Washington University School of Medicine, Department of Anatomy and Neurobiology, Box 8108, 660 S. Euclid Avenue, St Louis, MO, USA
* Author for correspondence (e-mail: bridgmap{at}pcg.wustl.edu)
Accepted 21 December 2002
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Summary |
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Key words: Actin, Nerve outgrowth, Motility
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Introduction |
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Growth cones, similar to other motile cells, exhibit retrograde flow of
actin, and beads applied to their surface move rearward
(Lin and Forscher, 1995;
Lin et al., 1996
). It has been
shown that inhibition of myosin activity results in disruption of retrograde
flow in growth cones (Lin et al.,
1996
). However, the identity of the myosins involved remains
unknown, although a recent report has suggested the involvement of myosin IC
(Diefenbach et al., 2002
). It
remains to be determined whether or not multiple types of myosin contribute to
retrograde flow. A number of different cell types have been used to study
retrograde flow in growth cones, but the myosins present have not been fully
identified. As a result of the data compiled from the human genome project, it
now appears that mammalian cells have three types of myosin II. Two types of
myosin II (A and B) have been identified in nerve growth cones
(Rochlin et al., 1995
), and it
is possible that a third type is also present
(Berg et al., 2001
). Myosin IIB
is the only isoform that appears to be enriched in neurons compared with other
cell types (Rochlin et al.,
1995
).
Recently we showed that neurons grown in cell culture from a myosin IIB
knockout mouse exhibited slowed outgrowth rates
(Tullio et al., 2001). Growth
cones showed altered actin organization, size and motility
(Bridgman et al., 2001
). In
addition, filopodia-dependent traction force, which is thought to contribute
to forward advance of the growth cone, was reduced. Since there is a close
relationship between traction force and retrograde flow, we have now tested
for differences in retrograde flow rates between growth cones from
myosin-IIB-knockout (KO) mice and their normal littermates. We find that the
retrograde flow rate is significantly increased more than two fold in the KO
growth cones compared with wild type (wt). KO growth cones also exhibited
reduced stability of lamellipodia, which may be a consequence of this
increased flow rate. In addition, microtubules penetrated a shorter distance
into filopodia. These results suggest that both myosin IIA and IIB contribute
to retrograde flow and traction force. In the absence of myosin IIB, myosin
IIA alone may take over these functions, and the increased rate of retrograde
flow mainly reflects the properties of this myosin. The increase in flow rate
may also adversely affect the microtubule-dependent maturation of filopodia.
This supports the idea that forward advance of the growth cone is myosin II
dependent and involves multiple myosin II isoforms.
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Materials and Methods |
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Microscopy and laser trapping
A laser tweezers was constructed using a SDL 500 mW diode laser emitting at
835 nm and an Olympus IX70 microscope as described by Fallman and Axner
(Fallman and Axner, 1997).
Cells were imaged with a Cooke Sensicam digital camera using DIC microscopy.
Time-lapse sequences were captured using Scanalytics IpLab. Measurements on
digital images were also performed using IpLab. A filopodium was defined as a
long narrow structure uniform in diameter or slightly tapering towards the tip
arising from the growth cone perimeter. A lamellipodium was defined as a broad
protrusive structure arising from the growth cone perimeter. Filopodium-like
structures were defined as long, narrow structures that contained small
branches or flaring tips. All structures were enriched in actin when
appropriately stained. We restricted our quantitative analysis to the more
labile population of protrusive structures at the leading edge (approximately
the distal one-third of the cone). A flow chamber was used for experiments to
allow delivery of nerve growth factor (NGF)coated beads. For most experiments,
cells were first equilibrated with low NGF medium prior to introducing
NGF-coated beads (Gallo et al.,
1997
).
Immunostaining
For comparison of microtubules, actin and myosin IIA, cells were fixed and
stained as previously described using method 1
(Rochlin et al., 1995).
Rhodamine phalloidin was used to stain actin. An affinity-purified polyclonal
antibody to rodent myosin IIA was used to detect myosin IIA
(Rochlin et al., 1995
). A rat
monoclonal antibody to tyrosinated tubulin was used to detect distal
microtubule segments.
