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The Journal of Neuroscience, November 1, 2002, 22(21):9358-9367
Growth Cone Turning Induced by Direct Local Modification of
Microtubule Dynamics
Kenneth B.
Buck and
James Q.
Zheng
Department of Neuroscience and Cell Biology, University of Medicine
and Dentistry of New Jersey, Robert Wood Johnson Medical School,
Piscataway, New Jersey 08854
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ABSTRACT |
Pathfinding by nerve growth cones depends on attractive and
repulsive turning in response to a variety of guidance cues. Here we
present direct evidence to demonstrate an essential and instructive role for microtubules (MTs) in growth cone steering. First, both growth
cone attraction and repulsion induced by diffusible cues in culture can
be completely blocked by low concentrations of drugs that specifically
inhibit dynamic microtubule ends in the growth cone. Second, direct
focal photoactivated release of the microtubule-stabilizing drug taxol
on one side of the growth cone consistently induces attraction (turning
toward the site of application). Using the focal pipette application
method, we also show that local MT stabilization by taxol induces
growth cone attraction, whereas local MT destabilization by the
microtubule-disrupting drug nocodazole induces repulsion (turning
away). Finally, the microtubule-initiated attractive turning requires
the participation of the actin cytoskeleton: local microtubule
stabilization induces preferential protrusion of lamellipodia before
the attractive turning, and the attraction can be abolished by
inhibition of either actin polymerization or the Rho family
GTPases. Together, these results demonstrate a novel steering mechanism
for growth cones in which local and selective modification of dynamic
microtubules can initiate and instruct directional steering. With the
subsequent concerted activity of the actin cytoskeleton, this
microtubule-initiated mechanism provides the growth cone with the
additional means to efficiently navigate through its environment.
Key words:
growth cone; microtubule; cytoskeleton; axon guidance; actin; Rho GTPase
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INTRODUCTION |
The establishment of specific
neuronal connections requires guided extension of developing axons to
their appropriate targets by a variety of diffusible and surface-bound
extracellular cues. The motile growth cones of elongating axons are
responsible for sensing spatially and temporally distributed guidance
signals that direct the growth cone to steer toward (attraction) or
away from (repulsion) the guidance source to reach their targets
(Tessier-Lavigne and Goodman, 1996 ). It is believed that asymmetric
activation of the surface receptors on the growth cone by guidance
molecules elicits a cascade of localized intracellular signaling events that carry the directional signal for growth cone steering. Presumably, the code for directional steering is maintained at each level of
intracellular signaling down to the cytoskeleton (Bentley and O'Connor, 1994; Jay, 1996; Zheng et al., 1996b). It has been
convincingly shown that cells undergoing chemotaxis use a similar
sensing mechanism (Parent and Devreotes, 1999). In nerve growth cones,
actin and microtubules (MTs) are the cytoskeletal components
responsible for locomotion and are the ultimate targets of directional
signaling. The actin cytoskeleton is found predominately in the
peripheral region (P-region) of the growth cone, where highly dynamic
lamellipodia and filopodia can be found (Smith, 1988). The actin-based
protrusive activity coupled with retrograde flow and attachment of the
actin network to the membrane and substrate is thought to generate the kinetic force necessary for growth cone motility (Lin et al., 1994;
Suter and Forscher, 2000). MTs are bundled together in the axonal shaft
and central region (C-region) of the growth cone, whereas individual
microtubules extend off the bundles and splay into the peripheral
region, where they occasionally invade the actin-rich areas. These
single MTs exhibit dynamic instability with characteristic rapid growth
and shrinkage (Tanaka and Kirschner, 1991). Although both cytoskeletal
components are fundamental for growth cone motility, it is commonly
believed that the actin cytoskeleton is primarily responsible for
initiating and directing growth cone steering, whereas MTs consolidate
and support the new extension initiated by the preferential actin
activity (Mitchison and Kirschner, 1988; Smith, 1988).
Recently, an increasing number of studies have suggested that dynamic
MTs in the growth cone may play a more critical role (Tanaka and
Kirschner, 1995; Tanaka et al., 1995; Challacombe et al., 1997; Mack et
al., 2000) for growth cone steering. In particular, dynamic MT ends
seen in the P-region of the growth cone were found to be essential for
repulsive turning of the growth cone at substrate borders (Tanaka and
Kirschner, 1995; Challacombe et al., 1997). Furthermore, interplay
between the actin and MT cytoskeleton has been shown to be critical for
axon branching (Dent and Kalil, 2001). Finally, a growing number of
signaling components that can target microtubules have been identified
(Gundersen and Cook, 1999). These findings raise an important question
of how local MT dynamics contribute to growth cone steering. It is not
clear from these studies whether microtubules play a supporting role to
consolidate directional growth initiated by the actin cytoskeleton or
an instructive role to initiate growth cone steering. In this study, we
present direct evidence that dynamic MT ends in the growth cone are
required for growth cone turning induced by diffusible cues, and that
local modification of the dynamics of MTs is sufficient to initiate and
direct the turning response.
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MATERIALS AND METHODS |
Cell culture. Dissociated cells from the neural tube
tissue of 1-d-old Xenopus embryos (Spitzer and Lamborghini,
1976) were plated on glass coverslips precoated with Con A (Chang et
al., 1998) (~1 µg/cm2; E-Y Labs). The
cultures were kept at 20-22°C in culture medium consisting of the
following (v/v): 50% Leibovitz L-15 medium (Invitrogen, Carlsbad, CA), 50% Ringer's solution (in
mM: 115 KCl, 2 CaCl2, 2.5 KCl, and 10 HEPES, pH 7.4), 1% fetal bovine serum (Invitrogen), and 50 ng/ml neurotrophin-3 (Regeneron Pharmaceuticals, Tarrytown, NY).
