenascin (Reichardt et al., 1990; Neugebauer et al., 1991; Reichardt and Tomaselli, 1991; Lochter and Schachner, 1993; Culp et al., 1997). The cadherins are calcium-dependent adhesion molecules and only N- and R-cadherin have been shown to promote neurite outgrowth (Bixby and Zhang, 1990; Redies and Takeichi, 1993). In addition, some Ig superfamily molecules mediate cell–cell adhesion and have also been shown to promote neurite outgrowth (Brummendorf and Rathjen, 1993; Burden-Gulley and Lemmon, 1995). L1 and NCAM are two examples of Ig superfamily CAMs that are abundantly expressed in the developing and adult nervous systems, and are capable of promoting neurite outgrowth (reviewed in Burden-Gulley and Lemmon, 1995; Brummendorf and Rathjen, 1998).

A number of receptor protein tyrosine phosphatases (RPTPs) resemble Ig superfamily CAMs such as NCAM, and appear to directly mediate adhesion or associate with other adhesion molecules (reviewed in Brady-Kalnay and Tonks, 1995; Streuli, 1996; Neel and Tonks, 1997; Brady-Kalnay, 1998). The coupling of CAM-like extracellular domains and phosphatase enzymatic activity within one molecule suggests that changes in the adhesive state of the phosphatase could potentially alter enzymatic activity. One such RPTP, PTPµ, is characterized by an extracellular segment that contains one MAM domain (meprins, A5, mu), one Ig domain, and four fibronectin type III repeats (Gebbink et al., 1991). Expression of PTPµ induced the aggregation of nonadhesive cells (Brady-Kalnay et al., 1993; Gebbink et al., 1993), and both the MAM domain (Zondag et al., 1995) and the Ig domain (Brady-Kalnay and Tonks, 1994a) have been shown to play a role in PTPµ-mediated cell–cell adhesion. Together, these studies demonstrated that the binding is homophilic (i.e., the "ligand" for PTPµ is an identical PTPµ molecule on an adjacent cell).

The juxtamembrane domain of PTPµ has 20% amino acid identity to the conserved intracellular domain of the cadherins (Brady-Kalnay and Tonks, 1994b). The cytoplasmic segment of cadherins binds to catenins that link this complex to the actin cytoskeleton (Gumbiner, 1995; Aberle et al., 1996). Previously, we showed that PTPµ associates with the cadherin/catenin complex (Brady-Kalnay et al., 1995; Hiscox and Jiang, 1998), specifically with N-, E-, and R-cadherins (also called cadherin-4; Brady-Kalnay et al., 1998). In addition, the intracellular segment of PTPµ was demonstrated to bind directly to the intracellular domain of E-cadherin (Brady-Kalnay et al., 1995), and the COOH-terminal 38 amino acids of E-cadherin were required for this interaction (Brady-Kalnay et al., 1998). However, a role for PTPµ in the regulation of cadherin function has not yet been shown.

The PTPµ protein is abundant in many parts of the central nervous system (Gebbink et al., 1991; Brady-Kalnay et al., 1995; Sommer et al., 1997; Brady-Kalnay, 1998) and is developmentally regulated in the retina. The retina is one of the best characterized and experimentally tractable systems for studying both cell–cell adhesion and development. The retina is comprised of a number of different cell types, and the molecular interactions of multiple CAMs are known to regulate retinal histogenesis and axonal pathfinding (Silver and Rutishauser, 1984; Matsunaga et al., 1988a,b; Brittis et al., 1995; Brittis and Silver, 1995). In the retina, PTPµ is primarily expressed on retinal ganglion cells (RGCs) whose sole function is to communicate via their neuronal processes with the brain. RGC axons migrate along the surfaces of neuronal and glial cells in the brain during development, thus using the CAMs on the surfaces of these cells as a substrate for neuronal migration.

Due to the homophilic binding nature of PTPµ, its developmental pattern of expression in the retina, and interaction with cadherins in other tissues, we tested the hypothesis that PTPµ regulates neurite outgrowth. In this report, we used an in vitro retina explant model system to study neurite outgrowth. We provide evidence that PTPµ is capable of promoting neurite outgrowth and cell migration when used as a culture substrate. The neurite outgrowth activity was specific to PTPµ since antibodies against PTPµ completely inhibited outgrowth on a PTPµ substrate. In addition, PTPµ was found in a complex with N-cadherin in retinal tissues and RGC neurites. To determine the physiological significance of an association between PTPµ and N-cadherin, the expression level and enzymatic activity of PTPµ were perturbed in retinal explants. Downregulation of PTPµ expression through antisense techniques resulted in a significant decrease in neurite outgrowth on an N-cadherin substrate, whereas there was no effect on laminin or L1-dependent neurite outgrowth. The overexpression of a catalytically inactive form of PTPµ significantly decreased neurite outgrowth on N-cadherin, thus illustrating the importance of the enzymatic activity of the PTPµ phosphatase in this process. These data indicate that PTPµ specifically regulates signals required for neurites to extend on an N-cadherin substrate, implicating reversible tyrosine phosphorylation in the control of N-cadherin function. Together, these results suggest that PTPµ plays a dual role in the regulation of neurite outgrowth during retinal development.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Antibodies and Reagents
Monoclonal antibodies against the intracellular (SK15, SK18) and extracellular (BK2, BK9) domains of PTPµ have been described (Brady-Kalnay et al., 1993; Brady-Kalnay and Tonks, 1994a). The MB4 monoclonal antibody was generated against the peptide CSH 338 (Brady-Kalnay and Tonks, 1994a) from the immunoglobulin domain of PTPµ (amino acids 231–256). The 494 polyclonal antibody generated against a PTPµ peptide (amino acids 42–60) has been described (Brady-Kalnay and Tonks, 1994a). A monoclonal pan-cadherin antibody (SMP) generated against the COOH terminus of chick N-cadherin was obtained from Sigma Chemical Co. The NCD-2 monoclonal antibody was generously provided by Dr. Gerald Grunwald (Thomas Jefferson University, Philadelphia, PA) from hybridoma cells generated by Dr. Masotoshi Takeichi (Hatta and Takeichi, 1986). A polyclonal antibody against N-cadherin (7873) was kindly provided by Dr. John Hemperly (Becton Dickinson Labs, Research Triangle Park, NC). Antibodies against chick NCAM (5e, RO28 and RO32) and L1 (RO21) were generously provided by Dr. Urs Rutishauser (Case Western Reserve University). Monoclonal antibodies against chick L1 (8D9), a bipolar neuron-specific antigen (3G3), and a Müller glia-specific antigen (5A7) were generously provided by Dr. Vance Lemmon (Case Western Reserve University). RPMI 1640 medium, laminin, and fetal bovine serum were obtained from GIBCO BRL. Aprotinin and leupeptin were obtained from Boehringer Mannheim Biochemicals. Tween-20 was obtained from Fisher Scientific Co. All other reagents were obtained from Sigma Chemical Co.

