Tài liệu Báo cáo khoa học: Cell surface nucleolin on developing muscle is a potential ligand for the axonal receptor protein tyrosine phosphatase-r ppt

Cell surface nucleolin on developing muscle is a potential
ligand for the axonal receptor protein tyrosine
phosphatase-r
Daniel E. Alete
1
, Mark E. Weeks
2
, Ara G. Hovanession
3
, Muhamed Hawadle
1
and
Andrew W. Stoker
1
1 Neural Development Unit, Institute of Child Health, University College London, UK
2 Molecular Oncology, CRUK, Barts and The London School of Medicine and Dentistry, John Vane Centre, UK
3 UPR 2228 CNRS, UFR Biomedicale-Universite Rene Descartes, Paris, France
Vertebrate nervous system development relies on a
multitude of guidance cues to stimulate axonal exten-
sion and stable synaptic contacts with targets such as
muscles. Interpretation of these environmental signals
by growth cones involves multiple receptor classes such
as cell adhesion molecules (CAMs) [1], DCC and neu-
Keywords
affinity chromatography; axon targeting;
nucleolin; RAP assay; receptor protein
tyrosine phosphatases
Correspondence
A. W. Stoker, Neural Development Unit,
Institute of Child Health, University College
London, 30 Guilford Street, London WC1N

1EH, UK
Fax: +44 207 78314366
Tel: +44 207 9052244
E-mail:
(Received 21 April 2006, revised 2 August
2006, accepted 15 August 2006)
doi:10.1111/j.1742-4658.2006.05471.x
Reversible tyrosine phosphorylation, catalyzed by receptor tyrosine kinases
and receptor tyrosine phosphatases, plays an essential part in cell signaling
during axonal development. Receptor protein tyrosine phosphatase-r has
been implicated in the growth, guidance and repair of retinal axons. This
phosphatase has also been implicated in motor axon growth and innerva-
tion. Insect orthologs of receptor protein tyrosine phosphatase-r are also
implicated in the recognition of muscle target cells. A potential extracellu-
lar ligand for vertebrate receptor protein tyrosine phosphatase-r has been
previously localized in developing skeletal muscle. The identity of this mus-
cle ligand is currently unknown, but it appears to be unrelated to the hepa-
ran sulfate ligands of receptor protein tyrosine phosphatase-r. In this
study, we have used affinity chromatography and tandem MS to identify
nucleolin as a binding partner for receptor protein tyrosine phosphatase-r
in skeletal muscle tissue. Nucleolin, both from tissue lysates and in purified
form, binds to receptor protein tyrosine phosphatase-r ectodomains. Its
expression pattern also overlaps with that of the receptor protein tyrosine
phosphatase-r-binding partner previously localized in muscle, and nucleo-
lin can also be found in retinal basement membranes. We demonstrate that
a significant amount of muscle-associated nucleolin is present on the cell
surface of developing myotubes, and that two nucleolin-binding compo-
nents, lactoferrin and the HB-19 peptide, can block the interaction of
receptor protein tyrosine phosphatase-r ectodomains with muscle and ret-
inal basement membranes in tissue sections. These data suggest that muscle

cell surface-associated nucleolin represents at least part of the muscle bind-
ing site for axonal receptor protein tyrosine phosphatase-r and that nucle-
olin may also be a necessary component of basement membrane binding
sites of receptor protein tyrosine phosphatase-r.
Abbreviations
AP, alkaline phosphatase; CAM, cell adhesion molecule; FGF, fibroblast growth factor; FITC, fluorescein isothiocyanate; HB-19,
5[Kw(CH
2
N)PR]-TASP; HSPG, heparin sulfate proteoglycan; PLAP, placental alkaline phosphatase; PTP, protein tyrosine phosphatase; RAP,
receptor affinity probe; RPTP, receptor protein tyrosine phosphatase; RTK, receptor protein tyrosine kinase.
4668 FEBS Journal 273 (2006) 4668–4681 ª 2006 The Authors Journal compilation ª 2006 FEBS
ropilins [2,3], and enzymes involved in phosphotyro-
sine signaling, such as the receptor protein tyrosine
kinases (RTKs) [4] and receptor protein tyrosine phos-
phatases (RPTPs) [5–7].
Evidence for the role of phosphotyrosine signaling
during axon growth and guidance has come from stud-
ies showing that signaling by fibroblast growth factor
(FGF) receptor, an RTK acting alongside neural
CAMs, promotes neurite growth [8,9]. Also, members
of the Eph family of RTKs regulate retinal axon guid-
ance in direct response to graded patterns of their lig-
ands, the ephrins [10–12]. It is therefore unsurprising
that the counterbalancing enzymes, the RPTPs, are
also implicated in many of these processes. There are
21 recognized human RPTPs [13], with homologs and
orthologs throughout the vertebrates and invertebrates.
RPTPs show strong developmental expression in the
central and peripheral nervous systems, coinciding with
significant events such as axonogenesis, target contact,

