Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, 303 East Chicago Avenue, Ward 11-145, Chicago, IL 60611, USA
Author for correspondence (e-mail:
r-goldman{at}northwestern.edu)
Accepted 1 April 2003
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Summary |
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Key words: Intermediate filaments, Peripherin, Dynein, Kinesin, Cytoskeleton
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Introduction |
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Recently, observations of neurons expressing either type IV IF GFP-tagged
neurofilament (NF) medium (GFP-NF-M) or heavy (GFP-NF-H) subunits, revealed
that short IF can move at rates up to 1.8 µm/second in vivo. However,
since these NF spend 73-80% of their time not moving, they remain categorized
as components of slow axonal transport
(Roy et al., 2000
;
Wang and Brown, 2001
;
Wang et al., 2000
). It has
also been shown that non-membrane bound particles containing non-filamentous
forms of NF proteins and kinesin can move rapidly along MT in cell-free
preparations of squid axoplasm (Prahlad et
al., 2000
). Similarly, in extracts of bovine spinal cord, NF have
been reported to move rapidly along MT in association with cytoplasmic dynein
and kinesin (Shah et al.,
2000
). However, since pause times were not determined in either of
these in vitro studies, it is possible that these movements may also reflect
stationary periods punctuated by rapid movements.
Direct observations of the motile properties of IF in non-neuronal cells
such as those expressing GFP-tagged vimentin, a type III IF protein, have
revealed a surprisingly wide range of movements
(Ho et al., 1998;
Prahlad et al., 1998
;
Yoon et al., 1998
). This was
especially evident in spreading cells in which large numbers of vimentin
particles move in both retrograde and anterograde directions at speeds >1.0
µm/second (Prahlad et al.,
1998
). These particles frequently form short filamentous
structures termed squiggles, which subsequently become incorporated into the
longer IF that typify fully spread fibroblasts. The rapid movements exhibited
by vimentin particles are dependent upon MT, conventional kinesin and
cytoplasmic dynein (Helfand et al.,
2002
; Prahlad et al.,
1998
).
On the basis of these observations, it was of interest to determine whether
similar types of rapidly moving IF particles and squiggles could provide a
mechanism for the timely turnover of IF subunits in the most distal regions of
axons. In this study we describe the transport of the type III IF protein
peripherin in differentiating PC12 cells, a widely used model for studies of
sympathetic neurons (Fujita et al.,
1989). Peripherin is the major IF protein present in PC12 cells
and small caliber, non-myelinated neurons of the PNS
(Brody et al., 1989
;
Escurat et al., 1990
;
Parysek and Goldman, 1988
;
Troy et al., 1990a
). Our
results demonstrate that non-membrane bound peripherin particles and squiggles
move bi-directionally along neurites as components of a rapid transit system
capable of delivering cytoskeletal proteins to all regions of neurons over
relatively short time periods. The results are discussed in light of various
models for the transport and turnover of neuronal cytoskeletal proteins.
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Materials and Methods |
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Antibodies
Rabbit anti-peripherin (Parysek and
Goldman, 1988), anti-dynein heavy chain (HC) and light
intermediate chains 1 and 2 [LIC 1 and 2; provided by Richard Vallee, Columbia
University (Helfand et al.,
2002
; Tynan et al.,
2000
)], Arp-1 [a gift of David Meyer, UCLA
(Troy et al., 1990b
)] and
anti-kinesin [PCP42; provided by Ron Vale, UCSF
(Prahlad et al., 1998
)] were
used. Mouse monoclonal anti-ß-tubulin (TU 27B, provided by Lester Binder,
Northwestern University), anti-dynein intermediate chain (IC; Chemicon
International, Inc.), anti-p150Glued and dynamitin (BD
Biosciences), and anti-kinesin heavy chain (H1, Chemicon) were also employed.
Other antibodies included mouse monoclonal anti-c-myc
(Evan et al., 1985
) and
anti-GFP (clones 7.1 and 13.1; Roche).
FITC-, lissamine-rhodamine and Cy5-conjugated goat anti-mouse and anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA) were employed for indirect immunofluorescence. Peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch) was used for immunoblotting. Immunoelectron microscopy was carried out by using 18 nm gold particles conjugated to goat anti-rabbit IgG (Jackson Immunoresearch) and 10 nm gold particles conjugated to goat anti-mouse IgG (Sigma).