Purification of PTPµ from Brain
Adult rat brains were minced and homogenized (Bellco Biotechnology) in a solution of 0.32 M sucrose in 50 mM Tris-HCl, 150 mM NaCl (TBS), pH 8.0, containing protease inhibitors 5 mM EDTA, 10 µg/ml turkey trypsin inhibitor, 2 mM benzamidine hydrochloride, and 200 µM phenylmethylsulfonylfluoride. The homogenate was layered onto a 0.8 M, 1.2 M sucrose gradient and centrifuged at 25,000 rpm for 45 min (SW28 rotor; Beckman Instruments, Inc.). The membrane layer was diluted with TBS and respun at 50,000 rpm for 30 min (Ti 70.1 rotor; Beckman Instruments, Inc.). The pellet was then extracted with 1% sodium deoxycholate in 50 mM Tris-HCl, pH 8.0. The membrane extract was respun at 50,000 rpm for 30 min and the supernatant was incubated overnight at 4°C with CNBr Sepharose 4B beads (Pharmacia LKB Biotechnology) that had been covalently coupled with the SK15 PTPµ antibody. The beads were washed extensively with 50 mM Tris-HCl containing 0.5% sodium deoxycholate and 0.5% NP-40, pH 8.0, followed by 10 mM Tris-HCl, pH 8.0. The protein was eluted from the column with 0.1 M diethylamine, pH 11.5, and neutralized with 2 M Tris-HCl, pH 3.6. For SDS-PAGE, sample buffer (4% SDS, 20% glycerol, 0.2 M dithiothreitol, bromphenol blue, 0.12 M Tris, pH 6.8) was added and boiled for 5 min at 95°C. L1 and N-cadherin were purified from chick brains using previously described procedures (Lemmon et al., 1989; Bixby and Zhang, 1990).

Protein purity was checked by two different methods. First, the eluted protein fractions were separated by 4–15% SDS-PAGE (gradient Tris-HCl gels; Bio-Rad Laboratories) and the gel was silver stained. In a second procedure, the eluted fractions were immunoblotted as previously described (Brady-Kalnay et al., 1995).

Dish Preparation and Culture of Retinal Explants and Dissociated Cells
35-mm tissue culture dishes (Falcon Labware) were coated with nitrocellulose in methanol (Lagenaur and Lemmon, 1987) and allowed to dry. A small amount of protein (2–4 µg) was spread across the center of the dishes, and they were incubated 30 min at room temperature. Remaining binding sites on the nitrocellulose were blocked with 2% BSA in PBS, and the dishes were rinsed with RPMI-1640 medium.

Retinal explant cultures were made according to a previously described procedure (Halfter et al., 1983; Drazba and Lemmon, 1990). In brief, embryonic day 8 (stage 32.5–33 according to Hamburger and Hamilton, 1951) White Leghorn chick eyes were dissected and the retina was flattened with the photoreceptor side down onto black nitrocellulose filters (0.45-µm pore size; Vanguard International, Inc.) that had previously been incubated in 0.05% concanavalin A to enhance attachment of the retina to the filter. The filter was then cut into 350-µm wide strips perpendicular to the optic fissure using a McIlwain tissue chopper. Strips were inverted onto substrate-coated culture dishes so that the ganglion cell layer was directly adjacent to the substratum. Cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum/2% chick serum/penicillin-streptomycin-fungizone and incubated at 37°C in 95% air/5% CO₂.

Neurite outgrowth on PTPµ, N-cadherin, and L1 substrates was inhibited by the addition of culture supernatant from the BK2 (anti–PTPµ), NCD2 (anti–N-cadherin), and 8D9 (anti–L1) hybridoma cells, respectively, into the medium at the time of plating retinal explants as described above. The medium was supplemented with fetal bovine serum and chick serum to maintain equivalent levels of growth factors to controls. Neurite outgrowth was examined at 24, 48, and 72 h after plating.

