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Volume 17, Number 14,
Issue of July 15, 1997
pp. 5455-5465
Copyright ©1997 Society for Neuroscience
Nerve Growth Factor Stimulates the Accumulation of  1 Integrin
at the Tips of Filopodia in the Growth Cones of Sympathetic Neurons
Peter W. Grabham and
Daniel J. Goldberg
Department of Pharmacology and Center for Neurobiology and
Behavior, Columbia University, New York, New York 10032
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Addition of nerve growth factor (NGF) to sympathetic neurons that
have been starved of it causes a rapid induction of growth cone
motility and the resumption of neurite growth. Using immunofluorescence staining, we show that within 10 min, NGF stimulated the accumulation of dense aggregates of  1 integrin [a receptor for extracellular matrix (ECM) proteins] at most of the tips of either newly extended or
preexisting filopodia. This effect occurred in the absence of ECM
proteins and in the presence of 1 mg/ml Arg-Gly-Asp-Ser peptide, which
blocks ECM binding to integrin, indicating that occupation of the
integrin receptor is not necessary for tip localization. In fact,
addition of either laminin or fibronectin caused a rapid withdrawal of
1 integrin aggregates from filopodial tips at a rate comparable to
that of the rearward flow of actin filaments in the periphery of the
growth cone. Surface labeling of the extracellular domain of 1
integrin while aggregated at the tips of filopodia or withdrawing in
response to ECM proteins showed that the receptor is positioned within
the membrane. The drug butanedione monoxime, an inhibitor of myosins,
blocked the accumulation of 1 integrin at the tips of filopodia
without inhibiting the formation of filo-podia, suggesting the
involvement of a myosin motor in 1 integrin transport. These results
provide the first evidence of NGF-mediated accumulation of ECM
receptors to sensory elements of the growth cone and suggest one
mechanism whereby soluble and substrate-bound cues coordinate to
produce directed neurite growth.
Key words:
nerve growth factor;
1 integrin;
sympathetic neuron;
growth cone;
filopodium;
extracellular matrix
INTRODUCTION
The rate and direction of neurite growth are
tightly regulated by external cues: positive and negative, soluble and
surface-bound, cellular and acellular (Baier and Bonhoeffer, 1994 ;
Goodman, 1996; Tessier-Lavigne and Goodman, 1996). The motile growth
cone at the end of the growing neurite is a prime target for these
cues; changes in growth cone behavior cause changes in neurite growth (Kapfhammer and Raper, 1987; O'Connor et al., 1990; Rivas et al., 1992; Lin and Forscher, 1995). The digitate filopodia, which can project many micrometers forward from the body of the growth cone, are
particularly important (Bentley and Toroian-Raymond, 1986; Caudy and
Bentley, 1986; Hammarback and Letourneau, 1986; Bandtlow et al., 1990;
Zheng et al., 1996).
Nerve growth factor (NGF) is a widely studied soluble promoter of
neurite growth. A member of the family of neurotrophins, it promotes
neurite growth from sympathetic neurons, some primary sensory neurons,
and, probably, from certain neurons in the CNS (Levi-Montalcini, 1987).
The promotion of neurite growth is effected by binding of NGF to the
trkA receptor, an integral membrane protein tyrosine kinase (Loeb et
al., 1991).
Laminin 1 [mouse Engelbroth-Holm-Swarm (EHS) laminin] and
fibronectin promote neurite growth as surface-bound proteins. They are
abundant in the extracellular matrix (ECM); ECM proteins can also be
found on the surface of certain cells that form pathways for neurite
growth (Rogers et al., 1986; Bixby et al., 1988; Reichardt and
Tomaselli, 1991). Laminin 1 and fibronectin on the substrate in culture
dishes promote neurite growth from a variety of vertebrate neurons,
including sensory and sympathetic neurons (Baron-van Evercooren et al.,
1982; Rogers et al., 1983), by binding to integrin receptors,
heterodimeric integral membrane proteins (Bozyczko and Horwitz, 1986;
Tomaselli et al., 1987; Reichardt and Tomaselli, 1991; Hynes, 1992).
The integrins that mediate the promotion of neurite growth by these ECM
proteins contain the 1 subunit (Bozyczko and Horwitz, 1986;
Tomaselli et al., 1987; Tomaselli et al., 1990; Reichardt and
Tomaselli, 1991).
Thus, process outgrowth from certain types of neurons, such as
sympathetic and primary sensory neurons, is stimulated both by the
soluble neurotrophins and the substrate-bound ECM proteins. The
mechanisms by which these molecules act rapidly and locally on the
growth cone to stimulate elongation seem to differ, however. Whereas
NGF stimulates actin-based protrusive and motile activities (Seeley and
Greene, 1983; Aletta and Greene, 1988), laminin 1 facilitates the
advancement of microtubules and membrane-bound organelles into
protrusive structures to begin their conversion into new neuritic
length (Rivas et al., 1992).
Growth cones are subject to multiple environmental cues as they
navigate (Goodman, 1996). There may be an element of redundancy to
provide a safety margin for correct guidance. But, also, there may be
combinatorial effects, which could greatly increase the information
supplied by a limited number of guidance molecules (Goodman, 1996).
Some long-term interactions between different types of cues have been
described. For example, treatment of PC12 cells with NGF increases the
expression of integrin at the surface over a period of days (Zhang et
al., 1993). We describe here a novel type of short-term interaction in
which NGF rapidly causes the accumulation of 1 integrin at the tips
of filopodia of sympathetic growth cones. Thus, the soluble
neurotrophin regulates the presentation of the ECM receptor to the
environment, moving it rapidly to where it may be able to interact more
effectively with surface-bound ligand.
MATERIALS AND METHODS
Materials. 2,3-Butanedione monoxime (BDM),
Arg-Gly-Asp-Ser (RGDS) peptide, fibronectin from bovine plasma,
cytochalasin D, anti-talin monoclonal antibody (8d4), and anti-1
integrin (W1B10) and anti-vinculin (human VIN-1) monoclonal antibodies
were obtained from Sigma (St. Louis, MO). NGF and laminin 1 (from mouse
EHS sarcoma) were from Boehringer Mannheim (Indianapolis, IN). Texas Red-phalloidin was obtained from Molecular Probes (Eugene, OR). Chickie II anti-1 integrin polyclonal antibody was a gift from Dr.
C. Buck (Wistar Institute, Philadelphia, PA), G2 anti-actin polyclonal
antibody was a gift from Dr. J. C. Bulinski (Columbia University, New
York, NY), and anti-growth-associated protein (GAP)-43 polyclonal
antibody was a gift from Dr. M. Willard (Washington University, St.
Louis, MO). ES66-8 rat hybridoma cells were generously provided by Drs.
K. M. Yamada (National Institutes of Health, Bethesda, MD) and A. F. Horwitz (University of Illinois, Urbana, IL). ES66 is a monoclonal
antibody directed against chicken 1 integrin, which does not block
function. The antibody was purified from hybridoma supernatants by
ammonium sulfate precipitation, followed by anion exchange
chromatography (Duband et al., 1988).