synaptogenesis and plasticity [14,15]. The type IIa sub-
family exemplifies these neural RPTPs. These enzymes
have two cytoplasmic phosphatase domains and an
extracellular region consisting of immunoglobulin-like
domains and fibronectin type III repeats, similar to the
NCAM family of cell adhesion molecules [15]. Mem-
bers of the type IIa RPTPs include vertebrate LAR,
PTPd and PTPr, leech hmLAR and Drosophila DLAR
and DPTP69D. Studies have implicated PTPd and
PTPr in retinal axon development in both chick and
Xenopus [16–18]. In Drosophila, DLAR and DPTP69D
have roles in photoreceptor and commissural axon
guidance [19–21]. Mouse LAR deficiency leads to
reduced size of basal forebrain cholinergic neurons and
diminished hippocampal innervation [22], whereas
PTPd deficiency causes impaired learning and
enhanced hippocampal long-term potentiation [23].
PTPr deficiency leads to the most extreme defects,
with hypomyelination of peripheral nerves, abnormalit-
ies in development of the hypothalamus and pituitary,
and ataxias [24,25].
Type IIa RPTPs have also been implicated in the
development of the neuromuscular system. In Dro-
sophila, DLAR and DPTP69D are required for guid-
ance of motor axons [26,27], and DLAR has also been
implicated in neuromuscular synaptic plasticity [28–
31]. LAR-related RPTPs also influence synaptogenesis
in muscles in other species [32,33]. A recent study of
mice with a double gene deficiency in PTPr and PTPd
demonstrated that these two RPTPs are critical for the

generation of a branched innervation pattern in the
diaphragm, and subsequent motor neuron survival
[34]. In the chick embryo, there is strong expression of
PTPr in vertebrate spinal and cranial motor neurons
[35,36], and there is evidence from affinity probe
assays of a potential PTPr ligand in developing mus-
cle [37]. Recent studies using gene knockdown in the
chick spinal cord have also shown that type IIa
RPTPs play a role in motor axon growth [38]. These
collective data show that vertebrate type IIa RPTPs,
like DLAR in the fly, are involved in neuromuscular
development.
Relatively little is known about the signaling mecha-
nisms of these type IIa RPTPs and how such signaling
is regulated. One way to understand these events might
be to identify the extracellular ligands of the RPTPs in
the neuromuscular system. For example, it is known
that PTPd and an isoform of LAR can interact
homophilically [39] and that LAR can also bind
heterophilically to the laminin–nidogen complex [40].
Heparin sulfate proteoglycans (HSPGs) have also been
identified as potential ligands for PTPr [41], and
recently syndecan and dallylike, both HSPGs, have
been reported as functional ligands for DLAR in Dro-
sophila motor axons and neuromuscular synaptogene-
sis [28,30,31]. Nevertheless, the potential ligand of
PTPr within developing muscle of the chick embryo
appears not be HSPG-related, and only interacts with
the short protein isoform of PTPr expressed in motor
neurons [37]. Given the interest in PTPr in neuromus-

cular development, we have undertaken the identifica-
tion of this potential muscle ligand using an affinity
chromatography approach. We report that chick nucle-
olin, expressed on the cell surface of developing muscle
cells, is a PTPr-binding protein. Nucleolin expression
correlates with the location of PTPr-binding sites on
developing muscle, and nucleolin-binding proteins and
peptides can perturb this PTPr binding. These data
demonstrate that cell surface nucleolin is likely to be
part of the PTPr-binding site in developing skeletal
muscles. We also show that nucleolin can be found in
retinal basement membranes and that this nucleolin
may also be necessary, alongside HSPGs, for PTPr
interactions.
Results
Nucleolin is a PTPr-binding protein
We have shown previously that PTPr binds to an
unidentified ligand, or ligand complex, in developing
muscle of the chick [37]. To identify PTPr-binding
proteins and potential ligands, an immobilized fusion
protein consisting of the first six subdomains of the
PTPr ectodomain fused to alkaline phosphatase (AP)
(termed FN3d–AP; Fig. 1A,B) was used to perform
affinity chromatography on solubilized muscle tissue
D. E. Alete et al. Nucleolin is a potential ligand for PTPr
FEBS Journal 273 (2006) 4668–4681 ª 2006 The Authors Journal compilation ª 2006 FEBS 4669
from 10-day-old (E10) chick embryos. To identify spe-
cifically retained proteins that interact with PTPr,we
performed (as a negative control) chromatography on
AP-conjugated sepharose. Detergent extracts of chick

muscle tissue were loaded onto these two columns, and
proteins were eluted using a high-salt buffer. Eluted
proteins were compared after SDS gel electrophoresis,
and this revealed a complex pattern of protein bands.
The only reproducible difference observed was a
95 kDa band identified as being present in the eluate
from the PTPr column, but absent in the control
eluate (Fig. 1C). For protein identification, multiple
affinity runs were performed, and eluates were con-
centrated, separated by SDS gel electrophoresis and
stained with Coomassie. The band of interest was
excised from the gel, digested with trypsin, and ana-
lyzed by tandem MS. As shown in Fig. 2A, 21 pep-
tides were sequenced and found to correspond to
chicken nucleolin (SwissProt accession number
P15771). All 21 peptides could be identified within the
C-terminal region of the nucleolin sequence (Fig. 2B),
with no peptide sequence tags being obtained from the
N-terminal part of nucleolin. This is probably due to
the clustering of glutamic acid residues within the
N-terminal region, preventing the formation of reason-
ably sized peptides for MS ⁄ MS analysis. The calcula-
ted mass of nucleolin, based on its sequence, is
76 kDa; however, it migrates at approximately
100 kDa in SDS gel electrophoresis, due to post-trans-
lational modifications and a high content of negatively
charged amino acids [42]. The identity of the 95 kDa
protein band as nucleolin was confirmed by immuno-
blotting eluates using antibody to nucleolin (Fig. 1D).
This revealed a band at approximately 95 kDa present

