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J Biol Chem, Vol. 273, Issue 50, 33354-33359, December 11, 1998
From the Department of Anatomy and Cell Biology, University of
Michigan, Ann Arbor, Michigan 48109-0616 and Neural cell adhesion molecules (CAMs) of the
immunoglobulin (Ig) superfamily mediate not only cell aggregation but
also growth cone guidance and neurite outgrowth. In this study we
demonstrate that two neural CAMs, L1-CAM and TAG-1, induce the
homophilic aggregation of Drosophila S2 cells but are
unable to interact with each other when expressed on different cells
(trans-interaction). However, immunoprecipitations from cells
co-expressing L1-CAM and TAG-1 showed a strong cis-interaction between
the two molecules in the plane of the plasma membrane. TAG-1 is linked
to the membrane by a glycosylphosphatidylinositol (GPI) anchor and
therefore is unable to directly interact with cytoplasmic proteins. In
contrast, L1-CAM-mediated homophilic cell adhesion induces the
selective recruitment of the membrane skeleton protein ankyrin to areas of cell contact. Immunolabeling experiments in which S2 cells expressing TAG-1 were mixed with cells co-expressing L1-CAM and TAG-1
demonstrated that the homophilic interaction between TAG-1 molecules
results in the cis-activation of L1-CAM to bind ankyrin. This
TAG-1-dependent recruitment of the membrane skeleton
provides an example of how GPI-anchored CAMs are able to transduce
signals to the cytoplasm. Furthermore, such interactions might
ultimately result in the recruitment and the activation of other
signaling molecules at sites of cell contacts.
The navigation of growth cones to their targets in the developing
embryonic nervous system is a critical step in the patterning of
neuronal projections. Substantial evidence suggests that axonal guidance depends in part on cell surface and on extracellular matrix
molecules, which are expressed along the path of the advancing growth
cones (1, 2). Axonal extension and pathway choices are influenced by
membrane receptors to these molecules, which are expressed on the
growth cone.
One group of such molecules are cell adhesion molecules
(CAMs)1 belonging to the
immunoglobulin (Ig) superfamily that are expressed by advancing growth
cones and are able to recognize and transduce environmental signals
(3). Ig domain CAMs have been implicated to act both as receptors as
well as substrates for growing axons (4, 5). For example, members of
the L1 family of neural CAMs, such as mammalian L1-CAMs and chicken
Ng-CAM, not only exhibit a strong Ca2+-independent
homophilic adhesive activity (6, 7), they also promote neurite
outgrowth in culture, probably by the activation of neuronal FGF
receptors (8). Through the interaction with the cytoplasmic linker
protein ankyrin, L1 family members are also connected to the membrane
skeleton (9). Human L1-CAM and the Drosophila L1 homologue
neuroglian both recruit ankyrin and other components of the membrane
skeleton to cell contact sites in Drosophila S2 cell
aggregates (10, 11). This interaction strictly depends on the
extracellular L1 adhesive activity, and ankyrin binding in turn
stabilizes the L1 adhesive interaction (12).
Members of another subgroup of Ig domain neural CAMs are anchored in
the plasma membrane by a glycosylphosphatidylinositol (GPI) moiety.
These include TAG-1 in mammalian species and its chicken homologue
axonin-1 (13, 14). TAG-1/axonin-1 has been shown to mediate homophilic
cell adhesion and is able to promote neurite outgrowth in culture (13,
15, 16). However, TAG-1-induced neurite growth is not only mediated by
but also requires other neuronal membrane proteins, such as L1- or
Materials and Antibodies--
A mouse polyclonal serum was
raised against a glutathione
S-transferase-Drosophila ankyrin fusion protein,
which has been previously described (21), and used at a dilution of
1:200 for indirect immunofluorescence microscopy. Rabbit anti-L1-CAM
was a gift from Dr. Vance Lemmon (Case Western Reserve University, Cleveland, OH), and the rabbit anti-TAG-1 serum was characterized previously (22, 23). Fluorescein isothiocyanate-conjugated goat
anti-mouse IgG, Texas Red-conjugated goat anti-rabbit IgG, and
horseradish peroxidase-conjugated goat anti-rabbit IgG were purchased
from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).