Cell culture. Sympathetic ganglia were dissected from
embryonic day 10-13 chick embryos, washed in HBSS, and dissociated by using 0.25% trypsin for 10 min at 37°C. After pelleting, cells were
plated onto coverslips precoated with 50 µg/ml
poly-D-lysine and 50 µg/ml poly-L-ornithine
in 0.1 M sodium borate, pH 8.0. To allow the use of larger
sample numbers with parallel conditions, multiwell dishes with
coverslips glued to the underside were used. Coverslips were
preincubated with RPMI 1640 medium containing 10% heat-inactivated
horse serum for 2 hr at 37°C, and cells were incubated in L15 medium
supplemented with 10% fetal bovine serum, glutamine, and 50 ng/ml NGF.
For cultures in which filopodia are already present after NGF
starvation, coverslips were preincubated in serum-free defined medium
(L15 supplemented with 2% BSA, glutamine, 10 µg/ml transferrin, 5 µg/ml insulin, 5 ng/ml sodium selenite, and 100 mM sodium
pyruvate), and cells were cultured in the same medium plus 50 ng/ml
NGF. After 13-15 hr of incubation at 37°C in 5%
CO2, quiescence of growth cones was induced by
removal of NGF and other serum factors. For both culture types, 5 ml of
medium was replaced every 30 min with serum-free defined medium for the first hour and then every hour for 4-5 hr (serum-supplemented cultures) or 6-8 hr (serum-free cultures), and then with L15 alone for
the last hour (both types of cultures). NGF and other reagents were
added to individual wells after aspiration of the last wash. All
experiments were performed at 37°C. Cultures were determined to be
>80% neurons as shown by immunoreactivity to GAP-43.
Immunocytochemistry. Cells were fixed by the addition of 5 ml of PBS, pH 7.4, containing 4% paraformaldehyde, 0.1%
glutaraldehyde, and 400 mM sucrose for 5 min at 37°C,
followed by one rinse and three 5 min washes in PBS and 0.15% Triton
X-100. After blocking with 10% normal goat serum (NGS) in PBS and
0.15% Triton X-100 for 30 min at room temperature (rt), cells were
incubated in either a 1:100 dilution (polyclonal antiserum) or a 5-10
µg/ml concentration (monoclonal antibodies) of primary antibody
diluted in PBS/1% NGS and 0.015% Triton X-100. Incubation times were
either 1 hr at rt (anti-actin and anti-GAP-43) or overnight at 4°C
(anti-1 integrin, anti-vinculin, and anti-talin). For double
staining, primary antibodies were applied consecutively. Cells were
then washed in PBS and incubated in a fluorescence-conjugated secondary antibody diluted 1:100 in PBS/1% NGS for 1 hr at rt. For most analyses, cells were double-stained with Chickie II anti-1 integrin followed by fluorescein-conjugated anti-rabbit IgG and 0.33 µM Texas Red-phalloidin. Controls included replacement
of the primary antibody with normal serum. To confirm staining of actin
and 1 integrin, we stained with the anti-1 integrin monoclonal
antibody (W1B10) and then the anti-actin polyclonal antibody (G2),
followed by a mixture of Texas Red-mouse IgG and fluorescein-rabbit
IgG. After a final wash in PBS, cells were mounted in 20 mg/ml propyl gallate in 90% glycerol/10% PBS. For surface staining of 1
integrin, cells were fixed without detergent extraction, quenched by
incubation in 0.1 M glycine/PBS for 5 min at rt, and then
stained for 1 integrin (using W1B10) as described above.
Analysis of formation of filopodia and integrin accumulation at
tips. Analyses were performed on cultures double-stained for 1
integrin and F-actin and viewed using filter sets that visualized fluorescein alone, Texas Red alone, or both simultaneously (Omega Optical, Brattleboro, VA). Tip staining of 1 integrin was seen as
green fluorescence at the tip of a Texas Red-phalloidin-stained filopodium. Any 1 integrin aggregates slightly behind the tips were
recognized against the F-actin stain and scored negative. To determine
whether tip staining was an artifact attributable to swelling of
filopodial tips, unconjugated Texas Red was used to label proteins
indiscriminately (Coates et al., 1992). More than 95% of tips were
seen to terminate without swelling. Tip staining was also assessed in a
blind study using silicon intensified target camera images. Digitized
images of growth cones stained for 1 integrin and Texas Red were
thresholded for pixel brightness with a lower limit that included all
lamellipodial regions. Those filopodia with tips but not shafts that
exceeded the threshold limit were scored positive. After 10 min of NGF
treatment, 87% of tips scored positive with anti-1 integrin
(Chickie II) staining compared with none with Texas Red. Threshold
counts were comparable to manual counts. Counts were therefore made
manually by traversing the stained culture and scoring both numbers of
filopodia and tip staining or tip staining alone (see Fig.
3A,B). For values per growth cone, at least 100 growth cones
were scored, and SDs were calculated. For values as percentages, at
least 200 filopodia were scored. For the determination of 1 integrin
aggregates in the distal and proximal halves of growth cones, at least
25 growth cones were scored. Statistical t tests using the
Bonferroni method for multiple comparisons (Wallenstein et al., 1980)
were performed when appropriate.
Fig. 3.
ECM proteins are not necessary for the
accumulation of 1 integrin at filopodial tips but cause aggregates
of 1 integrin in growth cones previously treated with 100 ng/ml NGF
to migrate rearward along filopodia. A, NGF causes the
accumulation of 1 integrin tip aggregates (0-10 min) in the
presence of 1 mg/ml RGDS peptide ( ), much the same as NGF alone
( ). Addition of 20 µg/ml fibronectin at 10 min
(arrowhead) stimulates a rapid decrease in aggregates of
1 integrin at filopodial tips in the absence but not the presence of
RGDS peptide. Results are representative of three separate experiments
(n = 200). B, Laminin 1 also causes a rapid delocalization of aggregates of 1 integrin from filopodial tips when added at a concentration of 20 µg/ml (indicated by the arrowhead). Results are representative of three separate
experiments (n = 200). C, Typical
growth cone treated with 100 ng/ml NGF for 10 min followed by 5 min of
treatment with 20 µg/ml laminin 1. Aggregates of 1 integrin,
visualized by immunofluorescence staining, can be seen situated along
filopodial shafts (arrow). Scale bar, 10 µm.
D, Laminin 1 causes rearward migration of aggregates of 1 integrin. Cells were treated as in C, and filopodia
were scored for aggregates of 1 integrin at the tips and in the
distal (excluding tips) and proximal halves of filopodia. Results are
representative of three separate experiments (n = 25). E, 1 Integrin aggregates are inserted in the
membrane while at the tips of filopodia and during rearward migration.
Cells were pretreated with NGF for 10 min, followed by 3 min of
treatment with fibronectin, surface-stained for 1 integrin, and then
scored as in D (n = 25). In
D and E, error bars show SDs.
Asterisks denote those values that are significantly different (p < 0.005) from control (0 min) values. Diamonds denote those values that are
significantly different (p < 0.05) from control values.
[View Larger Version of this Image (35K GIF file)]
RESULTS
Changes in the organization of actin and the localization of 1
integrin induced by readdition of NGF
Withdrawal and subsequent readdition of NGF to cultures of
sympathetic neurons produces a change in growth cones from a nonmotile, club-shaped phenotype to a highly motile structure with filopodia and
ruffling membranes (Seeley and Greene, 1983; Aletta and Greene, 1988).
In the present study, we used double immunofluorescence staining of
fixed chick embryonic sympathetic neurons to visualize changes in the
actin cytoskeleton and the distribution of 1 integrin during the
onset of motility. Although growth cones exhibit much variation within
a culture, NGF withdrawal concomitant with a sequential elimination of
other trophic factors on a
poly-D-lysine/poly-L-ornithine substrate
resulted in a synchronization of morphology that enabled us to
quantitate analysis of events after the readdition of NGF.
The distribution of F-actin and 1 integrin in a typical quiescent
growth cone is shown in Figure 1, A
and B. The growth cone is club-shaped and has few remaining
filopodia (mean number ± SD of filopodia per growth cone,
1.9 ± 1.9). F-actin, visualized by staining with Texas
Red-phalloidin, is mostly arranged diffusely in the peripheral region,
with some small dense aggregates. 1 Integrin, visualized by staining
with a polyclonal antibody, is distributed diffusely throughout the
growth cone. One minute after the readdition of 100 ng/ml NGF,
intensely stained patches of F-actin appeared close to the leading edge
of the growth cone (Fig. 1C). 1 Integrin was also
concentrated in some of these patches (Fig. 1D). Many
smaller aggregates of F-actin did not contain 1 integrin. By 3 min
after addition of NGF, numerous filopodia had extended, and F-actin
patches were larger and fused into a band filling the peripheral region
of the growth cone (Fig. 1E) Some aggregates of 1
integrin remained in this region, but much was distributed along newly
formed filopodia (Fig. 1F). Filopodia continued to extend during the next few min until, by 10 min after addition of NGF, growth cones had an average of 15.5 ± 2.6 filopodia, and 1 integrin was concentrated at the tips of most of
them (Fig. 1G-I). This was confirmed by additional
double staining with an antibody that recognizes actin (G2) and a
second anti-1 integrin antibody (W1B10). The focal adhesion
proteins, vinculin and talin, did not co-localize with 1 integrin at
the tips of filopodia, although we did detect these proteins
co-localized with the patches of 1 integrin that appeared in the
peripheral region after 1 min of NGF treatment (data not shown).
Fig. 1.
NGF rapidly induces the formation of filopodia and
the accumulation of 1 integrin at filopodial tips. Representative
growth cones were fixed at various times after addition of 100 ng/ml NGF and double-stained for detection of F-actin and 1 integrin by
fluorescence microscopy. Left panels show F-actin, and
right panels show 1 integrin. A, B,
The growth cone has few filopodia, and 1 integrin is distributed
unremarkably before NGF is added. C, D, One minute after
the addition of NGF, patches of F-actin and 1 integrin appear close
to the leading edge of the growth cone. E, F, Filopodia
have begun to extend by 3 min after the addition of NGF. 1 Integrin
can be found in extending filopodia. G, H, By 10 min,
filopodia have fully extended, and 1 integrin is concentrated at the
tips. Scale bar in A, 10 µm. I,
Quantitation of the number of filopodia (solid line) and
accumulation of 1 integrin (broken line) at
filopodial tips per growth cone after addition of NGF. The appearance
of tip aggregates lags somewhat behind the formation of filopodia.
Values are representative of three separate experiments. Sample
number = 100. Error bars show SDs. Asterisks denote
those values that are significantly different (p < 0.005) from control (0 min)
values.
[View Larger Version of this Image (54K GIF file)]
Accumulation of 1 integrin at filopodial tips is not dependent
on filopodial extension
A potential mechanism by which 1 integrin becomes localized to
filopodial tips is one in which aggregates of the receptor are situated
in the early (1 min) F-actin aggregates such that they can then
"ride" out on the tips of extending filopodia. We investigated this
possibility by exploiting a different manipulation of the culturing
conditions, which allows the retention of filopodia in the absence of
NGF. This was achieved by culturing cells in a serum-free medium.
Before removal of NGF, 53% of filopodia showed aggregates of 1
integrin. The distribution of F-actin and 1 integrin in a typical
growth cone several hours after removal of NGF is shown in Figure
2,A and B. There are numerous
filopodia, and F-actin is organized not only diffusely but also in
dense aggregates in the rear of the peripheral region and in the cores of some filopodia. 1 Integrin, however, is again distributed evenly
in the peripheral region of the growth cone, with some concentration at
the leading edge but no appreciable aggregation at filopodial tips.
Readdition of NGF resulted in the extension of veils of membrane rather
than filopodia (Fig. 2C); in fact, the mean number of
filopodia remained at ~30 per growth cone (Fig. 2E). Moreover, the appearance of patches of F-actin
and 1 integrin was not observed at the leading edge of the growth
cone 1 min after the addition of NGF (data not shown), as seen in cells
that had been initially cultured in serum. 1 Integrin did, however, rapidly accumulate at the tips of filopodia (Fig.
2D). Indeed, tip staining increased dramatically from
an average of 0.9 ± 2.5 to 22.8 ± 6.2 stained tips per
growth cone (Fig. 2E). It is possible that instead of
1 integrin accumulating at filopodial tips it is already present,
and NGF is causing an antigenic site on the ECM receptor to become
unmasked. However, additional double staining with the anti-actin
polyclonal antibody and two monoclonal antibodies that recognize
separate sites on the extracellular domain of 1 integrin (W1B10 and
ES66) showed a similar staining pattern, suggesting that this is not
the case.
Fig. 2.
Accumulation of 1 integrin at filopodial tips
is not dependent on the extension of filopodia. A, B,
Representative growth cone that had been cultured in serum-free
conditions, starved of NGF, and double-stained for F-actin
(A) and 1 integrin (B). Filopodia are present, and 1 integrin is distributed evenly along their length. C, D, Representative growth cone cultured
as in A and B, treated with 100 ng/ml NGF
for 10 min, and double-stained for F-actin (C)
and 1 integrin (D). Numerous veils have
extended (C, arrow), and filopodia show accumulation of
1 integrin at their tips. Scale bar in D, 10 µm.
E, Quantitation of the accumulation of 1 integrin at
filopodial tips. Although the number of filopodia per growth cone
remains constant, the number of tips with concentrations of 1
integrin increases after treatment with NGF. Results are representative
of three separate experiments (n = 100). Error bars
show SDs. Asterisks denote those values that are
significantly different (p < 0.005) from
control (0 min) values for tip staining.
[View Larger Version of this Image (64K GIF file)]
Although the localization of 1 integrin to tips was not dependent on
the formation of new filopodia, we found that it was sensitive to the
action of the F-actin-destabilizing drug cytochalasin D. Growth cones
were treated for 5 min before the addition of NGF with a concentration
of cytochalasin D (1 µM) that is sufficient to prevent
actin polymerization without greatly disrupting existing filopodia (Wu
et al., 1996). Under these conditions, only 9% of filopodia showed
integrin tip aggregates compared with 68% in control cultures.
Effect of ECM proteins on the accumulation of 1 integrin at
filopodial tips
The culture procedure described in the previous section permitted
the detection and quantitation of the accumulation of 1 integrin at
the tips of filopodia in the absence of added serum or purified ECM
proteins. We therefore used this paradigm to determine the effects of
ECM proteins on three aspects of 1 integrin relocalization: first,
to see whether ECM proteins can induce 1 integrin tip accumulation
independently of NGF; second, to confirm that ECM proteins are not
necessary for NGF to induce tip accumulation of 1 integrin; and
third, to determine the effect of ECM proteins on 1 integrin that
had previously accumulated at filopodial tips in response to NGF.
ECM proteins did not cause 1 integrin to accumulate at the tips of
filopodia. Addition of either 20 µg/ml fibronectin or 20 µg/ml
laminin 1 to NGF-starved neurons resulted in an accumulation of 1
integrin tip aggregates no greater than the control (<2% of the tips
had aggregates after 10 min, compared with 74% in paired dishes to
which NGF alone was readded) (data not shown).
Although there was no addition of serum or ECM proteins to the culture
medium or the substrate in this protocol, we could not be sure that ECM
proteins were not present, either in residual amounts from the
dissociation of the original ganglionic tissue or secreted by cells in
culture. We therefore examined NGF-mediated tip localization in the
presence of 1 mg/ml RGDS peptide, which blocks the binding of ECM
proteins such as fibronectin and vitronectin to 1 integrin. The rate
and extent of tip accumulation of 1 integrin were unaffected by the
RGDS peptide (Fig. 3A). After 10 min of NGF
treatment, 64% of filopodial tips showed 1 integrin aggregates,
compared with 67% in the presence of RGDS, providing more evidence
that occupancy of 1 integrin by ECM proteins is not necessary for
tip accumulation to be induced by NGF.
In parallel dishes of the same experiment, we added 20 µg/ml
fibronectin to cells that had been treated with NGF for 10 min. The ECM
protein stimulated a rapid reduction in the number of tip aggregates
(Fig. 3A). The percentage of filopodial tips with aggregates
of 1 integrin was nearly halved within 1 min, and, by 10 min, only
16% of the tips displayed aggregates. Interestingly, as the tip
staining reduced, we observed distinct aggregates of 1 integrin
situated proximally along the length of the filopodia. RGDS peptide
inhibited the action of fibronectin; in the presence of the peptide,
the percentage of tips with aggregates remained at around 60 for the 10 min after addition of fibronectin. Laminin 1 had the same effect as
fibronectin. Figure 3B shows the effect of laminin 1 on 1
integrin previously aggregated at filopodial tips. As seen with
fibronectin, laminin 1 caused a rapid reduction in tips with aggregates
of 1 integrin. In 1 min, the percentage of tips with aggregates
decreased from 60 to 25, and by 5 min it had reached a basal level of
11%.
The appearance of distinct aggregates of 1 integrin situated along
the length of filopodia (Fig. 3C) suggests that laminin 1 and fibronectin cause the aggregates of 1 integrin to migrate toward
the central region of the growth cone. We examined this possibility by
counting the number of aggregates of 1 integrin at the tip and in
the proximal and distal halves (excluding the tip) of each filopodium
during the first 5 min after addition of laminin 1. As the mean number
of tip aggregates per growth cone decreased from 9.0 ± 2.9 to
5.5 ± 1.4 during the first 2 min, the mean number of distal
aggregates increased from 1.3 ± 0.4 to 4.7 ± 1.2 (Fig.
3D). Between 2 and 5 min proximal aggregates became most
numerous, increasing from 0.9 ± 0.6 to 4.7 ± 1.1 per growth
cone. The mean length of filopodia did not change dramatically, and the
mean number of filopodia per growth cone remained constant throughout
(data not shown).
To determine whether 1 integrin is inserted in the membrane, we
performed a similar experiment on cells that had not been permeabilized. After fixing, staining was performed using the W1B10
integrin antibody, which is directed against the extracellular domain
of 1 integrin (Fig. 3E). The same pattern of integrin staining was observed both after NGF treatment, when aggregates are at
the tips of filopodia, and after subsequent addition of ECM protein (in
this case fibronectin), when aggregates migrate rearward along the
filopodia.
Pharmacological inhibition of myosin prevents formation of tip
aggregates of 1 integrin
The fact that the formation of aggregates of 1 integrin at the
tips of filopodia in response to NGF is rapid and can occur in
previously extended filopodia suggested that it results from the active
transport of 1 integrin. Because the peripheral region of the growth
cone, including filopodia, has an actin cytoskeleton, we thought the
accumulation might be myosin-driven. We therefore used BDM, a
pharmacological inhibitor of endogenous myosin ATPase activity (Cramer
and Mitchison, 1995), to test this possibility. To compare the
NGF-mediated formation of tip aggregates of 1 integrin with a
separate cellular response that might not involve a myosin motor, we
adopted the culture procedure used in Figure 1. Thus, we were able to
determine the effect of BDM on both the accumulation of tip aggregates
and the formation of filopodia. Pretreatment of neurons with 15 mM BDM for 5 min before the addition of NGF fully blocked
the accumulation of 1 integrin at the tips of filopodia, whereas the
formation of filopodia was only modestly inhibited. 20 min after the
addition of NGF the number of filopodia had increased from 2.5 ± 2.3 to 9.2 ± 2.4 per growth cone in the presence of BDM, compared
with an increase from 1.8 ± 1.8 to 14.4 ± 2.5 per growth
cone in the control. 1 Integrin tip aggregates, however, remained at
0.5 ± 0.7 per growth cone in the presence of BDM, compared with
an increase from 0.2 ± 0.4 to 11.1 ± 2.9 per growth cone
without BDM (Fig. 4,A and B). The
formation of the early (1 min) aggregates of F-actin and 1 integrin
at the leading edge of the peripheral region was, in contrast, observed to occur in the presence of BDM (data not shown).
Fig. 4.
Treatment of cells with 15 mM BDM for
5 min before the addition of 100 ng/ml NGF (A,
B) blocks the accumulation of 1 integrin at
filopodial tips. A, A representative growth cone treated
as above and stained for 1 integrin has numerous filopodia but no accumulation of 1 integrin at filopodial tips. B,
Counts of filopodia and tip aggregates per growth cone demonstrate
that, after treatment with BDM (as in A), the
accumulation of 1 integrin at filopodial tips is blocked, but the
formation of filopodia is only modestly reduced compared with parallel
control cultures (as shown). Error bars show SDs. Results are
representative of three separate experiments (n = 100). C, 1 Integrin aggregates remain either at the
tips or in the distal halves of filopodia when 15 mM BDM is
added to cells in which 1 integrin had previously accumulated at
tips in response to 100 ng/ml NGF. A typical growth cone shows that the
location of most 1 integrin aggregates is either at or just behind
the tip (arrow) 5 min after the addition of BDM. Scale bar, 10 µm. D, Counts of 1 integrin aggregates at
either the tip or in the distal (excluding the tip) and proximal halves
of filopodia show that, after 5 min of treatment, although BDM causes the number of tip aggregates to decrease, most remain in the distal half of filopodia. Results are representative of three separate experiments (n = 25). Error bars show SDs.
Asterisks denote those values that are significantly
different (p < 0.005) from control (0 mM) values.
[View Larger Version of this Image (70K GIF file)]
It is possible that we did not detect tip aggregates of 1
integrin not because BDM blocked the forward movement of 1 integrin, but rather because it prevented its retention at the tip. Thus, we did
another set of experiments in which tip aggregates were allowed to form
in response to NGF, and then BDM was added. Figure 4, C and
D, shows that, although 5 min of treatment with 15 mM BDM caused a significant reduction in tip aggregates,
this was attributable not to a disappearance of aggregates but to a
slight shift rearward from the tip. Although tip aggregates decreased from a mean value of 12.9 ± 2.2 to 8.7 ± 2.0 per growth
cone, distal aggregates increased from a mean value of 1.7 ± 1.4 to 6.4 ± 1.8, and proximal aggregates remained unchanged (Fig.
4D).
DISCUSSION
These studies of changes in the distribution of 1 integrin on
the surface of sympathetic growth cones in response to NGF and ECM
proteins are significant in two ways. First, they provide evidence of a
novel interaction between different types of environmental cues
regulating axon growth, one that occurs rapidly at the growth cone.
This type of interaction could be important in the turning of growing
axons toward the source of a chemoattractant, a means of guidance for
which the mechanism is not well understood. Second, in showing that the
binding of ligand links the receptor to a motor in the peripheral
region of the growth cone, these experiments provide evidence of a
particular mechanism of axon growth promotion by surface-bound ligands.
We discuss these two points below.
A neurotrophin regulates the distribution of a receptor for ECM
proteins on the growth cone
We find that NGF stimulates the rapid accumulation of 1
integrin at the tips of growth cone filopodia. Thus, a soluble
neurotrophin regulates the distribution of the receptor for ECM
proteins, which are typically surface-bound. By regulating the
distribution of 1 integrin on the growth cone, NGF may regulate the
sensitivity of the growth cone to ECM-type ligands. Filopodia are the
primary structures of the growth cone for interacting with
environmental cues (Bentley and Toroian-Raymond, 1986; Caudy and
Bentley, 1986) and thus serve as sensory elements of the growth cone.
The tip of the filopodium is not only a favorable site for encountering a cue, as when the grasshopper Ti1 filopodium touches a guidepost cell
(Caudy and Bentley, 1986; O'Connor et al., 1990). For 1 integrin,
at least, it may also be a preferred site for transducing the binding
of ligand into effects on the growth cone, because 1 integrin
attaches to the underlying actin cytoskeleton most readily at the
distal edges of the growth cone (Schmidt et al., 1995).
Chemoattraction, the turning of a growing axon toward a diffusible cue,
is an important mechanism of guidance in the developing nervous system
(Baier and Bonhoeffer, 1994; Goodman, 1996; Tessier-Lavigne and
Goodman, 1996). This turning is led by the growth cone, but the
mechanism of growth cone turning toward the source is unclear. Orientation of filopodia might be an early event (Gundersen and Barrett, 1980; Zheng et al., 1996). The effect we have described here
raises the possibility that the chemoattractant orients receptors for
the surface-bound cue that is promoting growth. If the relationship between chemoattractant and surface-bound cue was as described here for
NGF and ECM ligands, a gradient of chemoattractant across the growth
cone might be expected to concentrate receptors for the surface-bound
cue in protrusive structures (filopodia and veils) on one side of the
growth cone. These would thus interact more productively with the
surface-bound cue than would protrusions on the other side, and the
growth cone would turn. An interesting aspect of this mechanism is that
the surface-bound cue contributes to guidance while being homogeneously
distributed.
NGF causes redistribution of 1 integrin in the absence of
ECM protein
NGF affects the distribution of 1 integrin without needing the
receptor to have bound ECM protein. Accumulation of 1 integrin at
the tips of filopodia occurred rapidly in serum-free medium on a
substrate precoated only with poly-L-lysine (Fig. 2). It is
possible that small amounts of ECM proteins were either carried over
from tissue dissociation or synthesized in culture. However, the
NGF-stimulated accumulation of 1 integrin at filopodial tips was not
reduced by RGDS peptide (Fig. 3A), which blocks the
interaction of 1 integrin with fibronectin and vitronectin. In fact,
rather than the tip accumulation of 1 integrin being enhanced by
addition of ligand for 1 integrin (either laminin 1 or fibronectin),
it was greatly reduced (Fig. 3). Moreover, binding of fibronectin or
laminin in the absence of NGF failed to induce tip accumulation, indicating that there is a requirement for NGF in this process.
Recent work from Hotchin and Hall (1995) and Nobes and Hall (1995) has
shown that peptide growth factor can rapidly alter the surface
distribution of integrin in fibroblasts. We think the phenomenon we
have described here represents a different action of the growth factor,
an important distinction being its independence from ligand binding to
integrin. Platelet-derived growth factor causes the rapid formation of
aggregates of integrin at the periphery of fibroblasts only on a
substrate coated with an ECM protein such as fibronectin (Hotchin and
Hall, 1995; Nobes and Hall, 1995). In contrast to the tip aggregates we
have described here, these "focal complexes" do not form in
fibroblasts plated on poly-L-lysine. The focal complex also
differs from the tip aggregate in containing high concentrations of
several proteins that are found in classic focal adhesions, such as
F-actin, paxillin, and vinculin. We do not detect these in appreciable
amounts in the tip aggregates (Wu et al., 1996; this paper). Their
absence from the tip aggregates probably reflects the requirement for
binding of 1 integrin to ECM protein to recruit these other proteins
into complexes with integrin (Miyamoto et al., 1995a,b). We think that
the accumulation of 1 integrin at filopodial tips relies on the
ability of NGF to stimulate actin-based motility (see below), whereas
the formation of focal complexes may not. Thus, the elicitation of
focal complexes in fibroblasts is unaffected by cytochalasin D (Nobes
and Hall, 1995), which inhibits actin polymerization and causes F-actin to withdraw rapidly from the peripheral region of the growth cone (Forscher and Smith, 1988), whereas the accumulation of 1 integrin at filopodial tips in response to NGF is blocked.