in the PTPr eluate only. These data confirm that
nucleolin is a binding partner for PTPr under these
conditions.
PTPr can bind directly to nucleolin
To investigate whether nucleolin can bind directly to
PTPr, we carried out solid-phase binding assays using
A B C
D E
Fig. 1. Affinity chromatography isolation of protein tyrosine phosphatase-r (PTPr)-binding and lactoferrin-binding proteins. (A) Schematic dia-
gram of PTPr-derived proteins. Shown are the two main isoforms of PTPr, PTPr1 and PTPr2. The first six subdomains of the PTPr ecto-
domain were fused to placental alkaline phosphatase (AP) to generate the fusion construct FN3d–AP [41]. Circles, immunoglobulin-like
domains; squares, fibronectin type III domains; PTP, phosphatase catalytic domains; black dots, protease cleavage sites. (B) SDS ⁄ PAGE
separation of FN3d–AP purified from conditioned media using anti-placental alkaline phosphatase (PLAP) agarose. (C) SDS ⁄ PAGE and silver
stain of proteins isolated from AP sepharose (lane 1) and FN3d–AP sepharose (lane 2). A protein band of approximately 95 kDa is present
exclusively in the FN3d–AP eluate (arrowhead). (D) Immunoblot of proteins isolated from AP sepharose (lane 1) and FN3d–AP sepharose
(lane 2) using polyclonal rabbit anti-nucleolin serum. (E) Immunoblot of column eluates from blank sepharose (lane 1) and lactoferrin seph-
arose (lane 2) using polyclonal rabbit anti-nucleolin serum. A 100 kDa nucleolin band is present (arrow). Several lower molecular weight
bands corresponding to proteolytic fragments of nucleolin are also present.
Nucleolin is a potential ligand for PTPr D. E. Alete et al.
4670 FEBS Journal 273 (2006) 4668–4681 ª 2006 The Authors Journal compilation ª 2006 FEBS
recombinant, myc-tagged chick nucleolin purified from
transfected 293T cells. Purification of nucleolin is chal-
lenging, because it is accompanied by a high degree
of protein degradation (D. Alete & A. Hovanession,
unpublished results). Although our purified chick
nucleolin was similarly only around 20% intact (data
not shown), it was considered to be of sufficient qual-
ity for initial solid-phase overlay assays. The purified
nucleolin was immobilized on charged microtiter plates
and incubated with varying concentrations of condi-

tioned media containing FN3d–AP. The data revealed
significant binding of PTPr to nucleolin above that of
the BSA control (Fig. 3), demonstrating that PTPr
can bind to nucleolin directly. Nevertheless, this assay
was near the limit of detection for this interaction, as
successive dilution of the probe soon led to loss of sig-
nal. The low yields of the AP probe and of nucleolin,
together with the inevitable partial degradation of
nucleolin, mean that calculations of binding affinity
are unrealistic at this stage.
Nucleolin localization in muscle is analogous to
PTPr ligand localization
To determine whether the nucleolin identified by affin-
ity chromatography is potentially a muscle ligand for
PTPr, we compared the expression pattern of nucleo-
lin with the distribution of the muscle binding sites for
PTPr receptor affinity probes (RAPs) [43,44] (Fig. 4).
E10 cranial tissue sections were cut and stained using
FN3d–AP fusion protein (Fig. 4A,C) or antibody to
nucleolin (Figs 4B.D). The strongest binding of the
FN3d–AP fusion protein was localized to myotubes of
developing muscle (Fig. 4A and arrow in Fig. 4C),
A
B
Fig. 2. Tandem MS analysis of in-gel tryptic
digest of 95 kDa protein band isolated using
FN3d–AP sepharose. (A) Peptides deter-
mined by sequencing of relevant mass
peaks are shown. These were found to cor-
respond with a high degree of certainty to

chicken nucleolin (SwissProt accession
number P15771). Thirty-one per cent of the
chicken nucleolin sequence is covered by
MS ⁄ MS analysis. (B) Location of sequenced
peptides within the nucleolin sequence. The
primary structure of chicken nucleolin is indi-
cated by the single-letter amino acid code
sequence). Tryptic peptides are printed in
bold and underscored.
D. E. Alete et al. Nucleolin is a potential ligand for PTPr
FEBS Journal 273 (2006) 4668–4681 ª 2006 The Authors Journal compilation ª 2006 FEBS 4671
with binding also being observed in motor nerves and
other scattered cells and matrix. No AP signal at all is
generated when only soluble AP probes are used, or
when nonbinding forms of PTPr–AP are used [37,41].
Immunofluorescence staining using nucleolin antibody
produced a closely overlapping staining pattern to that
seen in the RAP assay, with most of the staining
localized to developing muscle (Fig. 4B). Increased
magnification showed that the most intense staining
was, as with the RAP stain, within the myotubes and
on myotube surfaces (arrow in Fig. 4D). Technical
limitations meant that we could not directly demon-
strate how much of the RAP stain and nucleolin fluor-
escence directly overlapped. Nevertheless, these data
demonstrate that the developmental expression of
nucleolin within developing muscle is largely coincident
with the location of the muscle binding sites for PTPr.
HB-19 pseudopeptide and lactoferrin bind
nucleolin and perturb PTPr binding to

developing muscle
To ascertain whether FN3d–AP binds to nucleolin in
chick muscle tissue, we tested whether the pentameric
pseudopeptide 5(Kw(CH
2
N)PR)-TASP (referred to as
HB-19 [45]) could perturb the binding of FN3d–AP.
HB-19 is a potent inhibitor of HIV entry into the cell,
acting by specifically binding to, and forming com-
plexes with, cell surface nucleolin [46–48]. It has been
shown to exert this effect independently of HSPGs, by
binding the C-terminal tail of nucleolin containing the
RGG domain, consisting of residues 656–707 [49]. HB-
19 (10 lm) or BSA (0.5 mgÆmL
)1
) were prebound to
chick sections and washed, and RAP analysis was then
performed as described earlier (Fig. 5). Prebinding of
Fig. 3. Solid-phase binding assay of protein tyrosine phosphatase-r
(PTPr) to purified nucleolin. Fifteen micrograms of BSA or affinity-
isolated nucleolin were bound to charged plates and incubated with
various concentrations of FN3d–AP supernatant. Binding of FN3d–
AP was determined by alkaline phosphatase (AP) activity measured
at 405 nm. The reaction rate (change in absorbance per min) over
1 h was used to quantify FN3d–AP concentrations. Filled circles
represent nucleolin; open circles represent BSA. Error bars show
standard error of the mean (n ¼ 3). *P £ 0.03, **P £ 0.005.
A
C
B

