The Journal of Cell Biology, Volume 158, Number 7, September 30, 2002 1219–1228
The Rockefeller University Press, 0021-9525/2002/09/1219/10 $5.00
Actin turnover is required to prevent axon retraction
driven by endogenous actomyosin contractility
Hal F. Yee, Jr.,
and Paul C. Letourneau
Department of Neuroscience, University of Minnesota, Minneapolis, MN 55455
Department of Medicine and Physiology, University of California Los Angeles, Los Angeles, CA 90095
rowth cone motility and guidance depend on the
dynamic reorganization of filamentous actin
In the growth cone, F-actin undergoes turnover,
which is the exchange of actin subunits from existing fila-
ments. However, the function of F-actin turnover is not
clear. We used jasplakinolide (jasp), a cell-permeable macro-
cyclic peptide that inhibits F-actin turnover, to study the
role of F-actin turnover in axon extension. Treatment with jasp
caused axon retraction, demonstrating that axon extension
requires F-actin turnover. The retraction of axons in response
to the inhibition of F-actin turnover was dependent on
myosin activity and regulated by RhoA and myosin light
chain kinase. Significantly, the endogenous myosin-based
contractility was sufficient to cause axon retraction, because
jasp did not alter myosin activity. Based on these observations,
we asked whether guidance cues that cause axon retraction
(ephrin-A2) inhibit F-actin turnover. Axon retraction in
response to ephrin-A2 correlated with decreased F-actin
turnover and required RhoA activity. These observations
demonstrate that axon extension depends on an interaction
between endogenous myosin-driven contractility and F-actin
turnover, and that guidance cues that cause axon retraction
inhibit F-actin turnover.
The extension and guidance of axons depends on the activity
of the growth cone. Growth cones are highly motile, actively
extending and retracting filopodia and lamellipodia. The
protrusion of lamellipodia and filopodia requires the poly-
merization of filamentous actin (F-actin)* at the leading
edge of the growth cone (for review see Suter and Forscher,
2000). After polymerization, F-actin is retrogradely transported
toward the center of the growth cone by a myosin-based
mechanism and is subsequently depolymerized (Lin et al.,
1996). In the growth cone, F-actin is dynamic and undergoes
turnover (Okabe and Hirokawa, 1990; Mallavarapu and
Mitchison, 1999). F-actin turnover is the result of poly-
merization at the barbed ends of filaments in conjunction
with depolymerization at the pointed ends, resulting in filament
“treadmilling” (Pollard et al., 2000). Estimates of the rate of
F-actin turnover in the lamellipodia of growth cones indicate
that F-actin is completely recycled by turnover within 3–5 min,
whereas filopodial actin bundles are more stable (Mallavarapu
and Mitchison, 1999). However, although the role of F-actin
polymerization in driving leading-edge protrusion is well
established, the significance of F-actin turnover after poly-
merization is not known.
Growth cones “pull” by generating contractile forces
(Lamoureux et al., 1989), and axon tension is an important
component of axon extension (Heidemann and Buxbaum,
1994). The generation of cellular contractile forces often
depends on actomyosin-based contractility. Antisense-mediated
down-regulation of myosin IIA and IIB has been shown to
inhibit neurite extension in neuro-2A cells (Wylie and
Chantler, 2001), and myosins are involved in the protrusion
of filopodia and lamellipodia (Jay, 2000; Bridgman et al.,
2001). Importantly, the retrograde transport of F-actin in
growth cones is driven by myosin motors (Lin et al., 1996).
These reports indicate that myosins are required for axon
Although myosins have been shown to be required for
axon extension, myosin-based contractility also has been
associated with the process of axon retraction. Myosin activity
drives the retraction of axons in response to the perturbation
of microtubule motor proteins (Ahmad et al., 2000). Similarly,
the activity of the GTPase RhoA and its downstream kinase
ROCK, which drive actomyosin-based contractility, cause
Address correspondence to Gianluca Gallo at his present address, Dept.
of Neurobiology and Anatomy, Drexel Univ. College of Medicine, 2900
Queen Ln., Philadelphia, PA 19129. Tel.: (215) 991-8288. Fax: (215)
843-9082. E-mail: Neurite@aol.com
*Abbreviations used in this paper: BDM, 2,3-butanedione monoxime;
CaRhoA, constitutively active RhoA; DRG, dorsal root ganglion; jasp,
jasplakinolide; lat-A, latrunculin-A; MLCK, myosin light chain kinase;
mRLC, myosin regulatory light chains; RGC, retinal ganglion cell; skMyo-
sin II, skeletal muscle myosin II.
Key words: jasplakinolide; RhoA; ephrin; cytoskeleton; myosin
1220 The Journal of Cell Biology
Volume 158, Number 7, 2002
neurite retraction (Kozma et al., 1997; Katoh et al., 1998).
Furthermore, axon guidance cues that cause retraction act
through a RhoA-dependent mechanism (Katoh et al., 1996;
Kranenburg et al., 1999; Wahl et al., 2000). Negative regu-
lation of RhoA or ROCK activity prevents the retraction of
established axons in vivo (Billuart et al., 2001). Collectively,
these studies demonstrate that myosin activity is required for
both axon extension and retraction. Thus, how myosin-
based contractility is regulated to contribute to axon exten-
sion versus retraction is an important issue in the biology of
axon extension and guidance.
In this paper, we demonstrate that pharmacological inhi-
bition of F-actin turnover causes myosin-dependent axon re-
traction and inhibition of axon extension. Additionally, we
provide evidence that the repellent guidance cue (ephrin-
A2) decreases the turnover of F-actin in retinal axons and
causes RhoA-dependent axon retraction. These data reveal
for the first time that axon extension and retraction depend
on an interaction between F-actin turnover and myosin-
Jasplakinolide (jasp) causes growth cone contraction
and axon retraction
To determine the response of growth cones to the inhibition
of F-actin turnover, we investigated the effects of jasp on ret-
inal and dorsal root ganglion (DRG) neuron cultures. jasp is
a cell-permeable macrocyclic peptide that binds to F-actin
and inhibits depolymerization at the pointed ends of fila-
ments, thereby inhibiting filament turnover. jasp has been
used to investigate actin turnover in a number of cell types
(Lee et al., 1998; Cramer, 1999; Ayscough, 2000; Lauter-
milch and Spitzer, 2000; Watanabe and Mitchison, 2002).