Dissociated retina cultures were prepared from E6 embryos using the procedure above, except that the retinas were incubated in 0.25% trypsin and 0.1% EDTA (Mediatech Cellgro) for 20 min at 37°C. The retinas were dissociated by trituration and resuspended in RPMI-1640/10% tryptose phosphate broth/4% fetal bovine serum/1% chick serum/gentamycin, and then cultured in 12-well plates (Falcon Labware). E6 retinas were used because a large percentage of the cells are still mitotic at this age, which is an important requirement for retroviral-mediated gene transfer. Expression of the exogenous gene was repressed by culturing infected cells in the presence of 3 µg/ml tetracycline (see below).

Immunolabeling of Retina Sections and Cultured Explants
For immunohistochemical labeling of retina sections, eyes from E8 chicks were fixed with 4% paraformaldehyde, 0.01% glutaraldehyde in PEM buffer (80 mM Pipes, 5 mM EGTA, 1 mM MgCl₂, 3% sucrose), pH 7.4. The tissue was rinsed and cryoprotected with 20% sucrose in PBS and frozen in OCT medium (EM Sciences). Cryostat sections were cut at 7-µm intervals, adhered to gelatin coated slides, and stored at –20°C. Sections were permeabilized and blocked with 1% Triton, 20% goat serum in PBS, and then incubated overnight at 4°C with primary antibodies diluted into block buffer (20% goat serum, 1% BSA, 0.5% saponin in PBS). After extensive rinsing, sections were incubated with fluorescein-conjugated secondary antibodies, rinsed extensively with PBS, and then coverslipped with IFF mounting medium (0.5 M Tris-HCl, pH 8.0, containing 20% glycerol and 1 mg/ml p-phenylenediamine). Immunolabeled sections were examined using a 40x objective on an Axiophot microscope (Carl Zeiss, Inc.), and images captured using a Hamamatsu-cooled CCD camera.

For immunocytochemical labeling of retinal explant cultures, the cultures were fixed as above, and then rinsed with PBS and incubated with block buffer. Cultures were incubated with primary antibodies in block buffer overnight at 4°C. After rinsing with PBS, the cultures were blocked with TNB reagent (supplied in TSA-direct kit; NEN Life Science Products) for 30 min, and then incubated with fluorophore or HRP-conjugated secondary antibodies diluted in TNB for 1 h at room temperature. After extensive rinsing with TNT buffer (0.1 M Tris, 0.15 M NaCl, 0.05% Tween-20), the cells were incubated with the tyramide-FITC reagent in 1x amplification diluent for 10 min to deposit FITC onto the HRP-conjugated secondary antibodies. After TNT rinses, the cultures were coverslipped with IFF mounting medium and examined using a 40x objective on a microscope (405M; Carl Zeiss, Inc.).

Antibody cross-linking ("patching") experiments were done by incubating live retinal cultures with a polyclonal antibody against the extracellular domain of PTPµ (494) for 40 min at 37°C. The cultures were then rinsed, fixed, and permeabilized as above. The cells were processed for immunocytochemistry as above using a monoclonal antibody against N-cadherin (NCD2) or NCAM (5e) and the appropriate secondary antibodies. The double-labeled samples were examined using a 100x Neofluar objective (1.3 numerical aperture) on a confocal microscope (LSM-410; Carl Zeiss, Inc.).

Immunoprecipitations
Antibodies were covalently coupled to CNBr Sepharose 4B (Pharmacia LKB Biotechnology) using the manufacturer's protocol, or Protein A beads using a previously described protocol (Brady-Kalnay et al., 1995). E8 retinas were homogenized with a tissue tearor (200; PRO Scientific Inc.) in lysis buffer (1% Triton, 20 mM Tris, pH 7.6, 2 mM CaCl₂, 150 mM NaCl, 1 mM benzamidine hydrochloride, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 1 mM sodium orthovanadate, 0.1 mM ammonium molybdate, 0.2 mM phenyl arsine oxide), and incubated on ice for 45 min. Triton-insoluble material was removed by centrifugation at 14,000 rpm, and the lysate was incubated with antibody-coupled beads overnight at 4°C. The beads were washed extensively with lysis buffer, and then boiled in sample buffer and separated by 6% SDS-PAGE. Proteins were transferred to nitrocellulose membrane and immunoblotted as described (Brady-Kalnay et al., 1995).

Construction and Expression of the PTPµ Retroviruses
The retroviral system used is a tetracycline-repressible promoter-based ("tet-off") system (Paulus et al., 1996). Using the pBPSTR1 vector generously provided by Dr. Steven Reeves (Harvard Medical School, Charlestown, MA), the following constructs were generated: antisense PTPµ, sense PTPµ, and a c s mutant form of PTPµ. A PTPµ antisense plasmid was generated that contained PTPµ coding sequence (base pairs 2449– 4358) in the opposite orientation to the promoter. The sense plasmid contained almost the entire coding sequence of PTPµ (base pairs 1–4350; i.e., it only lacked the last two amino acids and the stop codon). This was done to create an in-frame fusion with the green fluorescence protein (GFP) at the COOH terminus that we call PTPµGFP. This plasmid was generated by digesting full length PTPµ in bluescript (Brady-Kalnay et al., 1993) and ligating it into pEGFP-N3 from Clonetech. The mutant form of PTPµ containing the cysteine-to-serine mutation at residue 1095 was generated by PCR, and a BglII/BspE1 fragment was subcloned to replace that same fragment in the wild-type form of PTPµ in the vector PTPµ/pEGFP-N3. The resulting plasmid, c s mutant PTPµGFP, was sequenced to confirm that the single amino acid mutation was present. The c s mutation renders the phosphatase catalytically inactive (Barford et al., 1994). The pEGFP plasmids containing either wild-type or mutant PTPµ were subcloned into the tetracycline-regulatable retroviral vector, pBPSTR1. A replication-defective amphotrophic retrovirus was made by transfecting the PA317 helper cell line (CRL-9078; American Type Culture Collection) with the respective PTPµ-containing plasmids. Control virus was generated by transfecting PA317 helper cells with the pBPSTR1 plasmid alone. The virus was added to cells in the presence or absence of 3 µg/ml tetracycline. When tetracycline was added, expression of the viral gene was inhibited. Reduction in endogenous PTPµ expression was verified by immunoblotting lysates from infected cells with antibodies to PTPµ. The density of the bands on the immunoblot films was measured using the MetaMorph image analysis program (Universal Imaging Corp.).