For comparison of myosin IIA and myosin IC staining, two fixation methods were used. The first was 4% paraformaldehyde (EM grade) in 0.1 M cacodylate buffer, and the second was rapid immersion in cold methanol (20°C). The staining patterns were identical, although the first method gave a slightly higher diffuse background in thickened regions. The myosin IC monoclonal antibody (m2) was a gift of J. Albanesi.
Immunofluorescence images were taken using a slow scan CCD (Roper Scientific Series 300). Figures were prepared using Adobe Photoshop. Contrast in photographs was enhanced using the unsharp mask filter.
Preparation of beads
Carboxylated silica beads (1.5 µm, Bangs Laboratories) were linked to
NGF following published protocols (Gallo
et al., 1997).
Biolistics
A custom-designed gene gun (Bridgman et
al., 2003) was used to introduce a GFP-myosin IIA cDNA into
cultured control and KO SCG neurons. The GFP was fused to the N-terminus of
the myosin IIA heavy chain using the pEGFP-C3 vector (Clontech) as previously
described (Wei and Adelstein,
2000
), except that the pTRE promoter was replaced with the
cytomegalovirus promoter.
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Results |
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To determine the rate of retrograde bead movement, we took time-lapse images at 6.5 second intervals (Fig. 1A,B). We then tracked the bead centroid position over multiple frames. Graphs of net displacement over time indicated that the bead displacement in the peripheral domain was approximately linear over relatively short time periods in both wt and KO growth cones (Fig. 1C,D). Movement varied in rate depending on bead position, but in general progressively slowed as beads approached the central domain. Linear regression was used to calculate the slope of the bead displacement curve at its steepest point, and this value was used as the maximum rate of bead movement. Comparison of rates in wt and KO cones indicated that there was some variation in the rates of movement between different growth cones for both. However, the average maximum rate of movement in wt cones was significantly different from in KO cones (t-test, P<0.01). The rate of retrograde movement in KO cones was on average 2.1 times faster than the movement in wt cones (Fig. 1E).
|
Although there is good evidence that the rate of retrograde bead movement
on growth cones closely reflects the rate of retrograde flow of the actin
cytoskeleton, we wanted to verify this result in our system. To do this, we
expressed GFPmyosin-IIA in control and KO neurons. There is good
evidence that myosin II forms small bipolar filaments in lamellipodia of
motile cells, and the behavior of these minifilaments reflects the dynamics of
the actin cytoskeleton to which they are attached
(Svitkina et al., 1997).
GFPmyosin-IIA has been shown to incorporate into F-actin-rich
structures that normally are associated with myosin IIA in noneuronal cells,
such as stress fibers, indicating that it is correctly targeted
(Wei and Adelstein, 2000
)
(P.C.B., unpublished). In both wt and KO growth cones, GFPmyosin-IIA
appeared as small fluorescent spots (Fig.
2A). These spots were labile, they often appeared and disappeared
within minutes and also changed intensity. We assume that these spots
represent small bipolar filaments that have been observed in both noneuronal
cells and growth cones (Svitkina et al.,
1997
; Bridgman,
2002
). In peripheral regions of the growth cone, spots
consistently appeared and then (usually) underwent retrograde flow. The moving
spots often slowed their rate of movement, merged and accumulated into linear
arrays within the transition zone. We tracked a subset (those that could be
identified for at least three frames) of spots by time-lapse imaging to
determine their rate of movement (Fig.
2B). The average rates of spot movement for both wt and KO were
about the same as the average rates of bead movement (compare
Fig. 1C with
Fig. 2C). Again, as was
observed for bead movement, the retrograde movement of spots in KO growth
cones was little over twice (2.3x) the rate of movement in wt cones. The
difference was significant (t-test, P<0.001). This
indicates that the NGF-coated beads can act as an accurate marker for the
retrograde flow of the actin cytoskeleton and that KO growth cones have an
increased rate of retrograde flow.
