* Center for Neurologic Diseases, and Division of Experimental Medicine, Department of Medicine, Brigham and Women's
Hospital, Harvard Medical School, Boston, Massachusetts 02115; and § Mouse Biology Programme, European Molecular Biology
Laboratory, 00015 Monterotondo/Rome, Italy
Growth cones extend dynamic protrusions
called filopodia and lamellipodia as exploratory probes
that signal the direction of neurite growth. Gelsolin, as
an actin filament-severing protein, may serve an important role in the rapid shape changes associated with
growth cone structures. In wild-type (wt) hippocampal
neurons, antibodies against gelsolin labeled the neurite
shaft and growth cone. The behavior of filopodia in cultured hippocampal neurons from embryonic day 17 wt
and gelsolin null (Gsn) mice (Witke, W., A.H. Sharpe,
J.H. Hartwig, T. Azuma, T.P. Stossel, and D.J. Kwiatkowski. 1995. Cell. 81:41-51.) was recorded with time-lapse video microscopy. The number of filopodia along
the neurites was significantly greater in Gsn
mice and
gave the neurites a studded appearance. Dynamic studies suggested that most of these filopodia were formed
from the region of the growth cone and remained as
protrusions from the newly consolidated shaft after the
growth cone advanced. Histories of individual filopodia
in Gsn
mice revealed elongation rates that did not differ from controls but an impaired retraction phase that
probably accounted for the increased number of filopodia long the neutrite shaft. Gelsolin appears to function
in the initiation of filopodial retraction and in its
smooth progression.
NEURONAL growth cones are highly motile structures
that contain the mechanical elements necessary
to implement a repertoire of behaviors that determine the rate and direction of advance, as well as the
receptor elements that link environmental cues to the implementation of specific behaviors. (Trinkaus, 1984 Gelsolin is a good candidate for a Ca2+-sensitive factor
capable of regulating the dynamics of actin filaments in
growth cones. Gelsolin severs actin filaments in a Ca2+-
dependent manner and caps the plus ends of the severed
filaments, preventing the addition of actin monomers (Yin
and Stossel, 1979 This model of the function of gelsolin during motility is
supported by studies of non-neuronal cells. Overexpression of gelsolin in cultured NIH 3T3 fibroblasts results in
increased motility as assessed by tissue culture wound
healing and filter transmigration assays (Cunningham et
al., 1991 To determine the possible role of gelsolin during neurite
elongation, we compared the morphology and motility of
neuronal growth cones in cultures from Gsn Cell Culture
Hippocampal neuronal cultures were obtained from either wt (C57LB/6
or BALB/c) or Gsn Video Microscopy
Dissociated hippocampal neurons were observed with a 60× planapochromat oil immersion objective (1.4 NA), 100 W mercury light source under an inverted microscope (Diaphot 300; Nikon, Inc., Melville, NY). For
video-enhanced contrast differential interference contrast (VEC-DIC)
microscopy, a matching high resolution (1.4 NA) oil immersion condenser
was used. For time-lapse video microscopy, hippocampal cells cultured for
1 to 3 d were mounted in a Dvorak-Stotler chamber and kept at 37°C by
means of an air stream stage incubator (Nicholson Precision Instruments,
Gaithersburg, MD). A fiberoptic light scrambler (Knudsen Technical
Video, Woods Hole, MA), heat filters, and a monochromatic green filter
(546 nm) were used to achieve even illumination and reduce damage to
cells. DIC images were amplified with 4× relay lens and detected with a
Newvicon camera (C2400; Hamamatsu). Images were taken every 5 s,
summed by using Image-1 (Universal Imaging, West Chester, PA), and
stored on laser discs with an optical memory disc recorder (OMDR; LVR-5000A, Sony). For matching fluorescence and DIC images on fixed cells, images were magnified with a 2× relay lens, summed for 256 frames, and
the background subtracted. DIC images were captured with a Newvicon
camera, and fluorescence images were captured under regular fluorescence microscopy with a silicon-intensified target camera (C2400;
Hamamatsu). These images were stored in the computer discs directly.
Photographs were printed using Adobe Photoshop.