Infection of Retinal Explants with Retrovirus
Retroviral-mediated gene transfer requires that the infected cells are still mitotic in order to incorporate and express the retroviral gene. RGC neurons begin to drop out of cell division at E2–3 at a region just dorsotemporal to the optic fissure, and the wave of maturation continues outward from this region in a spatiotemporal fashion (Halfter et al., 1983; Prada et al., 1991). To infect the greatest number of cells for these experiments, retinas from viral-free E3.5–4 chicks (stage 20–23) (Spafas Inc.) were used. At this age, robust neurite outgrowth occurred on N-cadherin, laminin, and L1 substrates, but not on PTPµ. Therefore, PTPµ could not be used as a test substrate for these experiments. The dissection and plating procedure is as described above except that the retinas were cut at 250-µm intervals. Explant strips from each retina were laid in alternating fashion onto two similar substrate-coated dishes, with four explants per dish, then 28 µg of polybrene and 1 ml of virus-containing medium were added for a 6–18-h incubation at 37°C. After incubation in virus, the medium was exchanged with normal culture medium. Cultures were examined at 24 and 48 h after plating, and neurite outgrowth from each explant was photographed.

Quantitation of Neurite Outgrowth
To quantify the neurite outgrowth, the 35-mm negatives were scanned and the digitized images were analyzed using the MetaMorph image analysis program. Lengths of the five longest neurites per explant were measured perpendicular to the explant tissue. To measure the number of neurites per explant, the region of neurite outgrowth was outlined to define the region of interest, the neurites were highlighted using the threshold function, and the total number of highlighted pixels per region of interest was calculated. This method provided a means to compare density between control and test conditions on each substrate. The neurite length and density measurements were analyzed by Fisher's PLSD, Scheffe, and Student's t test (Statview 4.51; Abacus Concepts, Inc.), and similar results were obtained with each of these tests for each experiment. The data from all like experiments were combined and plotted (Cricketgraph III; Computer Associates International, Inc.).

	Results

Top Abstract Materials and Methods Results Discussion References

 
Expression of PTPµ in the Retina
During development, the retina becomes laminated such that cells of a particular type localize to distinct regions within the adult retina. A phase contrast image of an embryonic day 8 (E8) chick retina cross-section illustrates the limits of the neural retina and incomplete lamination at this stage of development (Fig. 1 A). Immunohistochemical labeling of E8 retina sections showed that the greatest level of PTPµ expression was present in the retinal ganglion cell layer and in their processes that make up the optic fiber layer (Fig. 1 B). RGC neurons were verified in this region by labeling with an antibody against chick L1 that specifically recognizes the RGC axons (Fig. 1 C). PTPµ was also expressed at high levels in a region directly adjacent to the pigmented epithelium, which is the outer limit of the neural retina and is thought to be populated in part by mitotic precursor cells (Fig. 1 B) (Mey and Thanos, 1992). In addition, PTPµ was observed in a region of the retina that labeled positively with the 3G3 antibody against bipolar neurons (Fig. 1 D). This immunolabeling pattern is consistent with that previously described for bipolar neurons (Tsui et al., 1992; Mi et al., 1998). Since the axons of RGCs form the optic nerve and are the sole output from the retina to the brain, the high expression of PTPµ on these cells is consistent with a putative role for PTPµ in neurite outgrowth.

View larger version (106K): [in this window] [in a new window] [Download PPT slide]   Figure 1 Expression of PTPµ in chick retina. E8 chick retina sections were immunohistochemically labeled with antibodies against (B) PTPµ, (C) L1, or (D) a bipolar neuron-specific antigen. (B) PTPµ labeling was present predominantly in regions that labeled positively for retinal ganglion cells (C) and bipolar neurons (D). Phase contrast image is shown in A. OFL, optic fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; PC, precursor cells; PE, pigmented epithelium. Scale bar, 50 µm.

View larger version (50K): [in this window] [in a new window] [Download PPT slide]   Figure 2 Purification of PTPµ from brain. PTPµ was purified from adult rat brain by immunoaffinity methods. (A) The eluted PTPµ protein was separated by 5–15% gradient SDS-PAGE and silver stained. Both the full-length protein (200 kD) and the proteolytically processed extracellular (105 kD) and intracellular (100 kD) fragments were detected (arrows). (B) The purified PTPµ protein was examined by immunoblotting to confirm that the preparation was not contaminated by other cell–cell adhesion molecules that promote neurite outgrowth. N-cadherin, NCAM, L1, and two different preparations of PTPµ were purified and equal amounts of the proteins were separated by 6% SDS-PAGE. Immunoblots using antibodies to PTPµ (top left), NCAM (top right), L1 (bottom left), or N-cadherin (bottom right) are shown.

