 |
 Previous Article | Next Article 
Volume 17, Number 18,
Issue of September 15, 1997
pp. 7025-7036
Copyright ©1997 Society for Neuroscience
Morphogenesis of the Node of Ranvier: Co-Clusters of Ankyrin
and Ankyrin-Binding Integral Proteins Define Early Developmental
Intermediates
Stephen Lambert,
Jonathan Q. Davis , and
Vann Bennett
Department of Cell Biology and the Howard Hughes Medical Institute,
Duke University Medical Center, Durham, North Carolina 27710
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
AnkyrinG 480/270 kDa and three ankyrin-binding integral
membrane proteins (neurofascin, NrCAM, and the voltage-dependent sodium channel) colocalize within a specialized domain of the spectrin-actin network found at axonal segments of nodes of Ranvier in myelinated axons. Before myelination in embryonic nerves, ankyrinG
480/270 kDa and the related ankyrin isoform ankyrinB 440 kDa are co-expressed along with NrCAM in an abundant, continuous
distribution along the length of axons. This study has resolved
intermediate stages in the developmental transition from a continuous
distribution of ankyrinG 480/270 kDa in all axons to a
highly polarized localization at the node of Ranvier in the developing
rat sciatic nerve. The first detected event is formation of clusters
containing the cell adhesion molecules neurofascin and NrCAM at sites
independent of myelin-associated glycoprotein (MAG)-staining Schwann
cell processes. Subsequent steps involve recruitment of
ankyrinG 480/270 kDa and the voltage-dependent sodium
channel to cluster sites containing cell adhesion molecules, and
elaboration of MAG-staining Schwann cell processes adjacent to these
cluster sites. Formation of the mature node of Ranvier results from the
fusion of asynchronously formed pairs of clusters associated with
MAG-positive Schwann cells flanking the site of presumed node
formation. Studies with the hypomyelinating mutant mouse
trembler demonstrate that the elaboration of compact
myelin is not required for the formation of these clustered nodal
intermediates. Clustering of neurofascin and NrCAM precedes
redistribution of ankyrinG 480/270 kDa and the
voltage-dependent sodium channel, suggesting that the adhesion molecules define the initial site for subsequent assembly of ankyrin and the voltage-dependent sodium channel.
Key words:
node of Ranvier;
cell adhesion molecules;
ankyrin;
voltage-dependent sodium channel;
myelination;
trembler
mutant
INTRODUCTION
The localization of ion channels, in
particular the voltage-dependent sodium channel, to gaps in the myelin
sheath known as the nodes of Ranvier, is crucial to the propagation of
saltatory action potentials in myelinated nerves. In the internodal
axonal membrane, the voltage-dependent sodium channel is found at
concentrations of 20-25 channels/µm2, compared
with concentrations ranging from 1000 channels/µm2
(Shrager, 1989 ) to 12,000 channels/µm2 (Ritchie
and Rogart, 1977) at the node of Ranvier. The asymmetric distribution
of ion channels in the myelinated axon and the regular spacing of these
nodes at ~100 times the axonal diameter (Rushton, 1951; Chiu, 1980)
are critical for the fast conduction velocities associated with these
axons and represent an interesting problem in cell polarity and
membrane differentiation.
A clue as to the molecular organization of the node comes from
observations that the voltage-dependent sodium channel co-purifies and
binds with high affinity to the peripheral membrane protein ankyrin
(Srinivasan et al., 1988). In addition, other ankyrin-binding proteins
are also localized at the node, including isoforms of neurofascin and
NrCAM (Davis et al., 1993, 1996), ankyrin-binding cell adhesion
molecules related to L1 (for review, see Hortsch, 1996). Ankyrin itself
is localized at the 1- to 2-µm-size nodal axonal plasma membrane
(Kordeli et al., 1990) and is a component of the electron dense
undercoating observed beneath the nodal membrane (Peters, 1966;
Ichimura and Ellisman, 1991). Ankyrins are a family of spectrin-binding
proteins that associate with diverse integral proteins, including ion
channels, calcium-release channels, and cell adhesion molecules, and
link these proteins to the spectrin-based membrane skeleton (for
review, see Bennett and Gilligan, 1993; Lambert and Bennett, 1993). The
isoforms of ankyrin localized at nodes of Ranvier have recently been
identified as 480 and 270 kDa alternatively spliced variants of the
ankyrinG gene (Kordeli et al., 1995). These ankyrin
isoforms are concentrated at axon initial segments and nodes of Ranvier
in the adult rat CNS and PNS (Kordeli et al., 1995), and to our
knowledge they represent the first unique cytoplasmic components of the
node to be identified. Nodal ankyrin isoforms are distinguished by a 46 kDa serine-threonine-rich domain glycosylated with O-GlucNAc residues
(Zhang and Bennett, 1996), and like other ankyrins associate with
integral proteins through a membrane-binding domain comprising 24 ANK
repeats. Biochemical studies of this domain (Michaely and Bennett,
1995a,b) suggest that ankyrin could facilitate lateral homo- and
heterocomplexes between integral membrane proteins.
The absence of suitable molecular markers has hindered elucidation of
the role of components of the electron-dense undercoating in the
differentiation of the nodal membrane. This study examines the
distribution of ankyrinG 480/270 kDa, the voltage-dependent sodium channel, neurofascin, and NrCAM during development in the myelinating sciatic nerve. AnkyrinG 480/270 kDa
polypeptides are highly expressed in a uniform distribution along
premyelinated axons at embryonic day 16 and redistribute to clusters
adjacent to the ends of Schwann cells in early postnatal development.
The voltage-dependent sodium channel also is localized in clusters, as
reported recently in developing and regenerating nerves
(Dugandzija-Novakovic et al., 1995; Vabnick et al., 1996), and is
colocalized at these sites with ankyrinG 480/270 kDa as
well as neurofascin and NrCAM. Clustering of neurofascin and NrCAM
precedes redistribution of ankyrinG 480/270 kDa and the
voltage-dependent sodium channel and also precedes early events in
myelin formation, as determined by the expression of myelin-associated
glycoprotein (MAG). These results and the finding of clusters in
hypomyelinating mice suggest that axonal adhesion molecules define the
initial site for subsequent assembly of ankyrin and the
voltage-dependent sodium channel into the differentiating nodal axonal
membrane and that these events are independent of the formation of
compact myelin.
MATERIALS AND METHODS
Preparation of antibodies. Antibodies against the
common tail region of rat ankyrinG 480 and 270 kDa were
raised in chickens for double-labeling studies. Chickens were immunized
with a purified recombinant polypeptide corresponding to the rat
equivalent of residues 1821-2337 of the human ankyrinG
sequence. This polypeptide was produced by expression in bacteria of
the rat cDNA using the pGemex expression vector (Promega, Madison, WI),
resulting in a fusion product with the viral gene 10 protein.
Antibodies were initially purified from chicken egg yolk using an Egg
Yolk Purification Kit (Pharmacia Biotech, Piscataway, NJ) and
affinity-purified against purified recombinant polypeptide immobilized
on Sepharose CL-6B (Pharmacia) after the previous depletion of
antibodies using immobilized gene 10 polypeptide. Antibodies against
neurofascin and NrCAM were generated in rabbits using purified
recombinant FNIII domains (residues 581-1020 of rat neurofascin and
residues 583-1018 of rat NrCAM) from these molecules as antigens.
These recombinant domains were prepared in bacteria as above.
Affinity-purified antibodies were then prepared from sera by
purification against immobilized native 186 kDa neurofascin and NrCAM.
Native proteins were purified from detergent extracts of adult rat
brain membranes as described previously (Davis et al., 1993).
Antibodies against neurofascin did not cross-react with purified native
NrCAM by immunoblot analysis and vice versa (Davis et al., 1996).
Antibodies against the voltage-dependent sodium channel were prepared
by immunization of rabbits with four multiple-antigen peptides
corresponding to residues 440-453, 482-492, 547-561, and 582-600 of
the  I subunit (Noda et al., 1986). These sequences are relatively
conserved in the three subunits expressed in the rat brain and are
in an area of the molecule proposed to be a cytoplasmic linker between the I and II transmembrane pore-forming units. An area of the rat I
subunit (corresponding to nucleotides 1292-2215) encompassing these
peptide sequences was cloned from a rat brain library using PCR. The
cDNA was subcloned into the pGemex expression vector, expressed in
bacteria, and the purified recombinant polypeptide was immobilized and
used in the affinity purification of the peptide antisera, as described
above. Preparation of affinity-purified antibodies against brain
spectrin has been described previously (Davis and Bennett, 1983).