Schneider's medium, penicillin/streptomycin stock solution, and fetal
calf serum were from Life Technologies, Inc.
Transfected Drosophila S2 Cell Lines--
Using Lipofectin (Life
Technologies, Inc.) Drosophila S2 cells were transfected
with pRmHa3 constructs. The pRmHa3 constructs expressing the neuronal
form of human L1-CAM and human TAG-1 protein, respectively, have been
described previously (11, 23). Subcloned cDNAs under the control of
the Drosophila metallothionein promoter (24) can be
expressed by the addition of 0.7 mM CuSO4 to
the cell culture medium. Co-transfection with the pPC4 plasmid was performed to confer Cell Aggregation Assays--
To discriminate between homophilic
and heterophilic cell adhesion mechanisms, mixing experiments were
performed using two transfected cell lines, one of which was labeled
with the vital fluorescent membrane dye DiI (Molecular Probes, Eugene,
OR) as described by Hortsch et al. (27). Labeled cells were
mixed with unlabeled cells at a ratio of 1:1 to a final cell
concentration of 3 × 106 cells/ml. Mixed cell
populations were induced overnight by the addition of 0.7 mM CuSO4 and aggregated for 4 h at room
temperature on a shaking platform at 200 rpm. Cell aggregates
containing at least 10 cells were analyzed by phase contrast and
epifluorescence microscopy on a Leitz Fluovert microscope for the
presence or absence of fluorescently labeled cells. Evaluating more
than 100 cell aggregates per experiment, the percentage of cell
clusters with five or more DiI-labeled cells was calculated for each
combination of labeled and unlabeled cells.
SDS-PAGE and Western Blot Analysis--
Transfected S2 cells
were pelleted and solubilized in SDS-containing buffer. Total cell
proteins were separated by electrophoresis in 10% SDS-polyacrylamide
gels and transferred onto nitrocellulose filters. Subsequently, the
blots were probed with specific primary and horseradish
peroxidase-conjugated secondary antibodies and developed with
3,3'-diaminobenzidine as described by Hortsch et al.
(28).
Immunofluorescence Staining and Confocal
Microscopy--
Immunocytochemistry of Drosophila ankyrin
distribution in S2 cells expressing different molecules was performed
as described previously (10). Briefly, cells expressing TAG-1 were
mixed with cells expressing both TAG-1 and L1-CAM at a ratio of 20:1 and incubated overnight with 0.7 mM CuSO4.
Subsequently, the cells were allowed to aggregate for 30 min on a
shaking platform before being attached to polylysine-coated microscope
slides. Attached cells were fixed with 2% paraformaldehyde for 10 min
and permeabilized for 10 min with 0.5% Triton X-100 in TBS buffer (10 mM Tris/HCl, pH 7.5, 0.15 M NaCl, and 5%
newborn calf serum). Rabbit anti-L1-CAM and mouse
anti-Drosophila ankyrin were used as primary antibodies, followed by an incubation with fluorescein isothiocyanate- or Texas
Red-labeled secondary antibodies. Slides were viewed with a Bio-Rad MRC
600 confocal scanning laser microscope.
Immunoprecipitation of L1-CAM and
TAG-1--
Immunoprecipitations were performed using a modification of
the protocol by Anderson and Blobel (29). For each immunoprecipitation, 1 × 107 cells expressing either L1-CAM or both L1-CAM
and TAG-1 were induced overnight with 0.7 mM
CuSO4. Without inducing cell aggregation by an incubation
step on a rotary shaker the cells were pelleted and solubilized in cold
dilution buffer (60 mM Tris/HCl, pH 7.5, 80 mM
NaCl, 1.25% Triton X-100, 6 mM EDTA, and a mixture of
protease inhibitors). The soluble fraction was incubated overnight with rabbit anti-TAG-1 antibodies. Supernatants were further incubated with
Protein A-Sepharose beads (Amersham Pharmacia Biotech) for 2 h,
and immunoprecipitates were eluted with SDS gel electrophoresis buffer
after three washing steps. After separation on 10% SDS-PAGE gels,
proteins were transferred to a nitrocellulose filter and then probed
with anti-L1-CAM antibody.