Videomicroscopic observations of both retinal and DRG
axons demonstrated that after treatment with jasp, growth
cone motility, defined as lamellipodial and filopodial exten-
sion, stopped and centripetal contraction of the peripheral
domain begun within 3–5 min after treatment (Fig. 1 A).
After growth cone contraction, axons started to retract 4–6
min after treatment with jasp (Fig. 1 B). The percentage of
axons retracting was maximal at 40 nM jasp (Fig. 1 C). Dur-
ing retraction, in response to 40 nM jasp, 73% of axons be-
came deformed and bent (Fig. 1 B). The response of growth
cones and axons to jasp was the same in embryonic chick
DRG cells and retinal ganglion cells (RGC), and embryonic
mouse–cerebellar granule cells (unpublished data). Addi-
tionally, axons retracted in response to jasp when cultured
on glass coated with either laminin or polylysine (unpub-
lished data), indicating that the effects of jasp are not medi-
ated by a disruption of integrin-based signaling. Treatment
of cultures with phalloidin, a peptide that stabilizes F-actin
through a mechanism similar to that of jasp, also caused
axon retraction (unpublished data).
We tested the effects of jasp treatment on long-term axon
growth. DRG explants were cultured for 24 h in the pres-
ence of 1–40 nM jasp, and the length of axons was com-
pared with vehicle-treated controls. jasp inhibited DRG
axon extension (Fig. 1 D). In addition to the dose-depen-
dent inhibition of axonal length by jasp, an inhibitory effect
on the number of axons extended from explants also became
evident at 10 nM (unpublished data). Cultures treated with
in individual panels indicate minutes relative to
the addition of jasp (40 nM at 0 min). The bars in
the leftmost panels denote 10 ?m. (A) Within 2–3
min after treatment with jasp, growth cones
became quiescent and underwent contraction
followed by axonal retraction. (B) Axons began
retracting 4–6 min after treatment with jasp. Axon
retraction often resulted in the formation of axonal
bends (indicated by arrowheads at 8 min). Note
that at 6 min after treatment, an additional retracting
growth cone becomes visible (arrow). (C) jasp
caused axon retraction during a 20-min observation
period in a concentration-dependent manner
(n ? 37 axons at each concentration). (D) Overnight
treatment of DRG explant cultures with jasp
caused a dose-dependent decrease in the length of
axon outgrowth (n ? 8 explants per concentration).
(E) Localized application of jasp to growth cones
causes contraction and axon retraction. Bends in
axons form distally after growth cone contraction
(arrow). (F) Application of jasp to axons does not
cause the formation of axon bends at the site of
application, but after prolonged application and
diffusion of jasp, axons retract with bends first
forming at the distal end of axons. The pipette was
removed before acquisition of the images from
which the montage is created. The pipette position
is denoted by the ? symbol. Bars, 10 ?m.
jasp induces axon retraction. Numbers
Actin turnover and myosin regulate axon extension |
Gallo et al. 1221
ence in the concentration dependence of the inhibitory ef-
fects of jasp on axon number relative to the inhibition of
axon length may be attributable to the observation that ax-
ons retract in response to the acute addition of jasp at con-
10 nM. Similar results were obtained using
retinal explants (unpublished data).
To determine the site of action of jasp responsible for
axon retraction, we investigated the effects of applying jasp
locally to growth cones or axons. Localized delivery of jasp
to growth cones caused them to contract and retract (Fig. 1
E). In these cases, bends first became evident along the dis-
tal most 20–40
m behind the growth cone as it started to
retract (Fig. 1 E, arrow). Conversely, localized application
of jasp to axons did not cause the formation of bends.
However, after prolonged delivery of jasp to axons, diffu-
sion of jasp to the distal growth cones was sufficient to
cause axon retraction and bending, which proceeded in a
distal-to-proximal manner, although the concentration of
jasp was greatest along the proximal portion of the axon
(Fig. 1 F). These observations indicate that jasp acts at the
growth cone to cause axon retraction. Pipette delivery of
medium with vehicle had no effect on growth cones and
10 nM jasp had only sparse axonal outgrowth. The differ-
jasp is rapidly internalized and inhibits F-actin
turnover in growth cones
We determined the role of F-actin in mediating jasp-in-
duced axon retraction. Cultures were treated first with la-
trunculin-A (lat-A), a drug that results in net depolymeriza-
tion of F-actin by binding cytosolic G-actin, rendering it
incompetent for polymerization. 5
90% of growth cone F-actin within 5 min of treatment
(unpublished data), resulting in growth cone collapse but
not axon retraction. Pretreatment of cultures with lat-A fully
inhibited jasp-induced axon retraction, demonstrating that
the effects of jasp treatment require F-actin (Fig. 2 A).
jasp binds F-actin at the same site as phalloidin (Bubb et
al., 1994). Therefore, internalization of jasp can be moni-
tored by first treating live growth cones with jasp, and subse-
quently determining the reduction in staining with fluo-
rochrome-labeled phalloidin. The relative percentage of
F-actin bound by jasp can be determined by measuring the
fluorescence intensity of phalloidin staining in jasp-treated
growth cones relative to the intensity in control growth
cones. A 10-min treatment with jasp was chosen because at
this time point axons are undergoing retraction. jasp bound
to 52, 85, 95, and 95% of growth cone F-actin at 5, 10, 20,
and 40 nM, respectively (Fig. 2, B and C). Because growth
M lat-A caused the loss
binds to F-actin, and inhibits its turnover.
(A) Treatment of DRG cultures with
40 nM jasp caused axon retraction
(?m/20 min). Treatment with lat-A
(LatA) did not cause axon retraction,
but blocked the effects of jasp (n ? 32
axons per group). (B) Phalloidin staining of
DRG axons treated with DMSO or 40 nM
jasp (C) for 10 min. Bar (B) 10 ?M.
