Howard Hughes Medical Institute and Departments of Cell Biology and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
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Abstract |
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AnkyrinG (/
) neurons fail to concentrate
voltage-sensitive sodium channels and neurofascin at
their axon proximal segments, suggesting that ankyrinG
is a key component of a structural pathway involved in
assembly of specialized membrane domains at axon
proximal segments and possibly nodes of Ranvier
(Zhou, D., S. Lambert, D.L. Malen, S. Carpenter, L. Boland, and V. Bennett, manuscript submitted for publication). This paper addresses the mechanism for restriction of 270-kD ankyrinG to axon proximal segments by evaluation of localization of GFP-tagged
ankyrinG constructs transfected into cultured dorsal
root ganglion neurons, as well as measurements of fluorescence recovery after photobleaching of neurofascin- GFP-tagged ankyrinG complexes in nonneuronal cells.
A conclusion is that multiple ankyrinG-specific domains, in addition to the conserved membrane-binding
domain, contribute to restriction of ankyrinG to the axonal plasma membrane in dorsal root ganglion neurons.
The ankyrinG-specific spectrin-binding and tail domains are capable of binding directly to sites on the
plasma membrane of neuronal cell bodies and axon
proximal segments, and presumably have yet to be
identified docking sites. The serine-rich domain, which
is present only in 480- and 270-kD ankyrinG polypeptides, contributes to restriction of ankyrinG to axon
proximal segments as well as limiting lateral diffusion
of ankyrinG-neurofascin complexes. The membrane-binding, spectrin-binding, and tail domains of ankyrinG
also contribute to limiting the lateral mobility of ankyrinG-neurofascin complexes. AnkyrinG thus functions
as an integrated mechanism involving cooperation
among multiple domains heretofore regarded as modular units. This complex behavior explains ability of
ankyrinB and ankyrinG to sort to distinct sites in neurons and the fact that these ankyrins do not compensate
for each other in ankyrin gene knockouts in mice.
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Introduction |
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CLUSTERING of voltage-gated sodium channels at the
axon proximal segments and nodes of Ranvier is
believed to be essential for production and propagation of action potentials in myelinated axons. The mechanisms responsible for concentrating sodium channels in
these specialized membrane domains in axons are not established, but are likely to involve specialized members of
the ankyrin family of spectrin-binding proteins. Ankyrins
are membrane-associated proteins that bind to certain ion
channels, including the voltage-sensitive sodium channel,
and have the potential to couple these channels to the
spectrin-actin network (Srinivasan et al., 1988; Bennett and Gilligan, 1993
; Bennett et al., 1997
). Ankyrins also
bind through their conserved membrane-binding domains
to cell adhesion molecules of the L1 cell adhesion molecule (CAM) family (Davis et al., 1993
, 1996; Davis and
Bennett, 1994
; Garver et al., 1997
), and have been proposed to form lateral complexes involving L1 CAMs and
ion channels (Michaely and Bennett, 1995a
,b; Lambert et
al., 1997
). Ankyrins are concentrated at physiological sites with a high density of voltage-sensitive sodium channels
including nodes of Ranvier, axon proximal segments, and
postsynaptic folds of the neuromuscular junction (Flucher
and Daniels, 1989
; Kordeli et al., 1990
). The isoforms of
ankyrin colocalized with voltage-sensitive sodium channels at nodes of Ranvier and axon proximal segments have
been identified as 480/270-kD alternatively spliced variants of ankyrinG (Kordeli et al., 1995
). An ankyrinG
polypeptide also has been identified at the neuromuscular
junction (Wood and Slater, 1998
). In addition to ankyrinG,
isoforms of neurofascin and NrCAM, which are members
of the L1 CAM family, have been identified at nodes of
Ranvier and axon proximal segments (Davis et al., 1996
).