D
Fig. 4. Immunohistochemical and receptor
affinity probe (RAP) analysis of chick E10
sections. (A,C) RAP staining using the
FN3d–AP fusion construct, demonstrating
protein tyrosine phosphatase-r (PTPr) affin-
ity for its ligand in the developing muscle.
(B, D) Immunofluorescence detection of
nucleolin expression using polyclonal rabbit
anti-nucleolin serum. Analogous muscle
staining patterns are observed in nucleolin
and RAP-stained serial sections. Both the
FN3d–AP and nucleolin antibody stain the
periphery of myotubes [arrows in (C) and
(D)]. DO, dorsal oblique eye muscle. Scale
bar ¼ 0.1 mm (A,B), and 25 lm (C,D).
Nucleolin is a potential ligand for PTPr D. E. Alete et al.
4672 FEBS Journal 273 (2006) 4668–4681 ª 2006 The Authors Journal compilation ª 2006 FEBS
HB-19 prevented subsequent FN3d–AP binding to
both muscle and basement membranes (Fig. 5A,B and
data not shown). We were surprised that HB-19
blocked the basement membrane sites, as these were
thought to be only HSPG-dependent. Nevertheless,
direct detection of nucleolin with a biotinylated form
of HB-19 revealed binding to basement membranes in
the retina (Fig. 5G). This may explain why HB-19
could block FN3d–AP binding to its HSPG ligands.
No nonspecific disruptive effects of HB-19 were
observed on the binding of antibodies to the antigens
laminin and myosin (data not shown).

Further perturbation experiments were carried out
using the protein lactoferrin. Lactoferrin, an iron-
binding protein of the transferrin family, is present
in external secretions and the secondary granules of
A
C
E
F
G
D
B
Fig. 5. HB-19 and lactoferrin perturb protein tyrosine phosphatase-r (PTPr) binding to its ligand in developing muscle. E10 cranial tissue sec-
tions were preincubated with 0.5 mgÆmL
)1
BSA (A,C,E), 10 lM HB-19 peptide (B) or 0.5 mgÆmL
)1
lactoferrin (D,F) before RAP assays using
FN3d–AP. (G) Fluorescent staining of retinal basement membrane using biotinylated HB-19 peptide. Preincubation with lactoferrin and HB-19
abolishes FN3d–AP binding to muscle tissue [asterisks in (A) and (B), and (C) and (D), respectively]. Lactoferrin and HB-19 prebinding also
perturbs retinal basement membrane staining [(E) and (F), and data not shown]. pe, Pigmented epithelium; nr, neural retina; vc, vitreous
chamber; is, interorbital septum; oc, optic chiasma. Scale bar ¼ 0.3 mm (A, B,C,D), and 0.15 mm (E,F,G).
D. E. Alete et al. Nucleolin is a potential ligand for PTPr
FEBS Journal 273 (2006) 4668–4681 ª 2006 The Authors Journal compilation ª 2006 FEBS 4673
polymorphonuclear leukocytes [50]. Lactoferrin is a
highly basic protein [51] that binds to, and is internal-
ized by, cell surface nucleolin, with the binding site
located within the C-terminal RGG domain of nucleo-
lin [52]. We preincubated sections of E10 chick
embryos with 0.5 mgÆmL
)1

lactoferrin. This preincuba-
tion with lactoferrin effectively blocked FN3d–AP
binding to muscle tissue (Fig. 5C,D). Like HB-19,
lactoferrin also perturbed PTPr binding to the base-
ment membrane of the retina (Fig. 5E,F). Lactoferrin
is also an HSPG-binding protein [50,53], and may
therefore be directly interfering competitively with
PTPr binding to basement membrane-associated
HSPGs. It is unlikely, however, that lactoferrin blocks
PTPr binding in muscles through an effect on HSPGs,
as our previous work has shown that the muscle bind-
ing site is not HSPG-related [41].
To confirm that lactoferrin can bind to nucleolin from
developing muscle, we carried out affinity chromatogra-
phy of muscle lysates using immobilized lactoferrin.
Nucleolin was specifically retained on the lactoferrin
column (Fig. 1E). Immunoblots using antibodies
against actin and myosin, as a control for nonspecific
binding, were negative (data not shown). Prebinding of
lactoferrin to muscle sections also showed no nonspe-
cific, disruptive effect on the binding of antibodies to
antigens such as laminin and myosin (data not shown).
Both HB-19 and lactoferrin bind to the C-terminal
RGG domain of nucleolin. Therefore, if PTPr also
binds to this domain, we would predict that an anti-
body raised to a sequence outside this domain might
have little effect on the RAP assay signal. This was
tested with an antibody to nucleolin raised against
amino acids 271–520. No effect on PTPr binding to
muscle was observed (data not shown). There is

currently no RGG-specific antibody to test directly
whether it can block PTPr binding.
These data demonstrate first that two known nucleo-
lin-binding components (HB-19 and lactoferrin) can
specifically inhibit PTPr binding to its muscle ligand.
Second, binding of PTPr to nucleolin is likely to be
mediated through the RGG domain of nucleolin.
Nucleolin is present on the surface of developing
muscle tissue
In order to function as a potential ligand for PTP r,
nucleolin must be present on the surface of the target
cells. Originally, nucleolin was reported to be exclu-
sively nuclear [54]; however, more recent studies have
shown that nucleolin is present on the surface of a
variety of cell lines [42,48,52,55,56] and on the surface
of endothelial cells [57,58]. To address whether nucleo-
lin is also present on the surface of developing muscle
cells, we carried out immunofluorescence analysis of
primary chick muscle cells isolated from E10 embryos
(Fig. 6). These were grown in 6% serum, as this has
been reported to promote the cell surface localization
of nucleolin in MDAMB-435 carcinoma cells [57].
Live, nonpermeabilized cells and fixed, semipermeabi-
lized cells were costained with antibodies to nucleolin
and myosin. Anti-nucleolin staining of the live nonper-
meabilized cells showed punctate patches on the out-
side of the cells (Fig. 6B). No myosin staining was
observed in nonpermeabilized cells, which confirms the
integrity of the membrane. By contrast, semipermeabi-
lized cells showed high levels of nucleolin staining

within the cytoplasm, with myosin staining also being
observed (Fig. 6A). As paraformaldehyde was used for
only partial permeabilization, we did not expect to see
nucleolar localization of nucleolin in these experi-
ments.
To determine more precisely if the staining observed
on the nonpermeabilized cells was present on myotubes,
the live cells were first incubated with anti-nucleolin
sera, and then fixed, permeabilized and treated with
myosin antibody (Fig. 6C,D). We observed punctate
staining of nucleolin on the surface of myotubes (arrow
in Fig. 6D), as well as on the surface of nonmyosin-
expressing cells. Some of the punctate localization also
occurred at the cell–cell interface between these cells and
myotubes (arrowheads in Fig. 6D). It is of interest that
a punctate pattern of nucleolin is also seen on the sur-
face of Hela cells after treatment of live cells with a
nucleolin ligand, midkine [59]. It is possible that the
punctate pattern in muscle cells is in part caused by clus-
tering of nucleolin by the antibodies in the live cells.
To investigate further which cells express surface
nucleolin in muscle, we performed confocal microscopy
on chick muscle sections costained with nucleolin and
myosin antibodies (Fig. 6E,F). In a flattened confocal
stack (Fig. 6F), intracellular overlap was observed as
yellow staining, but in addition we observed punctate
regions of nucleolin on myotube surfaces (arrows in
Fig. 6E). Furthermore, three-dimensional rendering of
the myotubes using the green channel (myosin) to differ-
entiate between intracellular nucleolin signal (yellow,

arrowhead in Fig. 6F) and surface nucleolin (red)
showed nucleolin patches on the surface of developing
myotubes (arrows in Fig. 6F). This form of nucleolin
should therefore be accessible to cell surface PTPr.
Discussion
Studies carried out previously have identified two dis-
tinct tissue binding sites for PTPr ectodomains, within
Nucleolin is a potential ligand for PTPr D. E. Alete et al.
4674 FEBS Journal 273 (2006) 4668–4681 ª 2006 The Authors Journal compilation ª 2006 FEBS
basement membranes [60] and in developing skeletal
muscle [37]. The basement membrane-associated lig-
ands have been identified as HSPGs. Here, using affin-
ity chromatography, tandem MS and RAP affinity
assays, we identified the multifunctional protein nucle-
olin as a potential new ligand present in developing
muscle. It was confirmed that nucleolin and the PTPr
ectodomain could directly interact. Furthermore, we
demonstrated for the first time that nucleolin is
expressed on the surface of developing myotubes, and
that its localization in muscle overlaps that of the pre-
viously characterized PTPr interactor.
Nucleolin was first described as a major nuclear pro-
tein consisting of a negatively charged N-terminal
domain, an RNA-binding domain and a C-terminal
domain rich in RGG motifs [54]. Nucleolin has been
reported to be involved in a diverse array of cellular
processes, including cell proliferation and growth,
cytokinesis, replication, embryogenesis and nucleogene-
sis [61]. More recently, numerous studies have reported
nucleolin as being present on the cell surface

[57,58,62,63] and to function as a ligand ⁄ receptor for a
number of different proteins, including lactoferrin [52],
pleiotrophin [48], achran sulfate [55], HIV [47], l-selec-
tin [42] and midkine [64]. Nucleolin does not have a
classic secretion signal and it is therefore not known
how it reaches the cell surface. The clustering of nucle-
olin on cell surfaces is nonetheless dependent on an
intact actin cytoskeleton, to which it must attach
through an unidentified, integral membrane protein
[56]. Nucleolin can also reach cell surfaces without
endogenous HSPG production [59], and nucleolin has
even been reported to function as a shuttle between
the cell surface and the nucleus [65].
Our study has now shown that nucleolin is found on
the surface of developing myotubes. Indeed, the overall
expression of nucleolin is specifically elevated in
embryonic muscle, although its non-nucleolar
AB
D
FE
C
Fig. 6. Nucleolin is on the cell surface of
developing myotubes. Immunofluorescence
staining of permeabilized (A) and live unper-
meabilized (B) chick primary muscle cultures
(nonconfocal images). Cells were stained
using anti-nucleolin (red), and anti-myosin
(green), and counterstained with DAPI
(blue). Cell surface staining is indicated in
the unpermeabilized cells [arrows in (B)].