Drosophila S2 Cells Which Express Human TAG-1 or L1-CAM Do Not Form
Mixed Aggregates--
Human L1-CAM and TAG-1 have both been shown to
induce the aggregation Drosophila S2 cells by a homophilic
mechanism (23, 27). Although initial experiments using the chicken
homologues of TAG-1 and L1-CAM, axonin-1 and Ng-CAM, coated to
Covaspheres, suggested that these molecules might also be able to
interact with each other when expressed on different surfaces (30),
follow-up experiments using axonin-1 and Ng-CAM expressed by tissue
culture cells indicated that these molecules are unable to engage in an adhesive trans-interaction (18).
To investigate whether human L1-CAM and TAG-1 are able to induce the
aggregation of cells by a heterophilic mechanism, populations of S2
cells expressing either L1-CAM, TAG-1, or the unrelated CAM
Drosophila fasciclin I were stained with the fluorescent dye DiI and mixed with unlabeled S2 cells expressing the same set of
homophilic adhesion molecules. Most cell aggregates were comprised of
several hundred cells, but no significant inclusion of cells expressing
TAG-1 into cell clusters expressing L1-CAM and vice versa
was observed (Fig. 2). As a result
approximately 50% of all cell clusters consisted of DiI-labeled cells
expressing one adhesive molecule, whereas the other clusters consisted
of unlabeled cells expressing the other CAM. Similar results were
obtained mixing induced S2:L1-CAM or S2:TAG-1 cells with S2 cells
expressing the unrelated CAM, fasciclin I. Drosophila
fasciclin I was used as a negative control in these experiments,
because it is not known to interact with L1-type molecules and a TAG-1
homologue has not been identified in Drosophila. In
contrast, more than 90% of all cell clusters contained labeled as well
as unlabeled S2 cells when both populations expressed either human
L1-CAM or TAG-1. This is in agreement with the homophilic adhesive
properties that have been demonstrated for both molecules (23, 27).
These results also indicate that cells expressing either human TAG-1 or
L1-CAM do not form mixed aggregates and that these two adhesive molecules do not engage in a measurable heterophilic trans-interaction with each other.
Human L1-CAM and TAG-1 Engage in a Cis-interaction within the Plane
of the Plasma Membrane--
Stable S2 cell lines expressing both
L1-CAM and TAG-1 were used to demonstrate that the two molecules
interact when expressed in the same cell. Immunoprecipitations with
anti-TAG-1 antibodies followed by immunoblotting with an anti-L1-CAM
antiserum indicated that L1-CAM and TAG-1 form stable complexes when
present in the same plasma membrane (Fig.
3, lane 2). As shown in
lane 1, no L1-CAM was immunoprecipitated from these cells
with non-immune rabbit serum. The observed co-immunoprecipitation of
L1-CAM with TAG-1 was also not caused by a cross-reactivity of the
anti-TAG-1 antiserum toward human L1-CAM, because this antiserum failed
to precipitate L1-CAM from cells that did not express TAG-1 (Fig. 3,
lane 3).
Homophilic, TAG-1-mediated S2 Cell Aggregation Does Not Induce
Ankyrin Recruitment at Cell Contact Sites--
Members of the L1
family induce the specific recruitment of ankyrin and other components
of the membrane skeleton to sites of cell contact in S2 cell aggregates
(10, 11). As shown for human L1-CAM in Fig. 4, A and
B, endogenous S2 cell ankyrin is specifically recruited to
cell contact sites by L1-CAM-mediated cell adhesion. No ankyrin
staining of the plasma membrane can be detected in non-contact areas or
in cells that have not joined cell aggregates. S2 cells expressing
human TAG-1 exhibit robust homophilic cell aggregation (Fig. 2).