Immunocytochemistry
Dissociated hippocampal cultures were fixed with 4% paraformaldehyde
and 3% sucrose in PHEM buffer solution (60 mM PIPES, 25 mM Hepes,
10 mM EGTA, 2 mM MgCl2, pH 6.9) at 37°C for 30 min and permeabilized with 0.1% Triton for 2 min. After blocking with 5% goat serum in
PBS buffer for 30 min, cells were incubated with primary antibodies in the
blocking solution overnight at 4°C. Cells were then washed three times at
5-min intervals and incubated with secondary antibodies diluted at 1:100
(Jackson ImmunoResearch Laboratory, West Grove, PA) for 1 h. Cells
were mounted on slides in Slow FadeTM mounting medium (Molecular
Probes, Inc., Eugene, OR) after three washes. The primary antibodies
used were affinity purified polyclonal antibody against recombinant murine gelsolin (kindly provided by Toshifumi Azuma, Brigham and
Women's Hopital, Boston, MA) at 1:250 dilution and monoclonal antibody against neuronal specific Quantification
Video sequences of cultured hippocampal neurons from either wt or Gsn The duration of filopodia were measured for each sequence. When >10
filopodia were present within the 10-µm segment of the neurite, only the
first 10 filopodia were measured. In a few cases, filopodia did not fully retract after 30 min, the time over which most of the sequences were recorded; the duration of these filopodia were entered as 30 min. Life histories for individual filopodia were obtained by measuring the length of
filopodia frame by frame and graphed as length versus time. The measurements for the elongation rate and retraction rate of filopodia were made
by using the KaleidaGraph program. Lamellipodial activity was measured
by determining the change in lamellipodial area at 5-s intervals.
Gelsolin Is Present in Neuronal Growth Cones of
Wild-Type Mice
To explore a possible role for gelsolin in neurite elongation, we first investigated the expression pattern of gelsolin in cultured embryonic hippocampal neurons from wt
mice. Hippocampal cells were cultured overnight on a
laminin substrate, which is found in the extracellular space
of the embryonic rat hippocampus (Gordon-Weeks et al.,
1989
Growth Cones and Neurites of
Gsn Embryonic day 17 hippocampal cells from wt and Gsn
Filopodia from 17 time-lapsed sequences of wt growth
cones and 18 sequences of Gsn
To investigate the cytoskeletal organization of filopodia
along neurites in Gsn
Delayed Retraction Accounts for the Increased
Numbers of Filopodia in Gsn Because we hypothesized that neurons lacking gelsolin
may have motility defects, the initial observations concentrated on rapid shape changes of lamellipodia and filopodia using time-lapse video enhanced contrast DIC microscopy. Measurements of neurite elongation did not reveal
significant differences in growth rates. The average rate of
neurite elongation in wt neurons was 24.00 ± 5.09 µm/h
(n = 17), and in Gsn To test this hypothesis, 17 sequences from wt mice and
18 sequences from Gsn
The life histories of 37 wt and 36 Gsn
Analysis of Lamellipodia
As described in invertebrate Aplysia neurons (Goldberg
and Burmeister, 1986 The analysis of area does not reflect the frequent protrusions and retractions in the vertical plane, because
filopodia that extend on the dorsal surface move out of focus over the time periods required to obtain their histories.
Therefore an accurate quantitation of filopodial extensions and retractions from the growth cone itself was not
technically possible. The difficulty in obtaining dynamic
motility measurements in three dimensions may obscure some quantitative differences between wt and Gsn The molecular basis for the complexities of neuronal
growth cone behavior is beginning to emerge. Highly complex motile phenomena such as turning clearly require ensemble behaviors in which signaling cascades implement
the engagement of actin filaments with microtubules as
they invade the distal region of the growth cone (Tanaka
and Sabry, 1995 The observation that neurites from Gsn Gelsolin is the founding member of a family of six mammalian genes/proteins (Schafer and Cooper, 1995 Given the filopodial retraction rates observed here and
the known rates of actin depolymerization, it is unlikely
that the retraction defect observed in the Gsn The tendency of the filopodia to remain along the consolidated neurite once the growth cone has advanced suggests a forward flow within the central cytoplasm while the