(C,D) Live unpermeabilized cells were incu-
bated with anti-nucleolin serum, permeabi-
lized, and then incubated with anti-myosin
serum. Cell surface nucleolin is shown on a
myosin-expressing cell [arrow in (C)], and at
the interface of a cell juxtaposed to a myo-
tube [arrowheads in (D)]. (E,F) Confocal ima-
ges of a tissue section through developing
chick skeletal muscle, stained for myosin
(green) and nucleolin (red). Regions of colo-
calization are shown in yellow. (E) Flattened
image stack through myotubes. Nucleolin
(red) present on the surface of myotubes is
indicated (arrows). (F) Three-dimensional
reconstruction of confocal images using
VOLOCITY software. Cell surface nucleolin
(red patches) is indicated (arrows). Scale
bars ¼ 20 lm.
D. E. Alete et al. Nucleolin is a potential ligand for PTPr
FEBS Journal 273 (2006) 4668–4681 ª 2006 The Authors Journal compilation ª 2006 FEBS 4675
localization was also observed in several other tissue
sites. The patch-like or punctate pattern of nucleolin
on myotubes in culture and also in muscle sections
may also indicate that nucleolin has a role at localized
areas of the developing muscle membrane. It is note-
worthy that several binding partners of nucleolin are
also found in a punctate pattern on cell surfaces, and
in one case pleiotrophin can copatch nucleolin [48].
Our previous data suggest that the PTPr ligand in
muscle does not localize selectively with developing

neuromuscular junctions [37]. Direct examination of
the localization of nucleolin and acetylcholine receptor
also indicates that nucleolin is not notably enriched in
neuromuscular junctions (D. Alete & A. Stoker,
unpublished data). It is possible that nucleolin is inter-
acting with motor axon-associated PTPr prior to neu-
romusclular junction formation, and it also cannot be
ruled out that nucleolin associates with PTPr on
sensory axons [66], in particular the mechanosensory
afferents.
In the blocking experiments with lactoferrin and
HB19, our data also showed a blockade of PTPr
interactions with other known ligand sites, in partic-
ular those of HSPGs in retinal basement membranes.
It is not clear what this means at present, as the
basement membrane interactions of PTP r are abso-
lutely dependent on HSPGs. If they are also depend-
ent on nucleolin, then this might invoke a receptor
complex containing both HSPGs and nucleolin, both
of which might be necessary for a functional interac-
tion with PTPr in the retinal inner basement mem-
brane. It is interesting to note that of the molecules
known to bind to surface nucleolin, pleiotrophin,
midkine, lactoferrin and PTPr also bind to HSPGs
[48,53]. For example, both midkine and PTPr have
been reported to bind to the HSPG agrin [41,67].
Although this may be coincidental, it suggests that
these molecules share some binding properties and
may therefore interact with nucleolin, or a complex
of nucleolin and HSPGs, in a similar fashion. Having

said this, the situation in muscle is still distinct, since
PTPr binding occurs independently of heparan sul-
fate [37]. Furthermore, nucleolin does not require
HSPGs to reach the cell surface, at least in CHO
cells [59].
The mechanism of molecular interaction between
PTPr and the muscle-associated nucleolin remains to
be determined. However, in light of the facts that HB-
19 and lactoferrin bind to the RGG domain at the
C-terminal tail of nucleolin, and that both these com-
ponents perturb the interaction between PTPr and its
muscle ligand, it is plausible to suggest that PTP r
interacts with the RGG domain of surface nucleolin.
The biological significance of the interaction between
PTPr and nucleolin in muscle has also yet to be eluci-
dated. Although both PTPr and PTPd influence motor
axon growth and branching within the target field
[34,38], nucleolin itself has not so far been implicated
in muscle or neuromuscular development. Further-
more, although nucleolin may act as a coreceptor for
HIV, for example, the normal molecular function of
cell surface nucleolin in any type of cell is still relat-
ively unclear. From the present study, we could hypo-
thesize that nucleolin might serve as part of a receptor
complex on the surface of developing muscle, recogni-
zing adhesive molecules such as PTPr present on
incoming growth cones of motor neurons or mechano-
sensory afferent axons. Recent studies have indeed
shown that cell surface nucleolin can function as a cell
adhesion molecule [68].

To address further the function of nucleolin in mus-
cles, function-blocking C-terminal antibodies would be
advantageous, and methods need to be developed for
isolating larger amounts of nondegraded, cell surface
nucleolin. If nucleolin is involved in an RPTP recogni-
tion complex, it will then be possible to test more
directly what the cellular and biochemical conse-
quences are of PTPr–nucleolin interactions.
Materials and methods
Fusion protein constructs and fusion protein
production
The FN3d–AP protein represents a truncated ectodomain
region of cPTPr1 (amino acids 1–597), fused at its C-termi-
nus to the placental AP gene in vector pBG as described
previously [41]. The FN3d–AP expression vector was trans-
fected into 293T cells (grown in, DMEM, 10% fetal bovine
serum, 1% penicillin ⁄ streptomycin mixture; Sigma Aldrich,
Gillingham, UK) using calcium phosphate. Conditioned
medium containing the secreted FN3d–AP fusion protein
was collected after 6–7 days, sterile filtered, buffered to
pH 7.4 with 20 mm Hepes, and stored at 4 °C. RAP assays
were carried out on unfixed tissue cryosections as described
previously [44].
Purification of fusion protein
One milliliter of anti-placental alkaline phosphatase-agarose
(anti-PLAP; Sigma) was packed into an FPLC column
(Amersham Biosciences, Chalfont St Giles, UK). Purifica-
tion of FN3d–AP was carried out using an AKTA FPLC
system (Amersham Biosciences). The column was equili-
brated using five column volumes of 0.05 m Tris and