480/270-kD ankyrinG colocalizes with sodium channel
clusters in amyelinated regions of peripheral nerves of the
dystrophic mouse (Deerinck et al., 1997), and in axons of
cultured retinal ganglion neurons in the absence of direct
contact with glial cells (Kaplan et al., 1997
). These results
imply targeting of channels and ankyrin to sites defined by
intrinsic polarity to the axon, independent of cell-cell contact, although soluble factors released by glial cells may
play a role (Kaplan et al., 1997
). 480/270-kD ankyrinG also
colocalized in the developing sciatic nerve in clusters containing sodium channels, neurofascin, and NrCAM (Lambert et al., 1997
). The earliest event observed in myelinating nerve, preceding expression of myelin-associated
glycoprotein (MAG) by Schwann cells, was clustering of
neurofascin and NrCAM, which were followed by ankyrin
and sodium channels. These observations led to the proposal that extracellular signals, perhaps related to the soluble factor described by Kaplan et al. (1997)
, induce clustering of neurofascin and NrCAM, which in turn recruit
ankyrinG and sodium channels (Lambert et al., 1997
).
Mice deficient in cerebellar ankyrinG polypeptides have
recently been developed by targeted gene disruption
(Zhou, D., S. Lambert, P. Melen, S. Carpenter, L. Boland,
and V. Bennett, manuscript submitted for publication).
Cerebellar neurons of these ankyrinG (/
) mice fail to
concentrate sodium channels as well as neurofascin at
axon proximal segments. These findings demonstrate that
480/270-kD ankyrinG is required for clustering of both sodium channels and neurofascin at axon proximal segments. Neurofascin and L1 CAMs therefore are not sufficient to provide the initial targeting signal for ankyrinG at
axon proximal segments, in contrast to expectations from
observations of development of the node of Ranvier (Lambert et al., 1997
).
This paper addresses the mechanism for restriction of 270-kD ankyrinG to axon proximal segments of cultured dorsal root ganglion (DRG)1 neurons. A conclusion is that multiple ankyrinG-specific domains, in addition to the conserved membrane-binding domain, cooperate in targeting and restriction of ankyrinG to axon proximal segments. Moreover, measurements of fluorescence recovery after photobleaching (FRAP) of green fluorescent protein (GFP)- tagged ankyrin constructs cotransfected with neurofascin in a nonneuronal cell line demonstrate that the membrane-binding, spectrin-binding, tail and serine-rich domains each contribute to immobilization of 270-kD ankyrinG-neurofascin complexes in the plasma membrane. These results provide strong evidence that association between the ankyrinG membrane-binding domain and neurofascin is not sufficient to restrict ankyrinG to axon proximal segments, and that other ankyrinG domains and yet to be defined protein interactions are also involved.
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Materials and Methods |
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DRG Culture
The culture of embryonic DRG was modified from methods previously
described (Kleitman et al., 1991). In brief, DRGs were obtained from E15
rat embryos and plated on laminin-coated dishes after dissociation with
0.02% collagenase and 0.25% trypsin-EDTA. The medium was changed
from normal medium (10% FBS in DME, 100 ng/ml NGF) to the defined
medium (1× N1 additive [Sigma Chemical Co., St. Louis, MO], 100 ng/ml
NGF, 10 µM FdU and 10 µM uridine in DME) 24 h after plating. The culture was kept in the defined medium for 2 d before changing back to normal medium for an additional 2 d. The antimitotic cycle was repeated
once to kill most of dividing nonneuronal cells. After 8 d in culture, DRG
neurons are ready for immunofluorescence and gene gun transfection.
Preparation of cDNA Constructs
Assembly of the full-length cDNA for 270-kD ankyrinG (Ank270) was
achieved by ligating the first half of membrane-binding domain, which was
isolated from adult rat brain 5'-stretch plus cDNA library (Clontech Laboratories Inc., Palo Alto, CA) and confirmed by DNA sequencing, with
construct M-Sb-Sr-T-C (Zhang and Bennett, 1996) through the EcoRI-NsiI sites. The COOH-terminal domain of 270-kD ankyrinG was PCR amplified and introduced into the SalI site of pEGFP-N1 vector (Clontech
Laboratories Inc.), while keeping in-frame with the downstream EGFP
protein (Ank-Ct). The full-length 270-kD ankyrinG with GFP tagged at its
COOH terminus (see Fig. 2, Ank270-GFP) was prepared by ligating the
EcoRI-EcoRV fragment of construct Ank270 into the EcoRI-EcoRV
sites of construct Ank-Ct. The cDNA construct for 190-kD ankyrinG (see
Fig. 2, Ank270[
SR,T]-GFP) was prepared similarly except using construct M-Sb-C (Zhang and Bennett, 1996
) instead of M-Sb-Sr-T-C. The
cDNA construct lacking the first half of the membrane-binding domain
(corresponding to the amino acids 0-393 of human ankyrinG [Kordeli et
al., 1995
]) was prepared by putting the EcoRV fragment of M-Sb-Sr-T-C
(Zhang and Bennett, 1996
) into the corresponding site of the construct Ank-Ct (see Fig. 2, Ank270[
M1,2]-GFP). The construct containing only
the membrane-binding and the spectrin binding domain (see Fig. 2,
Ank270[
SR,T,Ct]-GFP) was obtained by replacing the EcoRV-NotI fragment of construct Ank270[
SR,T]-GFP with a PCR-amplified EGFP sequence. The construct expressing the membrane-binding domain was
made by introducing a PCR-amplified sequence into the EcoRI-SalI sites
of pEGFP-N1 vector while keeping in-frame translation of the downstream EGFP (M-GFP). Constructs expressing the serine-rich (SR-GFP),
spectrin-binding (SB-GFP), tail (T-GFP), and COOH-terminal (Ct-GFP)
domains were prepared similarly. To make a construct with deletion of
the serine-rich domain (see Fig. 2, Ank270[
SR]-GFP), a PCR-amplified
fragment of the tail domain of the 270-kD ankyrinG was introduced into
the EcoRV site of construct Ank270(
SR,T)-GFP.
