Department of Molecular Biology and Microbiology, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106-4960
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 ![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 40× 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 1× 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 40× 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 100× 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
Purification of PTPµ
To determine whether PTPµ was capable of promoting
neurite outgrowth, it was purified from brain by immunoaffinity methods using a monoclonal antibody against
the intracellular domain of the protein (SK15). The PTPµ
purification strategy included stringent detergent washes
of the column before elution to exclude any associated proteins. The full length PTPµ protein from brain was
~200 kD, and two fragments of ~105 kD (predominantly
extracellular form) and 100 kD (the intracellular domain,
transmembrane region, and a short stretch of the extracellular domain) were observed (Fig. 2 A, arrows). These
fragments have been shown to be due to normal proteolytic processing of the protein into noncovalently associated extracellular and intracellular fragments, respectively (Brady-Kalnay and Tonks, 1994a). The eluted PTPµ
protein was examined by immunoblotting to confirm that
the preparation was not contaminated by other CAMs
that promote neurite outgrowth (Fig. 2 B). The samples
included purified proteins from brain: PTPµ (two different
preparations), N-cadherin, NCAM, and L1. N-cadherin,
NCAM, and L1 were detected in the appropriate purified
protein lanes, but were absent from the PTPµ preparations. PTPµ was only detected in the PTPµ preparations. These results clearly show that the PTPµ purified using
these stringent conditions was not contaminated with N-cadherin, L1, or NCAM, three CAMs that are highly expressed in brain and have been previously demonstrated
to promote neurite outgrowth (Rutishauser, 1983
; Lagenaur and Lemmon, 1987
; Bixby and Zhang, 1990
).
|
PTPµ Promotes Neurite Outgrowth
Retinal ganglion cell axons migrate along the surface of
neuronal or glial cells in the brain during development
(Mey and Thanos, 1992); therefore, CAMs expressed on
the surface of these cells can serve as a substrate for neuronal migration. Due to the complexity of the developing
brain, we have used an in vitro model system to gain a better understanding of the mechanisms that regulate axonal
growth. The in vitro system uses a purified protein-coated
dish as a substrate and measures the ability of neuronal
cells to extend neurites on that substrate. The homophilic binding activity of PTPµ and expression by RGC neurons
led us to test whether RGC neurons were capable of extending neurites on a PTPµ substrate. The purified PTPµ
or N-cadherin preparations from brain (Fig. 2) were individually coated as a substrate on dishes (Lagenaur and
Lemmon, 1987
) and used to culture E8 chick retinal explants (Fig. 3). Neurite outgrowth on a PTPµ substrate was observed within 2 d of plating, and the length and density of outgrowth increased over the following 2 d. Neurites grew out from explants derived from E6, 8, and 10 chick retinas, but not from E4 (data not shown), suggesting that PTPµ-dependent neurite outgrowth may be developmentally regulated. Alternatively, since the expression of PTPµ is very low at E4 (see Fig. 6), we may be
unable to detect neurite outgrowth in vitro at that stage. The most robust outgrowth occurred from E8 retinas and
this age was used for the experiments described here. On a
PTPµ substrate, the neurites tended to be somewhat fasciculated (Fig. 3, D and E). In addition, the growth cones
were small and spiky in nature, with multiple long filopodial processes and a small lamellipodial region (Fig. 3 F).
In contrast, outgrowth on N-cadherin was more robust and
was observed within 1 d of culture, suggesting a faster growth rate on N-cadherin than on PTPµ. Additionally,
the neurites were less fasciculated on N-cadherin (Fig. 3,
A and B), and the growth cones possessed a much larger
lamellipodial area and several short filopodial processes
(C). The lower density of neurite outgrowth on the PTPµ
substrate in comparison with the N-cadherin substrate may indicate that only a subset of RGC neurons are able
to respond to PTPµ and induce neurite outgrowth. However, PTPµ was expressed at equal levels by all neurites
growing on N-cadherin or PTPµ substrates (data not shown).
