* Instituto Mercedes y Martin Ferreyra-CONICET, 5000 Cordoba, Argentina; Departamento Quimica Biologica (CIQUIBIC),
Universidad Nacional Cordoba/CONICET, 5000 Cordoba, Argentina; and § Department of Neurology (Neuroscience), Harvard
Medical School, and Center for Neurological Diseases, Department of Medicine, Brigham and Women's Hospital, Boston,
Massachusetts 02115
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Abstract |
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In this study we have examined the cellular functions of ERM proteins in developing neurons. The results obtained indicate that there is a high degree of spatial and temporal correlation between the expression and subcellular localization of radixin and moesin with the morphological development of neuritic growth cones. More importantly, we show that double suppression of radixin and moesin, but not of ezrin-radixin or ezrin-moesin, results in reduction of growth cone size, disappearance of radial striations, retraction of the growth cone lamellipodial veil, and disorganization of actin filaments that invade the central region of growth cones where they colocalize with microtubules. Neuritic tips from radixin-moesin suppressed neurons displayed high filopodial protrusive activity; however, its rate of advance is 8-10 times slower than the one of growth cones from control neurons. Radixin-moesin suppressed neurons have short neurites and failed to develop an axon-like neurite, a phenomenon that appears to be directly linked with the alterations in growth cone structure and motility. Taken collectively, our data suggest that by regulating key aspects of growth cone development and maintenance, radixin and moesin modulate neurite formation and the development of neuronal polarity.
Key words: ERM proteins; growth cones; actin filaments; neurite formation; axonal elongation ![]() |
Introduction |
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DEVELOPING neurons often project elongated axons
toward their targets over relatively enormous distances. At the distal end of these projections is the
growth cone, a highly dynamic structure that contains cytoskeletal elements capable of generating a variety of navigational behaviors that determine the rate and direction of growth, as well as the distribution of membrane receptors that link environmental cues to implementation of
specific activities (Bray and Hollenbeck, 1988; Goodman
and Shatz, 1993
; Tanaka and Sabry, 1995
). Lamellipodial
veils and filopodial extensions are the most active processes, and are the sites at which growth cones undergo deformations such as elongation and retraction that are fundamental to movement. Currently the detailed molecular
assemblies that mediate and regulate growth cone morphology and activity are largely unknown. A crucial aspect
for solving this problem is the identification of proteins
that mediate interactions between cytoskeletal components and the plasma membrane. In this regard, the highly
related ezrin, radixin, and moesin proteins (the ERM1 proteins) are excellent candidates for molecules playing such a role (Bretscher et al., 1997
; Tsukita et al., 1997
). These
proteins have been localized to cleavage furrows, microvilli, ruffling membranes, and cell-cell/cell-matrix adhesion sites (Bretscher, 1983
; Tsukita et al., 1989
; Sato et al.,
1991
; Sato et al., 1992
; Berryman et al., 1993
) where they
appear to play a crucial role in modulating membrane protrusive activity (Takeuchi et al., 1994
; Henry et al., 1995
;
Martin et al., 1995
; Martin et al., 1997
). In accordance with
that, biochemical studies have established that the carboxyl termini of these proteins bind to actin filaments, while the amino terminus interacts with plasma membrane
proteins such as CD44 (Tsukita et al., 1994
; Hirao et al.,
1996
; for reviews see Bretscher et al., 1997
; Tsukita et al.,
1997
).
In the particular case of nerve cells, previous studies
have shown that an mAb designated 13H9 that recognizes
an epitope common to all members of the ERM family
(Winckler et al., 1994), and a polyclonal antibody against
radixin, strongly label growth cone actin-rich structures
such as radial striations, lamellipodial veils, and filopodial
extensions (Goslin et al., 1989
; Birgbauer et al., 1991
; DiTella et al., 1994
; Gonzalez-Agosti and Solomon, 1996
). In
addition, functional studies have established that in sympathetic neurons, growth cone collapse induced by NGF
deprivation is concomitant with a significant decrease of
the radixin staining of growth cones; interestingly, readding NGF, which induces rapid growth cone formation, is
accompanied by relocalization of radixin (Gonzalez-Agosti and Solomon, 1996
). Besides, the same authors showed
that when growth cones were subjected to an electrical field, radixin staining precisely localized to the leading
edges in the new direction of growth (Gonzalez-Agosti
and Solomon, 1996
). Taken collectively, these observations suggest that localization of radixin, and perhaps of
ezrin or moesin, may be essential for the normal expression of growth cone morphology and function.
To test this hypothesis directly, we examined the cellular functions of ERM proteins in mammalian neurons. To approach this problem, subcellular fractionation techniques in combination with Western blotting with specific antibodies were used initially to determine which type(s) of ERM proteins are present in growth cones. We then analyzed the pattern of expression, subcellular localization, and consequences of ERM suppression by antisense oligonucleotide treatment on the morphology, cytoskeletal organization, and dynamics of growth cones in primary cultured neurons. The results obtained suggest a key role for radixin and moesin in generating and maintaining the normal structural and functional organization of growth cones.