filopodia hold on to their attachment sites to create the
torque for the advancing growth cone. Because substrate
interactions dominate neuronal cell growth under tissue
culture conditions, neurons from Gsn; Bray
and Hollenbeck, 1988
; Goodman and Shatz, 1993
). The
lamellipodia and filopodia, which constantly protrude and
retract at the tips of incipient axons and dendrites (Goldberg and Burmeister, 1986
; Rivas et al., 1992
), are the most
active processes, and the detailed molecular assemblies
that mediate this activity are mostly unknown. Clearly, actin filaments are the major cytoskeletal component in
these structures and must be involved in lamellipodial and filopodial motility (Forscher and Smith, 1988
; Bridgman
and Dailey, 1989
; Lewis and Bridgman, 1992
). Disruption
of actin filaments by the depolymerizing agent cytochalasin abolished protrusive activity (Forscher and Smith,
1988
) and even retraction of both lamellipodia and filopodia (Marsh and Letourneau, 1984
; Bentley and Toroian,
1986
; Forscher and Smith, 1988
; Chien et al., 1993
). One
signaling molecule for actin polymerization is calcium ion (Ca2+), and many studies have demonstrated that increased intracellular Ca2+ due to depolarization, as well as
treatments with neurotransmitter or Ca2+ ionophore, alter
the morphology and motility of lamellipodia and filopodia
(Cohan and Kater, 1986
; Lankford and Letourneau, 1989
;
Cohan, 1992
; Grumbacher-Reinert and Nicholls, 1992
;
Rehder and Kater, 1992
; Neely, 1993
; Neely and Gesemann, 1994
; Zheng et al.,1996). Also within growth cones
are actin-binding proteins such as certain myosin isoforms,
tropomyosin,
-actinin, actin depolymerizing factor (ADF),1
filamin, and gelsolin (Bamburg and Bray, 1987
; Bridgman
and Dailey, 1989
; Letourneau and Shattuck, 1989
; Sobue
and Kanda, 1989
; Miller et al., 1992
; Tanaka et al., 1993
;
Tanaka and Sobue, 1994
). Although much is known about
the in vitro effects of these proteins on actin organization
and polymerization, how these actin-associated proteins
act in vivo to affect the motility of lamellipodia and filopodia is largely unknown.
; Yin et al., 1981
; Lamb et al., 1993
).
Binding of gelsolin to phosphoinositides (PPIs) causes the
release of gelsolin from the filament end, providing a site
for rapid actin monomer addition (Janmey et al., 1987
; Janmey and Stossel, 1987
, 1989
). Micromolar Ca2+ also activates gelsolin to nucleate actin filament growth from monomers, resulting in the formation of short actin filaments (Yin et al., 1981
; Janmey et al., 1985
). Thus, during
repetitive waves of high Ca2+/low PPIs alternating with
low Ca2+/high PPIs, gelsolin can disassemble an existing
actin filament architecture through its severing activity
and then lead to site-directed reformation of an alternative structure, contributing to the motile changes occurring
in cells.
). Moreover, gelsolin-null (Gsn
) mice, generated
by gene targetting methods (Witke et al., 1995
), have
grossly normal embryonic development and longevity but
defects in hemostasis, inflammation, and tissue remodeling. Cultured dermal fibroblasts from these Gsn
mice migrated more slowly in comparison to wild-type (wt) mice and had increased amounts of actin stress fibers.
and wt mice.
In high resolution time-lapse video microscopy images, considerably more filopodia were present along neurites
of Gsn
mice. A comparison of the behaviors of filopodia
in these mice leads to the observation that gelsolin has a
critical role in filopodial retraction.
Materials and Methods
mice (C57LB/6 or BALB/c; Witke et al., 1995
) at
embryonic day 17. Culture procedures generally followed those described
(Goslin and Banker, 1991
) with some minor modifications. The brief procedures are as follows. Brains were sterily removed from CO2-killed mice and placed in the sterile cold Hank's balanced salt solution without Ca2+
and Mg2+. Hippocampi were carefully dissected, and meninges were removed. Hippocampal cells were dissociated enzymatically with 0.25%
trypsin (Sigma Chemical Co., St. Louis, MO) for 15 min and mechanically by triturating with a fire-polished Pasteur pipette. The dissociated cells
were plated onto 24.5-mm circular glass coverslips (No. 1.5; German)
coated with laminin (20 µg/ml; Sigma Chemical Co.). The cells were cultured in a humidified 5% CO2 incubator at 37°C for up to 3 d.