Growth cone turning induced by extracellular gradients.
Microscopic gradients of chemicals were produced by the pipette
application method described previously (Lohof et al., 1992; Zheng et
al., 1996a). A standard pressure pulse of 3 psi was applied to a glass pipette (1 µm opening) at a frequency of 2 Hz, with durations of 20 and 5 msec for guidance molecules and MT drugs, respectively. The
direction of growth cone extension at the beginning of the experiment
was defined by the distal 20 µm segment of the neurite. The pipette
tip was positioned 45° from the initial direction of extension and
100 µm away for guidance cues or 50 µm away for MT drugs from the
center of the growth cone. The digital images of the growth cone at the
onset and end of the 30 min period were acquired and overlaid with
pixel-to-pixel accuracy, and the trajectory of new neurite extension
was traced using Adobe PhotoShop (Adobe Systems, San Jose, CA). The
turning angle was defined by the angle between the original direction
of neurite extension and a line connecting the positions of the growth
cone at the experiment onset and at the end of 30 min of exposure to
the gradient. Neurite extension was quantified by measuring the entire
trajectory of net neurite growth over the 30 min period. Only growth
cones extending  5 µm were scored for turning responses. For bath
application, drugs were added to the bath medium 20 min before the
onset of gradient application.
Microscopy and imaging for turning assay. All turning
experiments were performed in an open chamber on an inverted Nikon
(Tokyo, Japan) microscope equipped with differential interference
contrast (DIC) optics and a 40 × 1.3 numerical aperture (NA)
oil-immersion objective. A 0.5 inch CCD video camera (C2400-75i;
Hamamatsu, Bridgewater, NJ) was used for video imaging in conjunction
with an Argus-20 image processor (Hamamatsu) for image enhancement. The
video images were background subtracted, averaged over four video
frames, contrast enhanced in real time using Argus-20, and digitally
acquired using a personal computer (Wang and Zheng, 1998).
Focal laser-induced photolysis of caged taxol. Focal
laser-induced photolysis (FLIP) of caged taxol was achieved using the system described previously (Zheng, 2000). A nitrogen-pulsed laser (Laser Science, Franklin, MA) was focused to a spot of ~2 µm in diameter through a 40 × 1.3 NA oil-immersion objective (Nikon) for FLIP. Spatial restriction and efficiency of photoactivation were
evaluated by FLIP of photosensitive-caged fluorescein dextran as
described previously (Zheng, 2000). To allow the caged taxol (1 µM) to reach equilibrium inside the growth
cone, neurons were preincubated with caged taxol for 10 min before the
uncaging experiments. Each growth cone was positioned such that the
laser spot would be located in the P-region of the growth cone on one
side and be maintained throughout the entire experiment. Images of the growth cone at various times after the onset of repetitive FLIP were
digitally processed and acquired, and quantitative measurement and
analysis of the turning response were then performed.
Fluorescent staining. Xenopus neurons were
rapidly fixed with 4% paraformaldehyde and 0.25% glutaraldehyde in a
cacodylate buffer (0.1 M sodium cacodylate and
sucrose, pH 7.4) for 30 min and permeabilized with 0.1% Triton X-100
in Ringer's solution. Microtubules were labeled with a rat monoclonal
antibody against  -tubulin (Harlan Bioproducts for Science,
Indianapolis, IN) and a rabbit anti-rat IgG second antibody conjugated
with FITC (Sigma, St. Louis, MO); the same cells were then labeled with
1% rhodamine phalloidin (Molecular Probes, Eugene, OR) for the actin filaments.
Live fluorescent imaging of MTs. Rhodamine-conjugated
-tubulin proteins (Cytoskeleton, Denver, CO) were injected into one blastmere of Xenopus embryos at the two cell stage according
to the method described previously (Chang et al., 1998). Neurons cultured from these embryos were examined by fluorescence microscopy using a 100 × 1.3 NA objective. A cooled CCD camera (PXL1400; Roper Scientifics, Tucson, AZ) was used for image acquisition, and the
exposure time for each frame was 500 msec. To focally apply taxol, a
micropipette (1 µm opening) containing taxol solution was placed at
50 µm, 45° away from the growth cone. Four to five images of the
MTs of the growth cone were collected before the onset of the taxol
application. Because of photo bleaching and toxicity from the
high-resolution fluorescent imaging, we were only able to collect  20
frames at different times after the onset of focal taxol application.
Axon Imaging Workbench (Axon Instruments, Foster City, CA) was used for
imaging and recording.
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RESULTS |
Growth cone turning in diffusible guidance gradients requires
dynamic microtubule ends
To investigate the role of microtubules in growth cone steering,
embryonic Xenopus spinal neurons grown on coverslips coated with Con A (Chang et al., 1998) were used 6-10 hr after plating (hereafter referred to as 6 hr cultures) or 18-24 hr after plating (hereafter referred to as 18 hr cultures). Xenopus neurons
on Con A exhibited large and rapid-extending growth cones that allow spatially restricted manipulation of microtubule dynamics. Over a
period of ~30 min, the average lengths of growth cone extension were
~15 and 20 µm for 6 and 18 hr cultures, respectively. We first
examined the turning responses of the growth cones from 6 hr cultures
to gradients of glutamate (Zheng et al., 1996a) and netrin-1 (Kennedy
et al., 1994). A microscopic gradient of these molecules was created by
repetitive pulsatile ejection of the solution from a micropipette
placed 100 µm away from the growth cone at a 45° angle with respect
to the original direction of extension (Lohof et al., 1992; Zheng et
al., 1996a). Digital images of each growth cone at the onset and end of
the 30 min gradient application were collected and overlaid to
determine the response of the growth cone. Similar to our previous
observation (Zheng et al., 1996a), a gradient of glutamate (50 µM in pipette) consistently induced marked
attractive turning responses of Xenopus growth cones toward
the glutamate source within 30 min in 6 hr cultures (Fig.