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Materials and Methods |
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Cell Culture
Dissociated cultures of hippocampal pyramidal cells from embryonic rat
brain tissue were prepared as described previously (Cáceres et al., 1986;
Mascotti et al., 1997
). Cells were plated onto polylysine-coated glass coverslips (12 or 25 mm in diameter) at densities ranging from 5,000 to 15,000 cells per cm2, and were maintained with DMEM plus 10% horse serum for
1 h. The coverslips with the attached cells were then transferred to 60-mm
Petri dishes containing serum-free medium plus the N2 mixture of Bottenstein and Sato (1979)
. All cultures were maintained in a humidified 37°C
incubator with 5% CO2.
Antisense Oligonucleotides
Two groups of antisense phosphorothioate oligonucleotides (S-modified)
were used in this study. The first group consists of antisense oligonucleotides corresponding to positions 1-24 of the mouse ezrin- (As Ez1), radixin- (As Rx1), or moesin- (As Mo1) coding regions, and were identical
to those previously used by Takeuchi et al. (1994) to analyze the function
of ERM proteins in nonneuronal cells. The second group consists of antisense oligonucleotides corresponding to positions 1387-1404 of the mouse
ezrin (As Ez2), 1334-1348 of the mouse radixin (As Rx2), and 1463-1477
of the mouse moesin (As Mo2) coding regions. Analysis of a gene data bank base (Gene Bank) showed that the sequences selected have no significant homology with any other known sequence. The oligonucleotides were purchased from Quality Controlled Biochemicals (Hopkinton, MA);
they were purified by reverse chromatography, and were taken up in serum-free medium as described previously (Cáceres and Kosik, 1990
;
Cáceres et al., 1992
; Morfini et al., 1997
). For all the experiments the antisense oligonucleotides were preincubated with 2 µl of Lipofectin Reagent
(1 mg/ml; GIBCO BRL, Gaithersburg, MD) diluted in 100 µl of serum-free medium. The resulting oligonucleotide suspension was then added to
the primary cultured neurons at concentrations ranging from 0.5 to 5 µM.
Control cultures were treated with the same concentration of the corresponding sense-strand oligonucleotides or scrambled antisense oligonucleotides.
Primary Antibodies
The following primary antibodies were used in this study: an mAb against
tyrosinated -tubulin (clone TUB-1A2, mouse IgG; Sigma Chemical Co.,
St. Louis, MO) diluted 1:2,000; a mAb against mouse recombinant ezrin
(clone M11, rat IgG, a generous gift of Dr. Sh. Tsukita, Kyoto University,
Japan; see also Takeuchi et al., 1994
) used undiluted; an mAb against
mouse recombinant radixin (clone R21, rat IgG, a generous gift of Dr. Sh.
Tsukita, Kyoto University, Japan) used undiluted; an mAb against mouse
recombinant moesin (clone M22, rat IgG, a generous gift of Dr. Sh. Tsukita, Kyoto University, Japan; see also Takeuchi et al., 1994
) used undiluted; an mAb against human moesin (clone 38, mouse IgG; Transduction Laboratories, Lexington, KY) diluted 1:200; and an affinity-purified rabbit polyclonal antibody raised against a peptide corresponding to amino
acids 400-409 (KSAIAKQAAD) of mouse radixin (Research Genetics,
Huntsville, AL; see also Winckler et al., 1994
) diluted 1:50 or 1:100.
Immunofluorescence
Cells were fixed before or after detergent extraction under microtubule-stabilizing conditions, and were processed for immunofluorescence as previously described (Pigino et al., 1997). For some experiments a mild extraction protocol that preserves cytoskeletal-membrane interactions was
also used (Nakata and Hirokawa, 1987
; Brandt et al., 1995
; Pigino et al.,
1997
). Cells were washed in extraction buffer (80 mM Pipes, pH 6.8, 1 mM
MgCl2, 1 mM EGTA, 30% [vol/vol] glycerol, 1 mM GTP), incubated for
30 s with extraction buffer containing 0.02% saponin, and washed with extraction buffer. Cells were then fixed for 1 h at room temperature with 2%
(wt/vol) paraformaldehyde, 0.1% (vol/vol) glutaraldehyde in extraction
buffer, washed with PBS, permeabilized with 0.1% (vol/vol) Triton X-100
in PBS for 30 min, and finally washed in PBS. The antibody-staining protocol entailed labeling with the first primary antibody, washing with PBS,
staining with labeled secondary antibody (fluorescein- or rhodamine-conjugated) and washing similarly; the same procedure was repeated for the
second primary antibody. Incubations with primary antibodies were for 1 or 3 h at room temperature, while incubations with secondary antibodies
were performed for 1 h at 37°C. For some experiments, rhodamine-labeled phalloidin (Molecular Probes, Inc.) was included with the secondary antibody to visualize filamentous actin (F-actin). The cells were observed with an inverted microscope (Carl Zeiss Axiovert 35M) equipped
with epifluorescence and differential interference contrast (DIC) optics
using a 40×, 63×, or a 100× objective (Carl Zeiss). Fluorescent images
were captured under regular fluorescence microscopy with a silicon-intensified target camera (SIT-C2400; Hamamatsu Phototonics, Bridgewater, NJ). The images were digitized directly into a Metamorph/Metafluor Image Processor (Universal Imaging Corporation, West Chester, PA). Photographs were printed using Adobe Photoshop.