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The full-length rat neurofascin cDNA with a hemagglutinin (HA)
epitope at the NH2 terminus (Garver et al., 1997) was cut out by HindIII-NotI from PBluescript KS vector (Stratagene, La Jolla, CA), and subcloned into the corresponding sites of pEGFP-N1 vector (Clontech Laboratories Inc.). The resultant construct (see HA-NF in Fig. 2) does not
contain the EGFP sequence. The cytoplasmic domain-deleted neurofascin
(see HA-NF[
CD] in Fig. 2) was made by replacing the ScaI-NotI fragment of construct HA-NF with a PCR-amplified fragment containing the
sequence of the ScaI-ApaI fragment with a stop codon right after the
ApaI site. The extracellular domain-truncated neurofascin (see Fig. 2,
HA-NF[
EC]) was prepared through two steps. First, the 5'-untranslated
region of neurofascin with the start codon, the signal peptide, and the HA
tag was PCR amplified and subcloned into the BglII-HindIII sites of
pEGFP-N1 vector. Then the transmembrane and cytoplasmic domains
were introduced into the HindIII-NotI sites of the first-step construct.
Construct with the full-length neurofascin tagged with EGFP at its COOH terminus was obtained also by two steps (see HA-NF-GFP in Fig. 2). The
HindIII-ApaI fragment of neurofascin in pBluescript KS vector was transferred into the corresponding sites of pEGFP-N3 vector (Clontech Laboratories Inc.) so as to keep in-frame reading of EGFP with the introduced
neurofascin fragment. Then a PCR-amplified cytoplasmic domain of neurofascin was introduced into the ApaI site of the above construct.
cDNA Transfection and Immunofluorescence
DRG cultures were transfected using HeliosTM Gene Gun system following the manufacturer's instructions. 150-180 psi pressure was used to deliver cDNA-coated microcarriers into DRG cultures. 24 h after transfection, DRG cultures were fixed in 2% paraformaldehyde and then subjected to 0.5% Triton X-100 permeabilization before being stained with a polyclonal antibody specific for GFP (Clontech Laboratories Inc.). Immunofluorescence for endogenous proteins was performed similarly.
Human kidney 293 cells were cultured in 10% FBS and DME (GIBCO BRL, Gaithersburg, MD) and transfected by Lipofectamine according to the manufacturer's protocol (GIBCO BRL). 24 h after transfection, cultures were either subject to photobleaching (see below) or fixed in 2% paraformaldehyde and then stained with a monoclonal antibody specific for the HA epitope of neurofascin (Berkeley Antibody Co., Richmond, CA).
Chicken polyclonal antibody against 270/480-kD ankyrinG (Zhang and
Bennett, 1996) and rabbit polyclonal antibodies against ankyrinB (Chan et
al., 1993
), brain spectrin (Davis and Bennett, 1983
), and neurofascin
(Davis et al., 1996
) were described previously. TRITC-conjugated goat
anti-mouse and goat anti-rabbit, and FITC-conjugated goat anti-rabbit
antibodies were purchased from Rockland (Gilbertsville, PA). FITC-conjugated donkey anti-chicken was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).