III tubulin at 1:200 (Sigma Chemical Co.),
with secondary antibodies fluorescein-conjugated donkey anti-rabbit and
donkey anti-mouse, respectively. To label actin filaments, cells were incubated overnight at 4°C with 20 U of rhodamine phalloidin in the blocking
solution along with the primary antibody.
mice were obtained using a high resolution 60× lens and an additional 4×
relay lens. The images were quantified using the "measure curve length"
function in Image-1 software. The average time recorded for individual sequences was ~30 min. The interval between frames was 5 s, to ensure capturing the detailed dynamic behaviors of growth cones and neurites. 7-10
evenly spaced frames at 3-min intervals were measured for each sequence.
The length and number of filopodia along the neurite were measured
within 10 µm of the neurite from the neck of the growth cone. Using the
stated magnification, the width across the monitor screen was 45 µm,
making it possible to visualize both the growth cone and neurite for every
image quantified. To ensure the healthy status of sampled cells, only those
sequences that lasted for at least 20 min were quantified. The computation
and statistical analysis of the data were performed with Microsoft Excel 5.0.
Results
). The distribution of gelsolin was studied by immunostaining fixed, cultured hippocampal cells. In this study,
an affinity-purified polyclonal antibody raised against recombinant gelsolin was used. 1-d-old cultures from wt and
Gsn
mice were fixed and immunostained. Both neurons
and non-neuronal cells stained with the affinity-purified
polyclonal antibody against gelsolin. In neurons, gelsolin
immunoreactivity was present in the neurites and appeared concentrated in growth cones where it was expressed in the central domain as well as in the leading edge
of the lamellipodia and filopodia (Fig. 1). It co-localized with actin filaments as labeled by rhodamine phalloidin
(Fig. 1).
Fig. 1.
Fluorescence images of hippocampal neurons
stained with affinity-purified
polyclonal antibody to gelsolin from E17 wt mice (A) and
rhodamine phalloidin (B).
(C) Matching DIC image.
Note in wt mice (A), not only
the neurite but also the growth
cone stained and colocalized
with actin (B). Bar, 5 µm.
[View Larger Version of this Image (47K GIF file)]
Hippocampal Neurons
mice were dissociated and cultured on laminin-coated coverslips for 1 to 3 days. Hippocampal neurons from wt mice
cultured on laminin substrate typically had a small round
or oval cell body averaging 6-8 µm diam and usually had a
long neurite and several short processes. Growth cones at
the tips of neurites were ~7-10 µm across. Typically, the
region proximal to the growth cone of wt neurons was
fairly smooth with few filopodia, as shown in Fig. 2. Neuronal growth cones and neurites from Gsn
mice were
similar in size and morphology to those of wt mice. However, there were considerably more filopodia along the extending neurite of Gsn
mice than along those of wt mice,
as shown in Fig. 3. To quantitate this observation, a region
of the neurite 10 µm proximal to the neck of the growth
cone was analyzed.
Fig. 2.
A sequence of VEC-DIC video images of a hippocampal growth cone from E17 wt mice growing on laminin-coated substrate.
Images were recorded at 1 d in culture. The total length of the sequence shown is 24 min. Note growth cone extending smoothly with
few filopodia, especially few filopodia from the shaft of the neurite. Bar, 5 µm.
[View Larger Version of this Image (113K GIF file)]
Fig. 3.
A sequence of VEC-DIC video images of a hippocampal growth cone from E17 Gsn mice growing on laminin-coated substrate. The images were recorded at 1 d in culture. The total length of the sequence shown is 24 min. Note there were more filopodia in
Gsn
mice, especially filopodia on the shaft of the neurite. Bar, 5 µm.
[View Larger Version of this Image (120K GIF file)]
growth cones were quantitated. Filopodia along the neurite were quite dynamic,
making it important to sample data not only from different
neurons but also from a single neuron at different time points. For each sequence, the length and number of
filopodia along the neurite were measured in 7-10 frames,
taken in 3-min intervals. Results from these sequences
showed that the average number of filodopia in each
frame was significantly more (P < 0.001) in Gsn
mice
2.64 ± 0.15, n = 180) compared to wt mice (1.20 ± 0.11, n = 164; Fig. 4 A); however, the average length for a single
filopodia was not significantly longer in Gsn
mice than in
wt mice (P > 0.05; Fig. 4 B).
Fig. 4.
Graphs of length and number of filopodia along neurites from wt mice (black bars) and Gsn mice (hatched bars). In
A, Student t-test showed average numbers of filopodia along neurites were more in Gsn
mice (P < 0.001). B showed that the average length of individual filopodia along neurites in Gsn
mice
did not significantly differ from wt mice. Error bar represents standard error.