View larger version (157K): [in this window] [in a new window] [Download PPT slide]   Figure 3 PTPµ promotes neurite outgrowth from chick retinal explants. Neural retina explants from E8 chick embryos were cultured for 2 d on N-cadherin–coated dishes (A–C) or 4 d on dishes coated with PTPµ (D–F). The fields shown in B and E are higher power images of the same fields shown in A and D, respectively. Long neurites grew out onto each substrate, but were more fasciculated on the PTPµ substrate (D and E) as compared with the N-cadherin substrate (A and B). Growth cones on N-cadherin were largely lamellipodial with several short filopodial processes (C), whereas growth cones on PTPµ were typically small and spiky in appearance with multiple long filopodial processes and a small lamellipodial region (F). Scale bars: (A) 100 µm, (B) 50 µm, (C) 10 µm.

View larger version (53K): [in this window] [in a new window] [Download PPT slide]   Figure 6 PTPµ interacts with N-cadherin in chick retina. Lysates were made from embryonic day 4, 8, and 11 neural retinas. 5 µg total protein per lane was separated by 6% SDS-PAGE, transferred to nitrocellulose, and probed with antibodies against (A) PTPµ or (C) N-cadherin. (B and D) Immunoprecipitates from lysates of E8 retina were immunoblotted. Mouse IgG (lane 1), PTPµ (lanes 2 and 3) and N-cadherin (lane 4) antibodies were used for immunoprecipitation. The immunoprecipitates were subjected to 6% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies to (B) PTPµ or (D) N-cadherin.

View larger version (68K): [in this window] [in a new window] [Download PPT slide]   Figure 4 Neurite outgrowth on PTPµ is blocked by addition of antibodies against PTPµ. Neural retina explants from E8 chick embryos were cultured on a PTPµ (A–D), N-cadherin (E–G), or L1 (I–K) substrate in the presence of function-blocking antibodies against PTPµ (B, F, and J), N-cadherin (C and G), or L1 (D and K). The explants are on the left of each panel. The neurites in each dish were examined using dark-field optics. Antibodies against PTPµ inhibited neurite outgrowth on the PTPµ substrate (B), whereas they had no effect on neurite outgrowth on N-cadherin (F) or L1 substrates (J). Conversely, antibodies against N-cadherin inhibited neurite outgrowth on a N-cadherin substrate (G), but had no effect on neurite outgrowth on PTPµ (C). Similarly, antibodies against L1 inhibited neurite outgrowth on an L1 substrate (K), but had no effect on a PTPµ substrate (D). A dish coated with bovine serum albumin alone did not support neurite outgrowth (H), suggesting that, over time, the retinal explants did not secrete proteins that promote neurite outgrowth in this assay. Scale bar, 200 µm.

View larger version (128K): [in this window] [in a new window] [Download PPT slide]   Figure 5 PTPµ promotes migration of several cell types from chick neural retina. Neural retina explants from E8 chick embryos were cultured on a PTPµ substrate for 4 d. The cells were fixed and immunolabeled with antibodies against a bipolar-specific antigen (A and B), a Müller glia-specific antigen (C and D), or L1 (E and F). Phase contrast (A, C, and E) and fluorescent images (B, D, and F) are shown. The long neurites that extended on the PTPµ substrate were from RGC neurons as shown by the labeling with an antibody against L1 (E and F), which is restricted to RGC axons in retina. Bipolar neurons (A and B) and Müller glia (C and D) were also observed to migrate onto PTPµ. Scale bar, 50 µm.

To examine whether PTPµ associates with N-cadherin, the major cadherin in retina, immunoprecipitation experiments were done using E8 retina lysates. Similar results were obtained using E4 retinas (data not shown). Both the full-length (200 kD) and cleaved (100 kD) forms of PTPµ were immunoprecipitated by antibodies to PTPµ (Fig. 6 B, lanes 2 and 3), but not by mouse IgG (Fig. 6 B, lane 1). When immunoprecipitates of PTPµ were probed on immunoblots with antipancadherin antibodies, an association with N-cadherin was detected (Fig. 6 D, lanes 2 and 3). In the reciprocal experiment, N-cadherin was immunoprecipitated by antibodies to N-cadherin (Fig. 6 D, lane 4), but was not detected when mouse IgG was used for immunoprecipitation (lane 1). N-cadherin immunoprecipitates contained the 200-kD full-length form of PTPµ (Fig. 6 B, lane 4), and the 100-kD fragment of PTPµ that was also present in the PTPµ immunoprecipitates (lanes 2 and 3).

The results from the immunoprecipitation experiments indicated that PTPµ associates with N-cadherin. Since PTPµ is expressed at high levels within RGC neurons, it is likely that an association with N-cadherin occurs within these cells. To verify that PTPµ interacts with cadherins in RGC neurons, an antibody cross-linking experiment was performed using neurites from E8 chick retinal explants growing on the control substrate laminin. In Fig. 7, A–B and E–F, the cells were fixed and processed for immunocytochemistry using the 494 polyclonal antibody against PTPµ (A and E) and a monoclonal antibody against either N-cadherin (B) or NCAM (F). In the absence of PTPµ cross-linking, N-cadherin, NCAM, and PTPµ were present in a continuous fashion along the length of the axons and growth cones. For the cross-linking experiments, the explants were cultured for 24 h to allow significant RGC neurite outgrowth, and the live cultures were incubated with a polyclonal antibody against the extracellular domain of PTPµ (494) to permit clustering of PTPµ protein on the cell surface (patching; Fig. 7, C and G). The cells were then fixed and processed for immunocytochemistry with antibodies to N-cadherin (Fig. 7 D) or NCAM (H). The PTPµ protein was cross-linked into patches along the neurites (Fig. 7, C and G). Importantly, N-cadherin protein also became concentrated into the PTPµ patch sites (Fig. 7 D). Extensive colocalization was observed between PTPµ and N-cadherin, suggesting a stoichiometric association between these proteins in neurites. In contrast, another abundantly expressed cell adhesion molecule, NCAM, did not become concentrated into PTPµ patch sites (Fig. 7 H). The results of these experiments show that PTPµ and N-cadherin associate in the RGC neurites, and provide a basis for examination of the function of that association in neurite outgrowth.