For some experiments the relative intensities of tubulin, tau, -actinin,
cyclin-dependent kinase 5 (cdk5),
-gc (Mascotti et al., 1997
), ezrin, radixin, and moesin immunofluorescence as well as of phalloidin staining
were evaluated in fixed unextracted cells or in detergent-extracted cytoskeletons using quantitative fluorescence techniques as described previously
(DiTella et al., 1994
; DiTella et al., 1996
; Pigino et al., 1997
). To image-labeled cells, the incoming epifluorescence illumination was attenuated with glass neutral density filters. Images were formed on the faceplate of
the SIT camera set for manual sensitivity, gain, and black level; they were
digitized directly into the Metamorph/Metafluor Image Processor and
stored on laser discs with an optical memory disc recorder (OMDR, LQ-3031, Panasonic). Fluorescence intensity measurements were perfumed
pixel by pixel within the cell body and in neurites of identified neurons.
Using these data, we then calculated the average fluorescence intensity
within the cell body as well as the inner, middle, and distal third of identified neurites (either minor processes or axons), including the central and
peripheral regions of growth cones. In addition, in some cases the distribution of phalloidin staining or ERM immunofluorescence were analyzed
using high-resolution fluorescence microscopy and ratio image analysis
with the image processing menu of the Metamorph/Metafluor system.
Video Microscopy
For time lapse video-enhanced differential interference contrast microscopy (VEC-DIC), hippocampal cells were cultured for up to 3 d in special
Petri dishes prepared according to the procedures described by Dotti et al.
(1988). At different time points after plating, the dishes containing the attached cells were placed on the heated stage of the inverted microscope
and observed with a 100× DIC oil immersion objective. 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 a 1.6× relay lens, and were detected with a Newvicon camera (Dage-MTI, Inc.,
Michigan City, IN). Images were collected every 30 s, summed (32-frame
averaging) by using Metamorph software, and stored on a laser disc using
the OMDR. Photographs were then printed using Adobe Photoshop.
Subcellular Fractionation Techniques
Fetal rat brain (18 d of gestation) was fractionated according to Pfenninger et al. (1983; see also Quiroga et al., 1995) to obtain growth cone
particles (GCPs). In brief, the low-speed supernatant (L) of fetal brain homogenate (H) was loaded on a discontinuous sucrose gradient in which
the 0.75 M and 1 M sucrose layers were replaced with a single 0.83 M sucrose step. This procedure facilitated collection of the interface, and increased GCP yield without decreasing purity (Quiroga et al., 1995
). The
0.32 M/0.83 M interface, or A fraction, was collected, diluted with 0.32 M sucrose, and pelleted to give the GCP fraction. This was resuspended in
0.32 M sucrose for experimentation.
Western Blot Analysis of ERM Protein Expression
Changes in the levels of ezrin, radixin, and moesin during neuronal development were analyzed by Western blotting as previously described (Morfini et al., 1997; Pigino et al., 1997
). In brief, equal amounts of crude brain
homogenates, subcellular fractions, or whole cell extracts from cultured
cells were separated on SDS-PAGE and transferred to polivinylidene difluoride (PVDF) membranes in a Tris-glycine buffer, 20% methanol. The
filters were dried, washed several times with TBS (10 mM Tris, pH 7.5, 150 mM NaCl), and blocked for 1 h in TBS containing 5% BSA. The filters were incubated for 1 h at 37°C with the primary antibodies in TBS
containing 5% BSA. The filters were then washed three times (10 min
each) in TBS containing 0.05% Tween 20, and were incubated with a secondary horseradish peroxidase-conjugated antibody (Promega Corp.,
Madison, WI) for 1 h at 37°C. After five washes with TBS and 0.05%
Tween 20, the blots were developed using a chemiluminiscence detection kit (ECL; Amersham Life Science, Inc., Arlington Heights, IL). In addition, ERM protein levels were measured by quantitative immunoblotting
as described by Drubin et al. (1985; see also DiTella et al., 1996
). For such
a purpose, immunoblots were probed with the corresponding primary antibodies, followed by incubation with [125I]protein A. Autoradiography
was performed on X-omat AR film (Eastman Kodak Co., Rochester, NY)
using intensifying screens. Autoradiographs were aligned with immunoblots, and ERM protein levels were quantitated by scintillation counting
of PVDF blot slices.