Immunofluorescence
PC12 cells grown on laminin-coated coverslips were rinsed in PBS and fixed
in either methanol (Mallinckrodt;20°C) for 4 minutes or 3.5%
formaldehyde (Tousimis) at room temperature for 5 minutes at different times
during neurite extension. Following formaldehyde fixation, cells were
permeabilized with 0.05% NP-40 for 5 minutes. Cells were then washed with PBS
and processed for indirect immunofluoresence as previously described
(Prahlad et al., 1998;
Yoon et al., 1998
). Following
staining, coverslips were washed in PBS and mounted on glass slides in
gelvatol containing 100 mg/ml Dabco [1,4-diazabicyclo [2.2.2] octane; Aldrich
Chemical (Yoon et al., 1998
)].
In some preparations, membrane-bound organelles were stained with
DiOC6 or SP-DiOC18 (Molecular Probes) at a concentration
of 0.1 µg/ml for 20 minutes, and then cells were fixed and processed for
indirect immunofluorescence using peripherin antibodies
(Spector et al., 1997
).
Images of fixed, stained preparations were taken with a Zeiss LSM 510
microscope (Carl Zeiss) (Yoon et al.,
1998).
Statistical analysis
The immunofluorescence images of peripherin particles, kinesin and dynein
were subjected to statistical tests using a modification of our previously
published procedure (Prahlad et al.,
2000). These tests were carried out to make certain that these
associations were not random. To this end, the total number of pixels within
randomly selected cytoplasmic regions were determined in cells prepared for
double and triple fluorescence microscopy. Specifically, non-transfected cells
were fixed and processed for double label immunofluorescence using either
peripherin and kinesin antibodies or peripherin and dynein antibodies (see
above). For triple fluorescence observations GFP-peripherin-expressing cells
were fixed and stained with anti-dynein and anti-kinesin (see above). For each
double and triple fluorescence preparation, one cytoplasmic region from each
of 10 different cells (a total of 30 cells) was used for the statistical
analyses. After normalizing for differences in magnification, it was
determined that the average number of pixels per peripherin particle was
41.5±15.0 (n=300), the average number of pixels per kinesin
particle was 43.0±13.2 (n=200), and the average number of
pixels per dynein particle was 41.9±16.9 (n=200). For the
purposes of our calculations we assumed that the particles were circular and
that there was extensive overlap between or among the different antibody
staining patterns.
For double fluorescence images, the following formula was used to calculate
the expected number of peripherin particles (Edb) that would
coincide with either kinesin or dynein based on chance alone.
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The following formula was used for statistical analyses of triple
fluorescence images:
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The number of associations actually observed was also determined for each image. This was defined as the number of peripherin particles that were observed to associate with kinesin and/or dynein. Finally, a two-tailed Student's t-test was used to determine if the differences between the actual and expected values were statistically significant (P<0.001).
Transfection
Rat peripherin cDNA (provided by Linda Parysek, University of Cincinnati)
was amplified by PCR using primers that insert BamHI sites at the
5' and 3' ends. The resulting BamHI-BamHI
fragment was subcloned into the BamHI site of pEGFP-C1 (Clontech).
The preparation of GFP-vimentin cDNA and the myc-dynamitin construct has been
described elsewhere (Helfand et al.,
2002; Yoon et al.,
1998
). The GFP-peripherin construct was introduced into PC12 cells
by electroporation (Yoon et al.,
2001
), and myc-dynamitin cDNA was introduced by lipofectamine
delivery [Gibco (Yoon et al.,
1998
)]. In some experiments, PC12 cells were mock transfected with
the pCMV-myc vector (Clontech) as a control. Following electroporation, cells
were plated on laminin-coated coverslips in CM or DM (see above) and used for
live cell analysis within 48-72 hours of transfection. BHK-21 cells were also
analyzed 48-72 hours after transfection with GFP-vimentin cDNA as previously
described (Prahlad et al.,
1998
).
Live cell imaging
PC12 cells expressing GFP-peripherin were trypsinized, plated onto
laminin-coated coverslips, mounted on glass slides and sealed as previously
described (Yoon et al., 1998).