|
There is good evidence from experiments using neuroblastoma cells to
suggest that the dynamics of filopodia and lamellipodia result from the
balance between retrograde flow and actin polymerization
(Mallavarapu and Mitchison,
1999). Cessation of actin polymerization usually results in
retraction because of the action of retrograde flow. When the rates of actin
polymerization and retrograde flow are matched, no change occurs. However, if
the actin polymerization rate exceeds the rate of retrograde flow then
protrusion occurs. Actin polymerization and retrograde flow appear to be
independently regulated. Because the retrograde flow rate is increased in the
KO growth cones, we wanted to know if this had an affect on protrusion and
retraction rates. Therefore we used time-lapse images
(Fig. 3) to measure the rates
of both filopodia retraction and protrusion. Although the average rate of KO
protrusions was slightly less than that of wt (wt=3.3±0.34
µm/minute, n=16, KO=2.8±0.3 µm/minute, n=16,
s.e.m.), the difference was not significant. Similarly, the average rates of
retractions were the same (wt=9.8±1.7 µm/minute, n=16,
KO=10±2.2 µm/minute, n=16). Thus there is no detectable
difference in the average rates of protrusion or retraction of filopodia
between KO and wt.
|
Previously we observed an increased frequency of protrusion and retraction
in KO cones compared with wt (Bridgman et
al., 2001). This suggests that the lifetime of protrusions may
differ from KO to wt growth cones. Therefore we measured the lifetime of both
filopodia and lamellipodia. Filopodia form by extension, but disappear from
detection through several means. They can extend and then retract, extend and
be engulfed by advancing lamellipodia or extend and then move laterally to
fuse with adjacent filopodia. Occasionally, filopodia also fold back and fuse
with the cytoplasm proximal to their base. We only made comparisons of
filopodia that extended and then retracted to ensure that we were measuring
the lifetime of the actin bundle that forms the core of the filopodia.
Although wt filopodia had slightly longer average lifetimes than KO filopodia,
the difference was not significant (wt=202±1.7 seconds, n=19,
KO=169±2.4 seconds, n=23, s.e.m., t-test,
P>0.05). A similar comparison was done between lamellipodia,
except that we included all modes of lamellipodial disappearance (retraction,
lateral movement and filopodial extension) because they reflect a change in
the organization of the actin meshwork. Wt lamellipodia had significantly
longer lifetimes than KO filopodia (wt=239±2.8 seconds, n=18,
KO=102±1.1 seconds, n=18; s.e.m., t-test,
P<0.001). Notably, this is consistent with the observation that KO
cones are smaller and have fewer lamellipodia than wt cones in time-averaged
comparisons (Bridgman et al.,
2001
).
Retrograde flow has also been shown to affect the distribution of
microtubules in the peripheral regions of growth cones. Retrograde flow
transports extending microtubules rearward unless they are stabilized by
interactions with actin bundles (Zhou et
al., 2002; Schaefer et al.,
2002
). When they do interact with actin bundles the rate of
peripheral extension is the sum of the microtubule polymerization rate and the
retrograde flow rate (Schaefer et al.,
2002
). To determine if microtubule distribution was affected by
the increased rate of retrograde flow observed in KO growth cones, we triple
stained wt and KO growth cones for tyrosinated tubulin, F-actin and myosin IIA
(Fig. 4). In comparing images
from wt and KO growth cones stained for these cytoskeletal proteins, a general
impression was complicated by the difference in cone shapes and the apparent
lack of a distinct `transition zone' containing actin bundles in the KO.
However, upon close inspection, it was apparent that microtubules tended to
more fully fill the area normally associated with a transition zone in KO
cones compared with wt. Myosin IIA staining was associated with this region in
both KO and wt. By contrast, the actin-rich peripheral structures appeared to
contain fewer microtubule end segments in the KO. To obtain a
semi-quantitative assessment of the affect of retrograde flow on microtubule
distribution, we selected filopodia (or filopodia-like structures) in wt and
KO cones that were associated with or partially penetrated by microtubules and
then measured the distance from the microtubule tip to the distal tip of the
filopodium (Fig. 4C).