Morphometric Analysis of Neuronal Shape Parameters
Images were digitized on a video monitor using Metamorph/Metafluor
software. To measure neurite length or growth cone shape parameters,
fixed unstained or antibody-labeled cells were randomly selected and
traced from a video screen using the morphometric menu of the Metamorph as described previously (Cáceres et al., 1992; DiTella et al., 1994
;
DiTella et al., 1996
). All measurements were performed using DIC optics
at a final magnification of 768×. Differences among groups were analyzed
by the use of ANOVA and Student-Newman Keuls test.
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Results |
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Expression and Subcellular Distribution of ERM Protein in Developing Neurons
Previous studies have shown that antigen 13H9, which corresponds to an epitope common to all members of the
ERM family (Winckler et al., 1994), is expressed in cultured hippocampal pyramidal neurons (Goslin et al.,
1989
), cerebellar macroneurons (DiTella et al., 1994
) and
dorsal root ganglion neurons (Birgbauer et al., 1991
). Despite that, the pattern of expression and subcellular distribution of individual members of the ERM family during
neuronal development, both in situ and in vitro, has not
yet been established. Therefore, in the first set of experiments we used several antibodies specific for each of these
proteins to analyze their expression and subcellular distribution in brain homogenates and cell extracts by Western
blotting from developing and adult animals, as well as in
growth cone preparations obtained from E 18 rat embryos.
Fig. 1 A shows the monospecificity of the mAb M11 against mouse recombinant ezrin, of the affinity-purified rabbit polyclonal antibody raised against a peptide corresponding to amino acids 400-409 (KSAIAKQAAD) of mouse radixin, and of the mAb M22 directed against mouse recombinant moesin. Each of these antibodies labels a single band of 85 kD (anti-ezrin; Fig. 1 A, lanes 1 and 2), 82 kD (anti-radixin; Fig. 1 A, lanes 3 and 4), or 75 kD (anti-moesin; Fig. 1 A, lanes 5 and 6) in Western blots of whole tissue homogenates obtained from the cerebral cortex of E 18 rat embryos, or from cell extracts obtained from cultured hippocampal pyramidal neurons. A commercially available mAb against human moesin (clone 38) also strongly labeled a 75-kD band in embryonic brain extracts, and with a much lower intensity a protein specie migrating at the position of radixin (not shown).
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Fig. 1 B shows that in the cerebral cortex or hippocampus, expression of the ERM immunoreactive protein species is higher at late embryonic and early postnatal days, declining gradually but significantly until adulthood where the lowest levels are detected; an equivalent expression pattern was detected by quantitative Western blotting of cell extracts obtained from cultures of hippocampal pyramidal neurons (Fig. 1 C). In addition, Western blot analysis of GPCs obtained by subcellular fractionation of embryonic brain tissue revealed the presence of each of the ERM members in this compartment (Fig. 1 D). This analysis also showed that radixin is the most enriched ERM family member in GPCs, while ezrin is the less abundant.
In the next series of experiments, the subcellular distribution of ERM proteins in cultures of hippocampal pyramidal neurons was analyzed by fluorescence microscopy (Fig. 2). Both undifferentiated and neurite-bearing cells stained with the antibodies against ERM family members; however, significant differences in their staining patterns were detected. Staining with the anti-ezrin antibody (mAb M11) gave intense labeling of cell bodies, while neurites and growth cones stain only very faintly, becoming visible only when the mAb M11 was used at high concentrations. High power views of labeled cells revealed no apparent specific staining of growth cone components (data not shown).
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By contrast, radixin and moesin immunofluorescence
were not only detected in cell bodies, but also in neurites
and growth cones. High-resolution fluorescence microscopy revealed strong radixin staining of the actin-rich peripheral zone of growth cones; within that region, radixin
immunolabeling appears to be concentrated at the base of
short filopodial extensions that emerge from points all
along the growth cone perimeter (Fig. 2, A-E). On the
other hand, moesin immunofluorescence appears to be
concentrated along radial striations that extend from the
central growth cone region towards the peripheral lamellipodial veil (Fig. 2, F-J) with a pattern that closely resembles the distribution of antigen 13H9 (see also Goslin et al.,
1989; DiTella et al., 1994
) or F-actin. To test whether localization of radixin or moesin within growth cones involves a plasma membrane association, a mild extraction protocol using saponin was used. This procedure, which
selectively removes cytosolic proteins but retains cytoskeletal membrane interactions (Nakata and Hirokawa, 1987
;
Brandt et al., 1995
; see Materials and Methods), did not alter the distribution of either the radixin (Fig. 2, D and E)
or moesin immunolabeling (Fig. 2, I and J) when compared with that observed in cells fixed before detergent
extraction.
Phosphorothioate Antisense Oligonucleotides Inhibit Expression of ERM Family Members
The localization and staining patterns of radixin and
moesin are consistent with the possibility that they regulate important features of growth cone formation, maintenance, and/or dynamics. Therefore, to investigate this
possibility, we used antisense phosphorothioate oligonucleotides to inhibit expression of ERM proteins. Experimental conditions were optimized as follows. Neurons were cultured for 48 h in serum-free medium, during
which time the cells extend three to four minor processes
and a single axon; under control conditions all of these
neurites are tipped by well-defined growth cones (Cáceres
et al., 1986; Cáceres et al., 1992
; Mascotti et al., 1997
). Antisense, sense, or mismatched oligonucleotides were added
to the culture medium 24 h after plating in the presence of
Lipofectin, and were replenished every 6 h at concentrations ranging from 0.5 to 2 µM.