The culture medium used in these preparations was Leibovitz L-15 (Gibco/BRL)
containing 5% calf serum, 1 mM sodium pyruvate and 30 ng/ml NGF. Cells were
maintained at 37°C during microscopic examination with an air stream
incubator (NEVTEK). In some experiments cells were treated with colchicine (5
µg/ml; Sigma) in DM for 30-90 minutes. Under these conditions, no
microtubules could be detected by indirect immunofluorescence within 15-30
minutes (data not shown).
Time-lapse observations were made using a Zeiss LSM 510 confocal microscope
as previously described (Yoon et al.,
1998). Images were captured at
5 second intervals at a
resolution of 512x512 dots per inch with a scanning time of
1
second. Images were collected for
5-30 minute time periods. Analyses of
peripherin particle and squiggle motility were carried out in PC12 cells at
different times within 0.5-72 hours of trypsinization and replating in DM (see
above). Analyses of vimentin particle motility in BHK-21 cells were carried
out in the peripheral regions of interphase cells. In both cell types, rates
of translocation of particles and squiggles were obtained by monitoring
distance traveled during the 5 second intervals between capturing images using
Metamorph image analysis software (Universal Imaging Corp.) as previously
described (Yoon et al., 1998
).
Since the movements were discontinuous, pause times were also determined
(Wang et al., 2000
). The pause
time was defined as a 6 second interval during which a particle or squiggle
moved less than 0.5 µm.
Fluorescence recovery after photobleaching (FRAP) analyses were carried out
on the neurites of differentiated cells expressing GFP-peripherin at 48 hours,
using the Zeiss LSM 510 microscope as previously described
(Yoon et al., 1998). For this
purpose only one neurite was placed in the microscope field of view using a
100x oil immersion (1.4 NA, plan-apochromatic lens). In some
experiments,
8-10 µm2 areas were bleached along the long
axes of neurites and recovery was monitored. In other experiments, larger
regions (
35-50 µm2) were bleached. Owing to the size of the
bleach zone relative to the total area of the single fluorescent neurite
within the field of view, the gray-scale pixel values of the bleach zone were
normalized by dividing by the gray scale pixel values of the same sized
regions in control (unbleached) areas (the F.I. ratio) of the same neurite
using LSM510 imaging software. In order to analyze the details of fluorescence
recovery in these large bleach zones, the F.I. ratio was first determined for
the overall bleach zone and subsequently for 1 µm subdomains of the entire
area. In some FRAP experiments, colchicine was added at 5 µg/ml in DM 15
minutes prior to observation. In several experiments, cells were processed for
immunofluorescence with peripherin antibody (see above) on the microscope
stage to determine whether peripherin fibrils were present within the bleach
zone.
Neurite outgrowth was monitored after cells were plated in DM on locator coverslips (Bellco). Subsequently, phase images were taken of the same 100 cells at 6, 12, 24, 48 and 72 hours. Measurements of neurite length at each time interval were made with Zeiss LSM 510 imaging software.
IF-enriched cytoskeletal preparations
IF-enriched cytoskeletal preparations were made from subconfluent cultures
of transfected PC12 cells grown in DM for 24 hours as previously described
(Zackroff et al., 1982). These
preparations were analyzed by SDS-PAGE
(Laemmli, 1970
). The separated
proteins were transferred to nitrocellulose for immunoblotting
(Towbin et al., 1979
). All
antibody incubations were carried out in PBS containing 5% non-fat dry milk
(Sigma).
Platinum replica electron microscopy
PC12 cells were grown in DM for 24-48 hours. Two hours before processing,
cells were trypsinized and replated onto laminin-coated coverslips in DM.
Ultrastructural observations of cytoskeletal preparations were performed as
described elsewhere (Svitkina et al.,
1995). Briefly, cells on coverslips were extracted with PEM buffer
(100 mM PIPES, pH 6.9, 1 mM MgCl2, 1 mM EGTA) containing 1% Triton
X-100, 4% polyethylene glycol (PEG) for 5 minutes. In some experiments 10
µg/ml Taxol (Sigma) was added to the PEM buffer solution to preserve MT
integrity. In these experiments, actin was removed by adding 1 mg/ml DNase 1
to the PEM buffer and by incubating PC12 cytoskeletons with recombinant
gelsolin N-terminal domain [provided by Gary Borisy, Northwestern University
(Verkhovsky and Borisy,
1993
)]. In other experiments, 2 mM phalloidin (Molecular Probes)
was added to the PEM buffer to preserve actin structures. These preparations
were then fixed with 2% glutaraldehyde, labeled with gold-conjugated
antibodies, stained with 0.1% tannic acid/0.2% uranyl acetate and processed by
critical point drying/rotary shadowing as previously described
(Helfand et al., 2002
;
Svitkina et al., 1995
).