1b), whereas the control group
of growth cones showed no preferential response to the pipette
application of culture medium (Fig. 1a). The average turning
angle (in degrees) in the control group was 1.3 ± 3.6 SEM,
whereas that of growth cones exposed to glutamate was 9.4 ± 2.9 (p < 0.05; Kolmogorov-Smirnov test). The
slightly smaller average turning angle than that observed previously
for glutamate (Zheng et al., 1996a) likely resulted from the strong
adhesion of these growth cones on the Con A-coated surface, which could
limit the degree of steering.
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Figure 1.
Blocking of growth cone turning responses to
guidance gradients by inhibition of MT dynamics. a-e,
Growth cone turning induced in 6 hr cultures. A gradient of glutamate
(b) or netrin-1 (d) was
applied to the growth cone by repetitive pulsatile pressure ejection of
either 50 µM glutamate (GLU) or 5 µg/ml netrin-1 (N-1) solution from a micropipette (see
Materials and Methods). The control (a) was done
with the same pulsatile pipette application of culture medium
(CTRL) to the growth cone. Images of the growth cone
were collected at the onset (left image) and at the end
(right image) of a 30 min application period. The origin
is the center of the growth cone at the onset of the experiment.
Dotted lines indicate corresponding positions along the
neurite; dashed lines indicate the original direction of
extension. Superimposed traces on the
right (same magnifications as the growth cone images)
depict the trajectory of neurite extension during the 30 min period for
all of the growth cones in each group. Arrows indicate
the direction of the gradient. Bath application of 10 nM
nocodazole (+ Noc) abolished both attraction induced by
glutamate (c) and repulsion induced by netrin-1
(e). f, g,
Netrin-1-induced growth cone attraction in 18 hr cultures
(f) was also blocked by the presence of 10 nM nocodazole in the bath (g). Scale
bars: a-g, 10 µm. h, The distribution
of turning angles is presented to depict the overall responses under
different experimental conditions. For each condition, the percentage
value refers to the percentage of growth cones with the turning angle
less than or equal to a given angular value. Data shown are turning
responses induced by glutamate and netrin-1 in 6 hr (top
panel) and 18 hr (bottom panel)
cultures without and with the presence of the MT drugs nocodazole and
vinblastine in bath.
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We then tested the turning responses of Xenopus growth cones
to a gradient of the guidance molecule netrin-1 (generously provided by
Dr. M. Tessier-Lavigne, Stanford University, Stanford, CA) (Kennedy et
al., 1994). In 6 hr cultures, a gradient of netrin-1 induced clear
repulsion of the growth cones (Fig. 1d), resulting in an
average turning angle of  10.1 ± 2 (p < 0.01 compared with the 6 hr control; Kolmogorov-Smirnov test). In 18 hr cultures, however, the same gradient of netrin-1 induced significant
attraction of the growth cones (Fig. 1f). The average
turning angle of 9.3 ± 3.4 was significantly different from
0.2 ± 3.3 of the 18 hr control (p < 0.05; Kolmogorov-Smirnov test) (Table
1). This developmental switching of
turning responses to netrin-1 has been observed previously in
Xenopus growth cones grown on coverslips without any
substrate coating (Ming et al., 2001) and may be associated with a
change of intracellular levels of cAMP in these neurons (Wang and
Zheng, 1998; Ming et al., 2001). To better depict the overall responses of a population of growth cones to these guidance gradients, cumulative histograms of the distribution of turning angles (Fig. 1h)
as well as the turning scores [percentages of growth cones scored as
turning positively (+), negatively (), and having no turning response
(0)] (Table 1) are presented. As shown clearly, a gradient of
glutamate induced significant attraction, whereas a gradient of
netrin-1 induced clear repulsion in 6 hr cultures. For 18 hr cultures,
the majority of the growth cones turned positively toward the netrin-1
source. These turning results indicate that Con A coating did not alter
the responsiveness of cultured Xenopus neurons to these
guidance cues. Moreover, the fact that no significant change was
observed in the extension rate for these conditions (Table 1) indicates
that gradients of glutamate and netrin-1 exerted their effects
primarily on the direction of growth cone extension.
To determine the role of MTs in growth cone turning responses induced
by these diffusible gradients, we bath applied low concentrations of
membrane-permeant MT-specific drugs to inhibit the dynamic MTs in the
growth cone without causing significant MT depolymerization in the cell
(Tanaka et al., 1995; Rochlin et al., 1996; Challacombe et al., 1997).
The presence of a very low concentration (10 nM) of
nocodazole in the bath did not significantly affect the extension rate
of Xenopus growth cones but completely blocked both
attraction induced by glutamate (Fig. 1c) and repulsion
induced by netrin-1 (Fig. 1e) in 6 hr cultures. The average
turning angles induced by glutamate and netrin-1 gradients in the
presence of 10 nM nocodazole in bath were found
to be 2.5 ± 1.4 and 0.7 ± 3.2, respectively (Table 1).
Statistical analyses using a Kolmogorov-Smirnov test indicated no
preferential turning response induced by either gradient (p > 0.5 compared with the 6 hr control group;
p < 0.01 compared with their corresponding groups
without nocodazole in bath). Similarly, growth cone attraction induced
by netrin-1 gradients in 18 hr cultures was also abolished by 10 nM nocodazole in bath (Fig. 1g). The
average turning angle of 2.7 ± 2.9 was not different from that
of the 18 hr control (p > 0.5;
Kolmogorov-Smirnov test). The complete elimination of both attractive
and repulsive turning responses is better demonstrated by the
cumulative histogram of the distribution of turning angles (Fig.
1h) and the turning scores (Table 1). Similar abolition of
turning responses was also observed with a different MT-disrupting
drug, vinblastine. Vinblastine binds to a different site on
microtubules than nocodazole (Downing, 2000) and has been shown to
inhibit the dynamic instability of single MTs in the growth cone
without causing drastic MT depolymerization at low concentrations
(Tanaka et al., 1995; Challacombe et al., 1997). Bath application of 4 nM vinblastine completely abolished netrin-1-induced growth cone repulsion in 6 hr cultures and attraction in 18 cultures, although growth cone extension was partially inhibited (Fig. 1h, Table 1). To further confirm that dynamic MTs in
the growth cone are crucial, we used the MT-stabilizing drug taxol. Taxol at low concentrations has been shown to inhibit the dynamic instability of MTs and restrict MT distribution to the C-region of the
growth cone over time (Williamson et al., 1996; Challacombe et al.,
1997). Bath application of taxol at 7 nM slightly
inhibited growth cone extension but completely abolished both
attractive and repulsive responses induced by glutamate and netrin-1
gradients, respectively, in 6 hr cultures (Table 1). Similarly,
netrin-1-induced attraction in 18 hr cultures was also blocked (Fig.
1h, Table 1).
Double fluorescent staining of MTs and the actin cytoskeleton was
performed to confirm that these low concentrations of MT drugs
specifically affected dynamic MTs in the P-region of the growth cone.
Growth cones in control groups showed a typical pattern of these two
cytoskeletal structures similar to that observed in other types of
nerve growth cones (Smith, 1988). MT bundles were concentrated in the
neurite shaft and primarily terminated in the C-region of the growth
cone, with a number of single MTs splaying into the P-region (Fig.
2a). These free MT ends have been shown to undergo rapid growth and shrinkage (dynamic instability) (Tanaka and Kirschner, 1991; Tanaka et al., 1995). Bath application of
7 nM taxol only for 20 min caused the withdrawal
of many of these dynamic MTs from the P-region of the growth cone
without apparent effect on the overall MT organization in the C-region (Fig. 2b); the actin cytoskeleton was not altered by the low
concentration of taxol (Fig. 2b). For MT-disrupting drugs,
both 10 nM nocodazole (Fig. 2c) and 4 nM vinblastine (Fig. 2d) did not
affect the filamentous structures of the actin cytoskeleton in the
growth cone, and they did not significantly disrupt MTs in the C-region
of the growth cone and along the neurite. However, both drugs were
found to primarily remove dynamic MT ends in the P-region of the growth cone, with vinblastine being most effective (Fig.
2c,d). Live fluorescent imaging of MTs also
revealed that dynamic MT ends withdrew from the P-region of the growth
cone shortly after the addition of these MT-disrupting drugs (data not
shown). These results suggest that all three MT-specific drugs, at low
concentrations, blocked the turning responses to guidance gradients by
inhibiting the dynamic ends of MTs in the P-region of the growth cone.
Together, our findings demonstrate that dynamic microtubule ends in the growth cone, particularly in the P-region, are essential for
directional steering of the growth cone in response to extracellular
guidance cues.
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Figure 2.
Double fluorescent imaging of microtubules and
actin cytoskeleton in growth cones exposed to different MT-specific
drugs. a, Representative pair of fluorescent images of
the actin microfilaments (MFs) and MTs of a
control growth cone treated with medium only. b-d,
Representative pairs of MT and MF images of Xenopus
growth cones treated with (in nM) 7 taxol
(b), 10 nocodazole (c), and
4 vinblastine (d). All of the cells were fixed at
20 min after the treatment. For clarity, the margin of the growth cone
lamellipodia in MT staining has been outlined. Scale bar, 10 µm.
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Direct local modification of MT dynamics in the growth cone is
sufficient to induce steering
Dynamic MTs in the P-region of the growth cone are thought to play
an exploratory role, and preferential stabilization and growth are
believed to be involved in growth cone steering. To determine how local
microtubule dynamics in the growth cone contribute to growth cone
steering, we took advantage of the FLIP method (Zheng, 2000) to
directly stabilize MTs in a spatial and temporal manner in the P-region
of the growth cone where free MT ends were frequently observed (Fig.
3a). All subsequent
experiments were performed in 6 hr cultures. Neurons were incubated
with the membrane-permeant photosensitive caged taxol [1
µM paclitaxel,
2'-(4,5-dimethoxy-2-nitrobenzyl)carbonate; Molecular Probes]
for 10 min before focal photoactivation. FLIP of caged taxol in the
growth cone was performed in a small spot of ~2 µm in diameter on
one side of the growth cone (Fig. 3a-c, circles)
and repeated at a rate of one pulse every 3 sec (Zheng, 2000). A
majority of the growth cones (18 of 23) exposed to 30 min of repetitive
FLIP of caged taxol grew and steered toward the side of focal taxol
release (Fig. 3c,f). Growth cones in the two control groups, one with laser irradiation but without caged taxol
(Fig. 3b,d) and the other without laser but with
caged taxol (Fig. 3e), exhibited random turning responses.
Comparisons of the turning angles of the growth cones exposed
to FLIP of caged taxol and those of the two control groups showed that
local release of caged taxol induced significant turning of the growth
cones toward the side of taxol release (average turning angles:
11.7 ± 3.5, 0.3 ± 3.6, and 0.4 ± 3.9, respectively; p < 0.01; Kolmogorov-Smirnov test)
(Table 2). The cumulative histogram of
the distribution of turning angles and the turning scores of these
three groups (Fig. 3g, Table 2) clearly show that a greater
number of growth cones exposed to FLIP of caged taxol turned with a
positive angle than that of the two control groups. It should be noted
that caged taxol appeared to exert some inhibition on neurite
extension. The average length of neurite extension for growth cones
exposed to laser irradiation but without caged taxol was 19.1 ± 1.6 µm, whereas the average lengths for growth cones in the presence
of caged taxol with and without FLIP were 10.2 ± 0.8 and
10.3 ± 0.7, respectively (Table 2). This reduced extension may be
attributable to some residual activity of caged taxol or a small amount
of uncaging in response to ambient light. Nevertheless, spatially restricted application of taxol to one side of the growth cone clearly
caused the growth cone to turn positively, thus demonstrating that
local stabilization and possibly local promotion of MT polymerization of MTs are sufficient to induce a positive steering event (attractive turning).
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Figure 3.
Attractive turning of Xenopus
growth cones induced by repetitive FLIP of caged taxol.
a, Immunostaining of the actin cytoskeleton and
microtubules in the growth cone. For clarity, the margin of the growth
cone lamellipodia in MT staining has been outlined. The
circle indicates the location of the laser irradiation.
b, Control growth cone (no caged taxol) at the beginning
and end of 30 min of repetitive laser irradiation;
circles indicate the laser spot. The dotted
line indicates corresponding positions along the neurite;
dashed lines indicate the original direction of
extension. c, Growth cone loaded with caged taxol at the
onset and end of 30 min of repetitive FLIP. Numbers in
b and c represent minutes after the onset
of repetitive laser irradiation. d-f, Superimposed
traces of growth cone extension during the 30 min
experimental period in two control groups [d, focal
laser (fLaser) on but without caged taxol
(cTaxol); e, laser off but with
caged taxol] and in the group exposed to repetitive FLIP of caged
taxol (f). g, The
cumulative distribution illustrates the overall turning responses of
all of the growth cones examined in these three groups. Scale bars:
a-c, 10 µm; f, 5 µm.
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To confirm the above finding, we examined whether focal extracellular
application of the membrane-permeant taxol can also induce the growth
cone to turn positively. We modified the pulsatile ejection method used
for guidance gradients (Lohof et al., 1992; Zheng et al., 1996a) by
moving the pipette to a distance of 50 µm from the growth cone and
reducing the pulse duration from the standard 20 to 5 msec to create a
much greater concentration difference of taxol reaching the two sides
of the growth cone (Lohof et al., 1992). No turning response was
observed in the control group of growth cones exposed to focal
application of 1% DMSO (Fig.
4a). Among 30 growth cones
examined, random turning was observed, resulting in an average turning
angle of 1.3 ± 2.5 (Table 3).
This result indicates that this focal application method did not
produce any mechanical artifacts. When 5 or 50 µM taxol (paclitaxel; Molecular Probes) was
applied through the pipette to these growth cones, marked turning
responses toward the source of taxol were observed (Fig. 4b,
Table 3). The average turning angles were 11 ± 2.2 and 11 ± 3.2 for 5 and 50 µM taxol (in pipette),
respectively, and were significantly different from the DMSO control
(p < 0.01 and <0.05, respectively;
Kolmogorov-Smirnov test). Higher concentrations of
taxol, however, were found to inhibit the growth cone
extension, preventing further examination. When the taxol concentration
was reduced to 0.5 µM, no significant turning
was observed (p > 0.5; Kolmogorov-Smirnov
test). The cumulative histogram of the distribution of turning angles
(Fig. 4d) and turning scores (Table 3) better depict the
positive turning responses induced by focal taxol application. With the
modified pipette application method, the concentration of taxol
reaching the growth cone was estimated to be ~1/400 of the
concentration in the pipette using the theoretical analysis described
previously (Lohof et al., 1992). Therefore, the effective concentrations of taxol at the growth cone are ~10-100
nM for inducing attraction. Live imaging of
fluorescent MTs in the growth cone during taxol-induced turning showed
that local taxol application caused preferential growth and bundling of
MTs to the same side of the growth cone (Fig. 4e). These
results, along with those from FLIP of caged taxol, clearly demonstrate
that local stabilization and growth of MTs are sufficient to
instructively induce attractive turning of the growth cones.
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Figure 4.
Turning responses induced by focal application of
MT-specific drugs through a micropipette. a, In the
control, growth cones were exposed to focal application of 1% DMSO.
Representative images of a control growth cone at the onset
(left image) and at the end (right image)
of the 30 min application are shown together with the superimposed
traces of all of the growth cones (same magnifications
as the growth cone images). See the legend to Figure 1 for details.
b, Attractive turning induced by focal application of 5 µM taxol. c, Repulsive turning induced by
focal application of 100 µM nocodazole.