The distinct morphology of neurites on a PTPµ substrate
in comparison with N-cadherin suggests that PTPµ may
use a unique signaling mechanism to promote neurite outgrowth.
|
|
To ensure that the neurite outgrowth activity in the purified PTPµ preparation was mediated by PTPµ, retinal explants were cultured on a PTPµ substrate and N-cadherin or L1 control substrates in the presence or absence of function-blocking antibodies against PTPµ, N-cadherin, or L1 (Fig. 4). Neurite outgrowth on a PTPµ substrate was completely blocked by the addition of antibodies against the extracellular portion of PTPµ (Fig. 4 B), but was unaffected by the addition of antibodies against N-cadherin (C) or L1 (D). These results demonstrate that PTPµ specifically promotes neurite outgrowth. N-cadherin antibodies caused a significant inhibition in neurite outgrowth on N-cadherin (Fig. 4 G). Antibodies against the extracellular domain of PTPµ had no effect on neurite outgrowth on a N-cadherin substrate (Fig. 4 F). Similarly, the L1 antibodies used for these experiments completely blocked neurite outgrowth on an L1 substrate (Fig. 4 K), but antibodies against PTPµ had no effect (J). Therefore, the antibodies used for these experiments were specific. Due to the longer time course to achieve neurite outgrowth on a PTPµ substrate (2-4 d), it was a concern that the retinal explants may secrete factors over time that could deposit on the dish surface and promote neurite outgrowth. For the culture of retinal explants on purified protein substrates, the dishes were first coated with substrate protein, and then the remaining binding sites on the dish were blocked by the addition of BSA before plating. A control experiment was done in which retinal explants were cultured on dishes coated with BSA alone. In this situation, no neurites were observed to extend from any explants over the course of 4 d (Fig. 4 H), suggesting that the neurite outgrowth-promoting activity was due to the purified protein used as the substrate and was not due to a soluble factor originating from the retina tissue.
|
To determine which cell types extended neurites on
PTPµ, the cultures were fixed and processed for immunocytochemistry using antibodies against specific cell types
of the retina. All of the long neurites growing on PTPµ
were positively labeled with an antibody against L1 (8D9;
Fig. 5, E and F), which is only expressed by the RGCs
(Lemmon and McLoon, 1986), suggesting that the neurites on PTPµ were derived from RGC neurons. In addition,
other cells were observed to migrate from the explants
and were identified as bipolar neurons (based on reactivity to the 3G3 antibody; Fig. 5, A and B), or Müller glia
(based on reactivity to the 5A7 antibody; Fig. 5, C and D)
(Drazba and Lemmon, 1990
). Other cells from E8 retinas,
such as photoreceptor cells, were not observed to migrate on PTPµ. The cells that grew out onto the PTPµ substrate
all express PTPµ, suggesting that PTPµ may be acting homophilically to promote outgrowth.
|
PTPµ Interacts with N-Cadherin in Retina
PTPµ is abundant in chick retina, and its expression is developmentally regulated (Fig. 6 A). PTPµ expression is
observed by E4 (the earliest time-point examined), with
peak expression by E11. N-cadherin is also detected in retina at E4, and undergoes a similar increase in expression
with development (Fig. 6 C) (Inuzuka et al., 1991). The
molecular weight of PTPµ is altered with time, suggesting developmental regulation of glycosylation, proteolytic cleavage, and/or shedding. Similar modifications have been
observed for a related RPTP, LAR (Streuli et al., 1992
;
Serra-Pages et al., 1994
; Aicher et al., 1997
).
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.
|
PTPµ Is Required for N-Cadherin-dependent Neurite Outgrowth
Based on previous studies, we hypothesized that PTPµ
maintains a protein in the N-cadherin/catenin complex in a
dephosphorylated state that may be important for N-cadherin-mediated adhesion and/or neurite outgrowth. To
examine the role of PTPµ in N-cadherin-mediated adhesion events, a retrovirus encoding the antisense version
of the PTPµ cDNA sequence was used to downregulate
PTPµ expression in dissociated retinal cells. PTPµ is normally synthesized as a full length (200 kD) precursor that
can be expressed at the cell surface or cleaved into a 105 kD
form (predominately extracellular) or 100 kD form (the
intracellular domain, transmembrane region, and a short
stretch of the extracellular domain; Brady-Kalnay and
Tonks, 1994a; Gebbink et al., 1995
). Immunoblot analysis
and densitometric measurement of the gel bands that were
immunoreactive with an antibody against PTPµ demonstrated that the full-length protein (200-kD band) was reduced by 99% in cells infected with PTPµ antisense virus
when compared with cells infected with control virus (Fig.