ECM proteins cause rearward migration of 1 integrin
There is a steady rearward flow of actin filaments in the
peripheral region of the growth cone (Forscher and Smith, 1988; Okabe
and Hirokawa, 1991). It has been suggested that coupling of the
substrate to this flow could power advance of the growth cone or at
least generate tension within the growth cone (Mitchison and Kirschner,
1988; Goldberg et al., 1991; Lin and Forscher, 1995); recent evidence
supports this idea (Lin and Forscher, 1995). Because the actin
apparently flows continually rearward at a steady rate, whereas growth
and the generation of tension are irregular (Lamoureux et al., 1989),
it was suggested that attachment between the actin network and growth
cone receptors for substrate-bound molecules is intermittent (Mitchison
and Kirschner, 1988; Lin and Forscher, 1995). In fact, 1
integrin on the surface of the growth cone and other motile cells is
usually not attached to the rearward flow (Schmidt et al., 1995;
Felsenfeld et al., 1996).
We present evidence here that the binding of ECM protein to integrin
induces linkage to the rearward flow; recently, a similar finding was
reported for the lamellipodium of motile fibroblasts (Felsenfeld et
al., 1996). Addition of laminin 1 or fibronectin caused aggregates of
1 integrin that had formed at filopodial tips in response to NGF to
withdraw distoproximally along the filopodia. The withdrawal, which
occurs in the plane of the plasma membrane, was dependent on the
binding of ECM protein to the receptor, because the RGDS peptide
inhibited it. Although we cannot be sure that this rearward movement
was mediated by actin, it seems likely. Half of the aggregates had
withdrawn several micrometers (far enough to reach the proximal half of
the filopodium) within 5 min of addition of ligand, implying a rate of
recession in the range of the few micrometers per minute of rearward
flow of actin (Lin and Forscher, 1995). Actin is the major cytoskeletal
element of the filopodium (Letourneau and Ressler, 1983; Bridgman and Dailey, 1989), and no other mechanism for rearward transport of membrane proteins in the plane of the plasma membrane has been described for the peripheral region of the growth cone or of vertebrate non-neuronal motile cells.
Integrins are thought to work by binding to substrate-bound ligand
(Schwartz et al., 1995), as befits receptors that mediate processes
such as cell adhesion. Yet we find here that the binding of
non-substrate-bound ligand to 1 integrin is apparently sufficient to
engage the rearward flow mechanism. Some of the laminin 1 or fibronectin that we add in soluble form may well adhere to the substrate, but integrins in the growth cone membrane that bind to these
substrate-bound ligand molecules would remain stationary with respect
to the substrate rather than moving rearward; that is how tension would
be generated. Thus, the integrin molecules we see receding are
presumably bound to ligand that is not substrate-bound. Because
integrin cross-linking facilitates its interaction with the
cytoskeleton (Miyamoto et al., 1995a,b; Felsenfeld et al., 1996), the
ability of soluble laminin and fibronectin to induce rearward flow may
stem from their binding as a multimer. Consistent with this is the
inability of the monomeric ligand RGDS to induce rearward flow when
used at a concentration sufficient to occupy many of the receptors (as
judged by its ability to block the effect of fibronectin). Whatever the
mechanism, the results here provide evidence that substantial signaling
through integrin can be induced by soluble ligand.
Mechanism of delivery of 1 integrin to the tips
of filopodia
1 Integrin must form the tip aggregates by moving outward along
filopodia rather than riding out on the tips of newly forming filopodia, because the appearance of aggregates lagged behind the
formation of filopodia and occurred even in preexisting filo-podia. The distal edge of the growth cone and the lamellipodium of motile fibroblasts, particularly regions of tightly curved plasma membrane, can retain certain membrane proteins, including 1 integrin (Sheetz et al., 1990; Schmidt et al., 1993, 1995), so even diffusion of molecules of 1 integrin in the plane of the plasma membrane of the
filopodium might result in their accumulation at the tip. In this case,
NGF could promote the formation of aggregates by fostering the
retention of 1 integrin at the tip. We have previously presented
evidence suggesting that protein-tyrosine phosphorylation is essential
for the maintenance of the tip aggregates (Wu et al., 1996). NGF might
promote retention by its ability to stimulate tyrosine phosphorylation
(Maher, 1988). Alternatively, 1 integrin could reach the tip of the
filopodium by directed transport. In this case, NGF could promote the
formation of tip aggregates by stimulating this transport or by
promoting retention at the tip. Molecules of 1 integrin do undergo
directed forward movements in the plane of the plasma membrane in the
peripheral region of the growth cone (Schmidt et al., 1995). 1
Integrin could also be transported within filopodia in membrane-bound
vesicles and incorporated into the plasma membrane at the tips by
exocytosis, as has been suggested for non-neuronal motile cells
(Bretscher, 1996). However, vesicles have not been typically observed
in filopodia during observations of living growth cones with
video-enhanced contrast differential interference microscopy (Goldberg
and Burmeister, 1986; Forscher et al., 1987; Sheetz et al., 1990) or by
electron microscopic analysis of fixed specimens (Tosney and Wessells, 1983).
One piece of evidence for the importance of directed transport in the
formation of tip aggregates is the action of BDM. Aggregates did not
form in the presence of this drug. Clearly, BDM interfered with the
delivery, but not the retention, of 1 integrin, because aggregates
were retained at or near the tip when BDM was added after their
formation. Because F-actin is the major cytoskeletal constituent of the
peripheral region of growth cones and non-neuronal motile cells and
myosins are the only known actin-activated motor proteins, myosin is
the prime candidate for a transport motor in this region. The directed
forward transport in the plane of the plasma membrane of certain
proteins in growth cones was suggested to be powered by a myosin
(Sheetz et al., 1990). BDM strongly inhibits actin-activated ATPase
activity of some, and perhaps many or all, myosins (Cramer and
Mitchison, 1995). It is effective when applied to intact cells; for
example, it has been used to show an involvement of myosin in powering
the rearward flow of F-actin in the peripheral region of the growth
cone (Lin et al., 1996). It does not inhibit actin polymerization or
disrupt actin filaments (Cramer and Mitchison, 1995; Lin et al., 1996);
the present results showing nearly normal NGF-induced growth of
filopodia in the presence of BDM confirm that. Although the inhibition
of tip accumulation by BDM suggests an involvement of myosin,
additional work will be needed to confirm this, because BDM may not be
completely specific for myosins. (Zhu and Ikeda, 1993; Sellin and
McArdle, 1994).
A cycle for 1 integrin in the growth cone
The present results, in the context of previous work discussed
above, are consistent with the hypothetical scheme depicted in Figure
5. In this scheme, based on previous models of actin flow (Mitchison and Kirschner, 1988; Goldberg et al., 1991; Lin and
Forscher 1995), there is a cycle in which 1 integrin is transported to the distal edge (in the case under study, the tips of filopodia) and
binds ligand, which causes the integrin to link to the underlying network of actin filaments, which flows steadily rearward. If the
ligand is surface-bound, as is typical with ligands for 1 integrin
and as depicted in Figure 5, tension can be generated. Also, an
advancing growth cone will grow over the immobilized integrin. If the
ligand is soluble, the integrin will move rearward along the
filopodium, as seen in the present experiments. In this scheme, both
the forward and rearward transport of 1 integrin are powered by
actomyosin, although different species of myosin may be involved in the
two movements. The forward transport and probably the rearward as well
are stimulated by NGF. The key difference is that binding of ligand to
1 integrin is required for efficient coupling to the rearward, but
not the forward, transport. Thus, NGF turns on the transport machinery,
whereas the ECM protein is a coupling switch.