Antibodies against the MAG were purchased from Boehringer Mannheim
(Indianapolis, IN).
Gel electrophoresis and immunoblot analysis. Gel samples of
adult rat brain membranes were prepared as described previously (Kordeli et al., 1995) and fractionated on 3.5-17% exponential gradient gels before transfer to nitrocellulose (Davis and Bennett, 1983). Bound antibodies were visualized using 125I-labeled
protein A and autoradiography (Davis and Bennett, 1983).
Immunocytochemical procedures. Immunocytochemistry of frozen
sections from rodent sciatic nerves was performed essentially as
described (Kordeli et al., 1990). Animals of various ages were killed,
and the sciatic nerves were removed by dissection. Sciatic nerves were
immediately fixed in 2% paraformaldehyde either overnight at 4°C or
for 3 hr at 4°C (for experiments involving antibodies against the
voltage-dependent sodium channel) before cryopreservation in sucrose
and freezing in liquid nitrogen-cooled isopentane. Primary antibodies
bound on 4 µm cryosections were visualized with rhodamine-labeled
goat anti-rabbit Ig (Cappel, West Chester, PA) alone or in combination
with fluorescein-labeled mouse monoclonal anti-chicken Ig (clone CH31,
Sigma, St. Louis, MO) in double-labeling experiments. Confocal images
were collected on a Zeiss LSM 410 confocal microscope, and final
figures were prepared using Adobe Photoshop.
RESULTS
Characterization of antibodies against ankyrinG 480/270
kDa, the voltage-dependent sodium channel, neurofascin, and NrCAM
Figure 1 shows an immunoblot
analysis of total adult rat brain membranes (lane 1 ) probed
with antibodies raised against ankyrinG 480/270 kDa and the
following ankyrin-binding proteins: voltage-dependent sodium channel
(lane 2), neurofascin (lane 3), and NrCAM
(lane 4). Chicken antibodies against the common
"tail" domain of ankyrinG 480/270 kDa recognize two
polypeptides of 480 and 270 kDa as predicted. AnkyrinG 270 kDa arises from ankyrinG 480 kDa by alternative mRNA processing between nucleotides 7202 and 12446 of the
ankyrinG cDNA published sequence, removing 1748 amino acids
from the ankyrinG "tail" domain (our unpublished data).
Antibodies raised against the deleted sequence specific to
ankyrinG 480 kDa recognize nodes of Ranvier in adult
sciatic nerve (Zhang and Bennett, 1996) as well as nodal intermediates
in myelinating axons (not shown). Expression of ankyrinG
480 kDa precedes that of ankyrinG 270 kDa, which is
expressed only after day 10 in the developing nervous system.
Antibodies raised against the voltage-dependent sodium channel show a
single band representing the 260 kDa subunit, which migrates at 220 kDa in a continuous electrophoretic buffer system (Srinivasan et al.,
1988). These antibodies also strongly label nodes of Ranvier in adult
sciatic nerve (Davis et al., 1996), confirming earlier antibody
localization of the voltage-dependent sodium channel to the node
(Ellisman and Levinson, 1982). Antibodies against FNIII domains 1-4 of
neurofascin and NrCAM show three polypeptides of 186, 155, and 140 kDa
for neurofascin as described (Davis et al., 1996) and a single
polypeptide of 150 kDa for NrCAM. The three neurofascin polypeptides
are alternatively spliced products of the rat neurofascin gene (Davis
et al., 1996), whereas the 150 kDa NrCAM polypeptide represents the
extracellular domain of NrCAM (Kayyem et al., 1992; Davis and Bennett,
1994).
Fig. 1.
Immunoblot analysis of adult total rat brain
membranes with antibodies against (1)
ankyrinG 480/270 kDa, (2)
voltage-dependent sodium channel, (3)
neurofascin, and (4) NrCAM. Lane 1
shows the brain membrane preparation stained with Coomassie blue.
[View Larger Version of this Image (51K GIF file)]
AnkyrinG 480/270 kDa defines a specialized domain
within the spectrin-actin network at nodal axon segments of adult
nodes of Ranvier
Localization of ankyrinG with respect to spectrin was
examined in the adult sciatic nerve by confocal microscopy using
double-labeling with rabbit antibody against spectrin and a chicken
antibody against ankyrinG 480/270 kDa (Fig.
2). Spectrin (Fig. 2A)
stains continuously along the axon (Trapp et al., 1989), including the
nodal and paranodal areas, characterized by a reduction in axonal
caliber. Spectrin staining also occurs in the cytoplasmic process of
the Schwann cell on the outside edge of the myelin sheath. Antibody
against ankyrinG 480/270 kDa (Fig. 2B),
in contrast, is localized to a 1-2 µm zone comprising the nodal
membrane, in agreement with initial observations of
ankyrinG staining (Kordeli et al., 1995). The ankyrin
binding proteins NrCAM, neurofascin, and the voltage-dependent sodium
channel also are colocalized with ankyrinG 480/270 kDa at
the adult node of Ranvier (Davis et al., 1996).
Fig. 2.
Immunofluorescence localization of
ankyrinG 480/270 kDa with respect to spectrin at the node
of Ranvier. A 4 µm cryosection of an adult rat sciatic nerve was
double-labeled with an antibody against spectrin
(A) and an antibody against ankyrinG
480/270 kDa (B). The composite image
(C) was collected by confocal microscopy.
[View Larger Version of this Image (48K GIF file)]
AnkyrinG 480/270 kDa and ankyrinB 440 kDa
are co-expressed in an abundant, continuous distribution along
axons before myelination
The localization of ankyrinG 480/270 kDa and
ankyrinB 440 kDa were examined in premyelinated axons of
the developing rat nervous system. Extensive immunostaining for both
ankyrins was observed in axons of both the CNS and PNS in the embryonic
day 16 rat. Figure 3 shows the
colocalization (arrows) of ankyrinG 480/270 kDa
(B, D, F) with spectrin (A), NrCAM
(C), and the ankyrinB 440 kDa molecule
(E) in axons and bundles of axons emanating from dorsal root ganglia. AnkyrinG 480/270 kDa and
ankyrinB 440 kDa as well as the ankyrin-binding proteins
were abundantly expressed in these axons and localized continuously
along their length. Only faint levels of neurofascin immunostaining
were observed, confirming observations that suggest a predominantly
postnatal expression for this protein in mammals (Davis et al., 1993;
Mocosco and Sanes, 1995). No staining was detectable in these axons
using antibodies to the voltage-dependent sodium channel.
Fig. 3.
Colocalization of ankyrinG 480/270
kDa, spectrin, NrCAM, and ankyrinB 440 kDa in dorsal root
axons of the embryonic day 16 rat. Cryosections (4 µm) of the dorsal
roots from an embryonic day 16 rat were double-labeled with antibodies
to ankyrinG 480/270 kDa (B, D, F) and
spectrin (A), NrCAM (C), or
ankyrinB 440 kDa (E).
Arrows indicate the staining of axons or bundles of
axons emanating from the dorsal roots. Insets show a
single bundle of axons at 2× higher magnification.
[View Larger Version of this Image (159K GIF file)]
These results demonstrate a major difference between the distribution
of axonal ankyrins in early development and in adult nerve axons.
AnkyrinB 440 kDa, which apparently is present in some axons
before myelination, disappears from myelinated axons and is present in
unmyelinated axons in the adult CNS (Kunimoto et al., 1991; Chan et
al., 1993). AnkyrinG 480/270 kDa is co-expressed with
ankyrinB 440 kDa in embryonic nerve, but in adults it is absent from unmyelinated axons as well as regions of myelinated axons
in contact with myelin (Kordeli et al., 1995) (Fig. 2).
AnkyrinG 480/270 kDa redistributes to clusters
containing Na channel, neurofascin, and NrCAM localized at sites
adjacent to MAG-staining processes of Schwann cells during
myelination of peripheral nerve
The transition from a continuous distribution of
ankyrinG 480/270 kDa in axons to a highly polarized
localization at the node of Ranvier with low expression in unmyelinated
axons in the adult was studied in myelinating sciatic nerves during
early postnatal development of rats. Myelination in the sciatic nerve
occurs between postnatal days 2 and 13, and it occurs at different
rates and times depending on the axon. This heterogeneity allows
visualization of axons in various stages of myelination in a single
section of postnatal tissue, but it also presents a problem in
distinguishing early stages in myelination. Expression of the MAG
provided a marker for the onset of myelination and for Schwann cell
processes that contact axons. MAG is expressed by Schwann cells at
~1.5 turns of the Schwann cell around the axon and is initially
localized to the periaxonal interface (Martini and Shachner, 1986). As
myelination progresses, MAG becomes increasingly localized to areas of
uncompacted myelin such as the paranodal loops adjacent to the nodal
axon segment in adult nodes of Ranvier (Trapp et al., 1989).