8 A, lanes 1 and 3). In addition, the 100-kD band was reduced by 77%, whereas a 95-kD immunoreactive band
was unchanged (Fig. 8 A, lane 3). These results suggest
that PTPµ antisense expression inhibits the new synthesis
of PTPµ. Therefore, the full-length protein made before
infection has likely been cleaved over time to form the two
smaller fragments that may not turn over rapidly. The retrovirus used for these experiments is negatively regulated
by tetracycline. When tetracycline was present in the medium, PTPµ antisense virus had no effect on PTPµ expression level (Fig. 8 A, lane 4). In contrast, no change in
N-cadherin expression was detected in cells infected with
PTPµ antisense virus when compared with cells infected
with control virus (Fig. 8 B). The blot for PTPµ expression
was stripped and reprobed with antibodies to NCAM to
verify equal protein loading per lane (Fig. 8 C). These results demonstrate that the PTPµ antisense retrovirus was
able to infect the retina cells, resulting in a significant downregulation of PTPµ expression.
|
To determine whether PTPµ function is required for
N-cadherin-mediated neurite outgrowth, E4 retinas were
infected with PTPµ antisense or control retrovirus. Explants from the retinas were then cultured on N-cadherin
or control substrates and neurite outgrowth was examined
at 24 and 48 h after plating. E4 retinas were used for these
experiments because a large percentage of the cells are
still mitotic at this age, which is a requirement for retroviral-mediated gene transfer. Explants infected with PTPµ
antisense virus displayed a dramatic and significant decrease in neurite outgrowth on N-cadherin in comparison
with sister explants infected with control virus (Figs. 9, C-D,
and 10, A-B; Table I). A range of effects was detected, including no neurite outgrowth, some short neurites, and in
a few cases a small number of long neurites were observed.
After infection with antisense virus, there is an ~50%
reduction in PTPµ expression in the neurites when they
are immunolabeled with antibodies to PTPµ (data not shown). Therefore, there is a substantial reduction in the
PTPµ phosphatase overall, but some residual expression
of PTPµ in all of the neurites in the explant population.
The photograph shown (Fig. 9, C-D) is representative of
the median level of neurite outgrowth. Quantitation of all
of the results indicated that neurite lengths were reduced
by 53% and overall neurite density was reduced by 74% in
cultures expressing PTPµ antisense in comparison with cells expressing control virus. In contrast, PTPµ antisense
had no effect on either length or density of neurites growing on the control substrates laminin (Figs. 9, A-B, and 10, A-B), or L1 (Figs. 9, E-F, and 10, A-B; Table I). Since the
length and density of outgrowth on laminin and L1 in the
presence of PTPµ antisense virus was similar to that observed previously (Lemmon et al., 1992), it seems unlikely
that the PTPµ antisense virus altered RGC differentiation.
In addition, no difference in the level of axon fasciculation
was observed in cultures infected with control versus PTPµ
antisense virus on any substrate examined. More importantly, the ability of neurons to extend neurites on other
substrates suggests that the PTPµ antisense virus was not
toxic for the neurons themselves and did not have nonspecific effects on neurite outgrowth in general. These results
suggest that PTPµ is specifically involved in regulating N-cadherin-mediated neurite outgrowth.
|
|
The retroviral expression system used for these experiments is repressed in the presence of tetracycline (Paulus
et al., 1996). Retinal explants plated on N-cadherin in the
presence of both tetracycline and PTPµ antisense virus
showed no reduction in either neurite length or density
(Fig. 10, A-B; Table I) when compared with explants infected with control virus. These results demonstrate that
expression of PTPµ antisense is regulated by the presence of tetracycline. In addition, the observed decrease in neurite outgrowth on N-cadherin requires the expression of
antisense PTPµ.
|
PTPµ is capable of acting as both an adhesion molecule
and an enzyme; therefore, it was important to determine
which of these functions was necessary for the regulation of N-cadherin-mediated neurite outgrowth. To address this issue, we generated a PTPµ mutant that contained a single amino acid change (cysteine to serine) in
the catalytic domain of the phosphatase. This c s mutant, which encoded the full-length PTPµ protein tagged
at the COOH terminus with GFP, preserved the adhesive function of the extracellular segment but rendered
the phosphatase catalytically inactive. E4 retinas were infected with a retrovirus encoding the c
s mutant form
of PTPµ, and cultured on an N-cadherin substrate. Explants infected with the c
s mutant virus displayed a
dramatic and significant decrease (~50%) in neurite outgrowth on N-cadherin in comparison with sister explants
infected with control virus (Fig. 10, C-D; Table I). These
results were similar to those obtained in cultures infected
with PTPµ antisense virus (Fig. 10, A-B; Table I). Overexpression of full-length PTPµ coupled to GFP did not alter neurite outgrowth on N-cadherin (Fig. 10, C-D; Table
I). These results indicate that PTPµ tyrosine phosphatase
activity is a key regulatory component for proper N-cadherin function.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
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
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
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.
![]() |
Footnotes |
---|
Received for publication 15 June 1998 and in revised form 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.
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).
![]() |
Abbreviations used in this paper |
---|
CAM, cell adhesion molecule; GFP, green fluorescent protein; RGC, retinal ganglion cell; RPTP, receptor-type protein tyrosine phosphatase.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Aberle, H., H. Schwartz, and R. Kemler. 1996. Cadherin-catenin complex: protein interactions and their implications for cadherin function. J. Cell. Biochem. 61: 514-523 |
2. |
Aicher, B.,
M.M. Lerch,
T. Muller,
J. Schilling, and
A. Ulrich.
1997.
Cellular redistribution of protein tyrosine phosphatases LAR and PTP![]() |
3. |
Balsamo, J.,
T.C. Leung,
H. Ernst,
M.K.B. Zanin,
S. Hoffman, and
J. Lilien.
1996.
Regulated binding of a PTP1B-like phosphatase to N-cadherin: control of cadherin-mediated adhesion by dephosphorylation of ![]() |
4. |
Balsamo, J.,
C. Arregui,
T.C. Leung, and
J. Lilien.
1998.
The nonreceptor protein tyrosine phosphatase PTP1B binds to the cytoplasmic domain of N-cadherin and regulates the cadherin-actin linkage.
J. Cell Biol.
143:
523-532
|
5. | Barford, D., A.J. Flint, and N.K. Tonks. 1994. Crystal structure of human protein tyrosine phosphatase 1B. Science 263: 1397-1404 |
6. | Beggs, H.E., P. Soriano, and P.F. Maness. 1994. N-CAM-dependent neurite outgrowth is inhibited in neurons from fyn-minus mice. J. Cell Biol. 127: 825-833 [Abstract]. |
7. |
Behrens, J.,
L. Vakaet,
R. Friis,
E. Winterhager,
F. Van Roy,
M. Mareel, and
W. Birchmeier.
1993.
Loss of epithelial differentiation and gain of invasiveness correlates with tyrosine phosphorylation of the E-cadherin/![]() |
8. | Bentley, D., and T.P. O'Connor. 1994. Cytoskeletal events in growth cone steering. Curr. Opin. Neurobiol 4: 43-48 |
9. | Birchmeier, W.. 1995. E-cadherin as a tumor (invasion) suppressor gene. Bioessays 17: 97-99 |
10. | Bixby, J.L., and P. Jhabvala. 1992. Inhibition of tyrosine phosphorylation potentiates substrate-induced neurite growth. J. Neurobiol 23: 468-480 |
11. | Bixby, J.L., and R. Zhang. 1990. Purified N-cadherin is a potent substrate for the rapid induction of neurite outgrowth. J. Cell Biol 110: 1253-1260 [Abstract]. |
12. | Brady-Kalnay, S., A.J. Flint, and N.K. Tonks. 1993. Homophilic binding of PTPµ, a receptor-type protein tyrosine phosphatase, can mediate cell-cell aggregation. J. Cell Biol. 122: 961-972 [Abstract]. |
13. |
Brady-Kalnay, S., and
N.K. Tonks.
1994a.
Identification of the homophilic
binding site of the receptor protein tyrosine phosphatase PTPµ.
J. Biol.
Chem.