Fig. 5.
Hypothetical model for the coordinated action of
NGF and ECM proteins in a filopodium. A, In the
quiescent state, 1 integrin () is distributed evenly, and the
actin cytoskeleton (open triangles) is static.
B, NGF induces forward transport of 1 integrin to the
tip of the filopodium. It also promotes the polymerization of actin,
which can lead to protrusion. It is hypothesized that retrograde flow
of actin is also stimulated by NGF, but because 1 integrin couples
to this retrograde flow only in the presence of ECM ligand, 1
integrin accumulates at the tip. C, In the presence of
an ECM substrate (open circles) 1 integrin continues
to be transported forward. Aggregates located at the tip of the
filopodium bind to rearward flowing actin filaments probably via a
complex of other proteins (gray shapes) creating
tension and/or promoting growth.
[View Larger Version of this Image (35K GIF file)]
FOOTNOTES
Received Feb. 19, 1997; revised April 30, 1997; accepted May 8, 1997.
This work was supported by National Institutes of Health Grants NS25161
and GM32099. We thank Drs. C. Buck, C. Bulinski, and M. Willard for
their generous gifts of antibodies, Drs. K. Yamada and A. Horwitz for
the ES66-8 hybridoma cell line, Rachel Yarmolinsky and Eve Vagg for
image processing and photography, and Thu V. Chu for blind studies.
Correspondence should be addressed to Dr. Peter W. Grabham, Department
of Pharmacology, Columbia University, 630 West 168th Street, New York,
NY 10032.
REFERENCES
-
Aletta JM,
Greene LA
(1988)
Growth cone configuration and advance: a time-lapse study using video-enhanced differential interference contrast microscopy.
J Neurosci
8:1425-1435[Abstract].
-
Baier H,
Bonhoeffer F
(1994)
Attractive axon guidance molecules.
Science
265:1541-1542[Free Full Text].
-
Bandtlow C,
Zachleder T,
Schwab ME
(1990)
Oligodendrocytes arrest neurite growth by contact inhibition.
J Neurosci
10:3837-3848[Abstract].
-
Baron-van Evercooren A,
Kleinman HD,
Ohno S,
Marangos P,
Schwartz JP,
Dubois-Dalq ME
(1982)
Nerve growth factor, laminin and fibronectin promote nerve growth in human fetal sensory ganglia cultures.
J Neurosci Res
8:179-183[ISI][Medline].
-
Bentley D,
Toroian-Raymond A
(1986)
Disoriented pathfinding by pioneer neurone growth cones deprived of filopodia by cytochalasin treatment.
Nature
323:712-715[Medline].
-
Bixby JL,
Lilien J,
Reichardt LF
(1988)
Identification of the major proteins that promote neuronal process outgrowth on Schwann cells in vitro.
J Cell Biol
107:353-361[Abstract/Free Full Text].
-
Bozyczko D,
Horwitz AF
(1986)
The participation of a putative cell surface receptor for laminin and fibronectin in peripheral neurite extension.
J Neurosci
6:1241-1251[Abstract].
-
Bretscher MS
(1996)
Moving membrane up to the front of migrating cells.
Cell
85:465-467[ISI][Medline].
-
Bridgman PC,
Dailey ME
(1989)
The organization of myosin and actin in rapid frozen nerve growth cones.
J Cell Biol
108:95-109[Abstract/Free Full Text].
-
Caudy M,
Bentley D
(1986)
Pioneer growth cone steering along a series of neuronal and non-neuronal cues of different affinities.
J Neurosci
6:1781-1795[Abstract].
-
Coates TD,
Watts RG,
Hartman R,
Howard TH
(1992)
Relationship of F-actin distribution to development of polar shape in human polymorphonuclear neutrophils.
J Cell Biol
117:765-774[Abstract/Free Full Text].
-
Cramer LP,
Mitchison TJ
(1995)
Myosin is involved in postmitotic cell spreading.
J Cell Biol
131:179-189[Abstract/Free Full Text].
-
Duband J-L,
Nuckolls GH,
Ishihara A,
Hasegawa T,
Yamada KM
(1988)
Fibronectin receptor exhibits high lateral mobility in embryonic locomoting cells but is immobile in focal contacts and fibrillar streaks in stationary cells.
J Cell Biol
107:1385-1396[Abstract/Free Full Text].
-
Felsenfeld DP,
Choquet D,
Sheetz MP
(1996)
Ligand binding regulates the directed movement of 1 integrin on fibroblasts.
Nature
383:438-440[Medline].
-
Forscher P,
Smith SJ
(1988)
Actions of cytochalasins on the organization of actin filaments and microtubules in a neuronal growth cone.
J Cell Biol
107:1505-1516[Abstract/Free Full Text].
-
Forscher P,
Kaczmarek LK,
Buchanen JA,
Smith SJ
(1987)
Cyclic AMP induces changes in distribution and transport of organelles within growth cones of Aplysia bag cell neurons.
J Neurosci
7:3600-3611[Abstract].
-
Goldberg DJ,
Burmeister DW
(1986)
Stages in axon formation: observations of growth of Aplysia axons in culture using video-enhanced contrast-differential interference microscopy.
J Cell Biol
103:1921-1931[Abstract/Free Full Text].
-
Goldberg DJ,
Burmeister DW,
Rivas RJ
(1991)
Video microscopic analysis of events in the growth cone underlying axon growth and the regulation of these events by substrate-bound proteins.
In: The nerve growth cone (Letourneau PC,
Kater SB,
Macagno ER,
eds), pp 79-95. New York: Raven.
-
Goodman CS
(1996)
Mechanisms that control growth cone guidance.
Annu Rev Neurosci
19:341-377[ISI][Medline].
-
Gundersen RW,
Barrett JN
(1980)
Characterization of the turning response of dorsal root neurites towards NGF.
J Cell Biol
87:546-554[Abstract/Free Full Text].
-
Hammarback JA,
Letourneau PC
(1986)
Neurite extension across regions of low cell-substratum adhesivity: implications for the guidepost hypothesis of axonal pathfinding.
Dev Biol
117:655-662[ISI][Medline].
-
Hotchin NA,
Hall A
(1995)
The assembly of integrin adhesion complexes requires both extracellular matrix and intracellular rho/rac GTPases.
J Cell Biol
131:1857-1865[Abstract/Free Full Text].
-
Hynes RO
(1992)
Integrins: versatility, modulation, and signaling in cell adhesion.
Cell
69:11-25[ISI][Medline].
-
Kapfhammer JP,
Raper JA
(1987)
Collapse of growth cone structure on contact with specific neurites in culture.