Note the largely diminished staining in
jasp-treated axons. D and E are pseudo-
colored images of the same growth
cone double stained with phalloidin
and the antiactin antibody, respectively.
Note the almost identical staining
pattern demonstrating that using the
combined fixation-extraction protocol,
the antibody staining is reflective of
F-actin in growth cones. (F–I) Actin
antibody staining of growth cones
treated for 4 min with either DMSO
(F and G) or 40 nM jasp (H and I) and
then for an additional 2 min with either
DMSO (F and H) or 2 ?M lat-A (LatA;
G and I). Bars (D– I) 5 ?M. Note that
although lat-A causes the depoly-
merization of the majority of F-actin in
DMSO-treated growth cones, it has
only a partial effect on growth cones
treated first with jasp. jasp causes the
centripetal accumulation of growth
cone F-actin as revealed by actin
antibody staining. J and K are examples
of growth cones treated for 10 min with
DMSO or 40 nM jasp, respectively.
Note the accumulation of F-actin as
reflected by the presence of warmer
jasp is rapidly internalized,
colors (red and yellow) in the contracted growth cone treated with jasp. Bar (J), 10 ?M. L is an example of the deformations (red arrows)
of the axonal microtubule array that develop in axons undergoing jasp induced axon retraction (40 nM for 10 min).
1222 The Journal of Cell Biology
Volume 158, Number 7, 2002
cone contraction in response to jasp starts as early as 2 min
after treatment, we determined the binding of jasp to F-actin
after 2 min of treatment. Within 2 min of treatment, 40 nM
jasp was internalized and bound F-actin to a similar degree
(95%) as a 10-min treatment (unpublished data). Therefore,
jasp is rapidly internalized and binds to F-actin before
changes in growth cone motility and axon retraction.
To relate the effects of long-term treatment with jasp on
axon extension (Fig. 1 D) to the binding of jasp to F-actin,
we determined the decrease in phalloidin staining of growth
cones treated with 1–2.5 nM jasp for 24 h. Axon length was
decreased by 20 and 44%, which corresponds to jasp bind-
ing to 57 and 70% of available phalloidin binding sites on
F-actin, at 1 and 2.5 nM, respectively. These data indicate
that jasp can inhibit the long-term extension of axons by
binding to 57–70% of available binding sites on F-actin in
Depolymerization of F-actin by lat-A is dependent on the
rate of filament turnover. We tested the ability of lat-A to
cause the depolymerization of F-actin in growth cones
treated with jasp relative to control growth cones. Staining
F-actin with phalloidin is not possible in jasp-treated growth
cones (see previous section). Therefore, we used an antiactin
antibody to detect F-actin. Because these experiments sought
to detect only F-actin and not monomeric actin, we adopted
a combined fixation–extraction protocol previously used
to immunocytochemically visualize microtubules in growth
cones without interference from soluble tubulin (Gallo and
Letourneau, 1999). This protocol results in actin antibody
staining that exhibits the same staining pattern as phalloidin
(Fig. 3, A and B), and is thus representative of F-actin.
jasp inhibits growth cone F-actin turnover. Growth cones
were treated with 40 nM jasp or vehicle (DMSO) for 3 min
before treatment with 2
M lat-A for 2 min. In control
growth cones, a 2-min lat-A treatment depolymerized 85%
of F-actin. However, lat-A depolymerized only 40% of
F-actin in growth cones pretreated for 3 min with 40 nM
jasp (Fig. 2, F–I).
jasp causes reorganization of the growth cone
cytoskeleton without inducing F-actin polymerization
jasp inhibits F-actin turnover by preventing the recycling of
actin subunits through the inhibition of depolymerization at
the pointed ends of filaments. We investigated whether jasp
increased the amount of F-actin present in growth cones by
measuring the F-actin content of growth cones stained with
actin antibodies after combined fixation and extraction. Im-
portantly, jasp did not increase the F-actin content of
growth cones at a concentration of 40 nM that reliably
causes retraction of axons (10-min treatment, P
pared with vehicle-treated controls [
2, J to K; also compare Fig. 2, F to H), Thus, jasp-induced
axon retraction correlates with decreased F-actin turnover
without altering the F-actin content of growth cones.
To determine the effects of inhibiting F-actin turnover on
cytoskeletal organization, we stained cultures with antibod-
ies to actin and microtubules. Growth cone F-actin under-
went “clumping” after treatment with jasp (Fig. 2 K), which
correlated with the contraction of the growth cone. The mi-
crotubule cytoskeleton also underwent significant reorgani-
30]; compare Fig.
zation during jasp-induced axon retraction, which often in-
cluded development of many axonal bends (Fig. 1 B). These
bends represent curvatures in the axonal microtubule array
(Fig. 2 L). Lat-A treatment to depolymerize F-actin before
the addition of jasp prevented the development of axonal
bends (unpublished data).
Axon retraction in response to inhibition of F-actin
turnover requires myosin activity
Myosins have been involved in regulating both the extension
and retraction of axons (Introduction). The effects of myo-
sins on cell motility are mediated by an interaction with
F-actin, resulting in the development of contractile forces.
In response to the inhibition of F-actin turnover by jasp,
growth cones contracted, and axons underwent retraction.
This observation suggests the hypothesis that endogenous
myosin activity could be responsible for the retraction of ax-
ons in response to the inhibition of F-actin turnover. There-
fore, we tested this hypothesis by (1) determining whether
myosin activity was required for the retraction of axons in
response to jasp, (2) investigating the role of the regulation
of myosin activity through the RhoA and myosin light chain
kinase (MLCK) pathways in mediating the effects of jasp,
and (3) determining whether jasp treatment alters endoge-
nous levels of myosin activity.