Microtubules penetrated further towards the tip of filopodia in the wt because
the distance between microtubule ends and the tip of filopodia was
significantly reduced compared with the KO (wt=3.7±2.6 µm,
n=69; KO=4.6±2.9 µm, n=69; t-test,
P=0.05). This is consistent with the increased retrograde flow rate
of actin having an enhanced affect on distal microtubule segments associated
with actin bundles of filopodia in the KO. When actin bundles are largely
absent in the KO (i.e. the transition zone), the increased rate of flow has
minimal affect on microtubules, and microtubules extend to fill the
region.
|
A recent report from Dan Jay's laboratory
(Diefenbach et al., 2002)
presented data consistent with the idea that retrograde flow in chicken DRG
neurons is driven by myosin IC. Their data also suggest that myosin II has no
role in driving or regulating retrograde flow. To determine if myosin IC plays
a role in driving retrograde flow in mouse SCG neurons, we used the same
monoclonal antibody that was used in the Diefenbach study to stain SCG growth
cones (Fig. 5A). We could only
detect small amounts of myosin IC staining compared with myosin IIA staining
(independent of fixation method) in SCG growth cones. Most of the myosin IC
staining was confined to the neurite. To determine if this results from
differences in cell type as opposed to species differences, we also stained
mouse DRG neurons. DRG neurons stained for myosin IC were consistently
slightly brighter than SCG neurons (grown from the same mouse embryos and
cultured, fixed identically), but again the majority of the stain was confined
to the neurite with only a few small puncta in growth cones
(Fig. 5B). To determine if
myosin IC is upregulated in neurons from the KO mice, we also stained SCG and
DRG growth cones cultured from the KO mice. We could not detect any obvious
differences in the myosin IC stain pattern or intensity compared to wt
(Fig. 5). It seems unlikely
that this antibody, which was made to bovine adrenal myosin IC, is less
affective in detecting myosin IC in mouse compared to chicken. Thus, growth
cones of mouse peripheral nerves contain only small amounts of myosin IC. This
is consistent with two previous reports that showed myosin 1C protein levels
in mammalian nervous tissue are low compared to other tissues
(Wagner et al., 1992
;
Ruppert et al., 1995
). In
addition, myosin 1C (myr 2) does not appear to be upregulated in embryonic
rodent nervous tissue (Ruppert et al.,
1995
).
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Discussion |
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It has been suggested very recently that myosin IC is the motor responsible
for driving retrograde flow in growth cones
(Diefenbach et al., 2002).
However, mouse SCG growth cones contain little detectable myosin IC and show
no obvious sign of upregulation in KO growth cones. Although mouse DRG neurons
(wt and KO) stain slightly more intensely for myosin IC, the staining pattern
is similar to that observed in SCG neurons. The pattern indicates that most of
the myosin IC is associated with the neurite. It is unclear if the small
amount of myosin IC present at the base of the growth cone drives retrograde
flow. In contrast to the more limited distribution of myosin IC, myosin IIA is
relatively abundant in both mouse SCG and DRG neurons and their growth cones.
Its wide distribution within the cone, and its behavior when observed in
living cones is similar to that observed in noneuronal cells
(Svitkina et al., 1997
). The
distribution and dynamic behavior are consistent with a direct role in
retrograde flow. However, a direct test of a role for myosin IIA in retrograde
flow has not yet been done in mouse neurons.
In the Diefenbach study, micro-CALI of myosin II or IIB did not produce
significant affects on retrograde flow rates in chicken DRG neurons. However,
a negative result may be hard to interpret for the following reasons.
Micro-CALI of myosin II took minutes to produce easily detectable
morphological effects in growth cones. If a detectable effect on retrograde
flow also took several minutes, then many of the beads that were being
monitored during the laser irradiation probably moved a considerable distance
before being affected. This would affect the calculation of the average rate.