J Neurosci
7:201-212[Abstract].
-
Lamoureux P,
Buxbaum RE,
Heidemann SR
(1989)
Direct evidence that growth cones pull.
Nature
340:159-162[Medline].
-
Letourneau PC,
Ressler AH
(1983)
Differences in the organization of actin in the growth cones compared with the neurites of cultured neurons from chick embryos.
J Cell Biol
97:963-973[Abstract/Free Full Text].
-
Levi-Montalcini R
(1987)
The nerve growth factor 35 years later.
Science
237:1154-1162[Free Full Text].
-
Lin C-H,
Forscher P
(1995)
Growth cone advance is inversely proportional to retrograde F-actin flow.
Neuron
14:763-771[ISI][Medline].
-
Lin C-H,
Espreafico EM,
Mooseker MS,
Forscher P
(1996)
Myosin drives retrograde F-actin flow in neuronal growth cones.
Neuron
16:769-782[ISI][Medline].
-
Loeb DM,
Maragos J,
Martin-Zanca D,
Chao MV,
Parada LF,
Greene LA
(1991)
The trk proto-oncogene rescues NGF responsiveness in mutant NGF-nonresponsive PC12 cell lines.
Cell
66:961-966[ISI][Medline].
-
Maher PA
(1988)
Nerve growth factor induces protein-tyrosine phosphorylation.
Proc Natl Acad Sci USA
85:6788-6791[Abstract/Free Full Text].
-
Mitchison TJ,
Kirschner M
(1988)
Cytoskeletal dynamics and nerve growth.
Neuron
1:761-772[ISI][Medline].
-
Miyamoto S,
Akiyama SK,
Yamada KM
(1995a)
Synergistic roles for receptor occupancy and aggregation in integrin transmembrane function.
Science
267:883-885[Abstract/Free Full Text].
-
Miyamoto S,
Teramoto H,
Coso OA,
Gutkind JS,
Burbelo PD,
Akiyama SK,
Yamada KM
(1995b)
Integrin function: molecular hierarchies of cytoskeletal signaling molecules.
J Cell Biol
131:791-805[Abstract/Free Full Text].
-
Nobes CD,
Hall A
(1995)
Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia.
Cell
81:53-62[ISI][Medline].
-
O'Connor TP,
Duerr JS,
Bentley D
(1990)
Pioneer growth cone steering decisions mediated by single filopodial contacts in situ.
J Neurosci
10:3935-3946[Abstract].
-
Okabe S,
Hirokawa N
(1991)
Actin dynamics in growth cones.
J Neurosci
11:1918-1929[Abstract].
-
Reichardt LF,
Tomaselli KJ
(1991)
Extracellular matrix molecules and their receptors: functions in neural development.
Annu Rev Neurosci
14:531-570[ISI][Medline].
-
Rivas RJ,
Burmeister DW,
Goldberg DJ
(1992)
Rapid effects of laminin on the growth cone.
Neuron
8:107-115[ISI][Medline].
-
Rogers SL,
Letourneau PC,
Palm SL,
McCarthy J,
Furcht LT
(1983)
Neurite extension by peripheral and central nervous system neurons in response to substrate-bound fibronectin and laminin.
Dev Biol
98:212-220[ISI][Medline].
-
Rogers SL,
Edson KJ,
Letourneau PC,
McLoon SC
(1986)
Distribution of laminin in the developing nervous system of the chick.
Dev Biol
113:429-435[ISI][Medline].
-
Schmidt CE,
Horwitz AF,
Lauffenburger DA,
Sheetz MP
(1993)
Integrin-cytoskeletal interactions in migrating fibroblasts are dynamic, asymmetric, and regulated.
J Cell Biol
123:977-991[Abstract/Free Full Text].
-
Schmidt CE,
Dai J,
Lauffenburger DA,
Sheetz MP,
Horwitz AF
(1995)
Integrin-cytoskeletal interactions in neuronal growth cones.
J Neurosci
15:3400-3407[Abstract].
-
Schwartz MA,
Schaller MD,
Ginsberg MH
(1995)
Integrins: emerging paradigms of signal transduction.
Annu Rev Cell Biol
11:549-599[ISI][Medline].
-
Seeley PJ,
Greene LA
(1983)
Short latency actions of nerve growth factor at the growth cone.
Proc Natl Acad Sci USA
80:2789-2793[Abstract/Free Full Text].
-
Sellin LC,
McArdle JJ
(1994)
Multiple effects of 2,3-butanedione monoxime.
Pharmacol Toxicol
74:305-313[ISI][Medline].
-
Sheetz MP,
Baumrind NL,
Wayne DB,
Pearlman AL
(1990)
Concentration of membrane antigens by forward transport and trapping in neuronal growth cones.
Cell
61:231-241[ISI][Medline].
-
Tessier-Lavigne M,
Goodman CS
(1996)
The molecular biology of axon guidance.
Science
274:1123-1133[Abstract/Free Full Text].
-
Tomaselli KJ,
Damsky CH,
Reichardt LF
(1987)
Interactions of a neuronal cell line (PC12) with laminin, collagen IV, and fibronectin: identification of integrin-related glycoproteins involved in attachment and process outgrowth.
J Cell Biol
105:2347-2358[Abstract/Free Full Text].
-
Tomaselli KJ,
Hall DE,
Flier LA,
Gehlsen KR,
Turner DC,
Carbonetto S,
Reichardt LF
(1990)
A neuronal cell line (PC12) expresses two 1-class integrins-
 11 and 31-that recognize different neurite outgrowth-promoting domains in laminin.
Neuron
5:651-662[ISI][Medline]. -
Tosney KW,
Wessells NK
(1983)
Neuronal motility: the ultrastructure of veils and microspikes correlates with their motile activities.
J Cell Sci
61:389-411[Abstract].
-
Wallenstein S,
Zucker CL,
Fleiss JL
(1980)
Some statistical methods useful in circulation research.
Circ Res
47:1-9[Abstract/Free Full Text].
-
Wu D-Y,
Wang L-C,
Mason CA,
Goldberg DJ
(1996)
Association of 1 integrin with phosphotyrosine in growth cone filopodia.
J Neurosci
16:1470-1478[Abstract/Free Full Text].
-
Zhang Z,
Tarone G,
Turner DC
(1993)
Expression of integrin alpha 1 beta 1 is regulated by nerve growth factor and dexamethasone in PC12 cells. Functional consequences for adhesion and neurite outgrowth.
J Biol Chem
268:5557-5565[Abstract/Free Full Text].
-
Zheng JQ,
Wan JJ,
Poo M
(1996)
Essential role of filopodia in chemotropic turning of nerve growth cone induced by a glutamate gradient.
J Neurosci
16:1140-1149[Abstract/Free Full Text].
-
Zhu Y,
Ikeda SR
(1993)
2,3-Butanedione monoxime blockade of Ca2+ currents in adult rat sympathetic neurons does not involve "chemical phosphatase" activity.
Neurosci Lett
155:24-28[ISI][Medline].
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