To inhibit actomyosin contractility in growth cones, we
introduced nonfunctional skeletal muscle myosin II mole-
cules. We used a form of skeletal muscle myosin II (skMyo-
retraction. A and B show phalloidin-stained chicken embryonic
fibroblasts. (A) In control-loaded (BSA) fibroblasts, stress fibers form
at the circumference by 2 h after plating the cells (arrows). However,
in fibroblasts loaded with skMyosin II (SkMyo), stress fibers fail to
form. (C) Trituration loading of C3 toxin or SkMyo largely decreased
the percentage of fibroblasts with stress fibers 2 h after plating
(n ? 76 fibroblasts per group). (D) Trituration loading of DRG
neurons with SkMyo, or treatment with the myosin ATPase inhibitor
BDM (2 mM for 40 min), inhibits the distance axons retract after
treatment with 40 nM jasp for 20 min (n ? 43 axons per group).
Data is presented normalized to the distance control axons
retracted. (E) Example of BDM-treated growth cones before and 20
min after treatment with 40 nM jasp. Note that the growth cones do
not undergo contraction. CNT, control. Bars, 10 ?M.
Myosin activity is required for jasp-induced axon
Actin turnover and myosin regulate axon extension |
Gallo et al. 1223
sin II) that is not active in in vitro actin filament sliding as-
says (Cytoskeleton, Inc.). We reasoned that the introduction
of nonfunctional skMyosin II into cells could act as a domi-
nant negative. To test this, we trituration-loaded skMyosin
II into chicken embryonic fibroblasts and determined the
percentage of cells exhibiting stress fibers. It is well es-
tablished that formation and maintenance of stress fibers
strictly depends on myosin II activity (Kreisberg et al.,
1985). SkMyosin II decreased the percentage of fibroblasts
exhibiting stress fibers to a similar extent as treatment with
C3 toxin (Fig. 3, A–C), which inactivates RhoA and causes
disruption of stress fibers. Thus, the results of this bioassay
with fibroblasts are consistent with the idea that nonfunc-
tional skMyosin II acts as a dominant negative myosin II in
nonmuscle cells. Trituration loading of skMyosin II into
DRG neurons inhibited the distance axons retracted after
treatment with jasp (Fig. 3 D), indicating that myosin is re-
quired for axon retraction in response to jasp-induced inhi-
bition of F-actin turnover.
We further tested the role of myosin in mediating jasp-
induced axon retraction by treating DRG and RGC cultures
with 2,3-butanedione monoxime (BDM) before treatment
with jasp. BDM causes growth cone collapse at concentra-
tions that inhibit myosin activity (Ruchhoeft and Harris,
1997). Therefore, we determined the dose of BDM that col-
lapses 50% of growth cones and then tested the response of
the remaining growth cones to jasp. BDM inhibited jasp-
induced axon retraction in a manner similar to loading neu-
rons with skMyosin II (Fig. 3 D). Inhibition of myosin ac-
tivity by BDM also prevented growth cone contraction in
response to jasp (Fig. 3 E), and growth cones became immo-
tile but retained their original morphology. The addition of
BDM 4 min after treatment with jasp also decreased the rate
of axon retraction, indicating that myosin activity is contin-
uously required during the process of axon retraction (un-
Axon retraction in response to jasp requires F-actin.
Therefore, any experimental treatment that inhibits jasp-
induced axon retraction might act by decreasing F-actin
levels in growth cones. We controlled for this possibility by
measuring the F-actin content of growth cones under con-
ditions of inhibited myosin activity. SkMyosin II did not
affect the F-actin content of growth cones (Table I), ruling
out the alternative interpretation that interfering with myo-
sin activity inhibits jasp-induced axon retraction by decreas-
ing F-actin levels.
The activity of RhoA and MLCK regulates axon
retraction in response to inhibition of F-actin turnover
Myosin II activity is positively regulated in cells by phos-
phorylation of the myosin regulatory light chains (mRLC)
by MLCK. The RhoA pathway increases the phosphoryla-
tion state of mRLC via its effector kinase ROCK, which in-
activates mRLC phosphatase (Kimura et al., 1996). MLCK,
RhoA (Fig. 4 A), and ROCK (Wahl et al., 2000) are present
in growth cones. Therefore, we tested the roles of RhoA,
ROCK, and MLCK activity in mediating jasp-induced axon
RhoA activity was directly inhibited by trituration-loading
C3 into DRG neurons before treatment with jasp. C3 inhib-
ited the retraction of DRG axons in response to jasp (Fig. 4
B). Similarly, the ROCK inhibitor Y-27632 inhibited jasp-
induced growth cone contraction and axon retraction (Fig.
4, B and C). Inhibition of RhoA signaling by C3 did not al-
ter the F-actin content of growth cones (Table I). Collec-
tively, these results indicate that RhoA-driven ROCK kinase
activity is required for jasp-induced axon retraction.
To test whether experimentally increased levels of RhoA
activity potentiate jasp-induced axon retraction, DRG neu-
rons were loaded with a constitutively active form of RhoA
(caRhoA; L63 mutation). The F-actin content of growth
cones from neurons loaded with 1.5 mg/ml caRhoA was not
alter growth cone F-actin content
Altering the activity of myosin, RhoA and MLCK does not
TreatmentMean growth cone f-actin content
in arbitrary units
Dorsal root ganglion neurons were trituration loaded with protein or
peptide, cultured for 3 h and then fixed and stained with phalloidin. No
statistically significant differences from control trituration loaded growth
cones were observed.
retraction. (A) Immunocytochemical localization of RhoA and MLCK
in DRG growth cones. (B) Trituration-loaded C3 and the ROCK
inhibitor Y-27632 (10 ?M for 30 min) attenuate jasp-induced (40 nM
for 20 min) axon retraction in DRG cultures. Conversely, trituration
loading of constitutively active RhoA (CaRhoA) potentiates axon
retraction (n ? 52 axons per group). Data are presented normalized
to the distance control axons retracted. (C) Example of a Y-27632–
treated growth cone before and after treatment with jasp. Note that
the growth cone does not undergo contraction. (D) Trituration loading
of two separate MLCK-inhibitory peptides (MLCKp1 and p2) into DRG
neurons inhibits jasp-induced axon retraction. The pharmacological
MLCK inhibitor ML-7 (300 nM for 30 min) also inhibits the effects of
jasp. However, growth cones often underwent contraction (inset;
n ? 43 axons per group). Data are presented normalized to the
distance control axons retracted. CNT, control. Bars, 10 ?M.