However, no ankyrin recruitment to cell contacts or to other areas of
the plasma membrane was ever observed in TAG-1-expressing cell clusters
(Fig. 4, C and D). In some cells, especially
cells exhibiting no ankyrin recruitment to cell contact sites, ankyrin
staining appears in a punctate pattern. The reason for this punctate
intracellular distribution of ankyrin in S2 cells is unknown but has
been observed and described before (10, 12).
TAG-1 Homophilic Cell Adhesion Induces L1-mediated Ankyrin
Recruitment--
Because TAG-1 is unable to directly interact with the
intracellular membrane skeleton, we tested the possibility that it
might activate the ability of L1-CAM to bind ankyrin. No recruitment of
ankyrin to the plasma membrane was observed in single, non-aggregated S2 cells expressing L1-CAM as well as TAG-1 (not shown). However, when
these co-expressing cells were mixed and co-aggregated with S2 cells
expressing only TAG-1, a strong recruitment of ankyrin was observed at
cell contact sites between these two cell lines (Fig.
5, A-D). The example shown in
A depicts two cells that stain positive for L1-CAM
(left side) and therefore express both L1-CAM and
TAG-1. The corresponding micrograph on the right
side of A shows the distribution of ankyrin in
these cells. As indicated by the arrow ankyrin was recruited
to the cell contact between the two L1-CAM-positive cells. Ankyrin was
also recruited to contact sites these two cells had developed to cells
expressing only TAG-1 (marked by arrowheads). B,
C, and D show a range of other examples of
ankyrin recruitment to cell contact sites between the two different S2
cell types. A quantitative evaluation of these experiments indicates
that approximately 75% of such cell contacts exhibited a recruitment
of ankyrin (Table I).
Although we observed no trans-interaction between TAG-1 and L1-CAM in
our S2 cell aggregation experiment shown in Fig. 2, we considered that
a weak trans-interaction between TAG-1 and L1-CAM expressed by two
different cells might be responsible for the observed induction of
ankyrin binding to L1-CAM. We therefore determined the ankyrin
distribution in mixtures of cells expressing either TAG-1 or L1-CAM. By
analyzing a large number of immunostained slides, we identified rare
instances in which a TAG-1-expressing cell ended up in contact with a
cell expressing just L1-CAM (Fig. 5, E and F). In
none of the 29 examples we analyzed did we observe a recruitment of
ankyrin to these cell contact points (Fig. 5 and Table I). This
indicates that L1-CAM and TAG-1 must be co-expressed in the same cell
for TAG-1-mediated cell adhesion to induce ankyrin binding to
L1-CAM.
Complex homo- and heterophilic interactions between different
adhesion molecules within the plane of the plasma membrane and between
adjacent cells have been proposed to play an important role in
regulating the growth and guidance of axons during embryonic neurogenesis (5). Our present in vitro study addresses how two different CAMs, human TAG-1 and human L1-CAM, interact and functionally regulate each other. Some groups of CAMs are transmembrane proteins and therefore potentially able to directly influence intracellular processes, e.g. by activating second messenger
signaling cascades and/or by reorganizing components of the
cytoskeleton. However, other CAM families, such as the TAG-1/axonin-1
and the F3/F11 groups, are anchored in the plasma membrane by a GPI
moiety and therefore lack the means to interact with cytoplasmic
proteins without additional linker proteins. Nevertheless, GPI-anchored CAMs are also able to operate as signal-transducing molecules during
neuronal development. Although TAG-1/axonin-1 is fully functional as a
homophilic CAM without engaging in any heterophilic cis-interactions,
its ability to associate with other membrane proteins, especially
L1-CAM, appears to be essential for its neurite outgrowth-promoting
function (17, 31). Several models similar to the one displayed in Fig.