Figure 4 shows the localization of
ankyrinG 480/270 kDa (A, E) with respect to MAG
as a marker for Schwann cell processes (C, D) in the 2-d-old
rat sciatic nerve. Although ankyrinG 480/270 kDa was still
localized predominantly along the length of axons, 1- to 2-µm-size
clusters of staining (arrowheads) were observed. Clusters
were also occasionally observed in pairs ~5-10 µm apart. Ankyrin
clusters were localized to gaps between MAG staining, presumably
representing sites of node formation between myelinating Schwann cells.
Clusters were also observed at the ends of MAG-positive fibers.
Formation of ankyrinG 480/270 kDa clusters correlated with
the onset of myelination, as indicated by the expression of MAG. No
ankyrinG 480/270 kDa clusters were observed in areas of the
sciatic nerve with little or no apparent MAG staining.
Fig. 4.
Localization of ankyrinG 480/270 kDa
and ankyrinB 440 kDa with respect to MAG in the 2-d-old rat
sciatic nerve. Cryosections (4 µm) of the 2-d-old rat sciatic nerve
were double-labeled with antibodies to ankyrinG 480/270 kDa
(A), ankyrinB 440 kDa
(B), and MAG (C, D). Composite
images collected by confocal microscopy are shown in E
and F. Arrows denote the position of
ankyrinG 480/270 kDa clusters. Inset shows
one of these clusters at 3× higher magnification.
[View Larger Version of this Image (109K GIF file)]
The localization of ankyrinB 440 kDa with respect to MAG
was also examined in the 2-d-old rat sciatic nerve. Although
ankyrinB 440 kDa colocalized with ankyrinG
480/270 kDa in premyelinated axons of the embryonic nervous system
(Fig. 3E,F), it is expressed at much lower
concentrations in myelinating areas (i.e., MAG-positive areas) of the
2-d-old sciatic nerve (Fig. 4B,F). Figure
5 shows the localization of
ankyrinB 440 kDa (A) with respect to
ankyrinG 480/270 kDa (B) in the 2-d-old
sciatic nerve and indicates that these molecules colocalize in axons
lacking ankyrinG 480/270 kDa clusters. AnkyrinB
440 kDa also is colocalized with ankyrinG 480/270 kDa in
clusters, but it is not concentrated to the same degree as
ankyrinG 480/270 kDa. AnkyrinB 440 kDa also is
present in some but not all adult nodes of Ranvier (our unpublished
data). The two ankyrins thus may exhibit some functional overlap. The
remainder of this study will focus on ankyrinG 480/270
kDa.
Fig. 5.
Colocalization of ankyrinG 480/270 kDa
and ankyrinB 440 kDa in the 2-d-old rat sciatic nerve. A 4 µm cryosection of the 2-d-old rat sciatic nerve was labeled with
antibodies to ankyrinB 440 kDa (A)
and ankyrinG 480/270 kDa (B). The
composite confocal image is shown in C.
Arrows denote the position of ankyrinG
480/270 kDa clusters.
[View Larger Version of this Image (67K GIF file)]
Colocalization of ankyrinG 480/270 kDa and ankyrin-binding
proteins in developing nerve was assessed at the light level using double-labeling with respect to ankyrinG in the 2-d-old rat
sciatic nerve (Fig. 6).
AnkyrinG 480/270 kDa (D, E, F)
colocalized with the voltage-dependent sodium channel
(G), neurofascin (H), and NrCAM
(I) in paired clusters. In addition, single clusters
also exhibited this colocalization, and no clusters of
ankyrinG 480/270 kDa without ankyrin-binding proteins were
observed in 94 clusters examined. Inference of the colocalization of
these molecules is obviously limited to the resolution of the light
microscope in these experiments and awaits further confirmation by
double-labeling immunoelectron microscopy.
Fig. 6.
Co-clustering of ankyrinG and
ankyrin-binding proteins in paired cluster intermediates of the 2-d-old
rat sciatic nerve. Cryosections (4 µm) of the 2-d-old rat sciatic
nerve were double-labeled with antibodies against ankyrinG
(D-F) and the voltage-dependent sodium channel
(A), neurofascin (B), or
NrCAM (C). Composite confocal images of
individual double-cluster structures are shown in
G-I.
[View Larger Version of this Image (115K GIF file)]
The relationship of neurofascin-NrCAM clusters to MAG-staining Schwann
cell processes is shown at high magnification in the 5-d-old rat
sciatic nerve (Fig. 7). Neurofascin (Fig.
7A, red) is observed in paired clusters where
MAG-positive fibers (green) flank both sides of the
pair, as well as single clusters associated with asymmetrical MAG
staining. Overlap in neurofascin and MAG staining is clearly observed
(Fig. 7, arrowheads), although the highest density of
neurofascin clustering did not overlap with MAG staining and was
observed in front of the MAG-positive fiber. Because this antibody also
stains paranodal isoforms of neurofascin in adult nodes of Ranvier
(Davis et al., 1996), this result suggests that neurofascin might be
playing a role in establishment of glial-axonal junctions important to
the formation of the paranodal domain. Figure 7B shows
the localization of NrCAM (red) to both paired and single
cluster intermediates. In the paired intermediate, the unequal
accumulation of MAG staining (Fig. 7B, green) in
the developing paranodal area associated with the left-hand cluster suggests differing levels of myelination on either side of the developing node. The NrCAM clusters clearly localize outside of MAG
staining in front of the developing paranodal area, with no apparent
contact with the myelinating glial cell. A localization pattern similar
to that of NrCAM was observed for ankyrinG and the
voltage-dependent sodium channel (data not shown).
Fig. 7.
Immunolocalization of the ankyrin-binding cell
adhesion molecules neurofascin and NrCAM with respect to MAG in the
5-d-old rat sciatic nerve. Confocal micrographs show 4 µm
cryosections double-labeled with MAG (fluorescein) and neurofascin
(rhodamine in A) or NrCAM (rhodamine in
B). Arrowheads indicate areas of neurofascin and MAG overlap. Insets show areas of
interest at 2× higher magnification.
[View Larger Version of this Image (107K GIF file)]
Clustering of neurofascin and NrCAM precedes redistribution of
ankyrinG 480/270 kDa and the voltage-dependent Na
channel
The previous experiments demonstrate that ankyrinG
480/270 kDa and ankyrin-binding proteins concentrate in clusters
localized adjacent to Schwann cell processes as an initial event in
assembly of the node of Ranvier. Double-labeling experiments revealed
that all clusters that contained ankyrin also had ankyrin-binding
proteins (Fig. 6); however, these results do not answer the question of which protein arrives first at clusters. A careful examination of
colocalization of neurofascin and NrCAM with respect to
ankyrinG 480/270 kDa in the day 2 sciatic nerve revealed
the presence of neurofascin and NrCAM clusters without concomitant
clustering of ankyrinG 480/270 kDa (Fig.
8). In contrast, no examples were found
of clustered voltage-dependent sodium channel in the absence of ankyrin
(not shown). Figure 8 shows rhodamine-labeled clusters (arrows) of neurofascin (A) and NrCAM
(B) alone and double-labeled clusters of these
molecules with ankyrinG 480/270 kDa
(arrowheads). These data suggest that clustering of the cell
adhesion molecules neurofascin and NrCAM precedes that of
ankyrinG 480/270 kDa and the voltage-dependent sodium
channel in the myelinating sciatic nerve.
Fig. 8.
Colocalization of ankyrinG 480/270 kDa
(fluorescein in A and B) with respect to
neurofascin (rhodamine in A) and NrCAM (rhodamine in
B) in 4 µm cryosections of the 2-d-old rat sciatic
nerve. Co-clusters of ankyrinG 480/270 kDa with neurofascin
or NrCAM are denoted by arrowheads. Clusters of
neurofascin or NrCAM alone are denoted by
arrows.
[View Larger Version of this Image (79K GIF file)]
The possibility that NrCAM and neurofascin cluster before
ankyrinG 480/270 kDa is further supported by comparison of
the distribution of these molecules in the 2-d-old sciatic nerve (Fig.
9). AnkyrinG 480/270 kDa is
distributed continuously localized along the length of the axon with a
number of focused clusters (Fig. 9, arrows) of the molecule
(A). In contrast, neurofascin (Fig. 9B)
and NrCAM (C) are already highly clustered
(arrows), with very little staining observed along the rest
of the axon. Double-labeling of these sections with antibodies to MAG
(Fig. 9D,E,F) shows that many of these clusters are
associated with MAG-positive Schwann cells (arrows). In
addition, a number of neurofascin (Fig. 9E) and NrCAM (F) clusters (arrowheads) do not appear to
be associated with MAG-positive Schwann cells. Similar clusters were
not seen for ankyrinG 480/270 kDa. These observations
suggest that the cell adhesion molecules neurofascin and NrCAM are the
pioneer molecules in the assembly of clusters that subsequently contain
ankyrin and the voltage-dependent sodium channel.
Fig. 9.
Distribution of ankyrinG 480/270 kDa
(A, D), neurofascin (B, E), and NrCAM
(C, F) in 4 µm cryosections of the 2-d-old
sciatic nerve. Also shown is the localization of these proteins with
respect to MAG in the same sections (D-F).
Arrows denote clusters of proteins associated with
MAG-positive processes; arrowheads show clusters of
proteins not associated with MAG-positive processes.
[View Larger Version of this Image (129K GIF file)]
Formation of adult nodes of Ranvier occurs by fusion of paired
ankyrinG-sodium channel-adhesion molecule
clusters
The presence of ankyrin and ankyrin-binding proteins in
clusters early in myelination suggests that these clusters represent the initial intermediates in the assembly of a node of Ranvier. The
relationship between single clusters, paired clusters, and finally
mature nodes of Ranvier was analyzed by measurements of numbers of
single and paired clusters and distances between paired clusters at
different stages of development (Table
1). Single clusters are the predominant
form at day 2, whereas by day 5 the majority of ankyrinG
and ankyrin-binding protein clusters occur in pairs 5-10 µm apart,
and by day 7 very few single clusters are observed. These results
suggest that the first step is formation of single clusters, with a
second cluster subsequently forming at a later time. The asymmetric
formation of these cluster pairs may be related to differing levels of
myelination in the Schwann cells flanking the clusters. The observation
of differing levels of myelination on either side of the developing
node has been attributed to the partial autonomy of adjacent Schwann
cells (Allt, 1969).
Table 1.
Tabulation of nodal intermediates stained by
ankyrinG at different times during myelination of the rat
sciatic nerve
| Postnatal
day |
Number of nodes observed |
Relative % of single
clusters |
Relative % of paired clusters (5-10 µm) |
Relative % of paired clusters (<5 µm) |
Relative % of mature nodes
|
|
| 2 |
94 |
71 |
15 |
9 |
5
|
| 5 |
74 |
30 |
34 |
20 |
16
|
| 7 |
56 |
9 |
14 |
35 |
42 |
|
|
Cryosections (4 µm) were prepared from the same areas of
three separate sciatic nerves, at each time point, and double-stained for ankyrinG and MAG. No significant variation in staining
was noted between different nerves from the same time point. Single clusters were distinguished from mature nodes by the absence of MAG
staining on one side of the cluster.
|
|
Measurements of distances between ankyrin clusters at different stages
of development support the idea that pairs of clusters associated with
adjacent Schwann cells flanking the future nodal site fuse to form the
mature node of Ranvier. In the 7-d-old rat sciatic nerve a number of
paired intermediates are observed that are more highly localized and
closer together (<5 µm) than those seen previously in the 5-d-old
rat sciatic nerve. Figure 10 shows two
different 4 µm cryosections of the 7-d-old rat sciatic nerve labeled
with antibodies to ankyrinG 480/270 kDa. A number of
different nodal intermediates are visible along with a number of mature nodes. These different structures were also stained with antibodies to
NrCAM, neurofascin, and the voltage-dependent sodium channel (data not
shown). Some of the intermediates in Figure 10 (arrows) have
been numbered to reflect inferred stages in the formation of the node
of Ranvier. Individual intermediates from each potential stage are also
shown at higher magnification. These intermediates also clearly show
significant staining of ankyrinG 480/270 in the cytoplasm
during early stages of nodal formation and a predominant membrane
localization for this molecule during later stages.
Fig. 10.
Immunolocalization of ankyrinG
480/270 kDa to nodal intermediates in the 7-d-old rat sciatic nerve.
Cryosections (4 µm) of the 7-d-old rat sciatic nerve were labeled
with antibodies to ankyrinG 480/270 kDa.
Arrows indicate nodal intermediates and are numbered to
indicate progressive stages in node formation. Individual nodes from
each potential intermediate stage are shown below at higher
magnification.
[View Larger Version of this Image (98K GIF file)]
Formation of nodal intermediates does not require formation of
compact myelin
Peripheral myelin protein 22 (PMP-22) is expressed by
myelinating Schwann cells and incorporated into compacted regions of peripheral myelin. Mutations in this gene account for the trembler phenotype in mice, characterized by peripheral nerve hypomyelination and continuous Schwann cell proliferation into adulthood (Henry and
Sidman, 1988). In humans, PMP-22 mutations are responsible for
Charcot-Marie-Tooth (type 1A) syndrome (Valentijn et al., 1992), which
is characterized by decreased peripheral nerve conduction velocities,
segmental demyelination, and partial remyelination, along with Schwann
cell proliferation. To study how the inability to form compact myelin
affects formation of the node of Ranvier, sciatic nerves of 20-d-old
trembler mice were stained with antibodies against
ankyrinG, neurofascin, and the voltage-dependent
sodium channel. Figure
11A-C shows the
immunolocalization of these proteins in the sciatic nerves of wild-type
(++/++) trembler littermates. Interference contrast
microscopy (DIC) shows the presence of compact myelin (Fig.
11A), with mature nodes of Ranvier evident
as gaps in the myelin sheath (arrows). These nodes clearly
label with ankyrinG antibodies (arrows) in the
corresponding fluorescent micrograph (A). Wild-type
mature nodes also label with antibodies to neurofascin (Fig.
11B) and the voltage-dependent sodium channel (C). Only mature nodes were observed. In contrast,
DIC micrographs of the trembler sciatic nerve
(++/tr) show hypomyelination (Fig. 11D)
with an absence of clearly discernible nodal structures. Staining of these sciatic nerves with ankyrinG
antibodies, however, shows localization of
ankyrinG to structures resembling mature nodes or
potentially single-cluster intermediates (Fig. 11D,
arrows) and to structures resembling the paired-cluster
intermediates seen in developing sciatic nerves (large
arrowheads). A similar staining pattern was also observed with
antibodies to neurofascin (Fig. 11E) and the
voltage-dependent sodium channel (F).
Fig. 11.
Immunolocalization of ankyrinG
480/270 kDa, neurofascin, and the voltage-dependent sodium channel in
sciatic nerves from the hypomyelinated mutant mouse
trembler. Cryosections (4 µm) of sciatic nerves from
20-d-old wild-type (A-C) and
trembler (D-F) mice were stained
with antibodies against ankyrinG 480/270 kDa (A,
D), neurofascin (B, E), and the
voltage-dependent sodium channel (C, F). Nodes of
Ranvier are highlighted by arrows. Large
arrowheads indicate double-cluster structures in the
trembler mutant similar to the nodal intermediates observed
in the rat developing sciatic nerve. A and D
represent the corresponding DIC images of the immunofluorescent micrographs shown in A and D and
illustrate the lack of compact myelin in the trembler
mutant. Arrows (A) indicate the positions of
the nodes stained with the ankyrinG antibody in the
wild-type sciatic nerve.
[View Larger Version of this Image (75K GIF file)]
DISCUSSION
AnkyrinG 480/270 kDa and the three ankyrin-binding
integral membrane proteins neurofascin, NrCAM, and the
voltage-dependent sodium channel colocalize at the axonal membrane of
the adult node of Ranvier in a specialized membrane domain within the
spectrin-actin network (Kordeli et al., 1995; Davis et al., 1996)
(Fig. 2). Before myelination, ankyrinG 480/270 kDa and the
related ankyrin isoform ankyrinB 440 kDa are co-expressed
along with NrCAM in an abundant, continuous distribution along the
length of axons, whereas neurofascin and the voltage-dependent sodium
channel are present at barely detectable levels (Figs. 3, 4). This
study has resolved morphological intermediates in the developmental
transition from a continuous distribution of ankyrinG
480/270 kDa in all axons to a highly polarized localization at the node
of Ranvier. The first detected event is formation of clusters
containing the cell adhesion molecules neurofascin and NrCAM at sites
independent of MAG-staining Schwann cell processes. Subsequent steps
involve recruitment of ankyrinG 480/270 kDa and the
voltage-dependent sodium channel to cluster sites defined by the cell
adhesion molecules, and elaboration of MAG-staining Schwann cell
processes adjacent to these cluster sites. Single clusters with
associated Schwann cell processes then are joined by another
cluster-MAG-staining Schwann cell process to form a pair, and pairs of
clusters then fuse to form the mature node of Ranvier. In addition to
rearrangements of ankyrins, differences in expression of ankyrins also
occur during myelination: ankyrinB 440 kDa is downregulated
from myelinating axons soon after expression of MAG and is retained in
unmyelinated axons, whereas ankyrinG 480/270 kDa disappears
from unmyelinated axons.
AnkyrinG is a candidate to couple cell adhesion molecules
to the voltage-sensitive sodium channel during assembly of the node of
Ranvier. This idea is supported by observations that clusters of
neurofascin and NrCAM are joined by ankyrinG 480/270 kDa
and the voltage-dependent sodium channel during differentiation of myelinated axons, and biochemical data that suggest a multivalent ankyrin membrane-binding domain (Michaely and Bennett, 1995a,b). The
proposed ankyrinG-mediated link between adhesion molecules and ion channels would allow the cell adhesion molecules to direct the
localization of voltage-sensitive sodium channels in the axonal membrane. Additional interactions of adhesion molecules with the voltage-dependent sodium channel also are likely to occur. For example,
the  -2 subunit of the voltage-dependent sodium channel has homology
to the NrCAM-binding protein F11 and potentially could also associate
laterally with NrCAM (Isom et al., 1995).
The observations of this study of early and synchronous clustering of
ankyrin and the voltage-dependent sodium channel during differentiation
of myelinated axons disagree with the report that clustering of the
sodium channel precedes that of ankyrin in neuronal-Schwann cell
co-cultures (Joe and Angelides, 1992). This contradiction may result
from differences in in vivo development versus in
vitro cultures. Alternatively, ankyrin antibodies used in the
earlier study may not distinguish between ankyrinG 480/270
kDa and ankyrinB 440 kDa, both of which colocalize to
premyelinated axons (Fig. 3). The proposal of this study that formation
of the node of Ranvier results from the "fusion" of two cluster
intermediates composed of ankyrin, the sodium channel, and the cell
adhesion molecules is in agreement with the observations of Shrager and
colleagues, who have reported sodium channel clusters in single fibers
from remyelinating (Dugandzija-Novakovic et al., 1995) and myelinating (Vabnick et al., 1996) sciatic nerve axons.
Clustering of neurofascin and NrCAM could facilitate the subsequent
recruitment and enhance the local concentration of ankyrinG and the sodium channel by several mechanisms. The ankyrin
membrane-binding domain contains two binding sites for neurofascin as
well as additional sites for the anion exchanger (Michaely and Bennett,
1995a,b) and potentially could bind preferentially to dimers or higher oligomers of neurofascin. A second manner in which the clustering of
neurofascin and NrCAM might affect binding of ankyrin is through changes in intracellular signaling pathways. For example, tyrosine phosphorylation of neurofascin completely abolishes its ability to bind
to ankyrin (Garver et al., 1997), and local activation of a tyrosine
phosphatase could lead to recovery of neurofascin-ankyrin binding. In
support of these ideas, Dubreuil et al., (1996) demonstrated recently
that concentration of neuroglian, a Drosophila homolog of
L1, at sites of cell-cell contact is accompanied by recruitment of
Drosophila ankyrin in insect cells.
Recently, Ellisman and colleagues (Deerinck et al., 1997) observed that
aggregates of ankyrin and the voltage-dependent sodium channel form in
regions of nerves of dystrophic mice that lack Schwann cells,
demonstrating that direct Schwann cell-axon contacts are not required
for clusters. These findings contrast with the results of Shrager and
colleagues (Dugandzija-Novakovic et al., 1995; Vabnick et al., 1996),
who have demonstrated a role for the Schwann cell in the organization
and positioning of sodium channel clusters. The findings of this study
that clusters of neurofascin and NrCAM precede elaboration of
MAG-staining Schwann cell processes and that ankyrin and sodium
channels cluster in the trembler mutant is consistent with
the idea that the axon plays an active role in initial events in
myelination and node formation. A possible explanation for these
conflicting results is that Schwann cells are able to induce clusters
of ankyrin and ankyrin-binding proteins in the axonal membrane, which
then act as cues for the correct positioning of the Schwann cell during its adherence to the axonal membrane.
Mechanisms for the induction of ankyrin and ankyrin-binding protein
clusters might include the participation of secreted molecules produced
by Schwann cells or possibly other cells. Barres and coworkers (Kaplan
et al., 1997) have reported that oligodendrocyte-conditioned cell-free
medium can rapidly initiate aggregation of sodium channel and ankyrin
in axons of cultured rat retinal ganglion cells. Interestingly these
clusters appear to be spaced at the correct nodal interval, suggesting
that an intrinsic axonal factor might regulate nodal periodicity. An
example of a potential soluble factor is agrin, which is an
extracellular matrix protein expressed by Schwann cells as well as
other cells, including motor neurons (Ruegg et al., 1992). Agrin
induces sodium channel clustering on cultured muscle fibers (Sharp and
Caldwell, 1996) and is localized at nodes of Ranvier (Reist et al.,
1987).
This study, based on observations in the myelinating sciatic nerve and
experiments with the hypomyelinating mutant trembler, presents evidence that clustering of ankyrin and the voltage-dependent sodium channel precedes and does not require the elaboration of compact
myelin. Differentiation of the axonal membrane before the elaboration
of compact myelin has been noted in electron microscopy studies of
myelinating nerves (Waxman and Foster, 1980; Wiley-Livingston and
Ellisman, 1980). The localization of ankyrin and the voltage-dependent sodium channel in the trembler mutant contrasts with the
localization of the shaker K+ channel,
which does not localize to the paranodal area in these mutants (Wang et
al., 1995). A potential qualification in interpreting these results are
the cycles of demyelination and remyelination that the
trembler mutant exhibits, which may account for the presence of nodal intermediates in the sciatic nerves of these animals. The
presence of nodal intermediates in trembler, however, could be relevant to the pathophysiology of human equivalents of this mutation, such as Charcot-Marie-Tooth (type 1A) syndrome (Valentijn et
al., 1992). Formation of sodium channel clusters could still provide
electrophysiological benefits compared with totally dispersed distribution and could explain the relatively mild phenotype of this
disease. In this case, treatments with agents that promote clusters
could provide a potential therapeutic approach to ameliorate this
disease as well as other disorders of myelin, such as multiple sclerosis. A candidate for such a cluster-promoting factor is the
active component of oligodendrocyte-conditioned medium that induces
clustering of ankyrin and sodium channels (Kaplan et al., 1997).
The observation that ankyrinG is confined within a
specialized region of the spectrin-actin network in myelinated axons
implies that the spectrin-based membrane skeleton in internodal regions of myelinated axons involves ankyrin-independent attachment proteins. Spectrin does bind directly to brain membranes lacking ankyrin (Steiner
and Bennett, 1988), although the identity of these sites is not yet
known. An ankyrin-based mechanism for polarity in axons differs from
models for formation of basolateral domains of epithelial cells (Nelson
and Veshnock, 1987). In this system, the targeted recruitment of both
spectrin and ankyrin has been implicated in the polarized distribution
of the Na+/K+ ATPase in the
basolateral domain.
In conclusion, this study has used newly defined molecular components
of the node of Ranvier to discriminate early developmental stages in
myelination. The axonal clusters of neurofascin and NrCAM described
here are the earliest stage in assembly of the node of Ranvier yet to
be resolved. In addition to these findings, this work also presents a
new set of questions. The basis for initial clustering of cell adhesion
molecules and the role of extracellular signals versus intrinsic axonal
polarity is yet to be determined. The role of axonal vesicle transport
in the delivery of ankyrinG 480/270 kDa and the
voltage-dependent sodium channel to cell adhesion molecule clusters
remains to be elucidated. The negative signals that exclude
MAG-staining Schwann cell processes from the nodal axon segment also
are not understood. Answers to these questions may eventually
contribute to detailed understanding of the cellular events in
myelination and help develop effective strategies to promote
remyelination in clinical settings.
FOOTNOTES
Received March 20, 1997; revised May 29, 1997; accepted July 9, 1997.
This work was supported by the Howard Hughes Medical Institute and a
grant from National Institutes of Health (DK29808). Zhang Xu is
gratefully acknowledged for the preparation of the chicken anti-ankyrinG common "tail" antibody.
Correspondence should be addressed to Dr. Lambert at his present
address: Worcester Foundation for Biomedical Research, 222 Maple
Avenue, Shrewsbury, MA 01545.
REFERENCES
-
Allt G
(1969)
Ultrastructural features of the immature peripheral nerve.
J Anat
105:283-293[Web of Science][Medline].
-
Bennett V,
Gilligan DM
(1993)
The spectrin based membrane skeleton and micron scale organization of the plasma membrane.
Annu Rev Cell Biol
9:27-66[Web of Science].
-
Chan W,
Kordeli E,
Bennett V
(1993)
440kd ankyrinB, structure of the major developmentally regulated domain and selective localization in unmyelinated axons.
J Cell Biol
123:1463-1473[Abstract/Free Full Text].
-
Chiu SY
(1980)
Asymmetry currents in the mammalian myelinated nerve.
J Physiol (Lond)
309:499-519[Abstract/Free Full Text].
-
Davis JQ,
Bennett V
(1983)
Brain spectrin. Isolation of subunits and formation of hybrids with erythrocyte spectrin subunits.
J Biol Chem
258:7757-7766[Abstract/Free Full Text].
-
Davis JQ,
Bennett V
(1994)
Ankyrin-binding activity shared by the neurofascin/liter1/NrCAM family of nervous system cell adhesion molecules.
J Biol Chem
269:27163-27166[Abstract/Free Full Text].
-
Davis JQ,
McLaughlin T,
Bennett V
(1993)
Ankyrin-binding proteins related to nervous system cell adhesion molecules: candidates to provide transmembrane and intercellular connections in adult brain.
J Cell Biol
121:121-133[Abstract/Free Full Text].
-
Davis JQ,
Lambert S,
Bennett V
(1996)
Molecular composition of the node of Ranvier: identification of ankyrin-binding cell adhesion molecules neurofascin (mucin+/third FNIII domain
 ) and NrCAM at nodal axon segments.
J Cell Biol
135:1355-1368[Abstract/Free Full Text]. -
Deerinck TJ,
Levinson SR,
Bennett GV,
Ellisman MH
(1997)
Clustering of voltage-sensitive sodium channels on axons is independent of direct Schwann cell contact in the dystrophic mouse.
J Neurosci
17:5080-5088[Abstract/Free Full Text].
-
Dubreuil RR,
Macvicar R,
Dissanayake S,
Liu C,
Homer D,
Hortsch M
(1996)
Neuroglian-mediated cell adhesion induces assembly of the membrane skeleton at cell contact sites.
J Cell Biol
133:647-655[Abstract/Free Full Text].
-
Dugandzija-Novakovic S,
Koszowski AG,
Levinson SR,
Shrager P
(1995)
Re-clustering of Na+ channels and node of Ranvier formation in remyelinating axons.
J Neurosci
15:492-503[Abstract].
-
Ellisman MH,
Levinson SR
(1982)
Immunocytochemical localization of sodium channel distributions in the excitable membranes of Electrophorus electricus.
Proc Natl Acad Sci USA
79:6707-6711[Abstract/Free Full Text].
-
Garver T,
Davis J,
Ren Q,
Bennett V
(1997)
Tyrosine phosphorylation at a site highly conserved in the L1 family of cell adhesion molecules abolishes ankyrin-binding and increases lateral mobility of neurofascin.
J Cell Biol
137:703-714[Abstract/Free Full Text].
-
Henry EW,
Sidman RL
(1988)
Long lives for homozygous trembler mutant mice despite virtual absence of peripheral nerve myelin.
Science
241:344-346[Abstract/Free Full Text].
-
Hortsch M
(1996)
The L1 family of neural cell adhesion molecules: old proteins performing new tricks.
Neuron
17:587-593[Web of Science][Medline].
-
Ichimura T,
Ellisman MH
(1991)
Three-dimensional fine structure of cytoskeletal-membrane interactions at nodes of Ranvier.
J Neurocytol
20:667-681[Web of Science][Medline].
-
Isom LL,
Ragsdale DS,
DeJongh K,
Westenboek RE,
Reber BF,
Scheuer T,
Catterall WA
(1995)
Structure and function of the beta-2 subunit of brain sodium channels, a transmembrane glycoprotein with a CAM motif.
Cell
83:433-442[Web of Science][Medline].
-
Joe E-H,
Angelides K
(1992)
Clustering of voltage-dependent sodium channels depends on Schwann cell contacts.
Nature
356:333-335[Medline].
-
Kaplan MR,
Meyer-Franke A,
Lambert S,
Bennett V,
Duncan ID,
Levinson SR,
Barres BA
(1997)
Induction of sodium channel clustering by oligodendrocytes.
Nature
386:724-728[Medline].
-
Kayyem JF,
Roman J,
de la Rosa E,
Schwarz U,
Dreyer W
(1992)
Bravo/NrCAM is closely related to the cell adhesion molecules l1 and NgCAM and has a similar heterodimer structure.
J Cell Biol
118:1259-1270[Abstract/Free Full Text].
-
Kordeli E,
Davis J,
Trapp B,
Bennett V
(1990)
An isoform of ankyrin is localized at nodes of Ranvier in myelinated axons of central and peripheral nerves.
J Cell Biol
110:1341-1352[Abstract/Free Full Text].
-
Kordeli E,
Lambert S,
Bennett V
(1995)
AnkyrinG: a new ankyrin gene with neural specific isoforms localized at the axonal initial segment and node of Ranvier.
J Biol Chem
270:2352-2359[Abstract/Free Full Text].
-
Kunimoto M,
Otto E,
Bennett V
(1991)
A new 440 kDa isoform is the major ankyrin in neonatal rat brain.
J Cell Biol
115:1319-1331[Abstract/Free Full Text].
-
Lambert S,
Bennett V
(1993)
Anemia to cerebellar dysfunction: a review of the ankyrin gene family.
Eur J Biochem
211:1-6[Web of Science][Medline].
-
Martini R,
Shachner M
(1986)
Immunoelectron microscopic localization of neural cell adhesion molecules (L1, N-CAM and MAG) and their shared carbohydrate epitope and myelin basic protein in developing sciatic nerve.
J Cell Biol
103:2439-2448[Abstract/Free Full Text].
-
Michaely P,
Bennett V
(1995a)
The ANK repeats of erythrocyte ankyrin form two distinct but cooperative binding sites for the erythrocyte anion exchanger.
J Biol Chem
270:31298-31302[Abstract/Free Full Text].
-
Michaely P,
Bennett V
(1995b)
Mechanism for binding site diversity on ankyrin: comparison of binding sites on ankyrin for neurofascin and the Cl
 /HCO3 anion exchanger.
J Biol Chem
270:31298-31302. -
Mocosco LM,
Sanes JR
(1995)
Expression of four Ig superfamily adhesion molecules (L1, NrCAM/Bravo, Neurofascin/ABGP and N-CAM) in the developing mouse spinal cord.
J Comp Neurol
352:321-334[Web of Science][Medline].
-
Nelson WJ,
Veshnock PJ
(1987)
Ankyrin-binding to (Na+/K+)ATPase and implications for the organization of membrane domains in polarized cells.
Nature
328:533-536[Medline].
-
Noda M,
Ikeda T,
Kayano T,
Suzuki H,
Takeshima H,
Kurasaki M,
Takahashi H,
Numa S
(1986)
Existence of distinct sodium channel messenger RNAs in rat brain.
Nature
320:188-192[Medline].
-
Peters A
(1966)
The node of Ranvier in the central nervous system.
Q J Exp Physiol Cognit Med Sci
51:229-236[Abstract/Free Full Text].
-
Reist NE,
Magill C,
McMahan UJ
(1987)
Agrin-like molecules at synaptic sites in normal, denervated and damaged skeletal muscle.
J Cell Biol
105:2457-2469[Abstract/Free Full Text].
-
Ritchie JM,
Rogart RB
(1977)
Density of sodium channels in mammalian myelinated nerve fibers and nature of the axonal membrane under the myelinated sheath.
Proc Natl Acad Sci USA
74:211-215[Abstract/Free Full Text].
-
Ruegg MA,
Tsien KW,
Horton SE,
Kroger S,
Escher G,
Gensch EM,
McMahan UJ
(1992)
The agrin gene codes for a family of basal laminal proteins that differ in function and distribution.
Neuron
8:691-699[Web of Science][Medline].
-
Rushton WAH
(1951)
A theory of the effects of fibre size in medullated nerve.
J Physiol (Lond)
115:101-122.
-
Sharp AA,
Caldwell JH
(1996)
Aggregation of sodium channels induced by a postnatally upregulated isoform of agrin.
J Neurosci
16:6775-6783[Abstract/Free Full Text].
-
Shrager P
(1989)
Sodium channels in single demyelinated axons.
Brain Res
483:149-154[Web of Science][Medline].
-
Srinivasan Y,
Elmer L,
Davis J,
Bennett V,
Angelides K
(1988)
Ankyrin and spectrin associate with voltage-dependent sodium channels in brain.
Nature
333:177-180[Medline].
-
Steiner JP,
Bennett V
(1988)
Ankyrin-independent membrane protein-binding sites for brain and erythrocyte spectrin.
J Biol Chem
263:14417-14425[Abstract/Free Full Text].
-
Trapp BD,
Andrews SB,
Wong A,
O'Connell M,
Griffin JW
(1989)
Co-localization of the myelin-associated glycoprotein and the microfilament components F-actin and spectrin, in Schwann cells of myelinated nerve fibers.
J Neurocytol
18:47-60[Web of Science][Medline].
-
Vabnick I,
Novakovic SD,
Levinson SR,
Schachner M,
Shrager P
(1996)
The clustering of axonal sodium channels during development of the peripheral nervous system.
J Neurosci
16:4914-4922[Abstract/Free Full Text].
-
Valentijn LJ,
Baas F,
Wolterman RA,
Hoogendijk JE,
van den Bosch NH,
Zorn I,
Gabreels-Festen AW,
de Visser M,
Bolhuis PA
(1992)
Identical point mutations of PMP-22 in trembler-J mouse and Charcot-Marie-Tooth disease type 1A.
Nat Genet
2:288-291[Web of Science][Medline].
-
Wang H,
Allen ML,
Grigg JJ,
Noebels JL,
Tempel BL
(1995)
Hypomyelination alters K+ channel expression in mouse mutants shiverer and trembler.
Neuron
15:1337-1347[Web of Science][Medline].
-
Waxman SG,
Foster RE
(1980)
Development of the axon membrane during differentiation of myelinated fibres in spinal nerve roots.
Proc R Soc Lond [Biol]
209:441-446[Medline].
-
Wiley-Livingston CA,
Ellisman MH
(1980)
Development of axonal membrane specializations defines nodes of Ranvier and precedes Schwann cell myelin elaboration.
Dev Biol
79:334-355[Web of Science][Medline].
-
Zhang X,
Bennett V
(1996)
Identification of O-linked N-acetyl glucosamine modification of isoforms of ankyrin targeted to nodes of Ranvier.
J Biol Chem
271:31391-31398[Abstract/Free Full Text].
This article has been cited by other articles:
|
|
|
|
 
A. Brechet, M.-P. Fache, A. Brachet, G. Ferracci, A. Baude, M. Irondelle, S. Pereira, C. Leterrier, and B. Dargent
Protein kinase CK2 contributes to the organization of sodium channels in axonal membranes by regulating their interactions with ankyrin G
J. Cell Biol.,
December 15, 2008;
183(6):
1101 - 1114.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Zonta, S. Tait, S. Melrose, H. Anderson, S. Harroch, J. Higginson, D. L. Sherman, and P. J. Brophy
Glial and neuronal isoforms of Neurofascin have distinct roles in the assembly of nodes of Ranvier in the central nervous system
J. Cell Biol.,
October 22, 2008;
181(7):
1169 - 1177.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. J. P. Alix, A. C. Dolphin, and R. Fern
Vesicular apparatus, including functional calcium channels, are present in developing rodent optic nerve axons and are required for normal node of Ranvier formation
J. Physiol.,
September 1, 2008;
586(17):
4069 - 4089.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Susuki and M. N. Rasband
Spectrin and Ankyrin-Based Cytoskeletons at Polarized Domains in Myelinated Axons
Experimental Biology and Medicine,
April 1, 2008;
233(4):
394 - 400.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Dzhashiashvili, Y. Zhang, J. Galinska, I. Lam, M. Grumet, and J. L. Salzer
Nodes of Ranvier and axon initial segments are ankyrin G-dependent domains that assemble by distinct mechanisms
J. Cell Biol.,
June 21, 2007;
177(5):
857 - 870.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Eshed, K. Feinberg, D. J. Carey, and E. Peles
Secreted gliomedin is a perinodal matrix component of peripheral nerves
J. Cell Biol.,
May 7, 2007;
177(3):
551 - 562.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Maertens, D. Hopkins, C.-W. Franzke, D. R. Keene, L. Bruckner-Tuderman, D. S. Greenspan, and M. Koch
Cleavage and Oligomerization of Gliomedin, a Transmembrane Collagen Required for Node of Ranvier Formation
J. Biol. Chem.,
April 6, 2007;
282(14):
10647 - 10659.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Yang, Y. Ogawa, K. L. Hedstrom, and M. N. Rasband
{beta}IV spectrin is recruited to axon initial segments and nodes of Ranvier by ankyrinG
J. Cell Biol.,
February 12, 2007;
176(4):
509 - 519.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. S. Garbe, A. Das, R. R. Dubreuil, and G. J. Bashaw
{beta}-Spectrin functions independently of Ankyrin to regulate the establishment and maintenance of axon connections in the Drosophila embryonic CNS
Development,
January 15, 2007;
134(2):
273 - 284.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
O. W. Howell, A. Palser, A. Polito, S. Melrose, B. Zonta, C. Scheiermann, A. J. Vora, P. J. Brophy, and R. Reynolds
Disruption of neurofascin localization reveals early changes preceding demyelination and remyelination in multiple sclerosis
Brain,
December 1, 2006;
129(12):
3173 - 3185.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. C. Inda, J. DeFelipe, and A. Munoz
Voltage-gated ion channels in the axon initial segment of human cortical pyramidal cells and their relationship with chandelier cells
PNAS,
February 21, 2006;
103(8):
2920 - 2925.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. K. Huang, G. R. Phillips, A. D. Roth, L. Pedraza, W. Shan, W. Belkaid, S. Mi, A. Fex-Svenningsen, L. Florens, J. R. Yates III, et al.
Glial Membranes at the Node of Ranvier Prevent Neurite Outgrowth
Science,
December 16, 2005;
310(5755):
1813 - 1817.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Occhi, D. Zambroni, U. Del Carro, S. Amadio, E. E. Sirkowski, S. S. Scherer, K. P. Campbell, S. A. Moore, Z.-L. Chen, S. Strickland, et al.
Both Laminin and Schwann Cell Dystroglycan Are Necessary for Proper Clustering of Sodium Channels at Nodes of Ranvier
J. Neurosci.,
October 12, 2005;
25(41):
9418 - 9427.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. J. Devaux and S. S. Scherer
Altered Ion Channels in an Animal Model of Charcot-Marie-Tooth Disease Type IA
J. Neurosci.,
February 9, 2005;
25(6):
1470 - 1480.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. P. McEwen and L. L. Isom
Heterophilic Interactions of Sodium Channel {beta}1 Subunits with Axonal and Glial Cell Adhesion Molecules
J. Biol. Chem.,
December 10, 2004;
279(50):
52744 - 52752.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Corfas, M. O. Velardez, C.-P. Ko, N. Ratner, and E. Peles
Mechanisms and Roles of Axon-Schwann Cell Interactions
J. Neurosci.,
October 20, 2004;
24(42):
9250 - 9260.
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Yang, S. Lacas-Gervais, D. K. Morest, M. Solimena, and M. N. Rasband
{beta}IV Spectrins Are Essential for Membrane Stability and the Molecular Organization of Nodes of Ranvier
J. Neurosci.,
August 18, 2004;
24(33):
7230 - 7240.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Bonnon, L. Goutebroze, N. Denisenko-Nehrbass, J.-A. Girault, and C. Faivre-Sarrailh
The Paranodal Complex of F3/Contactin and Caspr/Paranodin Traffics to the Cell Surface via a Non-conventional Pathway
J. Biol. Chem.,
November 28, 2003;
278(48):
48339 - 48347.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. W. Custer, K. Kazarinova-Noyes, T. Sakurai, X. Xu, W. Simon, M. Grumet, and P. Shrager
The Role of the Ankyrin-Binding Protein NrCAM in Node of Ranvier Formation
J. Neurosci.,
November 5, 2003;
23(31):
10032 - 10039.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. L. Gatto, B. J. Walker, and S. Lambert
Local ERM activation and dynamic growth cones at Schwann cell tips implicated in efficient formation of nodes of Ranvier
J. Cell Biol.,
August 4, 2003;
162(3):
489 - 498.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Lemaillet, B. Walker, and S. Lambert
Identification of a Conserved Ankyrin-binding Motif in the Family of Sodium Channel {alpha} Subunits
J. Biol. Chem.,
July 18, 2003;
278(30):
27333 - 27339.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. J. Bailey, M. A. Stocksley, A. Buckel, C. Young, and C. R. Slater
Voltage-Gated Sodium Channels and AnkyrinG Occupy a Different Postsynaptic Domain from Acetylcholine Receptors from an Early Stage of Neuromuscular Junction Maturation in Rats
J. Neurosci.,
March 15, 2003;
23(6):
2102 - 2111.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Boiko, A. Van Wart, J. H. Caldwell, S. R. Levinson, J. S. Trimmer, and G. Matthews
Functional Specialization of the Axon Initial Segment by Isoform-Specific Sodium Channel Targeting
J. Neurosci.,
March 15, 2003;
23(6):
2306 - 2313.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. M. Curristin, A. Cao, W. B. Stewart, H. Zhang, J. A. Madri, J. S. Morrow, and L. R. Ment
Disrupted synaptic development in the hypoxic newborn brain
PNAS,
November 26, 2002;
99(24):
15729 - 15734.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. N. Rasband, E. W. Park, D. Zhen, M. I. Arbuckle, S. Poliak, E. Peles, S. G.N. Grant, and J. S. Trimmer
Clustering of neuronal potassium channels is independent of their interaction with PSD-95
J. Cell Biol.,
November 25, 2002;
159(4):
663 - 672.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Bouzidi, N. Tricaud, P. Giraud, E. Kordeli, G. Caillol, C. Deleuze, F. Couraud, and G. Alcaraz
Interaction of the Nav1.2a Subunit of the Voltage-dependent Sodium Channel with Nodal AnkyrinG. IN VITRO MAPPING OF THE INTERACTING DOMAINS AND ASSOCIATION IN SYNAPTOSOMES
J. Biol. Chem.,
August 2, 2002;
277(32):
28996 - 29004.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Gollan, H. Sabanay, S. Poliak, E. O. Berglund, B. Ranscht, and E. Peles
Retention of a cell adhesion complex at the paranodal junction requires the cytoplasmic region of Caspr
J. Cell Biol.,
June 24, 2002;
157(7):
1247 - 1256.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Gagelin, B. Constantin, C. Deprette, M.-A. Ludosky, M. Recouvreur, J. Cartaud, C. Cognard, G. Raymond, and E. Kordeli
Identification of AnkG107, a Muscle-specific Ankyrin-G Isoform
J. Biol. Chem.,
April 5, 2002;
277(15):
12978 - 12987.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. J. Arroyo, T. Xu, J. Grinspan, S. Lambert, S. R. Levinson, P. J. Brophy, E. Peles, and S. S. Scherer
Genetic Dysmyelination Alters the Molecular Architecture of the Nodal Region
J. Neurosci.,
March 1, 2002;
22(5):
1726 - 1737.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Mathis, N. Denisenko-Nehrbass, J.-A. Girault, and E. Borrelli
Essential role of oligodendrocytes in the formation and maintenance of central nervous system nodal regions
Development,
December 1, 2001;
128(23):
4881 - 4890.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. F. Ratcliffe, R. E. Westenbroek, R. Curtis, and W. A. Catterall
Sodium channel {beta}1 and {beta}3 subunits associate with neurofascin through their extracellular immunoglobulin-like domain
J. Cell Biol.,
July 23, 2001;
154(2):
427 - 434.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. I. More, F.-P. Kirsch, and F. G. Rathjen
Targeted ablation of NrCAM or ankyrin-B results in disorganized lens fibers leading to cataract formation
J. Cell Biol.,
July 9, 2001;
154(1):
187 - 196.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. Bennett and A. J. Baines
Spectrin and Ankyrin-Based Pathways: Metazoan Inventions for Integrating Cells Into Tissues
Physiol Rev,
July 1, 2001;
81(3):
1353 - 1392.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. V. Melendez-Vasquez, J. C. Rios, G. Zanazzi, S. Lambert, A. Bretscher, and J. L. Salzer
Nodes of Ranvier form in association with ezrin-radixin-moesin (ERM)-positive Schwann cell processes
PNAS,
January 30, 2001;
98(3):
1235 - 1240.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. M. Jenkins, K. Kizhatil, N. R. Kramarcy, A. Sen, R. Sealock, and V. Bennett
FIGQY phosphorylation defines discrete populations of L1 cell adhesion molecules at sites of cell-cell contact and in migrating neurons
J. Cell Sci.,
January 11, 2001;
114(21):
3823 - 3835.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Berghs, D. Aggujaro, R. Dirkx , Jr., E. Maksimova, P. Stabach, J.-M. Hermel, J.-P. Zhang, W. Philbrick, V. Slepnev, T. Ort, et al.
{beta}IV Spectrin, a New Spectrin Localized at Axon Initial Segments and Nodes of Ranvier in the Central and Peripheral Nervous System
J. Cell Biol.,
November 20, 2000;
151(5):
985 - 1002.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Tait, F. Gunn-Moore, J. M. Collinson, J. Huang, C. Lubetzki, L. Pedraza, D. L. Sherman, D. R. Colman, and P. J. Brophy
An Oligodendrocyte Cell Adhesion Molecule at the Site of Assembly of the Paranodal Axo-Glial Junction
J. Cell Biol.,
August 7, 2000;
150(3):
657 - 666.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. D. Trapp and G. J. Kidd
Axo-Glial Septate Junctions: The Maestro of Nodal Formation and Myelination?
J. Cell Biol.,
August 7, 2000;
150(3):
F97 - F100.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Bouley, M.-Z. Tian, K. Paisley, Y.-C. Shen, J. D. Malhotra, and M. Hortsch
The L1-Type Cell Adhesion Molecule Neuroglian Influences the Stability of Neural Ankyrin in the Drosophila Embryo But Not Its Axonal Localization
J. Neurosci.,
June 15, 2000;
20(12):
4515 - 4523.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. N Rasband and P. Shrager
Ion channel sequestration in central nervous system axons
J. Physiol.,
May 15, 2000;
525(1):
63 - 73.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Faivre-Sarrailh, F. Gauthier, N. Denisenko-Nehrbass, A. Le Bivic, G. Rougon, and J.-A. Girault
The Glycosylphosphatidyl Inositol-anchored Adhesion Molecule F3/Contactin Is Required for Surface Transport of Paranodin/Contactin-associated Protein (caspr)
J. Cell Biol.,
April 17, 2000;
149(2):
491 - 502.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. De Matteis and J. Morrow
Spectrin tethers and mesh in the biosynthetic pathway
J. Cell Sci.,
January 7, 2000;
113(13):
2331 - 2343.
[Abstract]
[PDF]
|
|
|
|
|
|
|
J. L. Dupree, J.-A. Girault, and B. Popko
Axo-glial Interactions Regulate the Localization of Axonal Paranodal Proteins
J. Cell Biol.,
December 13, 1999;
147(6):
1145 - 1152.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Zhou, S. Lambert, P. L. Malen, S. Carpenter, L. M. Boland, and V. Bennett
AnkyrinG Is Required for Clustering of Voltage-gated Na Channels at Axon Initial Segments and for Normal Action Potential Firing
J. Cell Biol.,
November 30, 1998;
143(5):
1295 - 1304.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Scotland, D. Zhou, H. Benveniste, and V. Bennett
Nervous System Defects of AnkyrinB (-/-) Mice Suggest Functional Overlap between the Cell Adhesion Molecule L1 and 440-kD AnkyrinB in Premyelinated Axons
J. Cell Biol.,
November 30, 1998;
143(5):
1305 - 1315.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
X. Zhang and V. Bennett
Restriction of 480/270-kD Ankyrin G to Axon Proximal Segments Requires Multiple Ankyrin G-specific Domains
J. Cell Biol.,
September 21, 1998;
142(6):
1571 - 1581.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. G. Koszowski, G. C. Owens, and S. R. Levinson
The Effect of the Mouse Mutation Claw Paw on Myelination and Nodal Frequency in Sciatic Nerves
J. Neurosci.,
August 1, 1998;
18(15):
5859 - 5868.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
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]
|
|
|
|
|
|
|
S. M. Jenkins and V. Bennett
Developing nodes of Ranvier are defined by ankyrin-G clustering and are independent of paranodal axoglial adhesion
PNAS,
February 19, 2002;
99(4):
2303 - 2308.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. M. Jenkins and V. Bennett
Ankyrin-G coordinates assembly of the spectrin-based membrane skeleton, voltage-gated sodium channels, and L1 CAMs at Purkinje neuron initial segments
J. Cell Biol.,
November 26, 2001;
155(5):
739 - 746.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Komada and P. Soriano
{beta}IV-spectrin regulates sodium channel clustering through ankyrin-G at axon initial segments and nodes of Ranvier
J. Cell Biol.,
January 21, 2002;
156(2):
337 - 348.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