RhoA and MLCK are required for jasp-induced axon
1224 The Journal of Cell Biology
Volume 158, Number 7, 2002
different from controls (Table I). Loading DRG neurons
with CaRhoA caused a 167% potentiation of the distance
the axon retracted in response to jasp (Fig. 4 B). This result
demonstrates that increased levels of RhoA activity potenti-
ate axon retraction that is induced by inhibition of F-actin
MLCK kinase activity was inhibited using two different
inhibitory peptides and ML-7, a pharmacological inhibi-
tor. DRG neurons were trituration-loaded with MLCK-
inhibitory peptides. The first peptide (MLCKp1; Kemp
et al., 1987; Kennelly et al., 1987) inhibits MLCK activity
by acting as a kinase pseudosubstrate. The second pep-
tide (MLCKp2; Kemp et al., 1987; Akasu et al., 1993)
blocks the required Ca
-calmodulin–mediated activation of
MLCK. Both peptides inhibited jasp-induced axon retrac-
tion to the same degree (Fig. 4 D). ML-7 also inhibited jasp-
induced axon retraction (Fig. 4 D). However, when MLCK
was inhibited, growth cones still underwent varying degrees
of contraction (Fig. 4 D, inset). Inhibition of MLCK activ-
ity with MLCKp1 did not affect the F-actin content of
growth cones (Table I).
Blocking ROCK and MLCK activity counters the
inhibitory effects of jasp on the rate of axon extension
We tested whether concentrations of jasp below those that
cause axon retraction in the acute treatment assay inhibited
axon extension over a longer time period. We found that 3
nM jasp reduced axon extension rate by 80% relative to the
control rate during a 40-min period (Fig. 5). Using the phal-
loidin binding assay we determined that, after a 40-min
treatment, 3 nM jasp bound 47% of available F-actin sites in
growth cones. Thus, attenuating F-actin turnover can re-
duce the mean rate of axon extension without causing axon
To determine if blocking ROCK activity could promote
axonal extension in the presence of jasp, cultures were
treated with 10
M Y-27632 for 30 min before exposure to
3 nM jasp. Under conditions of blocked ROCK activity,
axon extension was significantly less inhibited by 3 nM jasp
and continued at 67% of the control rate (Fig. 5). To inhibit
MLCK, MLCKp1 was introduced into established DRG ax-
ons using Chariot™ (see Materials and methods). Blocking
MLCK activity allowed axons to extend at 66% of the con-
trol rate in the presence of 3 nM jasp (Fig. 5). These data in-
dicate that the rate of axon extension is dependent on an
interaction between F-actin turnover and myosin-driven
jasp treatment does not alter endogenous myosin activity
The phosphorylation state of mRLC is the major determi-
nant of myosin II activity (Bresnick, 1999). The hypothesis
that endogenous myosin activity is responsible for axon re-
traction in response to jasp-mediated inhibition of F-actin
turnover demands that jasp treatment does not alter myosin
activity. To test whether the levels of myosin II activity are
changed when axons retract after treatment with jasp, we
monitored the phosphorylation state of mRLC. Urea-glyc-
erol PAGE separates the RLC by their charge, as well as size,
revealing the presence of phosphorylated forms as bands of
greater mobility (Fig. 6). Dissociated DRG neuron cultures
were treated with 40 nM jasp for 10 min, and the mRLC
were separated by urea-glycerol PAGE followed by Western
blotting with an antimyosin light chain antibody. jasp treat-
ment did not increase the level of mRLC phosphorylation
(Fig. 6). These data indicate that the effects of jasp on axons
and growth cones are not due to an increase in the levels of
F-actin turnover is decreased during RhoA-dependent
axon retraction in response to ephrin-A2
Negative guidance cues induce axon retraction. However,
little is know about the role of F-actin dynamics in guidance
cue–mediated axon retraction. We tested the hypothesis that
axon retraction in response to negative guidance cues could
utilize a similar mechanism based on actomyosin contractil-
ity and the inhibition of F-actin turnover. Ephrin-A2 is a
guidance cue for temporal retinal axons that in vivo has been
shown to mediate the retraction of inappropriately targeted
axons (O’Leary and Wilkinson, 1999). Thus, ephrin-A2 is a
valid model system for studying the cellular basis of axon re-
traction in response to biologically relevant guidance cues.
We determined whether F-actin turnover is altered by eph-
inhibition of axon extension. A 40-min treatment with 3 nM jasp
reduced axon elongation rate. The effects of 3 nM jasp on elongation
rate were attenuated by inhibition of ROCK with 10 ?M Y-27632 or
an MLCK inhibitory peptide (MLCKp1) delivered using Chariot™
(n ? 36 axons per group).
RhoA and MLCK activity are required for jasp-mediated
phosphorylation. Shown is a Western blot representative of myosin
light chains separated using urea-glycerol PAGE. This technique
separates the light chains by size and charge, thereby revealing the
extent of non, mono-, and di-phosphorylated chains. Note that
treatment with 40 nM jasp for 10 min did not change the profile of
light chain phosphorylation. The experiment was repeated three
times and produced consistent results. The slight di-phosphorylated
band was associated with both control and jasp-treated samples and
did not correlate with treatment across all experiments.
jasp does not alter levels of myosin light chain
Actin turnover and myosin regulate axon extension |
Gallo et al. 1225
rin-A2, and whether the activity of RhoA is required for
ephrin-A2–induced axon retraction.
In vitro, treatment of temporal retinal explants with eph-
rin-A2 caused axon retraction within 5–6 min (Fig. 7 A).