Arrows in a-c indicate the direction of
the gradient. d, Overall responses of
Xenopus growth cones to focal application of taxol and
nocodazole are shown in the cumulative distribution of turning angles.
e, Live imaging sequence of rhodamine-labeled MTs in a
growth cone exposed to focal taxol application. Arrows
indicate the direction of the focal taxol application.
Numbers represent times after the onset of taxol
application, with the first frame acquired 1 min before the onset of
taxol application. Scale bars, 10 µm.
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We then examined the effects of local destabilization and/or
depolymerization of MTs on growth cone turning. Because none of the
MT-disrupting drugs are available in caged form, we focally applied the
membrane-permeant MT-disrupting drug nocodazole to one side of the
growth cone through a micropipette. Focal application of 10 µM nocodazole (in pipette) did not produce any effect on the direction and rate of growth cone extension (average turning angle,
0.7 ± 2.7; p > 0.5 compared with the DMSO
control; Kolmogorov-Smirnov test) (Table 3). Focal application of 100 µM nocodazole, however, caused the growth cones
to steer away (Fig. 4c) (average turning angle, 11.4 ± 3; p < 0.05; Kolmogorov-Smirnov test). The
cumulative histogram of the distribution of turning angles (Fig.
4d) and the turning scores (Table 3) also confirm that focal
application of 100 µM nocodazole (in pipette)
caused a majority of the growth cones to steer away (repulsive
turning). We were unable to test higher concentrations of nocodazole
because of the inhibition of growth cone extension. Along with the
results from focal taxol application, we have demonstrated that
localized modification of MT dynamics on one side of the growth cone is
sufficient to instruct the growth cone to steer in a specific
direction. Local MT stabilization and growth initiate attractive
turning, whereas local MT destabilization and disassembly induce
repulsive turning.
The actin cytoskeleton participates in MT-initiated growth
cone attraction
Although our results have clearly established an essential as well
as instructive role for microtubules in directional steering of the
growth cone, we have also found evidence for the involvement of the
actin cytoskeleton in growth cone steering initiated by MTs. In growth
cone attraction induced by focal taxol application, time-lapse DIC
microscopy revealed preferential protrusion of lamellipodia on the side
of the growth cone exposed to focal taxol application, before the
actual turning of the growth cone (Fig. 5a, arrowheads).
Because the specificity of taxol on MTs has been well documented (Diaz
et al., 2000; Downing, 2000), this result suggests that local
stabilization and growth of MTs can initiate the actin-based protrusive
activity that may be important for directional steering of the growth
cone. To test this idea and examine the role of the actin cytoskeleton
in MT-initiated growth cone turning, we examined turning responses
induced by taxol when actin polymerization was inhibited by a low
concentration of cytochalasin D (CD). When 20 nM
CD was present in the medium, the growth cone exhibited reduced
actin-based motility as reviewed under high-resolution time-lapse DIC
microscopy. Both retrograde membrane ruffling and the protrusion of
lamellipodia and filopodia were partially inhibited. Furthermore,
growth cone filopodia were lost over time by CD treatment (Zheng et
al., 1996a) (see supplementary video clip at
http://www2.umdnj.edu/zhlabweb/video/CytoD.avi). However, considerable
extension of the growth cone was still observed. We found that
attractive turning of the growth cone induced by focal pipette
application of 5 µM taxol was completely
abolished by the presence of 20 nM CD in bath
(Fig. 5b, Table 3), leading to an average turning angle of
0.1 ± 4.4 (p > 0.5 compared with the
DMSO control; p < 0.01 compared with the 5 µM taxol group; Kolmogorov-Smirnov test). The
blockage of taxol-induced attraction is better depicted in the
cumulative distribution of the turning angles (Fig. 5d).
These results directly elucidate the involvement of the actin
cytoskeleton in growth cone steering induced by localized MT
activity.
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|
Figure 5.
Involvement of the actin cytoskeleton in
MT-initiated growth cone turning. a, Representative time
lapse sequence of the attractive turning response of a
Xenopus growth cone induced by focal taxol application.
Note the increased protrusion of lamellipodia on the side of the growth
cone facing the pipette before the actual turning of the growth cone
(arrowheads). b, Growth cone response to
focal application of 5 µM taxol in the presence of 20 nM cytochalasin D in bath. Representative images of a
growth cone at the onset (left image) and at the end
(right image) of the 30 min taxol application are shown
together with the superimposed traces of all of the
growth cones on the right (same magnifications as the
growth cone images). See the legend to Figure 1 for details.
c, Growth cone response to focal application of 5 µM taxol in the presence of 100 pg/ml toxin B in bath.
Scale bar, 10 µm. Arrows indicate the direction of the
taxol gradient. d, Overall responses of growth cones to
focal taxol application in the presence of toxin B (to inhibit the Rho
GTPases) and cytochalasin D (to inhibit the actin assembly) are shown
as the cumulative distribution of turning angles.
|
|
Microtubules have been shown to regulate lamellipodial dynamics both in
nerve growth cones (Tanaka and Kirschner, 1995; Gallo, 1998) and in
migrating fibroblasts (Mikhailov and Gundersen, 1998) and to initiate
actin-based intrapodia in motile growth cones (Rochlin et al., 1999).
Taxol has been shown to induce rapid microtubule growth to subsequently
produce lamellipodial protrusion in migrating fibroblasts
(Waterman-Storer et al., 1999). Recently, microtubule growth was found
to activate Rac1, a member of the Rho GTPases (Rho, Rac, and
Cdc-42) (Hall, 1998), to induce lamellipodial protrusion (Waterman-Storer et al., 1999). To test this possibility, we bath applied toxin B (from Clostridium difficile; Cytoskeleton)
to inactivate the Rho family of GTPases (Just et al., 1995). Toxin B at
100 pg/ml was found to completely abolish the attractive turning of
growth cones induced by focal taxol application (Fig. 5c,
Table 3), resulting in an average turning angle of 0.5 ± 3.1 (p > 0.5 compared with the DMSO control;
p < 0.01 compared with the 5 µM group; Kolmogorov-Smirnov test). The
blockage of taxol-induced attraction is also illustrated by the
cumulative distribution of the turning angles of the entire population
of growth cones examined, which is similar to that seen in the presence of CD (Fig. 5d); both CD and toxin B effectively blocked the
attraction induced by local taxol application. However, unlike CD,
toxin B exerted little effect on the actin-based motility of the growth cone. Both retrograde membrane ruffling and the protrusion of filopodia
and lamellipodia were not affected over the time period of the
recording, and the growth cone morphology was not significantly altered
(see the supplementary time-lapse video clip at
http://www2.umdnj.edu/zhlabweb/video/ToxinB.avi). These observations
suggest that toxin B did not block taxol-induced growth cone turning
through inhibition of the actin dynamics. Rather, it is likely that
toxin B blocked taxol-induced turning through its specific inhibition
of Rho GTPases signaling. These results demonstrate that the
MT-directed steering mechanism involves the actin cytoskeleton. Local
stabilization and growth of MTs on one side of the growth cone is
sufficient to initiate the steering event by inducing site-directed,
actin-based protrusion, possibly through the activation of the Rho
GTPases (e.g., Rac1), to complete the turning. With the existence of
molecules that are capable of interacting with both cytoskeletal
networks (Vega and Solomon, 1997; Waterman-Storer and Salmon, 1999), it
is conceivable that coordinated efforts between the microtubules and
actin cytoskeleton are fundamental for successful steering of the
growth cone in specific directions.
| |
DISCUSSION |
In this study, we have presented three pieces of direct evidence
to demonstrate an essential and instructive role of microtubules in
growth cone steering. First, we show that both attraction and repulsion
induced by diffusible guidance gradients can be blocked by inhibition
of the MT dynamics through low concentrations of MT-specific drugs. We
then demonstrate, to our knowledge for the first time, that local
modification of the MT dynamics in the growth cone is sufficient to
induce growth cone steering: local stabilization and growth induce
growth cone attraction, and local destabilization causes growth cone
repulsion. Finally, we show that the actin cytoskeleton actively
participates in MT-initiated growth cone turning; local MT
stabilization and growth likely activate the Rho family of GTPases to
induce the actin-based protrusive activities to complete the turning
process. These results indicate the existence of a novel steering
mechanism in which microtubules play a leading role for growth cone turning.
The actin filaments and microtubules are two fundamental cytoskeletal
components for growth cone motility (Mitchison and Kirschner, 1988).
There is ample evidence to support a major role for the actin
cytoskeleton in directional movement of nerve growth cones: (1) actin
filaments are concentrated at the peripheral region of the growth cone
that is tightly associated with motility (Smith, 1988; Lin and
Forscher, 1995), (2) disruption of the actin-based filopodia and
lamellipodia blocks growth cone turning induced by extracellular cues
(Bentley and Toroian-Raymond, 1986; Chien et al., 1993; Zheng et al.,
1996a), and (3) families of actin-associated proteins have been
identified and implicated in various signaling pathways to regulate the
dynamics of the actin cytoskeleton (Hall, 1998; Pollard et al., 2000).
The precise function of MTs and their associated proteins in growth
cone motility, particularly in directional growth cone steering, has
not been elucidated. Although neuronal processes are still capable of
extension when the actin cytoskeleton is disrupted by cytochalasins
(Marsh and Letourneau, 1984; Bradke and Dotti, 1999), it is generally
thought that the major function of MTs is to consolidate and provide
mechanical support to the new extension initiated by the actin
cytoskeleton. Local actin-based motility initiates and leads the
steering sequence that involves subsequent local stabilization of these
dynamic MTs followed by selective MT growth (Mitchison and Kirschner,
1988; Bentley and O'Connor, 1994). Recently, however, an increasing
body of evidence suggests that MTs may play a more active role in
directional motility of the growth cone (Tanaka and Kirschner, 1995;
Tanaka et al., 1995; Challacombe et al., 1997; Mack et al., 2000; Dent
and Kalil, 2001). Dynamic microtubule ends have been observed to
explore the actin-rich P-region of the growth cone, and inhibition of these dynamic MT ends in the growth cone was found to abolish repulsive
turning at the substrate border (Tanaka and Kirschner, 1995; Williamson
et al., 1996; Challacombe et al., 1997). In this study, we used low
concentrations of MT-specific drugs to test the role of MTs in growth
cone turning in response to diffusible cues. Low concentrations of
MT-stabilizing (taxol) or MT-destabilizing (nocodazole and vinblastine)
drugs inhibit free dynamic microtubule ends in the growth cone P-region
but do not inhibit axon extension, alter growth cone morphology, or
grossly disrupt the cytoskeletal architecture. Both attractive and
repulsive turning responses to guidance gradients were abolished by the
presence of these drugs in the medium. These results have further
substantiated the previous findings in growth cone repulsion at the
substrate boundary and have established the fundamental importance of
dynamic microtubule ends in both growth cone attraction and repulsion to diffusible guidance cues.
The most significant finding of this study is the demonstration of an
instructive role of microtubules in growth cone turning. Through direct
focal application of the MT-specific drugs taxol and nocodazole to one
side of the growth cone, we show that local modification of MT dynamics
in the growth cone is sufficient to induce directional steering of the
growth cone. Both FLIP and pipette techniques allowed us to introduce
MT-specific drugs in a spatially restricted manner within the growth
cone. When MTs are locally stabilized, the growth cone turns toward the
site of stabilization, whereas local destabilization results in turning away from the site of destabilization. It should be emphasized that
both taxol and nocodazole are applied in a local and pulsatile manner
either through photoactivation or through pipette application. These
MT-specific drugs are thus spatially restricted to only a small region
of the growth cone at low concentrations. For example, the peak taxol
concentration reaching the growth cone is estimated to be ~10
nM (corresponding to 5 µM in pipette) or less
on the application side but much lower on the distal side of the growth cone. Therefore, local and pulsatile application of these MT drugs is
most likely to produce only local effects on MT dynamics without grossly inhibiting MT dynamics in the entire growth cone, thus allowing
the growth and turning of the growth cone. Our live imaging of
fluorescent MTs showed that local taxol application promoted MT growth
to the side of taxol application. The effects of taxol certainly depend
on its concentrations as well as its spatial distribution; a high
concentration of taxol applied to the entire growth cone and the cell
would likely result in the inhibition of growth cone extension. The
live imaging of fluorescent MTs itself, however, appeared to exert some
adverse effect on dynamic MTs in the P-region, which prevented detailed
studies on the dynamics of MTs in the growth cone during turning
induced by local MT drugs. Such studies would be important for a better
understanding of how local MT dynamics regulate the growth cone
steering. In particular, with an improved dual-wavelength live-imaging
method of both cytoskeletal systems (Salmon et al., 2002), one would be
able to address how the local MT dynamics affect the local dynamics of
the actin cytoskeleton.
Our turning results were obtained using spatially restricted
MT-specific drugs. It is possible that intracellular signaling components downstream of guidance receptors use a similar strategy to
promote MT stabilization followed by local polymerization in a region
of the growth cone that will ultimately determine the direction of new
extension. Conversely, the observation that depolymerization of the
dynamic MTs on one side of the growth cone results in a turning
response in the other direction indicates that an alternate strategy
could be to inhibit local microtubule extension to promote a turn in
the opposite direction. Although our pipette application method does
not exclusively restrict the MT drugs to only one side of the growth
cone, the turning responses to focal taxol and nocodazole application
appeared to be as significant as those induced by guidance gradients of
netrin-1 and glutamine. During guidance, spatially and temporally
restricted signaling events could result in much more localized
modification of MT dynamics to effectively steer the growth cone. It
would be of great interest to examine whether and how some specific
intracellular signals (e.g., Ca2+)
influence local microtubule polymerization, depolymerization, or a
combination of both during turning responses to guidance cues.
Finally, the finding that the actin cytoskeleton participates in
MT-initiated growth cone turning reveals that the interplay between
these two cytoskeletal systems is critically important for directional
movement of the growth cone. Our conclusion is based on three
experimental observations: (1) high-resolution, time-lapse imaging
revealed preferential lamellipodial protrusion on the side of focal
taxol application, (2) complete blockade of taxol-induced growth cone
attraction by cytochalasin D, and (3) abolition of taxol-induced growth
cone attraction by toxin B, a toxin that specifically inactivates the
Rho family small GTPases (Just et al., 1995). These findings are
consistent with the emerging evidence on the importance of interaction
between these two cytoskeletal systems in cell motility (Dent and
Kalil, 2001; Salmon et al., 2002; Schaefer et al., 2002). Additionally, more recent findings directly demonstrate the interaction between microtubules and the Rho family small GTPases (Fukata et al., 2002;
Krendel et al., 2002), further suggesting a role for Rho GTPases in
microtubule and actin interaction. Together, our results indicate an
MT-initiated steering mechanism that requires coordinated activities
between these two cytoskeleton systems. The identification of signaling
pathways that interact with and/or target MTs (Gundersen and Cook,
1999) provides the potential for signals from the extracellular environment to regulate MT dynamics to control the directional motility
of growth cones. It is conceivable that different extracellular cues
may act through different signaling pathways that could target the
actin cytoskeleton and/or MTs independently, but the concerted efforts
between these two systems are required to enable the growth cone to
respond effectively to guidance cues. It is possible that signals
elicited by extracellular cues may act either as primary signals that
locally modify MTs as a first step in initiation or as reinforcing
signals in a feedback loop downstream of actin. Our data demonstrate
that at the very least, localized perturbations in microtubule dynamics
are indeed sufficient to initiate and direct new growth cone extension.
Thus, the next step is to identify and determine the role of some of
the signaling molecules that target MT dynamics in growth cone
guidance. Nevertheless, a two-way steering system in which either
microtubules or the actin cytoskeleton can take the driver's seat
clearly provides developing axons with advantages to effectively
respond to a wide variety of guidance cues.
| |
FOOTNOTES |
Received May 3, 2002; revised July 19, 2002; accepted Aug. 6, 2002.
This work was supported by a grant from the National Institutes of
Health. We thank Dr. Marc Tessier-Lavigne (Stanford University, Stanford, CA) for providing us the purified netrin-1 proteins and J. Gibney for her technical assistance throughout the entire project.
Correspondence should be addressed to Dr. James Zheng, Department of
Neuroscience and Cell Biology, University of Medicine and Dentistry of
New Jersey-Robert Wood Johnson Medical School, 675 Hoes Lane,
Piscataway, NJ 08854. E-mail: james.zheng{at}umdnj.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