[View Larger Version of this Image (15K GIF file)]
mice, neuronal cultures from Gsn
mice were fixed and double stained with an antibody to
neuron-specific
III tubulin to assess microtubules and
rhodamine phalloidin to assess actin filaments. Cells identified as neurons by DIC optics (Fig. 5 C) contained the
III isoform of tubulin in the neurite shaft (Fig. 5 B), similar to wt neurons (data not shown). Filopodia from growth
cones and neurites of Gsn
mice were labeled with
rhodamine phalloidin (Fig. 5 A) but did not generally contain microtubules. Therefore, filopodia along neurites in
Gsn
mice, like control mice, were mainly actin based.
Fig. 5.
Matching double
labeled fluorescence (A and
B) and DIC (C) images of a
hippocampal neurite and its
growth cone from Gsn mice
cultured on laminin substrate. The neuron was fixed
at 1 d in culture. A shows
rhodamine phalloidin fluorescence indicating actin filaments. B shows fluorescein
fluorescence derived from immunostaining with monoclonal antibody to neuron-specific
III tubulin. Note that the filopodia along the neurite seen in the matching DIC image contained actin filaments (A) but not microtubules (B). Also, the cell was positive for neuron-specific
III tubulin antibody (B), confirming the cell's
neuronal identity. Bar, 5 µm.
[View Larger Version of this Image (39K GIF file)]
Mice
mice was 29.85 + 3.91 µm/h (n = 18;
P > 0.05). In time-lapse sequences, most of the protrusive
activity arose from the growth cone, with far less activity in
the neurite shaft. Protrusive activity dropped off steeply
proximal to the neck of the growth cone. The increased
numbers of filopodia in Gsn
mice may be due to enhanced de novo elaboration of filopodia in the region of
the neurite just behind the growth cone or the retention of
filopodia, which failed to retract, after growth cone advance. Based upon time-lapse video data from wt and
Gsn
mice, only ~15-25% of the filopodia along the region of the neurite just proximal to the growth cone arose
de novo from the consolidated neurite. There was no significant difference in number of filopodia formed de novo
from the neurite shaft between wt and Gsn
mice. In contrast, the majority of neurite filopodia (75-85%) were
elaborated originally in the region of the growth cone and were retained on the neurite after the growth cone advanced. It was this population of filopodia, originally
formed from growth cones that contributed most to the increased number of filopodia along neurites in Gsn
mice.
These results suggested that the retraction of filopodia in
Gsn
mice might be delayed relative to wt mice.
mice were analyzed for the duration of filopodial persistence along neurites. The average
duration was 4.80 ± 0.55 min (n = 151) for Gsn
mice and
1.31 ± 0.15 min (n = 124) for wt mice (P < 0.001; Fig. 6
A). In wt mice, 62% of filopodia lasted 1 min or less. The
curve of filopodial persistence declined steeply: 91%
lasted <3 min and none lasted >10 min (Fig. 6 B). In contrast, only 23% of filopodia along neurites in Gsn
mice
retracted within 1 min, and the distribution curve exhibited a greater spread, with 12% of the filopodia persisting
for >10 min. Filopodia that persisted for >30 min (n = 5)
were assigned a 30-min duration, a strategy that would underestimate the number of long duration filopodia in Gsn
mice.
Fig. 6.
Duration of the extended phase of filopodia along neurites compared between wt mice (A, black; B, diamond) and
Gsn mice (A, hatched; B, square). Note the average duration
from Gsn
mice was significantly longer than that from wt mice
(P < 0.001 by Student t-test) in A. Error bar represents standard
error. In B, x axis represents the duration in minutes, y axis represents the percentage of neurite filopodia in wt mice (diamond),
and Gsn
mice (square) that remained extended for the corresponding time period. Note the marked difference in average,
median, and extreme values of filopodial duration in Gsn
versus
wt neurons.