F-actin turnover was studied after 4 and 10 min of treat-
ment with ephrin-A2. These time points reflect periods just
before and during axon retraction, respectively. Treatment
of control cultures with lat-A resulted in the depolymeriza-
tion of 85% of F-actin (Fig. 7, C and D). In contrast, lat-A
depolymerized only 50% of F-actin in axons treated with
ephrin-A2 for 4 min (Fig. 7 D). Similarly, after a 10-min
treatment with ephrin-A2, lat-A depolymerized only 35% of
F-actin (Fig. 7, C and D). These observations demonstrate
that ephrin-A2 reduces F-actin turnover.
In conjunction with the inhibition of F-actin turnover,
ephrin-A2 also caused a decrease in F-actin content. Relative
to controls, ephrin-A2 decreased F-actin levels by 36 and
56% at 4 and 10 min, respectively (Fig. 7 D). After lat-A
treatment, ephrin-A2–treated axons exhibited 100 and 76%
greater F-actin content, at 4 and 10 min, respectively, relative
to control axons treated only with lat-A (P
comparisons). Thus, relative to F-actin in control axons, the
F-actin present in ephrin-A2–treated axons is significantly less
sensitive to lat -A–induced depolymerization and does not
represent a lat-A–insensitive population of F-actin normally
present in control growth cones. These data demonstrate that
ephrin-A2–induced axon retraction correlates with depoly-
merization of F-actin and inhibition of F-actin turnover.
Next, we investigated whether ephrin-A2–induced, like
jasp-induced, axon retraction involves regulation of actomy-
osin contractility through RhoA. Similar to jasp, treatment
of cultures with lat-A before the addition of ephrin-A2 al-
most completely blocked axon retraction in response to eph-
rin-A2 (mean distance retracted of 1.9
0.001 for both
m/20 min; n
Ephrin-A2–induced axon retraction was blocked by 80–
90% when RhoA or ROCK were inhibited (Fig. 7, B and
E). Collectively, these data demonstrate that axon retraction
in response to ephrin-A2 correlates with decreased rates of
F-actin turnover and requires F-actin and RhoA activity, in-
dicating that biologically relevant signaling molecules can
cause axon retraction through a mechanism similar to that
operating in jasp-induced axon retraction.
In this work, we investigated the combined roles of F-actin
turnover and actomyosin contractility in the process of axon
extension and retraction. We demonstrate that when actin
filament turnover is inhibited, endogenous myosin-driven
forces result in axon retraction. These observations reveal for
the first time that axon extension and retraction depend on
an interaction between the degree of actin filament turnover
and endogenous levels of myosin-based contractility. Addi-
tionally, we report the new observation that F-actin turnover
is inhibited during axon retraction in response to a biologi-
cally relevant guidance cue, ephrin-A2.
jasp is rapidly internalized in growth cones and inhibits
growth cone F-actin turnover. Cramer (1999) used jasp to
demonstrate that polymerization of F-actin at the leading
edge requires G-actin released by filament turnover. Our
data are consistent with a similar requirement for F-actin
turnover in providing the actin monomers required for
growth cone–protrusive activity. First, treatment with jasp
alone caused growth cone contraction and cessation of pro-
trusive activity. Second, when myosin activity was inhibited,
growth cones did not contract in response to jasp, but re-
mained flattened. However, lamellipodial and filopodial
protrusion was inhibited. These observations indicate that
retraction that requires RhoA activity
and correlates with the presence of
stable F-actin. (A) Example of temporal
retinal axon retraction in response to 1
?g/ml ephrin-A2 (arrow shows distance
the axon retracted during a 20-min period;
?75 ?m). Bar (A) 10 ?M. (B) Example of
axons treated first with C3, and then
with ephrin-A2. Note that none of the
axons undergo retraction. (C) Phalloidin
staining of temporal retinal axons treated
for 10 min with 1 ?g/ml BSA or ephrin-A2,
and then for 2 min with DMSO or 2 ?M
lat-A (LatA). Note that lat-A caused
significant depolymerization of F-actin
in BSA-treated axons, but only partial
depolymerization in ephrin-A2–treated
axons. Bar (C), 5 ?M. (D) Quantification
of the F-actin content in the distal axons
of cultures treated with BSA or ephrin-A2
for 4 or 10 min before treatment with
DMSO or lat-A for 2 min (n ? 41 in
each group). (E) Inhibition of RhoA (C3)
or ROCK (Y-27632 and HA-1077) blocks
ephrin-A2–induced axon retraction
(n ? 35 in each group). CNT, control.
Ephrin-A2 induces axon
1226 The Journal of Cell Biology
Volume 158, Number 7, 2002
myosin activity is required for growth cone contraction in
response to the inhibition of F-actin turnover by jasp, and
that in the absence of myosin-based contractility, growth
cones become quiescent due to the lack of available G-actin
for polymerization at the leading edge. These observations
are consistent with the model proposed by Lin et al. (1996)
for the regulation of growth cone leading edge protrusion by
actin polymerization and retrograde flow.
The response of the growth cone to treatment with jasp,
which we refer to as contraction, differs from growth cone
collapse in response to pharmacological depolymerization of
F-actin (Letourneau et al., 1989) or guidance cues that cause
collapse (Fan et al., 1993; Ernst et al., 2000; Wahl et al.,
2000). Unlike during growth cone collapse, in response to
jasp, filopodia often remained attached to the substratum
while the growth cone lamellipodium contracted (Fig. 1 A).
Also, growth cones became phase dark and exhibited clump-
ing of F-actin without depolymerization (Fig. 2 K). The
demonstration that myosin and RhoA activity are required
for growth cone contraction also differentiates contraction
from collapse because inhibition of RhoA activity only par-
tially blocks collapse in response to repellant guidance cues
(e.g., ephrin-A5; Wahl et al., 2000). Thus, we interpret the
response of growth cones to jasp as a myosin-driven contrac-
tion of the lamellipodium.