6 have been proposed in which TAG-1/axonin-1 associates with other
membrane proteins expressed in the same plasma membrane in a
cis-interaction to form a signal-transducing, multimeric protein
complex (17, 18). A similar model has also been suggested to explain
the adhesion-dependent activation of neuronal FGF receptors
by L1-CAM, NCAM, and N-cadherin (8).
Although several molecular details of the TAG-1 interaction with L1-CAM
have been elucidated, its regulatory and functional aspects are not
well understood. L1-CAM/Ng-CAM not only can be co-immunoprecipitated
with TAG-1/axonin-1, but cross-linking experiments suggest that both
molecules directly bind to each other (18, 20). The part of the
axonin-1 molecule responsible for this cis-interaction has been mapped
to its amino-terminal Ig protein domains (32), whereas the homophilic
adhesive activity of TAG-1/axonin-1 is mediated by its fibronectin type
III domains (23). The protein domains of L1-CAM that mediate TAG-1
recognition and binding have not been identified yet. Also whether the
homophilic adhesive activities of L1-CAM and TAG-1 regulate their
cis-interaction is currently unknown. However, co-capping experiments
reported by Buchstaller et al. (18) and the quantity of
L1-CAM that co-immunoprecipitated with TAG-1 in our experiments suggest
that a significant fraction of L1-CAM molecules forms heterodimeric
complexes with TAG-1 before the two CAMs engage in cell adhesion and
L1-CAM binds to the membrane skeleton. After L1-CAM interacts with
ankyrin, it becomes resistant to Triton X-100 extraction as used in our
immunoprecipitation experiments and is unavailable for precipitation
with antibodies (10).
TAG-1/axonin-1 and L1-CAM/Ng-CAM are co-expressed in several locations
during nervous system development, suggesting that their interaction is
physiologically relevant (31, 33, 34). In cultures of chicken dorsal
root ganglion neurons Ng-CAM and axonin-1 protein are found in
overlapping areas on growth cone membranes, and axonin-1 expression by
these cells is required for neurite outgrowth on both axonin-1 and
Ng-CAM substrata (31). In contrast, in other developing neurons,
e.g. in the rat embryonic spinal cord, TAG-1 and L1-CAM
expression appear to be locally and temporally segregated (22, 35).
This finding indicates that TAG-1/axonin-1 and L1-CAM/Ng-CAM are not
obligatory co-receptors but are able to function independently. It
might mean either that the signaling capabilities of TAG-1/axonin-1 are
limited in certain areas of the developing nervous system or at certain
developmental time points or that TAG-1/axonin-1 associates with other
membrane proteins than L1-CAM/Ng-CAM to form functionally active
protein complexes.
Although the cis-activation of neuronal FGF receptors by L1-CAM is
thought to be the initial step in adhesion-induced neurite outgrowth,
processes such as the recruitment of other signaling molecules to cell
contact sites might also have an important, more indirect role. Our
findings presented here would support the hypothesis that TAG-1
homophilic adhesion might also activate neuronal FGF receptor activity
via L1-CAM. Other cellular changes that appear to be regulated by the
TAG-1/axonin-1 interaction with L1-CAM/Ng-CAM are the recruitment and
the release of several different protein kinase activities that are
associated with the two CAMs in chicken dorsal root ganglion neurons
(20). In the case of integrin- and cadherin-mediated cell adhesion,
interactions with cytoskeletal elements induce the assembly of
signal-generating and -processing multiprotein complexes at the
cytoplasmic aspect of the adhesion contact site. This makes the
observed assembly of membrane skeleton components in response to
TAG-1-mediated homophilic cell adhesion especially significant.
However, the most tantalizing aspect of the results reported here is
the observation that TAG-1 homophilic cell adhesion directly regulates
the functional status of L1-CAM. These findings suggest a mechanism
that explains how GPI-anchored CAMs might actively participate in
regulating the growth, organization, and differentiation of neuronal
cells during development.