[View Larger Version of this Image (17K GIF file)]
individual filopodia were studied frame by frame. These histories demonstrated that filopodia went through elongation/pause
phases and/or rapid linear retraction phases in both wt
(Fig. 7 A) and Gsn
(Fig. 7 B) mice. During the elongation
phase, the rate of elongation in Gsn
mice (0.048 ± 0.005 µm/s) did not significantly differ (P > 0.05) from wt mice
(0.051 ± 0.004 µm/s; Fig. 7 C). But during the linear retraction phase, the rate of retraction was twofold slower (P < 0.01) in the Gsn
mice (0.09 ± 0.01 µm/s) compared to
wt mice (0.16 ± 0.22 µm/s; Fig. 7 D). Furthermore, the dynamic pattern of filopodial retraction differed in Gsn
mice. In wt mice, most of the filopodia retracted in an "all or none" fashion (29/37, 78%); that is, once retraction began it continued until retraction was complete. Only 22%
(8/37) of the filopodia retracted in a "staircase" fashion,
e.g., cycles of elongation/pause phase followed by a rapid
retraction phase. In contrast, the majority of the Gsn
filopodia retracted in a "staircase" fashion (24/36, 67%);
the "all or none" behavior accounted for only 33% (12/36)
of the events. Even though some filopodia in Gsn
neurites were capable of retracting as fast as those of wt mice, the retraction of the majority of filopodia in Gsn
mice
was delayed. A statistical analysis showed that the filopodia from the Gsn
mice tended to pause for significantly
longer time periods than the wt mice (193.19 ± 43.87 s versus 49.32 ± 15.89 s; P < 0.01; Fig. 7 E).
Fig. 7.
Graphs of life histories of several filopodia along neurites from both wt (A) and Gsn mice (B). In the graphs, the x axis represents frames, which were 5 s apart, and the y axis represents the length of filopodia at any given time point. (C) The rate of elongation
during the linear elongation phase was similar in both wt (black bar) and Gsn
mice (hatched bar). (D) The rate of retraction during the
linear retraction phase was significantly slower (P < 0.01, Student t-test) in Gsn
mice (hatched bar) compared to wt mice (black bar).
(E) Further, the elongation/pause phase during retraction of filopodia in Gsn
mice (hatched bar) were significantly longer (P < 0.01, Student t-test) than those in wt mice (black bar). Error bar represents standard error.
[View Larger Versions of these Images (22 + 24K GIF file)]
), neurites gradually elongated by cycles of filopodial and lamellipodial protrusions, engorgement of the central domain of the growth cone, and consolidation of neurite. Both the engorgement of the central
domain of growth cones and the consolidation of neurites
did not appear to differ between wt and experimental mice. The protrusive and retractive activity of growth cone
lamellipodia from both wt and Gsn
mice was also analyzed. The changes in the area of lamellipodia at 5 s intervals in wt mice did not significantly differ (P > 0.05) from
that in Gsn
mice. However, the peripheral domain of hippocampal growth cones grown on a laminin substrate are
relatively loosely attached to the substrate compared to
the central domain and the shaft.
mice.
Nevertheless, there were so few filopodial extensions
from the neurite shaft, that markedly increased number of
filopodia along the region of the shaft under study had to
be a direct reflection of a filopodial retraction defect in the
growth cone.
Discussion
). Filopodia exhibit a considerably more
limited behavioral repertoire consisting of elongation and
retraction. These behaviors are mediated by a single cytoskeletal system: actin filaments. Actin-associated proteins are leading candidates to regulate length changes of
filopodia. Myosin is involved in the centripetal transport of actin filaments from the leading edge of the growth
cone (Lin et al., 1996
). In addition, myosin-V, a two-headed unconventional myosin present in growth cones
that possesses Ca2+-calmodulin-sensitive mechanochemical activities (Espreafico et al., 1992
; Cheney et al., 1993
)
appears to be involved in filopodial extension (Wang et
al., 1996
). The local obliteration of this molecule by chromophore-assisted laser inactivation in neuronal growth
cones from chick dorsal root ganglia decreased the rate of
filopodial extension but did not alter the rate of filopodial retraction, leading us to conclude that extension and retraction are independently regulated. Here we have demonstrated that the retraction phase is dependent on gelsolin.
mice contain
many more filopodia than those from wt mice could be explained not only by impaired retraction but also by increased filopodial extrusion. However, an alteration in
filopodia extrusion in the Gsn
is unlikely for several reasons. In general, filopodia tend to arise from the leading
edge of the growth cone and retract from the base (Lewis
and Bridgman, 1992
). As was apparent from the time-lapse video images, most of the filopodia on the consolidated segment under study here arose within the growth
cone, and as the growth cone advanced, the filopodia remained along the shaft of the newly consolidated neurite.