The effects of jasp on growth cones are similar to the re-
sponses of nonneuronal cells to jasp. Treatment with jasp
causes accumulation of F-actin in the central domain as the
growth cone undergoes contraction. In nonneuronal cells,
jasp causes the formation of F-actin aggregates (Lee et al.,
1998; Bubb et al., 2000). The formation of aggregates oc-
curs as F-actin filaments accumulate in the cytoplasm and is
Dictyostelium mutants lacking myosin II (Lee
et al., 1998). Similarly, when RhoA or myosins are inhib-
ited, growth cones do not undergo contraction. Thus, the
observations on growth cones provide evidence for the con-
servation of the myosin-based mechanism involved in the re-
sponses of both nonneuronal and neuronal cells to inhibi-
tion of F-actin turnover by jasp.
The observation that inhibiting F-actin turnover results in
myosin-dependent growth cone contraction and axon re-
traction, without altering myosin activity, demonstrate a
novel mechanism in the regulation of axon extension. In the
presence of “normal” rates of F-actin turnover, myosin activ-
ity drives retrograde flow, whereas actin filament polymer-
ization drives leading-edge protrusion. When filament turn-
over is inhibited, F-actin contracts centripetally through a
myosin-based mechanism. Thus, in growth cones, fast
F-actin turnover and endogenous myosin-based contractil-
ity combine to allow growth cone motility and axon ex-
tension to continue. When F-actin turnover is inhibited,
endogenous myosin-driven forces cause axon retraction.
Bradke and Dotti (1999) have suggested that local F-actin
turnover in growth cones specifies axonal versus dendritic
extension, and Bito et al. (2000) have shown that endoge-
nous RhoA activity limits axon formation. Hence, our ob-
servations may have relevance to the process of axonogenesis
as well as the extension of preestablished axons.
In this paper, we did not directly address the issue of
which myosin type drives jasp-induced axon retraction.
However, myosin II is the most likely candidate, given its es-
tablished role in the contractility of nonneuronal cells and
its presence in growth cones (Letourneau, 1981; Bridgman
and Dailey, 1989; Letourneau and Shattuck, 1989). In
growth cones, myosin IIB localizes to areas of the lamellipo-
dium undergoing retraction (Rochlin et al., 1995), provid-
ing additional evidence that myosin II may be responsible
for the retraction/contraction of the growth cone. Further-
more, MLCK and RhoA have well established roles in regu-
lating myosin II activity (Bresnick, 1999), and are required
for jasp-induced axon retraction.
We found that F-actin turnover in the distal axons of tem-
poral RGCs is decreased by treatment with ephrin-A2. Eph-
rin-A2 is a good model for axon guidance cues that cause
axon retraction, because of its in vivo role as a cue that pro-
motes the retraction of inappropriately targeted retinal axons
(Orioli and Klein, 1997; O’Leary and Wilkinson, 1999).
The relevance of axon retraction to neurodevelopment was
directly demonstrated by in vivo live imaging of axon path-
finding error correction in the projection of zebrafish retinal
axons (Hutson and Chien, 2002). It is at present unclear
whether all aspects of action retraction in vitro reflect retrac-
tion in vivo (e.g., axonal buckling). However, the mechanism
by which ephrin-A2 causes axon retraction shares similarities
with that underlying jasp-induced axon retraction. In partic-
ular, ephrin-A2–induced axon retraction requires RhoA ac-
tivity. The related ligand ephrin-A5 increases RhoA activity
in retinal neurons (Wahl et al., 2000). Although in our inves-
tigation we did not measure RhoA activity, it is likely that
ephrin-A2 also causes increased RhoA activity. It will be of
interest to investigate the role of GTPases in regulating F-actin
turnover during ephrin-A2–mediated axon retraction.
The experiments with ephrin-A2 indicate that axon re-
traction in response to guidance cues utilizes a similar mech-
anism as jasp-induced axon retraction. F-actin is required for
ephrin-A2–induced axon retraction. Contrary to this re-
quirement, ephrin-A2 caused the depolymerization of
of growth cone F-actin. However, F-actin depolymerization
coincided with decreased F-actin turnover. Thus, guidance
cues could induce axon retraction by activating myosin ac-
tivity through RhoA-ROCK while at the same time generat-
ing a favorable substrate for actomyosin contractility by in-
hibiting F-actin turnover. The molecular basis for the
coordinated depolymerization and stabilization of F-actin in
response to negative guidance cues will have to be investi-
gated further. However, the inactivation of cofilin by guid-
ance cues could result in the stabilization of F-actin (Aizawa
et al., 2001), whereas additional pathways could cause F-actin
depolymerization through filament severing or perhaps rap-
idly sequestering actin monomers making them unavailable
for recycling, or both.
Collectively, the results of the present study suggest a
model for the regulation of axon extension by an interac-
tion between myosin-based contractility and F-actin turnover
(Fig. 8 A). Endogenous actomyosin-based force generation in
axons can be a negative regulator of axon extension. RhoA
and MLCK contribute to the negative regulation of axon ex-
tension by myosin. Treatment of neurons with the RhoA-
inactivating enzyme C3 potentiates axon extension (Bito et
al., 2000). Similarly, blocking MLCK activity with a pseu-
Actin turnover and myosin regulate axon extension |
Gallo et al. 1227
dosubstrate peptide increases axon length by 27% (unpub-
lished data). While axons are extending, MLCK activity drives
the phosphorylation of mRLC and activates myosin. Endogenous
levels of RhoA activity also contribute to the regulation of
mRLC phosphorylation by acting through the RhoA effector
kinase ROCK, which is also capable of directly phosphorylat-
ing mRLC in addition to inhibiting the myosin phosphatase
(Bresnick, 1999). The finding that attenuation of F-actin
turnover inhibits axon extension and results in axon retrac-
tion in a manner dependent on RhoA and MLCK activity
demonstrates that the negative regulation of axon extension
by RhoA and MLCK is countered by the rapid turnover of
F-actin. In the absence of F-actin turnover, endogenous myo-
sin activity generates sufficient contractile force to produce
axon retraction. The mechanism by which the myosin activity
is either permissive or inhibitory for axon extension depend-
ing on F-actin turnover is unclear. However, under normal
conditions, myosin II is enriched in the central domain of
growth cones (Bridgman and Dailey, 1989) whereas F-actin
is localized mostly to the peripheral domain (Fig. 8 B). In the
absence of F-actin depolymerization, F-actin is expected to
undergo retrograde flow and accumulate in the central do-
main. Consistent with this expectation, we observed F-actin
clumping toward the central domain in response to jasp.