Fluorescence Recovery Measurement after Photobleaching
Photobleaching of the cytoplasm-localized Ank270-GFP was performed
essentially as described, using an Odyssey confocal imaging system
(Garver et al., 1997). Photobleaching of the plasma membrane-localized transfected proteins was performed in a Zeiss LSM 410 laser confocal system coupled with an UV laser (Carl Zeiss Inc., Thornwood, NY). A selected region enclosing the plasma membrane to be studied was photobleached using the UV laser beam for 60 ms. The radii of
photobleached regions range from 1 to 3 µm. The fluorescence recovery
inside the photobleached area was immediately recorded by a home-designed program containing several `Time Series' with different time intervals. The recorded data was analyzed using NIH Image software (Bethesda, MD). The recovered fraction was calculated as (Ie
Ii)/(Io
Ii). Io,
the average intensity of the selected region before photobleaching; Ii, the
average intensity of the selected region immediately after photobleaching;
and Ie, the average intensity of the selected region after 50 s of recovery. The recovery rate was calculated as w2/t, where w is the radius of the photobleached region, and t is the time in seconds required for the average intensity of the selected region to recover to the value of (Ie + Ii)/2.
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Results |
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Native AnkyrinG and Neurofascin but Not AnkyrinB and Spectrin Are Restricted to Axon Proximal Segments of DRG Neurons
480/270-kD ankyrinG (Fig. 1, A, D, and G) and neurofascin (Fig. 1 H) are concentrated at the axolemma of axon
proximal segments of cultured embryonic DRG neurons.
AnkyrinG also is associated with the plasma membrane in
cell bodies of neurons, although at a lower level than in the
axon proximal segment (Fig. 1, A, D, and G). AnkyrinB, in
contrast, is distributed along the length of axons and is
present in only low levels in axon proximal segments (Fig.
1 B). AnkyrinB also exhibits only minimal staining in the cell body (Fig. 1 B). Spectrin is distributed along axons in a pattern resembling ankyrinB (Fig. 1 E). These results demonstrate that cultured DRG neurons have a polarized distribution of native ankyrinG and neurofascin in axon proximal segments similar to localization of these proteins in
sections of intact brain tissue (Kordeli et al., 1995; Davis et
al., 1996
).
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AnkyrinG and ankyrinB both bind to neurofascin with
high affinity through their membrane-binding domains,
which share 74% amino acid identity (Davis et al., 1993;
Davis and Bennett, 1994
; Kordeli et al., 1995
; Zhang et al.,
1998
). Thus, enrichment of neurofascin and ankyrinG, but
not ankyrinB, at axon proximal segments of DRG neurons
(Fig. 1, C and I ) suggests a mechanism independent of
neurofascin for targeting ankyrinG specifically to the axolemma of axon proximal segments. The targeting protein(s) are not currently known spectrin polypeptides,
based on lack of concentration of spectrin at axon proximal segments (Fig. 1 F).
Unique Serine-rich and Tail Domains of 270-kD AnkyrinG Contribute to Restriction of AnkyrinG at Axon Proximal Segments
AnkyrinG is distinguished from ankyrinB by a unique
O-GlcNAc-glycosylated serine-rich domain and a highly
divergent tail domain (Kordeli et al., 1995; Zhang and
Bennett, 1996
). In addition, ankyrinG and ankyrinB differ
in sequence in portions of the spectrin-binding domain
and in the COOH-terminal domain. To identify the roles of the serine-rich and tail domains in targeting 480/270-kD
ankyrinG to axon proximal segments, we transfected DRG
neurons with cDNAs, each with a COOH-terminal GFP
tag, encoding 270-kD ankyrinG and its variants lacking the
serine-rich and/or tail domains (Fig. 2). The distribution of
transfected 270-kD ankyrinG (Fig. 2, Ank270-GFP) is visualized by immunostaining of the COOH-terminal GFP
tag, and is highly restricted to the axolemma of the proximal segment in a pattern similar to native 480/270-kD
ankyrinG (Fig. 3, A and B). The intensity of immunofluorescence attenuates abruptly beyond the proximal segment (Fig. 3 A and Fig. 4, A and B), which is usually tens
of micrometers in length in DRG neurons (Hsieh et al.,
1994
). Double labeling of cultures cotransfected with GFP-tagged 270-kD ankyrinG (Fig. 3 D) and HA-tagged
cytoplasmic domain-truncated neurofascin, HA-NF(
CD)
(Fig. 2 and Fig. 3 E), indicates the polarized distribution of
the transfected ankyrinG is not due to incomplete extension or partial optical sectioning of axons since the full
length of the axon is well displayed by mutant neurofascin
(Fig. 3 E). The relatively even distribution of cytoplasmic
domain-truncated neurofascin along the length of the
axon also suggests that the extracellular and transmembrane domains of neurofascin are not sufficient to achieve
polarized localization at the proximal segment. Some
GFP-tagged ankyrinG is also targeted to areas of the
plasma membrane of the cell body, at levels approximating those in the proximal segments (Fig. 3 C). This behavior is in contrast to the distribution of native ankyrinG and
could represent a consequence of high levels of expression of the transfected ankyrinG.
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Transfected 190-kD ankyrinG (refer to Fig. 2, Ank270
[SR,T]-GFP), which lacks the unique serine-rich and tail
domains of 270-kD ankyrinG (Kordeli et al., 1995
), is also
located at the axolemma of the proximal segment (Fig. 3,
F and G). However, 190-kD ankyrinG is much less restricted, and distributes well beyond the proximal segment
into the axon (Fig. 3 F and Fig. 4, A and B). The distance
required for the intensity of immunofluorescence to drop
to one-third of the intensity at the proximal segment is
more than twice as long for 190-kD ankyrinG than for 270-kD ankyrinG (Fig. 4 B). Insertion of the tail domain into
190-kD ankyrinG (refer to Fig. 2, Ank270[
SR-GFP]) improves, but does not completely restore, the restriction of
the transfected ankyrinG to the proximal area of the axon
(Fig. 3 I and Fig. 4, A and B). Ank270(
SR)-GFP distributes in a punctate pattern different from that of 270-kD
ankyrinG and 190-kD ankyrinG (Fig. 3, J and K). These
data suggest that the unique serine-rich domain and tail domain of 270/480-kD ankyrinG contribute to restriction of
ankyrinG at axon proximal segments.
Spectrin-binding and Tail Domains of AnkyrinG Contain Specific Plasma Membrane-binding Sites
To examine which domain(s) are sufficient for restriction
of ankyrinG at the axolemma of axon proximal segments,
different domains of 270-kD ankyrinG were individually
introduced into DRG neurons. The membrane-binding
domain (Fig. 5, E and F), the serine-rich domain (Fig. 5, G
and H) and the COOH-terminal domain (Fig. 5, I and J)
are predominantly distributed throughout the cytoplasm of the cell body and the axon, but are not concentrated at
the axolemma. The failure in recruiting the membrane-binding domain to the plasma membrane might be a consequence of over-expression and does not exclude the
possibility of a limited number of binding sites. The spectrin-binding domain (Fig. 5, A and B) and the tail domain
(Fig. 5, K and L), on the other hand, were targeted to the
axolemma of the proximal segment as well as the plasma
membrane of the cell body. The spectrin-binding domain
also distributes in a punctate pattern similar to that of
Ank270(SR)-GFP (Fig. 5, A and B). However, neither
the spectrin-binding nor tail domains are restricted to the
proximal segment as the case with transfected 270-kD
ankyrinG, and their intensity-drop profiles along axons are
similar to that of 190-kD ankyrinG (Fig. 6, A and B). These data suggest an important role for plasma membrane-binding sites in the spectrin-binding and the tail domain
for restriction of ankyrinG at axon proximal segments. The
restrictive effect of the serine-rich domain (refer to Fig. 3 I
and see Fig. 5, G and H) seems unrelated to targeting to
the plasma membrane, at least as resolved by these techniques.
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The transfected spectrin-binding domain of ankyrinB is not targeted to the axolemma and is distributed in the axoplasm and the cytoplasm of the cell body (Fig. 5, C and D). The spectrin-binding domain of ankyrinG thus contains targeting information lacking in the spectrin-binding domain of ankyrinB, and could contribute to specific restriction of ankyrinG to axon proximal segments.
AnkyrinG Domains Cooperate in Restricting the Lateral Mobility of AnkyrinG -Neurofascin Complexes
The serine-rich domain and possibly other domains of 270-kD ankyrinG could contribute to restriction of ankyrinG to
axon proximal segments through interactions limiting lateral mobility in addition to binding to membrane-targeting
sites. Activity of ankyrinG domains in immobilizing ankyrinG-neurofascin complexes was evaluated in a nonneuronal cell line (293 cells from human embryonic kidney) cotransfected with GFP-tagged ankyrinG and HA-tagged
neurofascin (Fig. 7). 270-kD ankyrinG transfected alone is
located throughout the cytoplasm of 293 cells (Fig. 7 A).
However, 270-kD ankyrin cotransfected with neurofascin
is recruited to the plasma membrane (Fig. 7, B and C).
Targeting of transfected ankyrinG in 293 cells to the
plasma membrane is solely governed by interactions between the membrane-binding domain and cotransfected
neurofascin, since the membrane-binding domain of ankyrinG alone is sufficient for membrane recruitment by co
transfected neurofascin (Fig. 7, D and E). Moreover, deletion of the first half of the membrane-binding domain
abolishes recruitment of ankyrinG to the plasma membrane even though the COOH-terminal, tail, serine-rich,
and spectrin-binding domains are still present (Fig. 7, F
and G). The structural requirements for activity of neurofascin in recruiting ankyrinG are explored in a separate
study (Zhang et al., 1998).
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Dynamic behavior of GFP-tagged ankyrinG-neurofascin
complexes were examined by measurements of FRAP of
the GFP signal (Fig. 8). Dynamic parameters were first determined for GFP-tagged 270-kD ankyrinG (Ank270-
GFP) alone, which was localized in the cytoplasm, and for
GFP-tagged neurofascin (HA-NF-GFP) alone. Cytoplasmic 270-kD ankyrinG exhibits rapid and nearly complete
recovery after photobleaching (91% recovery at a rate of
1.1 × 107 cm2/s), consistent with the diffusion rate of a
protein in solution (Fig. 8, A and B). GFP-tagged neurofascin (HA-NF-GFP) transfected in the absence of cotransfected ankyrinG exhibited a 72% recovery at a rate of
5.6 × 10 cm2/s (Fig. 8, A and C). This rate is comparable to
other membrane proteins that are freely diffusing in
plasma membranes (Jacobson et al., 1987
; Zhang et al.,
1991
). In contrast, plasma membrane-associated 270-kD
ankyrinG (Ank270-GFP cotransfected with HA-NF) displays a greatly reduced rate (4 × 10
10 cm2/s) as well as extent (10%) of FRAP (Fig. 8, A, B, and C). This dramatic
decrease of membrane lateral mobility is not due to lateral association of the extracellular domain of neurofascin with
itself or with other membrane-spanning proteins, since deletion of the extracellular domain of neurofascin has little
effect on membrane dynamic behavior of cotransfected
270-kD ankyrinG (Ank270-GFP with HA-NF[
EC] in
Fig. 8, A and B). Thus, 270-kD ankyrinG provides the major contribution to immobilization of ankyrin-neurofascin complexes in the plane of the plasma membrane.
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The role of ankyrinG domains in immobilizing neurofascin were evaluated using a range of ankyrinG constructs
(refer to Fig. 2, and see Fig. 8, A and C). The lateral mobility of the membrane-localized complexes formed by
cotransfected neurofascin (HA-NF) and the membrane-binding domain of ankyrinG (M-GFP) exhibits more than a 50% reduction of rate of recovery (2.6 × 109 cm2/s) and
a 30% decrease of extent of recovery (51%) compared
with behavior of GFP neurofascin (HA-NF-GFP) alone.
Addition of the spectrin-binding domain to the membrane-binding domain (Ank270[
SR,T,Ct]-GFP) results
in another 50% reduction in the rate of recovery (1.4 × 10
9 cm2/s) and a decrease in extent of recovery to ~25%
(refer to Fig. 4). Subsequent addition of the COOH-terminal domain (Ank270[
SR,T]-GFP), however, had no apparent effect. Addition of the tail domain (Ank270[
SR]- GFP) further decreased the rate (7 × 10
10 cm2/s) and the
extent (16%) of recovery. Finally, addition of the serine-rich domain (Ank270-GFP) had another reduction in rate to 4 × 10 cm2/s and extent of recovery to 10 percent.
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Discussion |
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The current paradigm for organization of ankyrin-membrane protein complexes derives from the erythrocyte
membrane, and envisions monomeric ankyrin forming
lateral complexes between integral proteins via the multivalent membrane-binding domain and spectrin via the spectrin-binding domain (Bennett, 1990; Michaely and
Bennett, 1995a
,b). This study provides evidence that a
simple erythrocyte-based model is not adequate to explain
ankyrinG organization at axon proximal segments. The
spectrin-binding, serine-rich, and tail domains all contribute to restriction of ankyrinG to the axonal plasma membrane in DRG neurons. Multiple ankyrinG domains also contribute to limiting the lateral mobility of ankyrin-neurofascin complexes in a nonneuronal cell. The ankyrinG-specific spectrin-binding and tail domains are capable of
binding directly to sites on the plasma membrane of axon
proximal segments and neuronal cell bodies, and presumably have yet to be identified docking sites. The serine-rich domain, which is present only in 480- and 270-kD ankyrinG
polypeptides, contributes to restriction of ankyrinG to
axon proximal segments as well as limiting lateral diffusion of ankyrinG-neurofascin complexes. AnkyrinG thus
functions as an integrated mechanism involving cooperation among multiple domains heretofore regarded as modular units. This complex behavior explains the ability of ankyrinB and ankyrinG to sort to distinct sites in neurons
and the fact that these ankyrins do not compensate for
each other in ankyrin gene knockouts in mice (Zhou, D.,
S. Lambert, P. Malen, S. Carpenter, L. Boland, and V. Bennett, manuscript submitted for publication) (Scotland,
P., D. Zhou, H. Benveniste, and V. Bennett, manuscript
submitted for publication).
The mechanism for restriction of dynamic behavior of
neurofascin by ankyrinG could result from self-association
of ankyrinG domains and/or low affinity interaction with
membrane or cytoskeletal proteins. For example, ankyrin
associates with micromolar affinity with microtubules
(Bennett and Davis, 1981). Association of transfected ankyrinG with endogenous spectrin does not seem likely,
since ankyrinG remained in the cytoplasm in the absence
of cotransfected neurofascin, whereas spectrin in these
cells is primarily localized at the plasma membrane
(Zhang et al., 1998
). As an alternative to specific protein
interactions, ankyrinG could be nonspecifically trapped in
the membrane-apposed cytoskeletal meshwork, according to the membrane-skeleton fence model (Kusumi et al.,
1993
; Jacobson et al., 1995
). Further analysis will be required to differentiate these possibilities.
Functions attributed so far to the spectrin-binding domain of ankyrin have been association with spectrin (Bennett, 1978; Davis and Bennett, 1984a
,b), and contact with
the Na+/K+ ATPase (Davis and Bennett, 1990
). Disruption of the spectrin network abolishes the polarized distribution of ankyrin as well as Na+/K+-ATPase at cell-cell
contact sites in cultured cells (Hu et al., 1995
). However,
antibody against total brain spectrin revealed no enrichment of spectrin at axon proximal segments of DRG neurons and thus, known spectrins are unlikely to provide a
sufficient mechanism to specifically enrich ankyrinG at the
sites. A similar lack of correlation between spectrin and
ankyrinG has been reported at nodes of Ranvier in sciatic
nerves, where spectrin is continuously distributed along
plasma membranes of axons and the myelinating Schwann
cell (Lambert et al., 1997
). Since different spectrin isoforms have different affinities for ankyrins (Howe et al., 1985
; Davis and Bennett, 1984a
), one explanation of these
results is the existence of a novel isoform of spectrin which
is targeted to axon proximal segments and nodes of Ranvier,
but is not identified by our current antibody. Alternatively, a novel ankyrinG-binding protein unrelated to spectrin could be targeted to axon proximal segments. In addition, a yet to be identified protein at the axolemma of axon
proximal segments provides a binding site for the tail domain of 270-kD ankyrinG.
The traditional interactions between membrane proteins and ankyrin mediated by the membrane-binding
domain of ankyrin may not provide the initial step in restriction of ankyrin at the axolemma of axon proximal segments. Instead, enrichment of ankyrin, which could be
achieved through combined influences of the spectrin-binding, tail, and serine-rich domains, may precede and direct assembly of membrane proteins at the sites. Failure of
transfected mutant neurofascin with a deleted cytoplasmic
domain to be concentrated at axon proximal segments
(Fig. 3 E) favors the possibility that the ankyrin-binding
activity, which is located in the cytoplasmic domain of neurofascin (Davis and Bennett, 1994) (Zhang, X., J.D. Davis,
S. Carpenter, and V. Bennett, manuscript in preparation), is required for restriction of neurofascin at these sites.
Moreover, the disrupted assembly of neurofascin as well
as sodium channels at axon proximal segments in ankyrinG
(
/
) mice further supports the role of ankyrin in directing membrane proteins to axon proximal segments (Zhou,
D., S. Lambert, P. Malen, S. Carpenter, L. Boland, and V. Bennett, manuscript submitted for publication).
The potential defining role of ankyrin in clustering of
membrane proteins at axon proximal segments is inconsistent with conclusions from analysis of morphogenesis of
nodes of Ranvier, suggesting that clusters of neurofascin/
NrCAM provide sites for subsequent assembly of ankyrin
and sodium channels (Lambert et al., 1997). The disparity
could be attributed to the potentially different mechanisms used in establishment of axon proximal segments
and nodes of Ranvier, despite similarities in their molecular organization (Kordeli et al., 1995
; Davis et al., 1996
;
Bennett et al., 1997
) and ultrastructure (Waxman, 1984
).
Clustering of ankyrin or sodium channels at axon proximal
segments does not require the presence of glial cells and
thus is most likely determined by an intrinsic polarity of
neurons (Fig. 3, A, D, and G) (Kaplan et al., 1997
). In contrast, clustering of ankyrin and sodium channels in the axonal region beyond the proximal segment, which is regarded as an early event for assembly of nodes of Ranvier,
requires signals from glial cells, although whether these
signals are diffusable or require direct glia-axon contact
has been debated (Salzer, 1997
; Dugandzija-Novakovic et
al., 1995
; Vabnick et al., 1996
, 1997
; Deerinck et al., 1997
;
Kaplan et al., 1997
). Clustering of neurofascin/NrCAM at
the initial sites of nodes of Ranvier provides a potential
mechanism to bridge glial cell signals to intracellular determinants, such as clustering of ankyrin, during the early
development of nodes of Ranvier. On the other hand, the
unique position of axon proximal segments might be sufficient to provide a cue to polarize the molecular composition at this region characterized by concentration of
ankyrin and subsequent recruitment of ankyrin-binding
membrane proteins.
In carefully timed neuronal Schwann cell cocultures, we
have observed that clustering of ankyrin precedes clustering of sodium channels. In addition, each MAG-positive
myelinating Schwann cell has two ankyrin clusters at each
end of this Schwann cell. These ankyrin clusters are covered by processes of the myelinating Schwann cell, suggesting axon-Schwann cell contact defines clustering as
well as the clustering sites of ankyrin (Zhang, 1998). These results indicate that ankyrin, as at axon proximal segments, is a candidate to direct assembly of sodium channels at the precursor sites of nodes of Ranvier, although
the role of glial cells may differ between nodes and proximal segments.
This paper suggests a complicated and cooperative scenario for establishment of the specialized membrane domain of axonal proximal segments, which may involve not
only interactions between membrane proteins and ankyrin
but also ankyrin and other yet to be identified proteins
which precede ankyrin at these sites. Isoforms of ankyrinG
also are targeted to sites of cell-cell contact in epithelial
tissues (Peters et al., 1995), and proteins similar to those of
axon proximal segments could also be required for epithelial polarity. A challenge for future work will be to fully elucidate the structural pathway leading to assembly of
ankyrin-based membrane domains.
![]() |
Footnotes |
---|
Received for publication 11 June 1998 and in revised form 7 August 1998.
L. Davis (Duke University, HHMI, Durham, NC) is gratefully acknowledged for contributions to culture of DRG neurons. H. Wilson (Duke
University) is thanked for advice on transfection with the Helios Gene
Gun.
This research was supported in part by a grant from the National Institutes of Health (DK 29808).
Address all correspondence to V. Bennett, Howard Hughes Medical Institute and Departments of Cell Biology and Biochemistry, Duke University
Medical Center, Durham, North Carolina 27710. Tel.: (919) 684-3538. Fax:
(919) 684-3590. E-mail: benne012{at}mc.duke.edu
![]() |
Abbreviations used in this paper |
---|
CAM, cell adhesion molecule; DRG, dorsal root ganglion; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; HA, hemagglutinin.
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