Quantitative immunofluorescence analyses indicate that antisense treatment reduced the levels of ERM proteins in a dose- and time-dependent manner (Fig. 3, A-C). By 1 d in either 1 or 2 µM of the antisense oligonucleotides specific for individual members of the ERM family, the levels of ezrin, radixin, or moesin had selectively diminished to <20% of the levels within parallel oligonucleotide-free cultures, or from equivalent ones treated with the corresponding sense or mismatched antisense oligonucleotides (Fig. 3, A-C). By 36 h, ezrin, radixin, or moesin were virtually undetectable in neurons exposed to 1 or 2 µM antisense, and the levels had fallen to <20% of the control levels in neurons exposed to 0.5 µM. Quantitative Western blotting confirmed these observations, and clearly revealed that the antisense oligonucleotides selectively and effectively reduced the levels of individual members of the ERM family (Fig. 3 D). Furthermore, adding the ezrin-radixin antisense mixture to the culture medium suppressed both ezrin and radixin expression, but did not affect that of moesin; conversely, the radixin-moesin antisense mixture suppresses both radixin and moesin expression without altering that of ezrin (Fig. 3, E and F). In this regard, it is worth noting that we could not find any clear cellular compensation for suppressing of one or two ERM family members, as determined by either quantitative fluorescence or Western blotting. In addition, the expression of ERM family members was totally suppressed in the presence of the ezrin-radixin-moesin mixture of antisense oligonucleotides (not shown).
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Inhibition of Radixin and Moesin Expression Alters Growth Cone Morphology and Cytoskeletal Organization
In the next series of experiments we analyzed the consequences of ERM suppression on growth cone morphology
and cytoskeletal organization. In control or sense-treated
neurons, growth cones at the tips of axons consist of a flattened expansion of the cytoplasm of ~10-15 µm across.
High-resolution fluorescence microscopy and digital image processing of growth cones from neurons double-stained with antibodies against tyrosinated -tubulin and
rhodamine phalloidin revealed a central microtubule-containing zone completely surrounded by a radially oriented
array of F-actin (e.g., actin ribs; see Weinhofer et al., 1997
)
from which numerous and short filopodial extensions
emerge (Fig. 4 A). None of the ezrin, moesin, or the pair of
ezrin-moesin (Fig. 4 B) or ezrin-radixin (Fig. 4 C) antisense oligonucleotides induced any significant alteration
in growth cone morphology, size, filopodial number, and/
or the pattern of distribution of microtubules and actin filaments (Fig. 4, B and C, and Fig. 5). In sharp contrast,
when neurons were cultured in the presence of the radixin-moesin antisense mixture (2 µM), a significant change
of growth cone shape was gradually induced. These alterations involved a dramatic reduction of growth cone area,
disappearance of radial striations, retraction of the growth cone lamellipodial veil, and a reduction in the number of
filopodia; the length of the remaining filopodia increases
significantly (Fig. 4, D-L; see also Fig. 5). Differences
were also detected in the staining pattern of rhodamine-
phalloidin. Thus, while in antisense-treated cells, F-actin
was concentrated at neuritic tips as in control cells, it was
not distributed following specific patterns (e.g., radial striations) but rather had a diffuse appearance filling the entire growth cone, including its central region where it colocalizes extensively with microtubules (Fig. 4, G-L). Ratio image analysis of growth cones from cells double-labeled
with rhodamine phalloidin and tyrosinated
-tubulin revealed that in neurons lacking radixin and moesin, F-actin
predominates over dynamic microtubules within the central region of growth cones (Fig. 4, G-L). Thus, a considerable reduction in the number of microtubules entering
neuritic tips was evident in cells lacking radixin and
moesin; besides, time-course analyses revealed that this
phenomenon parallels the reduction of growth cone size
observed in these cells. Our results also show that when
the medium is changed and replaced by a fresh one lacking
the radixin-moesin antisense mixture, the cells reexpress
radixin and moesin, a phenomenon paralleled by an increase in growth cone size, the appearance of actin ribs,
and lamellipodial veils (Table I).
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Inhibition of Radixin and Moesin Expression Alters Growth Cone Motility
Because of the alterations in growth cone shape and cytoskeletal organization detected in neurons with reduced
levels of radixin and moesin, we hypothesized that they
may have motility defects. Therefore, we decided to analyze rapid changes in lamellipodial and filopodial activity
in control and antisense-treated neurons using time-lapse
VEC-DIC microscopy. As expected, according to previous
observations from this and other laboratories (see Goldberg and Burmeister, 1986; Kleitman and Johnson, 1989;
Lu et al., 1997
), in control and sense-treated neurons, axons gradually elongate by cycles of lamellipodial protrusions and retractions, engorgement of the central domain
of the growth cone, and consolidation of the neurite. In
these cells the leading margins of lamellipodia advance
slowly and smoothly across the substratum at a rate of 0.08-0.15 µm/min (Fig. 6). Time-lapse sequences revealed
that after the leading edge of a lamellipodial veil have advanced 6-8 µm, a new axonal segment of ~4-6 µm in
length is formed at the base of the growth cone. A completely different picture was observed in neurons treated
with the radixin-moesin antisense oligonucleotide mixture; in these cells axonal tips, which lack a lamellipodial veil, advance 8-10 times slower than control growth cones;
this phenomenon is paralleled by a dramatic inhibition in
the rate of axonal elongation (Fig. 6).
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On the other hand, they display high filopodial protrusive activity (Fig. 7). This increased activity may be due to
a delayed retraction of filopodia and/or an enhanced rate
of filopodial extension. To test for these possibilities, sequences from control, sense-treated, and antisense-treated
cultures were analyzed for the duration of filopodial persistence or the rate of filopodial elongation. In nontreated
(Fig. 7, A-C), sense-treated (Fig. 7, D-F), or in cells
treated with a mixture of ezrin-radixin or ezrin-moesin antisense oligonucleotides (not shown), 75% of filopodia
lasted 1 min or less. The curve of filopodial persistence declined steeply: 85% lasted <3 min, and none lasted >10
min. In sharp contrast, more than 90% of growth cone
filopodia from cells with the double suppression of radixin
and moesin lasted for more than 20 min, with 55% failing
to retract after 30 min (Fig. 7, G-L). The life histories of
individual filopodia from control and antisense-treated neurons were studied frame by frame. These histories
demonstrate that in control cells, filopodia went to elongation/pause phases and/or rapid linear retraction phases.
Antisense-treated neurons also exhibited similar phases.
However, during the elongation phase, the rate of elongation of cells treated with the radixin-moesin antisense
mixture was 0.45 ± 0.08 µm/min (n = 100), which was significantly higher (P < 0.01) than that of nontreated cells (0.12 ± 0.02 µm/min, n = 50), radixin-moesin sense-treated (0.15 ± 0.04, n = 50) cells, or cells treated with an
ezrin-radixin antisense mixture (0.13 ± 0.03, n = 40).
Even more dramatic differences were observed when we
analyzed the retraction phase. Thus, the rate of retraction
was more than threefold slower in the radixin-moesin antisense-treated neurons than in control ones (Fig. 8 A).
Furthermore, the dynamic pattern of filopodial retraction
was also different. Thus, in control cells most of the filopodia retracted in an all-or-none fashion (see Lu et al., 1997);
that is, once retraction began it continued until completion. Only a small percentage (<10%) retracted in a discontinuous fashion, in which retraction was interrupted by
either a pause phase or an elongation/pause phase. On
the other hand, >90% of the radixin-moesin antisense-treated cells exhibit this behavior, with <5% accounting
for the all-or-none retraction pattern. This analysis revealed that during the retraction phase, filopodia from radixin-moesin-suppressed neurons tended to pause for significantly longer time periods than did filopodia from
control neurons (Fig. 8 B).
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Development of Neuronal Polarity in Radixin-Moesin-suppressed Neurons
The dramatic alterations in growth cone organization and motility observed in neurons lacking radixin and moesin raised the possibility of these cells having significant alterations in process formation. Therefore, we decided to examine the consequences of radixin and moesin suppression on neurite outgrowth and development of neuronal polarity. For such a purpose, cells were treated with the antisense oligonucleotides shortly after plating, when most of the cells lack neurites, and were examined 24 and 36 h later. The results obtained indicate that neither the single suppression of ezrin, radixin, or moesin, nor the double suppression of ezrin and radixin or ezrin and moesin alters the neurite outgrowth response of these cells when compared with the one observed in control or sense-treated cultures (Table II). Besides, in all cases these neurons follow a predictable temporal sequence of gross morphological changes that involves initial extension of a lamellipodial veil (Stage I) that is later replaced by three to four minor neurites (Stage II), one of which becomes the neuron's axon (Stage III).
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On the other hand, the mixture of radixin-moesin antisense oligonucleotides profoundly affects development of these neurons in a dose-dependent manner (Table II and Fig. 9). The most significant alterations, which were observed when the neurons were treated with 2 µM of the antisense mixture, involved: (a) a decrease in the length of minor processes and in the percentage of cells reaching stage III of neuritic development; (b) a reduction in growth cone area; and (c) the appearance of long filopodial extensions emerging from the cell bodies and neuritic tips (see Fig. 9, G-H). No such alterations were observed when the cells were treated with a mixture of the corresponding sense oligonucleotides (Table II).
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It has recently been shown that in cultured hippocampal
pyramidal neurons, it is possible to identify the prospective axon by the appearance of a large growth cone in one
of the multiple minor processes (Bradke and Dotti, 1997;
see also Morfini et al., 1994). Since the radixin-moesin antisense mixture significantly decreases the number of cells
entering stage III, it became of interest to determine
whether or not growth cone enlargement occurs in stage II
hippocampal pyramidal neurons treated with the radixin- moesin antisense oligonucleotide mixture. To address this
issue, growth cone surface area was measured in the neurites of stage II cells from control and antisense-treated
neurons. Frequency histogram analyses of growth cone
area revealed that in nontreated or sense-treated cultures,
more than 50% of stage II cells exhibit a single growth
cone that was significantly larger than the others (Figs. 9
and 10). By contrast, none of stage II cells from cultures treated with the radixin-moesin antisense mixture display
this pattern; most of the cells have small growth cones of
uniform size.
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Discussion |
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In nonneuronal cells, ERM proteins play crucial structural
and regulatory roles by stabilizing specialized plasma
membrane domains such as microvilli and membrane ruffles (see Bretscher et al., 1997; Tsukita et al., 1997
). The
present observations are fully consistent with this idea,
and provide the first set of direct experimental evidence
revealing the functional involvement of ERM family
members in neuronal morphogenesis. Several lines of evidence support this proposal. First, we found a high degree of spatial and temporal correlation between the expression and subcellular localization of radixin and moesin
with the morphological development of neuritic growth
cones. Second, and perhaps more importantly, double suppression of these proteins with a mixture of antisense oligonucleotides results in dramatic alterations of growth
cone shape, distribution of actin filaments, and locomotive activity of growth cones. The fact that we observed growth
cone alterations only after double suppression of radixin
and moesin, but not after ezrin-radixin or ezrin-moesin
suppression, is not unexpected. Treating nonneuronal cells
with mixtures of antisense oligonucleotides against ERM
family members has revealed that these proteins can be
functionally substituted (Takeuchi et al., 1994
). However,
this redundancy appears to be cell type-specific. Thus, in
thymoma and mouse epithelial cells, ezrin and radixin, rather than the combination of radixin-moesin, appear to
have redundant functions, while moesin has some synergetic functional interaction with the two other family
members (Takeuchi et al., 1994
). On the other hand, in
3T3 fibroblasts, displacement of moesin by exogenously
expressed radixin occurs without any apparent phenotype
such as alterations in spreading or cytokinesis, suggesting that in these cells radixin and moesin can be functionally
substituted (Henry et al., 1995
). Differences in the use of
ERM family members among different cell types may reflect the very distinct and restricted patterns of expression
of these proteins in cell tissues, and/or variations in their
subcellular distribution (Amieva et al., 1994
; Amieva and
Furthmayr, 1995
; Berryman et al., 1993
; Takeuchi et al.,
1994
; Schwartz-Albiez et al., 1995
; Bretscher et al., 1997
). In agreement with that, we showed by quantitative immunofluorescence of cultured neurons and Western blotting
of growth cone fractions that only radixin and moesin are
concentrated in growth cones; ezrin was mainly restricted
to the cell body.
Recent observations of living growth cones using Helisoma neurons as a model system have established that
growth cone formation is characterized by a three-stage
process involving sequential appearance of a terminal
swelling, extension of radial striationsa chevron arrangement of actin ribs
and formation of a lamellipodial veil. After a continuous lamellipodium has surrounded the
terminal swelling, growth cones continue to increase in
size as a result of lateral and centrifugal extensions (Welnhofer et al., 1997). Since disappearance of radial striations
and lamellipodial veils and reduction of growth cone size
are early and typical features of neuritic tips from neurons
lacking radixin-moesin, it follows that these proteins appear to regulate key stages of growth cone morphogenesis.
Such a role is fully consistent with current views about the
involvement of ERM family members in regulating cortical structures. Administration of antisense oligonucleotides to suppress ERM protein expression results in loss
of cell surface microvilli and cell contacts (Takeuchi et al.,
1994
). On the other hand, overexpression of the COOH-terminal domains of ezrin or radixin, which may sequester
a substantial portion of F-actin and associated proteins, results in dramatic reorganization of the cortical cytoskeleton (Henry et al., 1995
; Martin et al., 1995
; Martin et al.,
1997
) while overexpression of the NH2-terminal domain,
acting as a negative dominant mutant by inactivating
COOH-terminal domains, reduces microvilli formation
(Crepaldi et al., 1997
). On the basis of these and related
observations, it has been proposed that ERM family members working in conjunction with actin cross-linkers may
promote assembly of actin-rich surface structures by linking the sides of actin filaments to plasma membrane proteins (Tsukita et al., 1989
; Roy et al., 1997
; Bretscher et al.,
1997
; Tsukita et al., 1997
). In support of this we found that
in neurons expressing reduced levels of radixin and
moesin, growth cone alterations were paralleled by a dramatic disorganization of F-actin.
Inhibition of radixin and moesin expression also result
in the appearance of several long filopodial processes at
the tip of neurites; these structures appear to arise by impaired retraction and increased filopodial extrusion. While
the reason(s) for this phenotype are not clear, at present
several possibilities should be considered. For example,
long filopodial extensions may arise if ERM proteins also
contribute to the regulation of actin assembly by capping
actin filament ends at restricted sites (Mitchison and Kirschner, 1988; Forscher et al., 1992
). In this regard it is worth
noting that radixin was originally isolated as a barbed-end capping protein (Tsukita et al., 1989
), and that recent studies suggest that the nature of the interaction of ERM family members with actin may be far more complex than
originally expected (Berryman et al., 1995
; Bretscher et al.,
1997
). Alternatively, these long filopodial processes may
not simply represent the consequences of suppressing radixin and moesin, but rather arise as a result of an imbalance in the activity of other actin-associated proteins involved in regulating length changes of filopodia, such as
myosin-V (Lin et al., 1996
). Clearly, additional studies are
required for distinguishing between these and related possibilities.
Analysis of the phenotype of neurons expressing low
levels of radixin and moesin also revealed an inhibition of
their neurite outgrowth response. It is likely that this negative response is directly linked with alterations in growth
cone structure and motility. In fact, there is a considerable
body of evidence indicating that growth cone formation is
essential for proper neurite extension and differentiation
(Goldberg and Burmeister, 1986; Weinhofer et al., 1997
).
Besides, according to current models, the cycle of lamellipodial protrusion, extension, and adhesion to the substrate will promote generation of tension to move the growth
cone and its content (Joshi et al., 1985
; Mitchison and Kirschner, 1988
; Letourneau, 1997
). In support of this finding, it
has been shown that fully developed lamellipodial growth
cones translocate several times faster than either neuritic
tips containing only filopodial extensions or blunt ends
(Kleitman and Johnson, 1987
). The disappearance of radial striations and lamellipodial veils may therefore significantly affect process extension by reducing the surface
membrane at the leading edge, and hence decreasing the
force generation capability of growth cones. Additional
events may also account for the reduced rate of elongation. For example, since growth cones are the preferred sites for adding newly synthesized membrane proteins during elongation (Pfenninger and Mayle-Pfenninger, 1981
;
Lockerbie et al., 1991
; Craig et al., 1995
; Bradke and Dotti,
1997
), preventing advance of microtubules not only into
the peripheral domain of the leading edge, but also into
its central domain, may affect process extension by decreasing the membrane vesicle content of neuritic tips. In favor
of this view, we found that actin disorganization was accompanied by a significant decrease in the number of
microtubules entering the growth cones and in the bodipy-ceramide staining of neuritic tips (Cáceres and Paglini, unpublished observations); besides, time-lapse video
microscopy revealed no evidence of engorgement of these
neuritic tips.
In cultured hippocampal pyramidal neurons, neuronal
polarization occurs at the transition between stages II and
III, when one of the multiple minor neurites initiates a
phase of rapid elongation to become the neuron's axon
(Dotti et al., 1988). It has recently been shown that one of
the earliest events preceding axonal formation is the appearance of a large growth cone in one of the minor neurites (Bradke and Dotti, 1997
). No such increase in growth
cone size was detected in stage II cells from cultures treated with the mixture of radixin-moesin antisense oligonucleotides. All growth cones display reduced size, and
lack radial striations and lamellipodial veils. Besides, the
percentage of cells reaching stage III of neuritic development was dramatically reduced. These phenotypes are not
simply the consequence of suppressing any growth cone
actin-regulatory protein, since for example, hippocampal pyramidal neurons from gelsolin-null mice that display delayed filopodial retraction extend axons and elongate neurites at a rate similar to that of their counterparts in wild-type animals (Lu et al., 1997
). Therefore, our results also
suggest that radixin and moesin modulate the development of neuronal polarity by regulating key aspects of
growth cone development and maintenance.
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Footnotes |
---|
Received for publication 30 April 1998 and in revised form 10 September 1998.
The authors express their deep gratitude to Dr. Sh. Tsukita (Kyoto University, Japan) for providing some of the antibodies used in this study.
Address all correspondence to Alfredo Cáceres, Instituto Mercedes y
Martín Ferreyra, Casilla de Correo 389, 5000 Córdoba Argentina. Tel.: 54-51-681465. Fax: 54-51-695163. E-mail: acaceres{at}immf.uncor.edu
This work was supported by grants from CONICET (PICT-PIP 0052), CONICOR, Fundación Perez-Companc, Fundación Antorchas, and a Fogarty International Collaborative Award (FIRCA). It was also supported by a Howard Hughes Medical Institute grant to A. Cáceres (HMMI 75197-553201) awarded under the International Research Scholars Program. G. Paglini and P. Kunda are fellows from the National Council of Research from Argentina (CONICET).
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Abbreviations used in this paper |
---|
DIC, differential interference contrast; ERM, ezrin, radixin, and moesin; VEC-DIC, video-enhanced differential interference contrast.
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