Only rarely did filopodia arise from the shaft. Thus, even if
increased filopodial extrusion did occur within the growth
cone, these Gsn
filopodia failed to coordinate their retraction with the forward advance of the growth cone.
Filopodia from Gsn
mice clearly remained extended for
longer times than wt controls. The tendency of Gsn
mice
to undergo a stuttering rather than a smooth retraction as
well as a less steep slope of retraction (but normal rates of
extension) also indicated that a retraction deficit existed. Although Gsn
mice had many more filopodia within the
segment under study, the mean lengths of the individual
filopodia did not differ from wt mice. Therefore, even
though retraction was impaired, after achieving a certain
length, filopodia did not continue to elongate. Therefore,
filopodial growth is an independent process that is limited
by factors other than the onset of retraction.
) with
similar domain structure that are expressed in diverse cell
types. All have the ability to bind to actin, and several
have actin filament severing activity. Adseverin has the
most structural and functional similarity to gelsolin, with
Ca2+-regulated actin filament severing activity, and its actin filament binding is inhibited by PPI, as well as phosphatidylinositol and phosphatidylserine (Maekawa and
Sakai, 1990
). Moreover, adseverin has been reported to be
expressed in mammalian brain tissues, though its precise
cellular localization is unknown (Tchakarov et al., 1990
).
Those facets of growth cone motility that are normal in the
Gsn
mice may be due to the presence of adseverin in
these regions or the actin regulatory protein ADF (Bamberg and Bray, 1987). ADF has complex effects on actin
filaments that are dependent upon conditions of pH and
ionic strength (Moon and Drubin, 1995
), so that it may
function to sever actin filaments and regulate actin dynamics in neuronal growth cones.
mice is due
to a dysfunction involving an effect of gelsolin on the depolymerization of actin filaments. Filopodial retraction occurs at rates from 0.09 to 0.16 µm/s, which corresponds to
32-59 monomers/s, based on the fact that 1 µm of polymerized actin contains 370 actin monomers (Hanson and
Lowy, 1963
). Since the most rapid off rate of actin monomers from an actin filament end is 0.18 monomers/s (Pollard, 1986
), simple monomer dissociation cannot explain
filopodia retraction. More likely, the nature of the dysfunction resulting in impaired filopodial retraction is due
to a loss of the actin filament-severing activity of gelsolin.
One potentially critical functional site may be the base of
the filopodia where gelsolin may sever active filaments
and thereby allow a coordinately engaged myosin motor
to slide the filaments through the proximal actin mesh. In
this model, filopodia remain extended as long as actin filaments at their base remain intact and prevent sliding of filaments into the shaft or growth cone. Few studies directly visualize well-preserved actin filaments in neuronal filopodia. In one study, negatively stained and freeze-etched EM
of permeabilized growth cone from rat superior cervical
ganglia explants contained tightly packed actin bundles in
the core of their filopodia (Lewis and Bridgman, 1992
). In
quiescent filopodia, the filament bundles ended at the base
of the filopodia; in contrast, in putatively active regions
the bundles extended into the lamellar region. Although
our experiments do not directly address filopodia protrusions from the growth cone itself, our model would predict
that gelsolin severs actin filaments at the point where they
extend into the lamella. Gelsolin may also release actin
from sites of tension along the filopodia, where actin is
linked to molecules such as vinculin and talin, which indirectly interact with the substrate. These explanations are
consistent with the stuttering retraction pattern seen in the
Gsn
mice and with the variation in retraction times,
which may arise due to the quite different detailed organizational patterns of actin filaments around individual
filopodia.
mice, placed in tissue culture, may enhance a phenotype that is less apparent
in vivo. The Gsn
mice do not display any obvious neurologic deficits, but they have not been subjected to detailed
anatomic and behavioral examination. The approach taken
here of knocking out specific genes encoding growth cone
proteins, may allow the systematic assignment of function to the many components of the growth cone.
Received for publication 12 March 1997 and in revised form 8 July 1997.
Please address all correspondence to Dr. Kenneth S. Kosik, Center for Neurological Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115; Tel.: (617) 525-5230; Fax: (617) 525-5252.We would like to thank Adriana Ferreira for assistance with some of the early observations and Philip G. Allen for helpful discussions.
ADF, actin depolymerizing factor; DIC, differential interference contrast; PPI, phosphoinositides; VEC, video enhanced contrast; wt, wild type.
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