Controls for these preparations involved all of the various steps and
incubations described above using either no antibodies or secondary
gold-coupled antibodies alone.
Microinjection
PC12 cells growing in DM on locator coverslips (Bellco) were selected using
a Zeiss axiomat inverted microscope. Antibodies directed against kinesin heavy
chain (H1; Chemicon) or control preimmune IgG (5 mg/ml) were dialyzed into
microinjection buffer (20 mM Tris, pH 7.5 in 75 mM NaCl), clarified by
centrifugation and then microinjected into the selected cells
(Prahlad et al., 1998).
Injected cells were fixed and processed for immunofluorescence within 0.5-4
hours.
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Results |
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Platinum replica immunogold electron microscopic analyses revealed clusters of gold corresponding to the peripherin particles. These were most obvious in growth cones in close association with the dense actin networks found in these regions as well as the actin bundles in filopods (Fig. 2D-E). Short and longer linear arrays of gold-conjugated antibodies corresponding to the peripherin squiggles and longer IF observed by immunofluorescence were present within the central region of growth cones and in neurites (Fig. 2F-G; and data not shown). No gold labeling was observed in control cells (see Materials and Methods; data not shown). These observations confirm the presence of the different forms of peripherin seen by light microscopy.
We also determined whether the same IF patterns were seen during neurite
outgrowth in GFP-peripherin-expressing PC12 cells grown in DM for periods up
to 72 hours. Direct observations of GFP-peripherin fluorescence patterns in
these cells was indistinguishable from those seen at the same time intervals
in non-transfected cells (e.g., 2 hours;
Fig. 1J-L and data not shown).
Further evidence supporting the incorporation of GFP-peripherin into
endogenous peripherin structures was derived from SDS-PAGE and immunoblotting
of IF-enriched cytoskeletal preparations made from cultures containing
70% GFP-peripherin-transfected PC12 cells
(Zackroff et al., 1982
). The
results showed that peripherin and an 85 kDa band corresponding to
GFP-peripherin were present in the endogenous IF system
(Fig. 3).
|
Fast transport of peripherin
Since non-transfected and GFP-peripherin transfected PC12 cells exhibited
indistinguishable patterns and assembly states of IF protein, we next
determined the properties of peripherin in vivo. Live cells expressing
GFP-peripherin were observed at time intervals after trypsinization and
replating into DM. At early time points (0.5-6 hours), peripherin particles
and squiggles were observed to move in all regions of the cytoplasm including
cell bodies, neurites and growth cones (see
Fig. 4A-D and Movie 1).
Numerous peripherin particles could also be seen using phase contrast
microscopy (see arrowheads in Fig.
2A-C).
|
The rates of peripherin particle translocation were determined in cells
0.5-2 hours after replating in DM (see Materials and Methods), prior to the
formation of distinct neurites. Approximately 72% of these particles moved
(n=50; see Table 1).
Of these particles, almost half exhibited `reversals'; that is, an individual
particle moved first in one direction and then in the opposite direction (see
Table 1). However, the majority
of the movements (75%) were directed towards the cell surface
(anterograde) at rates ranging from 0.08-1.45 µm/second (averaging
0.34±0.21 µm/second; Table
1). Particles moving towards the nucleus (retrograde) moved at
rates ranging from 0.08-1.20 µm/second (averaging 0.34±0.24
µm/second; Table 1).
Calculation of pause times (see Materials and Methods) revealed that the
particles moved
46% of the time. These movements were similar to vimentin
particle motility in peripheral regions of spread BHK-21 cells
(Table 1).
|
Peripherin squiggles were also studied in PC12 cells within 0.5-2 hours in
DM. Overall their movements were similar to those described for particles at
the same time points (Fig. 4F;
Table 1). