Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612
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Abstract |
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Nerve growth depends on the delivery of cell body-synthesized material to the growing neuronal processes. The cellular mechanisms that determine the topology of new membrane addition to the axon are not known. Here we describe a technique to visualize the transport and sites of exocytosis of cell body- derived vesicles in growing axons. We found that in Xenopus embryo neurons in culture, cell body-derived vesicles were rapidly transported all the way down to the growth cone region, where they fused with the plasma membrane. Suppression of microtubule (MT) dynamic instability did not interfere with the delivery of new membrane material to the growth cone region; however, the insertion of vesicles into the plasma membrane was dramatically inhibited. Local disassembly of MTs by focal application of nocodazole to the middle axonal segment resulted in the addition of new membrane at the site of drug application. Our results suggest that the local destabilization of axonal MTs is necessary and sufficient for the delivery of membrane material to specific neuronal sites.
Key words: exocytosis; axon; microtubule; dynamic instability; diffusion ![]() |
Introduction |
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THE addition of new membrane to nerve processes is
an essential step in nerve growth and differentiation
(Craig and Banker, 1994). New membrane components are synthesized in the cell body and delivered to the
plasmalemma by fast axonal transport (Allen et al., 1982
;
Nakata et al., 1998
). Inhibition of the new membrane supply, using either pharmacological treatments or the optical tweezers technique, rapidly arrests axonal growth (Martenson et al., 1993
; Jareb and Banker, 1997
; Chang et al.,
1998
). The topology of new membrane insertion into the
growing nerve processes remains controversial. Insertion
of new membrane may occur in the vicinity of the cell
body, at the growth cone area, or along the growing neurites (Bray, 1970
; Futerman and Banker, 1996
). Two different experimental strategies, previously used to determine the sites of new membrane addition to the growing
axons, have focused on the different aspects of membrane
addition. In the first approach, neurites are tagged with
plasma membrane-attached microparticles, or reporter
molecules, and movement of these markers is monitored using video microscopy. This method has been designed to
detect the sites of bulk membrane insertion into the axon;
it does not allow for discrimination between exo- and endocytic contributions to the net addition of the new membrane. The second approach focuses specifically on the
exocytic component and attempts to visualize the sites of
addition of the newly synthesized plasma membrane proteins or lipids to the plasmalemma.
In the attempts to determine the sites of membrane
insertion using the first approach, plasma membrane
markers were found to be stationary (Bray, 1970), to move
retrogradely (Koda and Partlow, 1976
), or to move anterogradely (Zheng et al., 1991
; Okabe and Hirokawa, 1992
).
The translocation of the markers was taken as a measure
of the net insertion, or retrieval, of plasma membrane material along the axon proximal to the site of tagging. However, the plasma membrane probes used in these experiments, such as large membrane-associated particles, are
likely to be anchored to the axonal cytoskeleton (Abercrombie et al., 1970
; Peng and Chang, 1982
; Okabe and Hirokawa, 1992
; Lin et al., 1996
). In this case, the movement
of the probes would reflect the translocation of unidentified cytoskeletal elements and may not be related to the
movement of plasma membrane lipids, as is the case for
nonneuronal translocating cells (Abercrombie et al., 1970
;
Sheetz et al., 1989
; Lee et al., 1990
). To overcome this
technical limitation, attempts have been made to specifically detect the translocation of plasma membrane lipids
(Popov et al., 1993
; Dai and Sheetz, 1995
). In Xenopus spinal cord neurons grown on laminin-coated coverslips, bulk
anterograde lipid flow has been reported (Popov et al.,
1993
), suggesting membrane addition along the neurite. In
contrast, in chick DRG neurons, rapidly diffusing latex microbeads were found to translocate retrogradely (Dai and
Sheetz, 1995
), indicating preferential membrane addition
to the growth cone region and bulk membrane endocytosis
at the cell body. The efforts to directly visualize the sites of
addition of new proteins or lipid to the growing axon using
the second approach have also led to conflicting results
(Feldman et al., 1981
; Griffin et al., 1981
; Pfenninger and
Maylie-Pfenninger, 1981
; Craig et al., 1995
; Harel and Futerman, 1996
; Vogt et al., 1996
). Thus, strong evidence has
been obtained both supporting and opposing the concept
of preferential addition of the newly synthesized plasma
membrane components to the growth cone region.
In this study, we directly visualized the traffic of new membrane material in the Xenopus embryo neurons by locally labeling the cell body-derived vesicles with the fluorescent lipid analogue 1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanide (DiIC12).1 The anterograde transport of new membrane carriers was detected by digital fluorescent microscopy. The new membrane was transported by the cell body-derived tubovesicular organelles, which were delivered to and preferentially inserted into the distal axon. We found that modulation of axonal microtubule dynamics had a dramatic effect on the pattern of membrane addition to the axonal plasmalemma. Thus, the dynamics of axonal microtubules may serve as a basic regulator of the topology of the new membrane addition to the nerve processes.
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Materials and Methods |
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Cell Culture
Xenopus embryo neuronal cultures were prepared as previously reported
(Anderson et al., 1977). In brief, the neuronal tube of embryos at stages
19-24 was dissociated in Ca2+- and Mg2+-free solution (115 nM NaCl, 2.6 mM
KCl, 10 mM Hepes, 0.4 mM EDTA, pH 7.6). Dissociated cells were plated
on glass coverslips precoated with concanavalin A (0.1-1.0 µg/cm2; Sigma
Chemical Co., St. Louis, MO). The cultures were kept at 20°C in culture
medium consisting of (vol/vol) 50% Leibovitz L-15 medium (GIBCO BRL;
Life Technologies, Gaithersburg, MD), 49% Ringer's solution (115 mM
NaCl, 2 mM CaCl2, 2.5 mM KCl, 10 mM Hepes, pH 7.6), 1% fetal bovine
serum (GIBCO BRL), and 50 ng/ml neurotrophin-3. The neurons were
used for experiments 24-48 h after plating.
Local Labeling of the Cell Body-derived Vesicles
DiIC12 (Molecular Probes, Eugene, OR) was prepared as a 1 mg/ml stock
solution in methanol. Local perfusion of the cell body was performed according to previously reported methods (Popov et al., 1993). In brief, two
pipettes were placed opposite to each other at a distance of 20-40 µm
from the neuronal surface. The first pipette (inner diameter 2-3 µm) was
filled with 10 µg/ml of DiIC12, which was dissolved immediately before experiments in the solution containing 57.5 mM NaCl, 60 mM KCl, 2 mM
CaCl2, and 10 mM Hepes, pH 7.6. A small hydrostatic pressure applied to
the pipette resulted in a constant outflow of the intrapipette solution. The
second pipette with tip opening of ~10 µm was used for removal of the superfusion solution from the culture medium, thereby reducing the affected
area to ~30 µm. The pipettes were withdrawn from the soma 30-60 s after
the onset of perfusion.
Image Acquisition and Data Analysis
Cells were observed using an inverted microscope (model IX 50; Olympus America, Inc., Melville, NY) equipped with differential interference contrast optics, a 60× Fluorite objective (NA 1.2), and a 100-W mercury lamp. The light passed through an infrared-blocking filter, neutral density filters, and a rhodamine wide-band filter. Images were acquired with a charge-coupled device (CCD) camera (model ImagePoint or SenSys; Photometrics, Tucson, AZ) driven by IPLab (Signal Analytics Corp., Vienna, VA) imaging software. Exposure time was 0.2-1 s. Images were processed with IPLab and Photoshop (Adobe Systems, Mountain View, CA). Quantitation of data was performed using IPLab software. Typically, the distribution of fluorescence intensity along the axon was obtained by measuring the intensity within individual pixels along the length of neurite or along individual filopodia. The background fluorescence, measured in cell-free areas ~10 µm from the axon, was subtracted from the fluorescence at the neurite.
Microinjection of Cy3-tubulin into Xenopus Embryos
Cy3-tubulin was a generous gift of Dr. Gary Borisy (University of Wisconsin, Madison, WI). Details of Cy3-tubulin preparation can be obtained
from http://borisy.bocklabs.wisc.edu. Xenopus embryos were injected with
10-25 nl of 10 mg/ml Cy3-tubulin as described before (Chang et al., 1998).
The embryos were allowed to develop to stages 19-24 and were then used
for the preparation of neuronal cultures.
Detergent Extraction
Neurons labeled with Cy3-tubulin were extracted in a microtubule (MT)- stabilizing buffer (60 mM Pipes, 1 mM MgCl2, 5 mM EGTA, 0.1% Triton X-100, 10 µM taxol, pH 6.8) for 5 min and examined under a fluorescent microscope. For each axon, the fluorescence intensity was compared before and after extraction.
Immunocytochemistry
The cells were permeabilized with 0.1% Triton X-100 in solution containing 10 µM taxol, 60 mM Pipes, 1 mM EGTA, 4% polyethylene glycol, and
1 mM MgCl2, pH 6.9, and fixed with 0.5% glutaraldehyde in PBS for 20 min. Glutaraldehyde was quenched with two changes of 2 mg/ml sodium
borohydride in PBS. Cells were incubated with a primary and then with a
secondary antibody for 1 h at room temperature. All antibody solutions
were prepared in PBS containing 2 mg/ml bovine serum albumin. The primary antibody used was a mouse monoclonal antibody to -tubulin (Amersham Corp., Arlington Heights, IL), and the secondary antibody was
rhodamine-conjugated anti-mouse IgG (Jackson ImmunoResearch, West
Grove, PA).
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Results |
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Staining of Cell Body-derived Vesicles with Fluorescent Lipid Analogue DiIC12
The cultures of Xenopus embryo neurons were prepared
on a concanavalin A-coated substrate in the presence of
the neurotrophic factor neurotrophin-3 (Chang et al.,
1998). Under these culture conditions, neurites form ~4-6 h
after plating and grow at a the rate of ~30-40 µm/h for a
few days; these neurites are usually referred to as "axons"
(Reinch et al., 1991
; Zheng et al., 1994
; Tanaka et al.,
1995
). Experiments were performed 24-48 h after cell culture preparation on axons that were 1.2-2.0 mm in length and were free of contact with other cells.
We used a local superfusion technique (Popov et al.,
1993; Engert and Bonhoeffer, 1997
) to stain the plasma
membrane at the cell body with fluorescent lipid analogue
DiIC12 (Fig. 1 A). Within 2-3 min after the onset of perfusion, the soma was brightly labeled with DiIC12 molecules.
Initially, the staining was largely localized to the cell body.
With time, plasma membrane staining could be detected at
progressively greater distances from the soma (Fig. 1 B),
consistent with lateral diffusion of DiIC12 molecules along the axonal plasmalemma (Popov et al., 1993
). In the first
20 min after cell body labeling, no plasma membrane
staining was observed at the distal axonal segment, in
agreement with the rapid drop in the rate of diffusional
transport with increasing distance. However, fluorescent
microscopy of the axon ~1 mm from the soma, well beyond the reach of diffusional transport, revealed brightly stained tubovesicular organelles (Fig. 1 C). As judged by
the fluorescent microscopy, these organelles ranged from
~0.2 µm (the diffraction limit of the light microscopy) to
~4 µm in length. The DiIC12-stained organelles could be
detected with a delay of ~10-15 min after the onset of
soma labeling. The organelles were transported in an anterograde direction at the rate of 2.93 ± 0.78 µm/s (mean ± SEM of 137 vesicles in 15 neurons), in agreement with previously reported rates of the fast axonal transport (Allen et al., 1982
; Nakata et al., 1998
). Occasionally, we observed some "hesitation" in this anterograde transport; however,
none of the organelles changed direction of movement
(total of 1,578 vesicles in 26 neurons; average observation
distance ~150 µm). This result suggests that the polarity
of axonal MTs in growing Xenopus neurites is uniform.
Staining of neuronal cultures with rhodamine-123, a mitochondria-specific fluorescent dye, indicated that the movement of mitochondria from the soma to the axon was a
very rare event. The majority of mitochondria frequently
changed their direction of movement and did not translocate over long distances along the axon (data not shown).
Moreover, DiIC12-stained tubovesicular organelles were
able to fuse with the plasmalemma (see below). Accordingly, we conclude that the overwhelming majority of
DiIC12-stained vesicles detected along the axon are distinct from mitochondria.
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No fluorescent vesicles could be detected at the distal
axon within 1 h after the labeling of the cell body if the
longer hydrocarbon chain DiIC12 analogue, DiIC18, was
used. These data are consistent with the scenario in which
the lypophilic long-chain DiIC18 molecules are restricted
to the plasma membrane. Because of the low level of constitutive endocytic activity at the soma region (Bunge,
1977), the rate of internalization of DiIC18 molecules is
low. Therefore, the appearance of DiIC12-labeled vesicles
at the distal axon reflected a redistribution of DiIC12 molecules from the plasma membrane to the cytoplasm, and
nonspecific staining of all lipid-containing organelles in
the cell body. These organelles were transported anterogradely, and at sufficiently large distances from the soma,
they could be visualized in the absence of plasma membrane staining.
Cell Body-derived Vesicles Are Transported to the Growth Cone Region and Fuse with the Plasma Membrane
Within 10-20 min after the staining of the cell body, we
observed an accumulation of DiIC12-labeled vesicles at the
central cytoplasmic domain of the growth cone (Fig. 2). Individual vesicles could be resolved as brightly stained
puncta both along the axon (Fig. 1) and at the growth cone
region (Figs. 2 and 3). In parallel with accumulation of
DiIC12-labeled vesicles at the distal axon, we observed a
progressive increase in the diffuse staining of the peripheral growth cone region (Fig. 2). In agreement with previously published data (Forscher and Smith, 1988), the cell body-derived vesicles were largely excluded from the peripheral growth cone lamella and from the filopodia (Figs.
2 and 3). Therefore, we interpret the diffuse staining of the
axon as incorporation of DiIC12 molecules into the plasma
membrane. This diffuse staining gradually spread from the
growth cone towards the cell body (Fig. 2), consistent with
lateral diffusion of DiIC12 molecules along the plasmalemma.
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Since no plasma membrane staining at the middle axonal segment could be detected (Fig. 4 A), the staining of
the peripheral growth cone was not due to the diffusion of
DiIC12 molecules from the soma along the plasmalemma.
Hence, the diffuse staining of the growth cone reflected
the fusion of DiIC12-labeled vesicles with the plasma membrane at the distal axon. To test this model, before the
staining of the soma with DiIC12, we pretreated neuronal cultures for 1 h with brefeldin A (10 µg/ml), a drug that inhibits the supply of the new membrane to the axon (Lippincott-Schwartz et al., 1989; Jareb and Banker, 1997
). As
expected, the number of DiIC12-labeled vesicles at the distal axon was dramatically reduced, and no plasma membrane staining could be detected (Fig. 4 C). No fluorescent
vesicles could be detected at the distal axon after the treatment of neuronal cultures with 5 µg/ml nocodazole, a drug
that promotes MT disassembly (Fig. 4 D). This is consistent with the idea that the transport of the cell body-
derived vesicles from the soma crucially depends on the
integrity of axonal MTs. Cytochalasin D (5 µM), a drug
that inhibits actin polymerization, had no obvious effect
on organelle delivery to, and incorporation into, the distal
axon (Fig. 4 E). Similar results were obtained with latrunculin A (5 µM), a drug that induces depolymerization of
actin filaments (data not shown). To investigate whether
the drugs used in this study induced significant disassembly or assembly of axonal MTs, we loaded fluorescently labeled tubulin into neurons by embryo injection (Reinch et
al., 1991
; Chang et al., 1998
) and examined the neurons
with fluorescent microscopy. Detergent extraction of the
neurons in an MT-stabilizing buffer revealed that 76.6 ± 4.4% (mean ± SEM, data from 15 axons) of the total tubulin was in the polymer form. 1 h after the treatment of
neuronal cultures with brefeldin A (10 µg/ml) or with cytochalasin D (5 µM), this value was not significantly different from control (Table I). On the contrary, 1 h after the
treatment with nocodazole (5 µg/ml), the fraction of the
tubulin in polymer form decreased to ~11% (Table I).
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Taken together, these results suggest that, in growing axons, the newly synthesized plasma membrane components are transported in the form of tubovesicular organelles along axonal microtubules. The new membrane material is delivered to the growth cone region where it is inserted into the plasmalemma.
Inhibition of Dynamic Instability of Axonal Microtubules Prevents Insertion of New Membrane at the Growth Cone Region
MTs at the growth cone region display a complex pattern
of behavior and appear to be significantly more dynamic
than MTs along the axon (Bamburg et al., 1986; Tanaka
and Kirschner, 1991
; Tanaka et al., 1995
). To test whether
the high rate of MT turnover at the distal axon contributes
to the preferential insertion of the cell body-derived vesicles at the growth cone region, we determined the effects
of low concentrations of taxol and vinblastine on the pattern of membrane insertion into the growing axons. Taxol and vinblastine are antimitotic drugs that, in micromolar
concentrations, stabilize and disrupt MTs, respectively. In
nanomolar concentrations, both drugs decrease the dynamic instability of MTs (Jordan et al., 1992
, 1993
). In
nanomolar concentrations, vinblastine and taxol did not
affect the polymerization of MTs in Xenopus neurons (Table I). Neither taxol (7 nM) nor vinblastine (3 nM) affected the delivery of cell body-derived vesicles to the distal axon, as evidenced by the accumulation of the DiIC12-stained organelles at the growth cone region (Fig. 5, A and
B). However, the staining of the plasma membrane, and
thus insertion of new membrane into the growth cone region, were dramatically inhibited. The quantitative analysis of the plasma membrane staining (Fig. 5 E) was facilitated by the fact that under the cell culture conditions used
in this study, axons possess a rich net of filopodia that
spans the entire length of the neurite, including the growth
cone. Since DiIC12-labeled vesicles were largely excluded
from the filopodia, the sampling areas were chosen along
the length of individual filopodia at the growth cone. Similar results were obtained when the sampling areas were
chosen at the lamellipodium region of the growth cone. In
a series of control experiments, neuronal cultures were
pretreated with 3 nM vinblastine for 30 min to allow accumulation of the fluorescent vesicles at the growth cone
region, after which the concentration of vinblastine was
increased to 1 µM. This treatment resulted in the disassembly of axonal MTs (data not shown), and rapid staining
of the plasma membrane at the distal axon (Fig. 5 C).
Hence, inhibition of membrane insertion by nanomolar
concentrations of vinblastine is not related to a nonspecific
effect of the drug on vesicular fusion.
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Local Disassembly of Axonal Microtubules Leads to Insertion of Cell Body-derived Vesicles into the Plasma Membrane along the Axon
Under the cell culture conditions used in this study, the
majority of DiIC12-stained vesicles are transported all the
way down to the growth cone region, where the vesicles
fuse with the plasma membrane. The insertion of cell
body-derived vesicles along the axon may be inhibited
because of the limitations imposed by the axonal cytoskeleton. Alternatively, the plasma membrane along the
axon may lack the appropriate membrane receptors
(t-SNAREs), which are necessary for vesicular targeting
and/or fusion (Rothman, 1994; Calacos and Scheller, 1996
;
Hanson et al., 1997
). To distinguish between these possibilities, we locally applied nocodazole (5 µg/ml) to the
middle axonal segment located at least ~500 µm away both from the soma and the growth cone. This distance
was sufficient to exclude the lateral diffusion of DiIC12
molecules from the soma or the growth cone region to
the superfused site. 30 min after the onset of perfusion
with nocodazole, we stained cell body-derived vesicles
with DiIC12 molecules as described above. Within 10-20
min after the staining of the soma, we observed the accumulation of cell body-derived organelles 50-80 µm
proximal to the perfusion site, local staining of the
plasma membrane, and depolymerization of MTs at the
perfusion site (Fig. 6). Thus, plasma membrane components for vesicular exocytosis are likely to be present
throughout the whole axonal surface. Local disruption of MTs along the axon is sufficient to induce the fusion
of cell body-derived vesicles with the plasma membrane.
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Discussion |
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New membrane components are synthesized in the cell
body and conveyed along MTs by the kinesin superfamily
of proteins (Brady and Sperry, 1995; Block, 1998
; Hirokawa, 1998
). To determine the sites of new membrane
insertion into the growing neurites, we developed a technique for the acute labeling of all membrane structures in
the cell body with the fluorescent lipid analogue DiIC12.
In sufficiently long neurites, we were able to visualize the
anterograde transport of individual cell body-derived
tubovesicular organelles. The movement of these organelles occurred at the rates of fast axonal transport and
required the integrity of axonal MTs. The number of the
stained vesicles along the axon was dramatically diminished after pretreatment of neuronal cultures with brefeldin A. These results along with previously reported data
(Nakata et al., 1998
) suggest that the cell body-derived
tubovesicular organelles that we observe are likely to be
involved in the constitutive delivery of newly synthesized
membrane material from the Golgi complex to the plasma
membrane. There are three major findings in this report:
(a) under cell culture conditions used in this study, the vesicles were delivered all the way down to the growth cone
region, where they fused with the plasma membrane; (b)
local disruption of MTs at the middle axonal segment resulted in the insertion of new membrane along the axon;
and (c) inhibition of dynamic instability of axonal microtubules impaired the insertion of new membrane material
into the distal axon.
Preferential insertion of some of the newly synthesized
plasma membrane proteins into the growth cone region
has been observed in the past (Craig et al., 1995; Vogt et al.,
1996
). Our results provide direct evidence for the idea that
the growth cone is the primary site of new membrane addition to the plasmalemma (Futerman and Banker, 1996
).
Exocytosis of DiIC12-stained vesicles along the axon would
be expected to result in the disappearance of the fluorescent signal because of the rapid diffusion of DiIC12 molecules along axonal plasmalemma. We were not able to
detect such fusion events. Although we cannot exclude
the possibility that some of the small vesicles escape detection and do fuse with the plasmalemma along the axon,
the vast majority of the vesicles appeared to be transported all the way down to the growth cone. This conclusion was further supported by the staining of the
plasma membrane at the growth cone region, but not at
the middle axon (Fig. 4). Interestingly, the staining of
the plasma membrane at the distal axon was preceded by
an accumulation of DiIC12-labeled vesicles at this region. The observed delay in the insertion of new membrane
into the plasmalemma is consistent with the idea that a
number of intermediate steps (such as vesicular docking
and priming) are required for the exocytotic fusion to
proceed (Sudhof, 1995
). It should be noted that only cell body-derived vesicles were stained in our experiments. We have not attempted to determine the contribution of local synthesis of plasma membrane lipids
along the neurite (de Chaves et al., 1995
) to the generation of new axonal structure.
Although the idea of preferential insertion of new membrane components into the distal axon is now widely accepted, a simple estimate based on the diffusion equation
indicates that in sufficiently long axons, new membrane is
likely to be inserted along the axon. Indeed, the coefficient
of lateral diffusion of the plasma membrane proteins or lipids (D) does not exceed 108 cm2/s (Tank et al., 1982
). Assuming that the newly synthesized membrane material is
inserted exclusively into the growth cone region, the characteristic time of diffusional transport from the growth cone
region (t) can be estimated as t
L2/D, where L is the distance from the growth cone. Even for relatively short distances (L = 1 mm), this time is ~10 d. Thus, in the absence of anterograde (Popov et al., 1993
) or retrograde (Dai and
Sheetz, 1995
) plasma membrane flow, intercalation of
plasma membrane proteins and lipids along the axon is the
only feasible mechanism for new material delivery to the
axonal shaft (Bloom and Goldstein, 1998
). Rigorous quantitative analysis supports the idea that, in sufficiently long axons, axonal plasma membrane components should be inserted at sites all along the axon (Khanin et al., 1998
).
This model predicts that the molecular components
that are required for vesicular exocytosis are distributed
throughout the whole axonal surface. In agreement with
this prediction, in hippocampal cultures, both immunocytochemical data (Galli et al., 1995; Garcia et al., 1995
)
and reports on the constitutive membrane recycling along
the axon (Matteoli et al., 1992
) imply the presence of
t-SNAREs throughout the whole axonal surface. In Xenopus embryo neurons, constitutive secretion of acetylcholine can be detected throughout the whole neuronal
surface; moreover, similar to hippocampal cultures, immunoreactivity for syntaxin and SNAP-25 can be detected
along the axon (Antonov et al., 1998
). Taken collectively,
these results suggest that axonal shaft is competent to support vesicular exocytosis. Our experiments with local application of nocodazole to the middle axon directly confirm this idea. We found that local disruption of axonal
MTs led to the accumulation of cell body-derived vesicles at the site of nocodazole application and insertion of the
vesicles into the plasmalemma (Fig. 6). Thus, local disruption of axonal MTs is sufficient for the insertion of new
membrane material to specific neuronal sites.
These experiments suggest a mechanistically simple
model in which association of a vesicle with an MT limits its
access to the plasma membrane. The movement of individual vesicles along MTs is driven by the members of kinesin
superfamily of proteins (Vale et al., 1996; Block, 1998
; Hirokawa, 1998
). Kinesin remains bound to MTs while undergoing multiple rounds of activity (Vale et al., 1996
;
Block, 1998
). The average distance traveled by a single kinesin molecule after a binding to an MT is about 600 nm in
a motility assay (Vale et al., 1996
). Obviously, if the movement of the vesicle is driven by more than one kinesin
molecule associated with the same MT, the average travel
distance along the MT will be larger. However, MTs are
much shorter than axonal length, and the vesicle is expected to "fall off" the MT upon reaching its plus (directed towards the growth cone) end. We speculate that
because of the high density of MTs in the axonal shaft, the dissociated vesicle is likely to reattach rapidly to another
(or to the same) MT. Therefore, the probability of vesicular exocytosis along the axon remains low.
It remains to be established what properties of the growth cone region allow exocytosis to proceed. Since none of cell body-derived vesicles that we detected was transported retrogradely, the vesicles are expected to accumulate at the distal axon. Unique molecular composition of the growth cone region may contribute to trapping of the vesicles in the cytomatrix and/or facilitate their translocation to the plasma membrane. Unexpectedly, we found that inhibition of dynamic instability of MTs dramatically impaired insertion of new membrane into the distal axon. The easiest interpretation of these data is that dynamic instability of MTs at the growth cone region increases the probability of dissociation of a vesicle from an MT. However, we cannot exclude the possibility that dynamic MTs at the distal axon are essential for the proper organization of the apparatus for the delivery of the vesicles to the plasma membrane sites specialized for fusion.
Treatment of neuronal cultures with low doses of vinblastine or taxol is known to inhibit axonal elongation
(Tanaka et al., 1995) and growth cone turning (Challacombe et al., 1997
). These results are believed to reflect
the essential role of dynamic MTs in the rearrangement of
MTs during growth cone migration and in the generation
of new axonal structure (Tanaka et al., 1995
; Challacombe
et al., 1997
). Our results suggest that dynamic instability of
microtubules at the distal axon may also contribute to the release of vesicles from the MTs and insertion of new
membrane into the growth cone region. Therefore, the effect of low doses of taxol and vinblastine on growth cone
migration, at least in part, may be related to the inhibition
of new membrane addition to the plasmalemma.
Tubulin dimers must contain GTP to polymerize. GTP
is rapidly hydrolyzed upon addition of dimers to the growing MT. This creates a small cap of GTP-tubulin at the
growing end of an MT, while MT lattice is primarily composed of GDP-tubulin (Desai and Mitchison, 1997). Both
kinetochores (Inoue and Salmon, 1995
; Severin et al., 1997
) and membrane organelles (Blocker and Griffiths,
1998
) recognize MT plus ends (presumably through their
association with GTP cap), and their movement can be
powered by MT dynamics (Desai and Mitchison, 1995
;
Lombillo et al., 1995
). Therefore, dynamic changes in MT length may directly contribute to intracellular motility
(Desai and Mitchison, 1995
; Inoue and Salmon, 1995
).
Moreover, experimental evidence suggests that motors
may not easily detach from MTs when they reach their
ends (Verde et al., 1991
; Hyman and Karsenti, 1996
). Our
data suggest that dynamic instability of axonal MTs can increase the probability of vesicle dissociation from the MT
and regulate the number of cell body-derived vesicles
available for exocytosis. Thus, dynamics of axonal MTs
may serve as a basic regulator of the topology of the new
membrane addition to the nerve processes.
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Footnotes |
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Received for publication 16 July 1998 and in revised form 30 September 1998.
Address all correspondence to Sergey Popov, Department of Physiology
and Biophysics M/C 901, University of Illinois at Chicago, 835 S. Wolcott
Ave., Chicago, IL 60612. Tel.: (312) 413-5682. Fax: (312) 996-1414. E-mail:
spopov{at}uic.edu
We thank Gary Borisy, Vladimir Gelfand, Leonid Margolis, and Mark Rasenick for the helpful discussion and comments and Michael O'Donoghue for help with the manuscript. We are grateful to Regeneron Pharmaceuticals Inc. for providing NT-3 and to Gary Borisy for providing Cy3-tubulin.
Supported by The National Institutes of Health (NS 33570).
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Abbreviations used in this paper |
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DiIC12, 1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanide; MT, microtubule.
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