In addition, the inactivation by microCALI may be incomplete. We note that
microCALI of myosin IIB did produce an increase in the average rate of bead
movement compared with the IgG control
(Diefenbach et al., 2002), but
the increase was not significant. If residual myosin IIB activity was present,
it would presumably act to slow retrograde flow to a reduced extent. Thus a
rate increase may be hard to detect. Further experiments in this system are
needed to determine whether or not myosin II plays a role in retrograde flow
of chicken neurons. It is possible that the myosin dependence of retrograde
flow is species specific or utilizes multiple myosin types. In any case, still
unexplained is the observation that retrograde flow rates exceed the actin
filament sliding speeds of any of the myosins present in non-muscle cells. The
dynamic contraction model (Svitkina et
al., 1997
) is the only current model that has the potential to
explain this discrepancy (Brown and
Bridgman, 2003
).
The increased retrograde flow rate observed in the KO mouse SCG growth
cones did not have a detectable affect on protrusion and retraction rates.
This is not surprising considering that actin polymerization in filopodia and
lamellipodia appears to independently regulate and respond to changes in flow
rate over a wide range (Mallavarapu and
Mitchison, 1999). Thus, the growth cone can compensate for the
increased flow rate by increasing the actin polymerization rate. It is unclear
whether or not retraction rates are dependent on retrograde flow. Similar to
observations on neuroblastoma cells
(Mallavarapu and Mitchison,
1999
), we observed slow and fast rates of filopodia retraction in
both wt and KO growth cones. Fast retraction might involve only the
contractile activity of myosin IIA. Alternative mechanisms such as F-actin
severing, might also contribute. Slow retraction is more likely to be
influenced by the rate of retrograde flow. However, we did not observe a
difference between the wt and KO in the distribution of fast and slow
retractions. We also did not observe a difference when we only compared slow
retraction rates.
The lifetimes of lamellipodia, but not filopodia, were shorter in the KO
compared with the wt. This is consistent with the overall morphology of the
growth cones from wt and KO. KO growth cones are about half the area of wt and
are more irregular in shape owing to multiple filopodial structures around
their periphery (Bridgman et al.,
2001; Tullio et al.,
2001
). This suggests that the increased rate of retrograde flow
may destabilize lamellipodial structures, but not filopodia. KO growth cones
have fewer transverse actin bundles in the transitional and central zones
(Bridgman et al., 2001
).
Normally transverse bundles form in the periphery and are transported rearward
by retrograde flow (Danuser and Oldenbourg,
2000
). This suggests that myosin IIB is required to crosslink
actin in lamellipodia to form these bundles. In their absence, lamellipodia
are less stable, and this is reflected in their decreased lifetimes in the KO.
A consequence of this decreased stability is less persistent growth and
forward advance because normal-sized areas of lamellipodia fail to form at the
leading edge (Bridgman et al.,
2001
). The decreased formation of large protrusive structures in
the direction of growth, combined with decreased traction force of filopodia
(Bridgman et al., 2001
), may
contribute to the slower rate of outgrowth observed in the KO neurons. The
parameters that have been tested in this and other studies that contribute to
the slowed outgrowth rates are qualitatively summarized in
Table 1.
|
Consistent with the observation that retrograde flow is increased in KO
growth cones, microtubules penetrate into actin-rich filopodia less than in
the wt. It has been shown that microtubules in this region are influenced by
retrograde flow and that their extension into the periphery reflects a balance
between their polymerization rate and the rearward transport by retrograde
flow (Schaefer et al., 2002).
When microtubules interact with actin bundles in the growth cone periphery
they can influence the direction of growth
(Zhou et al., 2002
).
Presumably the interaction between microtubules and actin bundles leads to an
increased maturation of the local cytoplasm that increases its stability and
dynamics. This may include stabilization or maturation of adhesive contacts,
increased actin polymerization rates and the transport of new materials to the
region (Gomez et al., 1996
;
Renaudin et al., 1999
;
Kaverina et al., 2002
). Thus,
if this process is inhibited, outgrowth will also be inhibited. This is
consistent with the reduced formation of new protrusions that persist in the
direction of growth and reduced outgrowth rates observed in the myosin IIB
KO.
In conclusion, multiple isoforms of myosin II may act through several distinct but related activities to drive and regulate growth cone advance. These activities will ultimately also be important for determining the direction of growth when encountering targets that stimulate or repel the growth cone during pathfinding.
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Acknowledgments |
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