We thank Dr. Vance Lemmon for generously
providing the rabbit anti-L1 antiserum and the human L1 cDNA and
Drs. Stephen Ernst and Robert Chandler for a critical reading of the
manuscript. D. K. would also like to thank Dr. Joseph Papamatheakis
for his support.
*
This work was supported by National Institutes of Health
Grant HD29388 and a grant from the Spinal Cord Research Foundation (to
M. H.) and by European Union Grant BMH4-CT95-0524 and Greek Secretariat for Research and Technology Grant PENED 95 No. 451 (to
D. K.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Tel.: 734-647-2720;
Fax: 734-763-1166; E-mail: Hortsch{at}umich.edu.
The abbreviations used are:
CAM, cell adhesion
molecule; DiI, 1,1'-dioctadecyl-3,3,3'-tetramethylindocarbocyanine
perchlorate; FGF, fibroblast growth factor; GPI, glycosylphosphatidylinositol; Ig, immunoglobulin; NCAM, neural cell
adhesion molecule; Ng-CAM, neuron-glia cell adhesion molecule; TAG-1, transiently expressed axonal surface glycoprotein-1; PAGE, polyacrylamide gel electrophoresis.
Cis-activation of L1-mediated Ankyrin Recruitment by TAG-1
Homophilic Cell Adhesion*
,, and Institute of
Molecular Biology and Biotechnology, Foundation for Research and
Technology and the Department of Basic Sciences, University of Crete
Medical School, Heraklion 71110, Crete, Greece
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1 integrin-type molecules (17, 18). TAG-1/axonin-1
interacts with a number of different heterophilic binding partners,
including several members of the L1 family, NCAM, nervous
tissue-specific chondroitin sulfate proteoglycans, and several
extracellular matrix molecules (18, 19). It has been suggested that
some of these heterophilic interactions might enable TAG-1 to induce or
influence intracellular signaling processes (5). Axonin-1 and Ng-CAM
expressed in the same plasma membrane engage in a strong
cis-interaction, forming larger multimeric complexes that are also
associated with several intracellular protein kinase systems (18, 20).
This cis-interaction between TAG-1/axonin-1 and L1-type molecules is
essential for the stimulation of neurite outgrowth on TAG-1/axonin-1
substrates in culture, and homophilic TAG-1 cell adhesion appears to
activate TAG-1-associated L1-CAM molecules (16, 17). Although several
lines of evidence suggest that the interaction of TAG-1 with L1-CAM is
an important link in TAG-1-initiated signal transduction, a direct
demonstration that TAG-1 is capable of altering the functional state of
L1-CAM has been missing. In our present study we demonstrate that
TAG-1-mediated homophilic cell adhesion induces an intracellular
restructuring of the membrane skeleton by the cis-activation of human
L1-CAM.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-amanitin resistance as a selectable marker to
transfected cells (25). Detailed methods for establishing cloned S2
cell lines using soft agar cloning have been previously reported in
detail (26). Individual cell clones were induced overnight and analyzed
by Western blotting for high expression of the transfected cDNAs.
Selected lines, designated S2:L1-CAM, S2:TAG-1, and S2:L1/TAG-1,
expressed either the neuronal form of human L1-CAM, human TAG-1, or
both proteins (Fig. 1).
View larger version (70K):
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Fig. 1.
Western blot of human L1-CAM and human TAG-1
expression in Drosophila S2 cells. Transfected S2
cells were induced overnight and solubilized in phosphate-buffered
saline containing 2% SDS. Proteins were separated by SDS-PAGE on 10%
polyacrylamide gels and blotted onto nitrocellulose filters. Blot A was
incubated with a rabbit anti-L1-CAM antiserum and blot B with a rabbit
anti-TAG-1 antiserum. Lanes 1 contain total proteins from
untransfected S2 cells; lanes 2 from S2 cells expressing
human TAG-1; lanes 3 from S2 cells expressing human L1-CAM;
and lanes 4 from S2 cell lines expressing both human L1-CAM
and TAG-1. Each lane contains approximately 50 µg of
protein.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (45K):
[in a new window]
Fig. 2.
Human L1-CAM and TAG-1 do not cause
heterophilic cell adhesion. Combinations of two S2 cell
populations expressing either human L1-CAM, human TAG-1, or
Drosophila fasciclin I were mixed at a ratio of 1:1 and were
allowed to aggregate on a shaking platform for 4 h. One of the
cell populations was fluorescently labeled with the lipophilic dye DiI.
Cell aggregates containing at least 10 cells were analyzed for the
presence or absence of fluorescently labeled cells, and the percentage
of cell clusters with five or more DiI-labeled cells was calculated for
each combination of S2 cell lines. Per experiment at least 100 cell
aggregates were analyzed for each combination of cells. Results shown
are the mean ± S.D. of three independent experiments.
View larger version (35K):
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Fig. 3.
Co-immunoprecipitation of L1-CAM and TAG-1
protein from S2 cell protein extracts. S2 cells expressing either
human L1-CAM or both human L1-CAM and human TAG-1 were induced
overnight with 0.7 mM CuSO4 and solubilized
with a Triton X-100-containing buffer. As indicated at the
bottom of the figure immunoprecipitations from these
extracts were performed using either a rabbit non-immune serum or a
rabbit antiserum against TAG-1. Immunoprecipitated proteins were
separated on 10% SDS-PAGE gels and transferred to nitrocellulose
filters, which were probed with a rabbit anti-L1-CAM antiserum.
View larger version (130K):
[in a new window]
Fig. 4.
Ankyrin is recruited to cell contact sites in
S2 cells expressing human L1-CAM but not in cell aggregates expressing
human TAG-1. After protein induction from the transfected cDNA
constructs S2 cells expressing human L1-CAM (A and B) or
human TAG-1 (C and D) were allowed to aggregate, fixed, and
fluorescently stained using a mouse anti-Drosophila ankyrin
antiserum. Scale bar is 25 µm.
View larger version (96K):
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Fig. 5.
Ankyrin recruitment to human L1-CAM is
induced by homophilic TAG-1-mediated cell adhesion. S2 cells
expressing human TAG-1 protein were mixed at a ratio of 20:1 with S2
cells co-expressing human TAG-1 and human L1-CAM (A-D) or
with S2 cells expressing only human L1-CAM (E and F). This
ratio of cells was selected to maximize the number of cell contacts
between cells expressing both adhesion molecules with cells expressing
only TAG-1, rather than contacts between cells expressing L1-CAM and
TAG-1. After protein induction cells were briefly allowed to aggregate
and subsequently processed for double immunofluorescence using a rabbit
anti-L1-CAM antiserum (left panels) and a mouse
anti-Drosophila ankyrin antiserum (right
panels). The arrow in A marks a cell
contact between two cells expressing L1-CAM as well as TAG-1. Cell
contacts between cells expressing TAG-1 with cells expressing both
adhesion molecules are indicated by arrowheads. Scale
bar is 25 µm.
Quantification of ankyrin recruitment to cell contact sites involving
cells expressing TAG-1 and cells expressing either human L1-CAM or
human L1-CAM and TAG-1
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (29K):
[in a new window]
Fig. 6.
Model of homo- and heterophilic interactions
of TAG-1. The homophilic trans-interaction between TAG molecules
expressed by different cells is mediated by its fibronectin type III
domains (23), whereas the Ig domains of TAG appear to be responsible
for its heterophilic cis-interaction to L1-type proteins (32). The
protein domains of L1-CAM that are involved in binding to TAG-1 are
currently unknown. The two TAG-1 molecules are drawn in a
horseshoe-shaped domain arrangement, which has been proposed for the
chicken homologue of TAG-1, axonin-1 (32).