View larger version (84K): [in this window] [in a new window] [Download PPT slide]   Figure 7 PTPµ colocalizes with N-cadherin in retinal neurites. Neural retina explants from E8 chick embryos were cultured overnight on laminin. The cells were fixed and double labeled with a polyclonal antibody against PTPµ (A and E) and a monoclonal antibody against either N-cadherin (B) or NCAM (F). These results show that PTPµ, N-cadherin, and NCAM are expressed in a continuous fashion along neurites and into growth cones. Live cells were incubated with a polyclonal antibody against PTPµ to cluster the PTPµ protein on the surface of the neurites into "patches" (C and G), and then the cells were fixed and double labeled with a monoclonal antibody against N-cadherin (D) or NCAM (H). N-cadherin (D), but not NCAM (H), colocalized to the PTPµ patch sites, suggesting an interaction with PTPµ within the neurites. As a control for nonspecific fluorescence from the secondary antibodies, fixed retina cultures were processed for immunocytochemistry as above except that the primary antibodies were omitted (I and J). The image shown in I is a control for images shown in A, C, E, and G, whereas the image shown in J is a control for images shown in B, D, F and H. The control images (I and J) were collected using identical settings on the confocal microscope as those used for the corresponding experimental images (A–H). Scale bar, 20 µm.

View larger version (36K): [in this window] [in a new window] [Download PPT slide]   Figure 8 PTPµ expression is reduced in retinal cells infected with antisense PTPµ retrovirus. Dissociated chick retinal cells infected with control virus (lanes 1 and 2) or PTPµ antisense virus (lanes 3 and 4) in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 3 µg/ml tetracycline were harvested 72 h after infection. 7.5 µg of each lysate was separated by 6% SDS-PAGE, transferred to nitrocellulose, and probed with antibodies to (A) PTPµ, (B) N-cadherin, or (C) NCAM. (A) A substantial reduction in PTPµ expression was detected in lysates from PTPµ antisense– infected cells in the absence of tetracycline (lane 3) in comparison with lysates from control virus–infected cells (lane 1). Densitometric measurement of the PTPµ bands showed a 99% reduction of the 200-kD band and a 77% reduction of the 100-kD band, whereas a 95-kD band was unchanged. No difference in PTPµ expression was observed when cells infected with PTPµ antisense were cultured in the presence of tetracycline (lane 4). (B) In contrast, no difference in N-cadherin expression was detected in lysates from control virus (lanes 1 and 2) or PTPµ antisense virus (lanes 3 and 4) infected cells. (C) The blot probed for PTPµ expression was stripped and reprobed for a nonspecific protein, NCAM, to verify equal protein loading per lane.

View larger version (110K): [in this window] [in a new window] [Download PPT slide]   Figure 9 PTPµ is required for N-cadherin–dependent neurite outgrowth. E4 chick retina explants were infected with control (A, C, and E) or PTPµ antisense (B, D, and F) virus and cultured on laminin (A and B), N-cadherin (C and D), or L1 (E and F) substrates for 48 h. The neurites in each dish were examined using dark-field optics. No difference in neurite length or density was observed in cultures infected with vector or PTPµ antisense virus when cultured on laminin (A and B) or L1 (E and F) substrates. In contrast, PTPµ antisense resulted in a dramatic reduction of both neurite length and density in cultures on a N-cadherin substrate (C and D). Scale bar, 400 µm for images shown in A and B. Scale bar, 200 µm for images shown in C–F.