Thus, the accumulation of F-actin in the myosin II–rich cen-
tral domain may increase the ability of myosin II to interact
with F-actin and generate contractile forces.
Materials and methods
jasp was purchased from Molecular Probes, Inc. and dissolved in DMSO.
Exozyme C3, constitutively active (L63) RhoA, and skMyosin II were pur-
chased from Cytoskeleton, Inc. and dissolved in cell-loading buffer. ML-7
was purchased from BIOMOL Research Laboratories, Inc. and dissolved in
DMSO. Y-27632 and HA-1077 were obtained from BIOMOL Research
Laboratories, Inc. and prepared in water. MLCKp1 and MLCKp2 were ob-
tained from BIOMOL Research Laboratories, Inc. and Calbiochem, re-
spectively; both were resuspended in cell loading buffer. Brain-derived
neurotrophic factor and neurotrophin-3 were gifts of Dr. J. Cantello (Re-
generon Pharmaceuticals, Inc., Tarrytown, NY). NGF was purchased from
R&D Systems. All other reagents were purchased from Sigma-Aldrich.
Embryonic chick DRG (E9) and retinal explants (E7) were cultured as de-
scribed previously (Ernst et al., 2000) in defined Ham’s F12 medium
(GIBCO BRL) containing additives and 20 ng/ml brain-derived neu-
rotrophic factor for DRG, and grown on laminin (Invitrogen) or polylysine-
coated (Sigma-Aldrich) glass coverslips (both at 25
videomicroscopy, glass coverslips were affixed using aquarium sealant to
plastic dishes with holes (22-mm diameter) drilled in the center.
g/ml). For live
Cell trituration and Chariot™-based delivery
Proteins and peptides were trituration-loaded into dissociated DRG neu-
rons as described in Jin and Strittmatter (1997) in cell loading buffer con-
taining 5 mg/ml of protein or peptide. All experiments with trituration-
loaded cells were performed within 3–5 h after plating the cells. In our
culturing system, DRG neurons exhibit axons that are between 50 and 200
m long 3–5 h after plating.
The Chariot™ peptide (Active Motif LLC) method was used to load pep-
tides into neurons. 1.2
l Chariot™ peptide stock solution was mixed with
g MLCK peptide (BIOMOL Research Laboratories, Inc.), or 6
iot™ peptide with 1
g C3 for 30 min at RT as per the manufacturer’s direc-
tions. BSA was used as an inert control protein. The Chariot™ peptide–pro-
tein complex was added to cultures for 3–4 h before use in experiments.
Growth cones and axons were visualized with an inverted microscope
(model IX70; Olympus) using phase-contrast optics (20–60
Time lapse sequences were obtained using a Spot camera (model 2.1.0;
Diagnostic Instruments) driven by digital image acquisition software
(MetaVue™ 4.6r7; Universal Imaging Corp.). The procedure for quantifica-
tion of fluorescence was essentially the same as in Ernst et al. (2000). The
integrated pixel intensity of the distal 10
if one was present, was obtained by subtracting the background intensity.
jasp was applied locally by placing a pipette
m-diam tip, filled with medium containing 40
jasp to diffuse onto growth cones or axons.
m of axon plus the growth cone,
m above growth cones
?M jasp) and allowing
Phalloidin staining was used to visualize F-actin as described previously
(Gallo and Letourneau, 1998; Molecular Probes, Inc.). For antibody stain-
ing of F-actin, cultures were simultaneously fixed and extracted using
0.2% glutaraldehyde and 0.1% TX-100 in PHEM buffer and stained as de-
scribed in Gallo and Letourneau (1999). Omission of the primary antibody
(1:20 in 10% normal goat serum [NGS]; Sigma-Aldrich) gave no staining.
Microtubule staining was performed exactly as described in Gallo and Le-
For determination of RhoA and MLCK localization, cultures were fixed
with 4% PFA and ?20?C methanol for 15 min, respectively, and then TX-
100 treated. Primary antibodies in NGS were applied for 1 h (1:400 of rab-
bit anti-RhoA [Santa Cruz Biotechnology, Inc.] or 1:400 of monoclonal
mouse anti-MLCK [Sigma-Aldrich]). Secondary antibodies were applied for
1 h in NGS (1:400 of rhodamine-labeled goat anti–rabbit and goat anti–
mouse, respectively; Cappel). Omission of primaries gave no staining.
Urea-glycerol PAGE was used to monitor the phosphorylation state of my-
osin light chains as described in Yee et al. (2001). Proteins from purified,
dissociated DRG neurons (Gallo and Letourneau, 1999) from a total of 100
ganglia per sample were used. Cells were scraped off the dish in 10% TCA.
Protein samples were then run on urea-glycerol PAGE followed by West-
ern blotting with an antibody raised to a conserved sequence in myosin
The authors wish to acknowledge Andrew C. Melton (UCLA) for excellent
This research was supported by National Institutes of Health grants to
P.C. Letourneau (HD19950) and to H.F. Yee (DK02450 and DK57532).
Submitted: 25 April 2002
Revised: 16 July 2002
Accepted: 22 August 2002
F-actin turnover and myosin-driven contractility. (A) Regulation of
axon extension by the combination of F-actin turnover and actomyosin
contractility. (B) Diagram of relationship between F-actin (blue) and
myosin II (red) in the peripheral (P) and central (C) domains of
growth cones. Under “normal” conditions, F-actin is depolymerized
at the interface between the P- and C-domains and the monomers
are turned over (arrow). In the presence of jasp, F-actin is not
depolymerized and filament turnover is blocked. F-actin undergoes
retrograde transport and accumulates in the myosin II–enriched
C-domain resulting in increased actomyosin contractility.
Working model for the regulation of axon extension by
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