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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R. Islam, S.-Y. Wei, W.-H. Chiu, M. Hortsch, and J.-C. Hsu Neuroglian activates Echinoid to antagonize the Drosophila EGF receptor signaling pathway Development, May 15, 2003; 130(10): 2051 - 2059. [Abstract] [Full Text] [PDF]
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K. Kyriakopoulou, I. de Diego, M. Wassef, and D. Karagogeos A combination of chain and neurophilic migration involving the adhesion molecule TAG-1 in the caudal medulla Development, March 3, 2003; 129(2): 287 - 296. [Abstract] [Full Text] [PDF]
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A. Bizzoca, D. Virgintino, L. Lorusso, M. Buttiglione, L. Yoshida, A. Polizzi, M. Tattoli, R. Cagiano, F. Rossi, S. Kozlov, et al. Transgenic mice expressing F3/contactin from the TAG-1 promoter exhibit developmentally regulated changes in the differentiation of cerebellar neurons Development, January 1, 2003; 130(1): 29 - 43. [Abstract] [Full Text] [PDF]
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Y. Nakai and H. Kamiguchi Migration of nerve growth cones requires detergent-resistant membranes in a spatially defined and substrate-dependent manner J. Cell Biol., December 23, 2002; 159(6): 1097 - 1108. [Abstract] [Full Text] [PDF]
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 J. D. Malhotra, M. C. Koopmann, K. A. Kazen-Gillespie, N. Fettman, M. Hortsch, and L. L. Isom Structural Requirements for Interaction of Sodium Channel beta 1 Subunits with Ankyrin J. Biol. Chem., July 12, 2002; 277(29): 26681 - 26688. [Abstract] [Full Text] [PDF]
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M. Traka, J. L. Dupree, B. Popko, and D. Karagogeos The Neuronal Adhesion Protein TAG-1 Is Expressed by Schwann Cells and Oligodendrocytes and Is Localized to the Juxtaparanodal Region of Myelinated Fibers J. Neurosci., April 15, 2002; 22(8): 3016 - 3024. [Abstract] [Full Text] [PDF]
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M. Denaxa, C.-H. Chan, M. Schachner, J. G. Parnavelas, and D. Karagogeos The adhesion molecule TAG-1 mediates the migration of cortical interneurons from the ganglionic eminence along the corticofugal fiber system Development, November 15, 2001; 128(22): 4635 - 4644. [Abstract] [Full Text] [PDF]
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H. D. Moulding, R. L. Martuza, and S. D. Rabkin Clinical Mutations in the L1 Neural Cell Adhesion Molecule Affect Cell-Surface Expression J. Neurosci., August 1, 2000; 20(15): 5696 - 5702. [Abstract] [Full Text] [PDF]
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H. Kamiguchi and V. Lemmon Recycling of the Cell Adhesion Molecule L1 in Axonal Growth Cones J. Neurosci., May 15, 2000; 20(10): 3676 - 3686. [Abstract] [Full Text] [PDF]
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J. D. Malhotra, K. Kazen-Gillespie, M. Hortsch, and L. L. Isom Sodium Channel beta Subunits Mediate Homophilic Cell Adhesion and Recruit Ankyrin to Points of Cell-Cell Contact J. Biol. Chem., April 6, 2000; 275(15): 11383 - 11388. [Abstract] [Full Text] [PDF]
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 S. Kenwrick, A. Watkins, and E. D. Angelis Neural cell recognition molecule L1: relating biological complexity to human disease mutations Hum. Mol. Genet., April 1, 2000; 9(6): 879 - 886. [Abstract] [Full Text] [PDF]
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M. Koroll, F. G. Rathjen, and H. Volkmer The Neural Cell Recognition Molecule Neurofascin Interacts with Syntenin-1 but Not with Syntenin-2, Both of Which Reveal Self-associating Activity J. Biol. Chem., March 30, 2001; 276(14): 10646 - 10654. [Abstract] [Full Text] [PDF]
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