0.5 m NaCl (pH 8.0) at a flow rate of 0.5 mLÆmin
)1
, and
Nucleolin is a potential ligand for PTPr D. E. Alete et al.
4676 FEBS Journal 273 (2006) 4668–4681 ª 2006 The Authors Journal compilation ª 2006 FEBS
flow-through absorbance was measured at 280 nm. Condi-
tioned media was centrifuged at 400 g for 5 min on a
Sorvall Legend RT with a 7500 6445 rotor, and the
supernatant was recovered and loaded onto the column.
The column was then washed with five column volumes of
the equilibration buffer to remove unbound components.
Bound components were eluted using a mixture containing
0.05 m glycine and 0.5 m NaCl (pH 2.8). Fractions
(500 lL) were collected directly into tubes containing 50 lL
of a 1.0 m Tris ⁄ HCl (pH 9.0) solution. Purified fusion
constructs (determined by the absorbance at 280 nm) were
pooled, and desalted using a PD10 desalting column
(Amersham Biosciences), and their purity was determined
by gel electrophoresis.
Affinity chromatography
Limb and chest muscle tissue (1 g) was dissected from E10
chick embryos and homogenized with a glass homogenizer
in a mixture containing 10 mL of 4% Chaps, 100 mm
KH
2
PO
4
(pH 7.5), 5% glycerol, and protease inhibitor
cocktail (Roche, Lewes, UK). The lysate was vortexed for
1 h at 4 °C and centrifuged at 2000 g on a Sorvall Legend

RT with a 7500 6445 rotor, and the supernatant was recov-
ered and diluted 1 : 2 in NaCl ⁄ P
i
and incubated with affin-
ity matrix (1 mL of CnBr sepharose covalently coupled to
2 mg of FN3d–AP, AP or lactoferrin) overnight at 4 °C.
Chromatography was carried out on an AKTA FPLC sys-
tem (Amersham Biosciences) under the following condi-
tions. The column was washed with five column volumes of
NaCl ⁄ P
i
containing 0.1% Tween and 30 mm EDTA. Bound
components were eluted using NaCl ⁄ P
i
containing 0.5 m
NaCl (2 mL) into 200 lL fractions, separated by PAGE
(6% gel) under reducing conditions, and visualized by silver
staining [69].
Protein identification by tandem MS (LC-MS
⁄
MS)
Bands from a Coomassie-stained gel were cut out and sub-
jected to digestion with trypsin as follows. Gel pieces were
washed three times in 30 lL of 50% CH
3
CN with agita-
tion. The gel pieces were dried in a vacuum centrifuge for
10 min, and reduced with a mixture containing 10 mm
dithiothreitol and 10 mm NH
4

HCO
3
(pH 8.0) (15 lL) for
45 min at 50 °C; this was followed by alkylation with
50 mm idoacetamide and 10 mm NH
4
HCO
3
for 1 h at
room temperature in the dark. Gel pieces were washed
three times in 30 lL of 50% CH
3
CN and vacuum-dried
before being reswollen with 50 ng of modified trypsin
(Promega, Southampton, UK) in 5 lLof10mm
NH
4
HCO
3
. The pieces were then overlaid with 10 mm
NH
4
HCO
3
(10 lL) and incubated for 16 h at 37 °C. The
samples were centrifuged at 16 000 g on a Heraeus Biofuge
and the supernatant was recovered. Peptides were further
extracted twice with 10 lL of 5% trifluoroacetic acid in
50% CH
3

CN and the supernatants were pooled. Peptide
extracts were vacuum-dried and resuspended in 5 lLof
double-distilled H
2
O containing 20 mm NH
4
HPO
4.
Diges-
ted peptide mixtures were separated by nanoHPLC (Ulti-
mate; LC Packings, Amsterdam, Holland) equipped with a
PepMap column (75 lm · 15 cm; LC Packings) at a flow
rate of 300 nLÆmin
)1
. Eluting peptides were analysed by
ESI-MS ⁄ MS in a quadrupole ⁄ orthogonal acceleration
time-of-flight (Q-TOF) mass spectrometer (Micromass,
Wythenshaw, Manchester, UK), using a nanoelectrospray
ion source and ESI emitters with a 15 lm tapered end
(New Objective, Woburn, MA, USA). Proteins were identi-
fied using the SwissProt database with the MASCOT search
engine (). A parent ion toler-
ance of ± 3 m ⁄ z, a peptide ion tolerance of ± 3 m ⁄ z, one
missed cleavage, fixed carbamidomethylation of cysteines
and variable oxidation of methionines were specified.
Purification of nucleolin
A myc-tagged chick nucleolin expression vector [gift from
E. A. Nigg, Swiss institute for experimental cancer research
(ISREC), Epalinges, Switzerland] was transfected into 293T
cells. Cells were cultured for 2 days, washed in cold

NaCl ⁄ P
i
and solubilized in a mixture containing 4% Chaps,
100 mm KH
2
PO
4
(pH 7.5), 5% glycerol, and protease
inhibitor cocktail (Roche). The lysate was centrifuged at
1500 g on a Sorvall Legand RT with a 7500 6445 rotor,
and the supernatant was recovered. Myc-tagged nucleolin
was purified using anti-myc agarose (Sigma), as described
for fusion protein purification.
Solid-phase binding assay
Fifteen micrograms of nucleolin and BSA were immobi-
lized on a 96-well microtiter plate for 2 h at room tem-
perature. The remaining binding sites were saturated by
overnight incubation in NaCl ⁄ P
i
containing 2% goat
serum (Dako, Glostrup, Denmark). Wells were incubated
for 3 h at room temperature with conditioned media con-
taining AP fusion proteins (FN3d). After four washes in
NaCl ⁄ P
i
and one in SEAP buffer (0.5 m m MgCl
2
,1m
diethanolamine, pH 9.8), the bound AP activity was
determined by adding 200 lL of SEAP buffer containing

10 mm p-nitrophenyl phosphate. Progress curves were
recorded for 1 h at room temperature, at 405 nm, using
a Dynex MRX microplate reader (Dynex, Worthing,
UK).
Immunoblot analysis
Affinity-purified samples were separated on a 6% Tris ⁄ gly-
cine gel by SDS ⁄ PAGE and transferred onto PVDF mem-
brane for 40 min at 120 V. Membranes were blocked with
D. E. Alete et al. Nucleolin is a potential ligand for PTPr
FEBS Journal 273 (2006) 4668–4681 ª 2006 The Authors Journal compilation ª 2006 FEBS 4677
NaCl ⁄ P
i
containing 5% milk powder for 1 h and incubated
with rabbit polyclonal anti-nucleolin serum (1 : 5000; Santa
Cruz Biotechnology, Santa Cruz, CA, USA) in NaCl ⁄ P
i
,
5% milk and 0.05% Tween-20 overnight at 4 °C. After
extensive washing, the membranes were incubated with per-
oxidase-coupled goat anti-rabbit serum (1 : 10 000; Dako),
and bound antibody was detected using ECL (Amersham
Biosciences).
Immunohistochemistry and immunofluorescence
analysis
E10 chick embryo heads were frozen in Tissue-Tek OCT
(optimal cutting temperature) compound (Sakura Finetek,
Torrance, CA, USA), cryosectioned (10–12 lm) and moun-
ted on superfrost plus slides (VWR, Lutterworth, UK). For
immunohistochemistry, sections were fixed for 5 min with
)20 °C methanol and then blocked with 1% goat serum

and 0.05% Tween in NaCl ⁄ P
i
for 30 min at room tempera-
ture. Primary antibodies used were rabbit anti-nucleolin
(Santa Cruz) at 1 : 100 dilution, and mouse anti-myosin
(F59; Developmental Studies Hybridoma Bank) used at
1 : 100. Antibodies were diluted in NaCl ⁄ P
i
containing 1%
goat serum and 0.05% Tween-20, and incubated on sec-
tions for 1 h at room temperature. Sections were then
washed three times in NaCl ⁄ P
i
and 0.05% Tween-20, and
secondary antibodies [goat anti-rabbit biotin conjugated,
1 : 100 (Dako); goat anti-mouse fluorescein isothiocyanate
(FITC) conjugated 1 : 100 (Dako)] were incubated for 1 h
at room temperature. After three final washes, FITC-labe-
led sections were mounted with Vectashield Hardset
TM
mounting medium with 4’-6-diamidino-2-phenylindole
(DAPI) (Vector Labs, Burlingame, CA, USA). For biotin-
labeled sections, slides were incubated for a further 30 min
in NaCl ⁄ P
i
and 0.05% Tween, containing streptavidin-con-
jugated Cy3 (1 : 400; Amersham Biosciences), and washed
and mounted as described. For HB-19 peptide staining,
biotinylated HB-19 peptide [49] was diluted in NaCl ⁄ P
i

containing 1% goat serum and 0.05% Tween-20 to a final
concentration of 10 lm and incubated for 1 h at room
temperature. Sections were washed and incubated with
streptavidin-conjugated Cy3 as described above. Sections
were analyzed using an Axiophot fluorescence micro-
scope (Zeiss, Welwyn Garden City, UK) and photographed
with a Leica DC500 digital camera (Leica, Milton Keynes,
UK).
Myotube cultures
Embryonic myotube cultures were established from E10
chick muscle tissue. E10 trunk tissue was dissociated enzy-
matically and plated at 10
6
cells per 60 mm plate on fibro-
nectin-coated glass coverslips in a mixture containing
DMEM, 2% chick serum, 4% fetal bovine serum and 1%
penicillin ⁄ streptomycin (Sigma) for 72 h. For cell surface
staining, primary antibodies [rabbit anti-nucleolin, 1 : 100;
mouse anti-myosin (F59), 1 : 100] were diluted in growth
medium and incubated with the cells for 30 min at 4 °C.
Cells were then washed three times with cold NaCl ⁄ P
i
and
fixed with 4% paraformaldehyde for 15 min at 4 °C. Secon-
dary antibodies (anti-rabbit biotin conjugated, anti-mouse
FITC conjugated) were diluted 1 : 100 in a mixture con-
taining NaCl ⁄ P
i
, 1% goat serum and 0.05% Tween-20, and
incubated with the cells for 1 h at room temperature. Cells

were washed, mounted and analyzed as described above.
To semipermeabilize cells for intracellular staining, cells
were incubated in 4% paraformaldehyde for 30 min at 4 °C
in the first instance and then stained as described above.
Confocal microscopy and imaging
E10 chick head sections were stained as described earlier
with antibodies against myosin and nucleolin with FITC
and Cy3 secondary antibodies, respectively. The sections
were examined using a Leica TCS 4D laser scanning confo-
cal microscope. The 488 nm line of the laser was used to
visualize the FITC–myosin and the 568 nm line was used
for the Cy3–nucleolin. With use of these wavelengths,
separation of the fluorescent signals from the two fluoroph-
ores was almost complete. A series of optical sections 1 lm
apart were taken through a depth of 20 lm. All images
were stored digitally, and three-dimensional reconstruction
and visualization were carried out using the volocity soft-
ware (Improvision Ltd, Coventry, UK). The parameters
were set as follows: green channel 100% density, red chan-
nel 1% density, medium noise filter.
Acknowledgements
We thank Clare Faux and Juan Pedro Martinez-Barb-
era for critical reading of the manuscript. The research
was funded by the Wellcome Trust (071418).
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