View larger version (43K): [in this window] [in a new window] [Download PPT slide]   Figure 10 Quantitation of the effect of PTPµ perturbation on neurite outgrowth. E4 chick retina explants were infected with control virus (gray bars) or PTPµ antisense virus (black bars) and cultured on laminin, N-cadherin, or L1 substrates for 48 h. PTPµ antisense virus caused a significant reduction in neurite length (A) and density (B) on N-cadherin, but not on laminin or L1 substrates. The retrovirus used is negatively regulated by tetracycline (tet-off system). Retinal explants plated on N-cadherin in the presence of both tetracycline and PTPµ antisense virus showed no reduction in either neurite length (A) or density (B) when compared with explants infected with control virus. For statistical analysis, see Table I. E4 chick retinas were infected with control virus (gray bars) or test virus encoding the c s (C to S) mutant of PTPµ (cross-hatched bars) or PTPµGFP sense control (striped bars), and cultured on N-cadherin for 48 h. The c s mutant caused a significant reduction in neurite length (C) and density (D), whereas the sense control had no effect on either length or density. For statistical analysis, see Table I.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
To gain an understanding of the function of PTPµ in the developing nervous system, we examined the ability of embryonic chick retinal neurons to extend neurites on a PTPµ substrate. PTPµ promoted neurite outgrowth from RGC neurons, and the migration of bipolar neurons and Müller glia from E8 retinal explants. Neurite outgrowth on a PTPµ substrate was blocked by the addition of antibodies against PTPµ, indicating that the neurite outgrowth activity in the purified PTPµ preparation was specific to the PTPµ protein. The morphology of neurites growing on PTPµ was unique from that observed on other purified CAMs, suggesting PTPµ may use a distinct signaling mechanism to promote neurite outgrowth. Within the retina, the expression of PTPµ is developmentally regulated and increases over time in a pattern similar to N-cadherin expression (Matsunaga et al., 1988; Inuzuka et al., 1991). Previous studies have demonstrated that cadherins and PTPµ associate with one another (Brady-Kalnay et al., 1995, 1998; Hiscox and Jiang, 1998). In this study, we demonstrated that PTPµ and N-cadherin form a complex in retinal tissue. An association between PTPµ and N-cadherin was also demonstrated in RGC neurites, suggesting that PTPµ may be involved in the regulation of N-cadherin– mediated neurite outgrowth. The downregulation of PTPµ expression through antisense techniques resulted in a decreased ability of RGC neurites to extend on a N-cadherin substrate, but did not affect neurite outgrowth on laminin or L1 substrates. Overexpression of a catalytically inactive form of PTPµ also inhibited N-cadherin–mediated neurite outgrowth, thus providing further evidence that a component of the N-cadherin/catenin complex may be a substrate of PTPµ. Together, these results provide evidence that PTPµ is capable of promoting neurite outgrowth individually, and specifically regulating neurite outgrowth mediated by N-cadherin.

Axonal pathfinding, fasciculation, target recognition, and synapse formation are all processes that require contact-mediated recognition of cell surface cues. The diversity of the CAMs and other molecules involved in axonal pathfinding reflects the staggering array of decisions an individual axon must make along the way to its target. Many of the guidance molecules are members of the immunoglobulin superfamily. These include CAMs like L1 and NCAM, tyrosine kinases such as Eph family members and FGF receptors and even some RPTPs; for example, DLAR, DPTP69D, and now PTPµ. Presumably, these molecules mediate specific recognition events at different points during axonal outgrowth and pathfinding. CAMs are not solely involved in adhesion of neurons to one another; they also participate in signal transduction. The interaction of a growth cone with a particular CAM can lead to rapid and specific changes in growth cone morphology (Burden-Gulley et al., 1995). This implies the adhesion molecules are sending signals that result in a transient change in the underlying cytoskeleton (Burden-Gulley and Lemmon, 1996) that guide a neuron toward its target (Lin and Forscher, 1993; Bentley and O'Connor, 1994).

RPTPs Are Involved in Axonal Guidance
Previous inhibitor studies suggested that tyrosine phosphatases are involved in the control of neurite outgrowth in general and on CAM substrates (Bixby and Jhabvala, 1992; Beggs et al., 1994; Ignelzi et al., 1994). Recent studies suggest that regulation of tyrosine phosphorylation by RPTPs affects axonal growth possibly by "steering" growth cones along the appropriate pathway (Desai et al., 1997). In Drosophila, two CAM-like RPTPs are expressed in the central nervous system, and knockout experiments have demonstrated that they play critical roles in development. Mutant embryos for the Drosophila RPTPs, DLAR and DPTP69D, display an inability of specific motorneurons to recognize guidance cues that allow them to innervate appropriate target muscles (Desai et al., 1996; Krueger et al., 1996). In addition, a LAR homologue in leech was shown to accumulate in a subset of axonal growth cones and play a guidance role during outgrowth of these axonal processes (Gershon et al., 1998). Together with the present study, these data provide the first evidence that RPTPs could be directly involved in axonal pathfinding and suggest that tyrosine phosphorylation is a key regulator of axonal guidance and choice point recognition.

In the present study, we demonstrated that PTPµ plays a role in neurite outgrowth at physiological levels of protein expression. This data is important because previous studies on the ability of PTPµ to mediate aggregation were performed when PTPµ was massively overexpressed (Brady-Kalnay et al., 1993). We have now demonstrated that PTPµ promotes neurite outgrowth from RGC neurons, presumably through a homophilic binding mechanism. PTPµ may have several important roles in nervous system development. First, it may act as a cell–cell adhesion molecule necessary for maintenance of nervous system integrity. This could occur through homophilic binding of PTPµ on the surfaces of two apposing axons to promote axon fasciculation, a process required for nerve formation. A similar role has been suggested for other CAMs such as L1 (Lemmon et al., 1989; Tang et al., 1992) and NCAM (Rutishauser et al., 1978). Second, PTPµ may act as a permissive molecule for axonal growth. The expression of PTPµ by axons, astrocytes (our unpublished data) and other nonneuronal cells makes this role a likely possibility. A contact attraction role for PTPµ is a third possibility, such that PTPµ actively guides axons during pathfinding. For example, PTPµ may be expressed at specific choice points where axons must choose the appropriate pathway. RGC growth cones encounter several choice points during outgrowth to their target, the optic tectum (Tessier-Lavigne, 1995). The mechanisms regulating this stereotypical innervation pattern are only partly understood, but involve tyrosine phosphorylation (Cox et al., 1990; Drescher et al., 1997). A fourth possible function of PTPµ is as a sensor molecule. For example, changes in the adhesive state of the extracellular environment may be transmitted through PTPµ via regulation of its catalytic domain. PTPµ may then directly regulate the phosphorylation state of a number of cytosolic proteins, including components of the cadherin/catenin complex. This idea is supported by the inhibition of N-cadherin–mediated neurite outgrowth when RGC neurons overexpress a catalytically inactive form of PTPµ.

Cadherins Are Regulated by Tyrosine Phosphorylation
A likely target of the PTPµ enzyme is a component of the N-cadherin complex, which was previously postulated to be a substrate of PTPµ (Brady-Kalnay et al., 1995, 1998). The association of cadherins and catenins with receptor and nontransmembrane PTPs has now been observed by many groups (Brady-Kalnay et al., 1995; Balsamo et al., 1996, 1998; Fuchs et al., 1996; Kypta et al., 1996; Aicher et al., 1997; Cheng et al., 1997; Brady-Kalnay et al., 1998; Hiscox and Jiang, 1998). The juxtamembrane domains of PTP (Fuchs et al., 1996) and PCP2 (Cheng et al., 1997) interact with β catenin. A LAR-like RPTP associates with the cadherin/catenin complex in PC12 cells (Kypta et al., 1996) and the intracellular domain of LAR was shown to bind directly to β catenin and plakoglobin in vitro (Aicher et al., 1997). The PTP1B cytoplasmic phosphatase was shown to interact with the N-cadherin/catenin complex and dephosphorylate β catenin (Balsamo et al., 1996), a process required for N-cadherin–mediated adhesion and actin linkage (Balsamo et al., 1998). Therefore, it is likely that regulation of the cadherin/catenin complex by PTPs will be an important mechanism of control in many cell types, including neurons.

Tyrosine phosphorylation of the cadherin/catenin complex correlates with suppression of cadherin-mediated adhesion (Matsuyoshi et al., 1992; Behrens et al., 1993; Hamaguchi et al., 1993), adherens junction disassembly/ loss of cytoskeletal association (Warren and Nelson, 1987; Volberg et al., 1991, 1992), invasion and malignant progression (Kemler, 1993; Birchmeier, 1995). Tyrosine phosphorylation of the cadherin/catenin complex can be catalyzed by pp60src, EGF receptor, c-erbB2, or hepatocyte growth factor receptor (Tsukita et al., 1991; Hoschuetzky et al., 1994; Ochiai et al., 1994; Shibamoto et al., 1994). These data indicate that one of the mechanisms the cell uses to regulate the function of the cadherin/catenin complex is tyrosine phosphorylation. In the present study, PTPµ associated with N-cadherin in lysates from retina as demonstrated by immunoprecipitation techniques. A similar association was demonstrated in RGC neurites through antibody cross-linking and immunocytochemistry techniques. In addition, the ability of neurites to migrate on N-cadherin was significantly impaired when PTPµ expression was downregulated. These results provide evidence that N-cadherin–mediated neurite outgrowth requires functional PTPµ. The inhibition of N-cadherin–mediated neurite outgrowth due to overexpression of the catalytically inactive form of PTPµ further supports the idea that cadherins or their associated proteins need to be dephosphorylated to function in adhesion (Brady-Kalnay et al., 1995, 1998). Therefore, PTPµ tyrosine phosphatase activity is a key regulatory component of the N-cadherin/catenin complex. In contrast, PTPµ downregulation did not alter neurite outgrowth on L1 or laminin control substrates. These results suggest that the effects of PTPµ downregulation were specific to N-cadherin–mediated neurite outgrowth and not due to general alterations in cellular phosphotyrosine that could nonspecifically affect neurite outgrowth.

Perspectives on the Developmental Role of PTPµ in the Retinotectal System
Our data demonstrate that PTPµ can promote neurite outgrowth, which is consistent with a role for PTPµ in neuronal pathfinding. This promotion of neurite outgrowth could be mimicking the ability of certain neurons to respond to signaling events initiated by PTPµ. The inability of PTPµ to promote neurite outgrowth from retinas earlier than E6 suggests that a threshold level of PTPµ expression on axons may be required for PTPµ to independently promote neurite outgrowth through a homophilic mechanism. At earlier ages, PTPµ may play other specific roles; for example, regulation of N-cadherin–dependent adhesion that is required for morphogenetic movements or axonal pathfinding events. N-cadherin is one of the key molecules involved in many aspects of retinal function from histogenesis and lamination to neurite outgrowth and synapse formation (Matsunaga et al., 1988; Redies and Takeichi, 1993; Fannon and Colman, 1996). PTPµ may regulate N-cadherin function by modulating signals that allow neurons to respond to N-cadherin–mediated adhesion.

	Acknowledgments

 
This work was supported by Research Oncology Training Grant CA-59366-04 to S. Burden-Gulley, and a grant from the American Cancer Society, Ohio Division, Cuyahoga County Unit, and National Institutes of Health grant 1RO1-EY12251 to S. Brady-Kalnay. Additional support was provided by the Visual Sciences Research Center Core Grant from the National Eye Institute (PO-EY11373).

Submitted: 15 June 1998

Revised: 3 February 1999

Address correspondence to Susann Brady-Kalnay, Department of Molecular Biology and Microbiology, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106-4960. Tel.: (216) 368-0330. Fax: (216) 368-3055. E-mail: smb4{at}po.cwru.edu

A number of individuals provided assistance with this study, and their efforts were greatly appreciated. These include Dr. Leif Stordal, who generated the retroviral plasmids used for the antisense experiments, and Tracy Mourton and Ferdouse Sartawi, who provided expert technical assistance. The many contributions of Dr. Vance Lemmon throughout the course of this study are greatly appreciated. In addition, we thank Drs. Lloyd Culp, Lynn Landmesser, Gary Landreth, Vance Lemmon, and Robert Miller for helpful comments on the manuscript.

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