269:
28472-28477
|
14. | Brady-Kalnay, S.M., and N.K. Tonks. 1994b. Protein tyrosine phosphatases: from structure to function. Trends Cell Biol. 4: 73-76 . |
15. | Brady-Kalnay, S.M., D.L. Rimm, and N.K. Tonks. 1995. The receptor protein tyrosine phosphatase PTPµ associates with cadherins and catenins in vivo. J. Cell Biol. 130: 977-986 [Abstract]. |
16. | Brady-Kalnay, S.M., and N.K. Tonks. 1995. Protein tyrosine phosphatases as adhesion receptors. Curr. Opin. Cell Biol. 7: 650-657 |
17. |
Brady-Kalnay, S.M.,
T. Mourton,
J.P. Nixon,
G.E. Pietz,
M. Kinch,
H. Chen,
R. Brackenbury,
D.L. Rimm,
R.L. Del Vecchio, and
N.K. Tonks.
1998.
Dynamic interaction of PTPµ with multiple cadherins in vivo.
J. Cell Biol.
141:
287-296
|
18. | Brady-Kalnay, S. 1998. Ig superfamily phosphatases. In Ig Superfamily Molecules in the Nervous System. Vol. 6. P. Sonderegger, editor. Harwood Academic Publishers, Amsterdam, Netherlands. 133-159. |
19. | Brittis, P.A., V. Lemmon, U. Rutishauser, and J. Silver. 1995. Unique changes of ganglion cell growth cone behavior following cell adhesion molecule perturbations: a time-lapse study of the living retina. Mol. Cell. Neurosci 6: 433-449 |
20. | Brittis, P.A., and J. Silver. 1995. Multiple factors govern intraretinal axon guidance: a time-lapse study. Mol. Cell. Neurosci 6: 413-432 |
21. | Brummendorf, T., and F.G. Rathjen. 1993. Axonal glycoproteins with immunoglobulin- and fibronectin type III-related domains in vertebrates: structural features, binding activities, and signal transduction. J. Neurochem. 61: 1207-1219 |
22. | Brummendorf, T., and F. Rathjen. 1998. Molecular interactions involving immunoglobulin superfamily adhesion proteins. In Ig Superfamily Molecules in the Nervous System. Vol. 6. P. Sonderegger, editor. Harwood Academic Publishers, Amsterdam, Netherlands. 23-56. |
23. | Burden-Gulley, S.M., and V. Lemmon. 1995. Ig superfamily adhesion molecules in the vertebrate nervous system; binding partners and signal transduction during axon growth. Semin. Dev. Biol. 6: 79-87 . |
24. | Burden-Gulley, S.M., and V. Lemmon. 1996. L1, N-cadherin and laminin induce distinct distribution patterns of cytoskeletal elements in growth cones. Cell Motil. Cytoskelet 35: 1-23 |
25. | Burden-Gulley, S.M., H.R. Payne, and V. Lemmon. 1995. Growth cones are actively influenced by substrate-bound adhesion molecules. J. Neurosci. 15: 4370-4381 [Abstract]. |
26. |
Cheng, J.,
K. Wu,
M. Armanini,
N. O'Rourke,
D. Dowbenko, and
L.A. Lasky.
1997.
A novel protein-tyrosine phosphatase related to the homotypically adhering ![]() |
27. | Cox, E.C., B. Muller, and F. Bonhoeffer. 1990. Axonal guidance in the chick visual system: posterior tectal membranes induce collapse of growth cones from the temporal retina. Neuron 4: 31-37 |
28. | Culp, L.A., K.L. O'Connor, and R. Lechner. 1997. Extracellular matrix adhesion: biological, molecular and pathogenic mechanisms. In Principles of Medical Biology. Vol. 7B. JAI Press Inc., Greenwich, CT. 573-607. |
29. | Desai, C., J.G. Gindhart, L.S.B. Goldstein, and K. Zinn. 1996. Receptor tyrosine phosphatases are required for motor axon guidance in the Drosophila embryo. Cell 84: 599-609 |
30. | Desai, C.J., Q. Sun, and K. Zinn. 1997. Tyrosine phosphorylation and axon guidance: of mice and flies. Curr. Opin. Neurobiol. 7: 70-74 |
31. | Drazba, J., and V. Lemmon. 1990. The role of cell adhesion molecules in neurite outgrowth on Müller cells. Dev. Biol. 138: 82-93 |
32. | Drescher, U., F. Bonhoeffer, and B.K. Muller. 1997. The Eph family in retinal axon guidance. Curr. Opin. Neurobiol 7: 75-80 |
33. | Fannon, A.M., and D.R. Colman. 1996. A model for central synaptic junctional complex formation based on the differential adhesive specificities of the cadherins. Neuron 17: 423-434 |
34. |
Fuchs, M.,
T. Muller,
M.M. Lerch, and
A. Ulrich.
1996.
Association of human
protein-tyrosine phosphatase ![]() |
35. | Gebbink, M., I. van Etten, G. Hateboer, R. Suijkerbuijk, R. Beijersbergen, A. van Kessel, and W. Moolenaar. 1991. Cloning, expression and chromosomal localization of a new putative receptor-like protein tyrosine phosphatase. FEBS Lett. 290: 123-130 |
36. |
Gebbink, M.F.B.G.,
G.C.M. Zondag,
R.W. Wubbolts,
R.L. Beijersbergen,
I. van Etten, and
W.H. Moolenaar.
1993.
Cell-cell adhesion mediated by a receptor-like protein tyrosine phosphatase.
J. Biol. Chem.
268:
16101-16104
|
37. | Gebbink, M.F.B.G., G.C.M. Zondag, G.M. Koningstein, E. Feiken, R.W. Wubbolts, and W.H. Moolenaar. 1995. Cell surface expression of receptor protein tyrosine phosphatase RPTPµ is regulated by cell-cell contact. J. Cell Biol 131: 251-260 [Abstract]. |
38. |
Gershon, T.R.,
M.W. Baker,
M. Nitabach, and
E.R. Macagno.
1998.
The leech
receptor protein tyrosine phosphatase HmLAR2 is concentrated in growth
cones and is involved in process outgrowth.
Development (Camb.)
125:
1183-1190
|
39. |
Gumbiner, B.M..
1995.
Signal transduction by ![]() |
40. | Halfter, W., D.F. Newgreen, J. Sauter, and U. Schwarz. 1983. Oriented axon outgrowth from avian embryonic retinae in culture. Dev. Biol 95: 56-64 |
41. | Hamaguchi, M., N. Matsuyoshi, Y. Ohnishi, B. Gotoh, M. Takeichi, and Y. Nagai. 1993. pp60v-src causes tyrosine phosphorylation and inactivation of the N-cadherin catenin cell adhesion system. EMBO (Eur. Mol. Biol. Organ.) J. 12: 307-314 [Abstract]. |
42. | Hamburger, V., and H. Hamilton. 1951. A series of normal stages in the development of the chick embryo. J. Morphol. 88: 49-92 . |
43. | Hatta, K., and M. Takeichi. 1986. Expression of N-cadherin adhesion molecules associated with early morphogenetic events in chick development. Nature 320: 447-449 |
44. | Hiscox, S., and W.G. Jiang. 1998. Association of PTPµ with catenins in cancer cells: a possible role for E-cadherin. Int. J. Oncology 13: 1077-1080 . |
45. |
Hoschuetzky, H.,
H. Aberle, and
R. Kemler.
1994.
![]() |
46. | Ignelzi, M.A., D.R. Miller, P. Soriano, and P.F. Maness. 1994. Impaired neurite outgrowth of src-minus cerebellar neurons on the cell adhesion molecule L1. Neuron 12: 873-884 |
47. | Inuzuka, H., S. Miyatani, and M. Takeichi. 1991. R-cadherin: a novel Ca2+ dependent cell-cell adhesion molecule expressed in the retina. Neuron 7: 69-79 |
48. | Kemler, R.. 1993. From cadherins to catenins: cytoplasmic protein interactions and regulation of cell adhesion. TIG (Trends Genet.) 9: 317-321 . |
49. | Krueger, N.X., D. Van Vactor, H.I. Wan, W.M. Gelbart, C.S. Goodman, and H. Saito. 1996. The transmembrane tyrosine phosphatase DLAR controls motor axon guidance in Drosophila. Cell 84: 611-622 |
50. | Kypta, R., H. Su, and L. Reichardt. 1996. Association between a transmembrane protein tyrosine phosphatase and the cadherin-catenin complex. J. Cell Biol. 134: 1519-1529 [Abstract]. |
51. | Lagenaur, C., and V. Lemmon. 1987. An L1-like molecule, the 8D9 antigen, is a potent substrate for neurite extension. Proc. Natl. Acad. Sci. USA 84: 7753-7757 [Abstract]. |
52. | Lemmon, V., S.M. Burden, H.R. Payne, G.J. Elmslie, and M.L. Hlavin. 1992. Neurite growth on different substrates: permissive versus instructive influences and the role of adhesive strength. J. Neurosci 12: 818-826 [Abstract]. |
53. | Lemmon, V., K. Farr, and C. Lagenaur. 1989. L1 mediated axon outgrowth occurs via a homophilic binding mechanism. Neuron 2: 1597-1603 |
54. | Lemmon, V., and S. McLoon. 1986. The appearance of an L1-like molecule in the chick primary visual pathway. J. Neurosci 6: 2987-2994 [Abstract]. |
55. | Lin, C.H., and P. Forscher. 1993. Cytoskeletal remodeling during growth cone- target interactions. J. Cell Biol. 121: 1369-1383 [Abstract]. |
56. | Lochter, A., and M. Schachner. 1993. Tenascin and extracellular matrix glycoproteins: from promotion to polarization of neurite outgrowth in vitro. J. Neurosci. 13: 3986-4000 [Abstract]. |
57. | Matsunaga, M., K. Hatta, A. Nagafuchi, and M. Takeichi. 1988a. Guidance of optic nerve fibres by N-cadherin adhesion molecules. Nature 334: 62-64 |
58. | Matsunaga, M., K. Hatta, and M. Takeichi. 1988b. Role of N-cadherin cell adhesion molecules in the histogenesis of neural retina. Neuron 1: 289-295 |
59. | Matsuyoshi, N., M. Hamaguchi, S. Taniguchi, A. Nagafuchi, S. Tsukita, and M. Takeichi. 1992. Cadherin-mediated cell-cell adhesion is perturbed by v-src tyrosine phosphorylation in metastatic fibroblasts. J. Cell Biol. 118: 703-714 [Abstract]. |
60. |
Mey, J., and
S. Thanos.
1992.
Development of the visual system of the chick![]() |
61. | Mi, Z., W. Weng, M.H. Hankin, V. Narayanan, and C.F. Lagenaur. 1998. Maturational changes in cell surface antigen expression in the mouse retina and optic pathway. Dev. Brain Res. 106: 145-154 |
62. | Neel, B.G., and N.K. Tonks. 1997. Protein tyrosine phosphatases in signal transduction. Curr. Opin. Cell Biol. 9: 193-204 |
63. | Neugebauer, K.M., C.J. Emmett, K.A. Venstrom, and L.F. Reichardt. 1991. Vitronectin and thrombospondin promote retinal neurite outgrowth; developmental regulation and role of integrins. Neuron 6: 345-358 |
64. | Ochiai, A., S. Akimoto, Y. Kanai, T. Shibata, T. Oyama, and S. Hirohashi. 1994. c-erbB-2 gene product associates with catenins in human cancer cells. Biochem. Biophys. Res. Commun. 205: 73-78 |
65. | Paulus, W., I. Baur, F.M. Boyce, X.O. Breakfield, and S.A. Reeves. 1996. Self-contained, tetracycline-regulated retroviral vector system for gene delivery to mammalian cells. J. Virol. 70: 62-67 [Abstract]. |
66. | Prada, C., J. Puga, L. Perez-Mendez, R. Lopez, and G. Ramirez. 1991. Spatial and temporal patterns of neurogenesis in the chick retina. Eur. J. Neurosci. 3: 559-569 |
67. | Redies, C., and M. Takeichi. 1993. N- and R-cadherin expression in the optic nerve of the chicken embryo. Glia 8: 161-171 |
68. | Reichardt, L.F., B. Bossy, S. Carbonetto, I. de Curtis, C. Emmett, D.E. Hall, M.J. Ignatius, F. Lefcort, E. Napolitano, T. Large, et al. 1990. Neuronal receptors that regulate axon growth. In Cold Spring Harbor Symposia on Quantitative Biology. Vol. 55. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 341-350. |
69. | Reichardt, L.F., and K.J. Tomaselli. 1991. Extracellular matrix molecules and their receptors: functions in neural development. Annu. Rev. Neurosci. 14: 531-570 |
70. | Rutishauser, U.. 1983. Molecular and biological properties of a neural cell adhesion molecule. Cold Spring Harbor Symp. Quant. Biol 48: 501-514 |
71. | Rutishauser, U., J.-P. Thiery, R. Brackenbury, and G.M. Edelman. 1978. Adhesion among neural cells of the chick embryo. III. Relationship of the surface molecule CAM to cell adhesion and the development of histotypic patterns. J. Cell Biol 79: 371-381 [Abstract]. |
72. |
Serra-Pages, C.,
H. Saito, and
M. Streuli.
1994.
Mutational analysis of proprotein processing, subunit association, and shedding of the LAR transmembrane protein tyrosine phosphatase.
J. Biol. Chem.
269:
23632-23641
|
73. |
Shibamoto, S.,
M. Hayakawa,
K. Takeuchi,
T. Hori,
N. Oku,
K. Miyazawa,
N. Kitamura,
M. Takeichi, and
F. Ito.
1994.
Tyrosine phosphorylation of ![]() |
74. | Silver, J., and U. Rutishauser. 1984. Guidance of optic axons in vivo by a preformed adhesive pathway on neuroepithelial endfeet. Dev. Biol 106: 485-499 |
75. |
Sommer, L.,
M. Rao, and
D.J. Anderson.
1997.
RPTP![]() ![]() |
76. | Streuli, M.. 1996. Protein tyrosine phosphatases in signaling. Curr. Opin. Cell Biol. 8: 182-188 |
77. | Streuli, M., N. Krueger, P. Ariniello, M. Tang, J. Munro, W. Blattler, D. Adler, C. Disteche, and H. Saito. 1992. Expression of the receptor-linked protein tyrosine phosphatase LAR: proteolytic cleavage and shedding of the CAM-like extracellular region. EMBO (Eur. Mol. Biol. Organ.) J. 11: 897-907 [Abstract]. |
78. | Tang, J., L. Landmesser, and U. Rutishauser. 1992. Polysialic acid influences specific pathfinding by avian motoneurons. Neuron 8: 1031-1044 |
79. | Tessier-Lavigne, M.. 1995. Eph receptor tyrosine kinases, axon repulsion, and the development of topographic maps. Cell 82: 345-348 |
80. |
Tessier-Lavigne, M., and
C.S. Goodman.
1996.
The molecular biology of axon
guidance.
Science
274:
1123-1133
|
81. | Tsui, H.T., W.B. Pope, C.S. Kim, and W.L. Klein. 1992. Transient expression of adheron molecules during chick retinal development. J. Neurobiol. 23: 720-738 |
82. | Tsukita, S., K. Oishi, T. Akiyama, Y. Yamanashi, T. Yamamoto, and S. Tsukita. 1991. Specific proto-oncogene tyrosine kinases of src family are enriched in cell-to-cell adherens junctions where the level of tyrosine phosphorylation is elevated. J. Cell Biol. 113: 867-879 [Abstract]. |
83. | Volberg, T., B. Geiger, R. Dror, and Y. Zick. 1991. Modulation of intercellular adherens-type junctions and tyrosine phosphorylation of their components in RSV-transformed cultured chick lens cells. Cell Regul. 2: 105-120 |
84. | Volberg, T., Y. Zick, R. Dror, I. Sabanay, C. Gilon, A. Levitzki, and B. Geiger. 1992. The effect of tyrosine-specific protein phosphorylation on the assembly of adherens-type junctions. EMBO (Eur. Mol. Biol. Organ.) J. 11: 1733-1742 [Abstract]. |
85. | Warren, S.L., and W.J. Nelson. 1987. Nonmitogenic morphoregulatory action of pp60v-src on multicellular epithelial structures. Mol. Cell. Biol. 7: 1326-1337 |
86. |
Zondag, G.,
G. Koningstein,
Y.P. Jiang,
J. Sap,
W.H. Moolenaar, and
M. Gebbink.
1995.
Homophilic interactions mediated by receptor tyrosine phosphatases µ and ![]() |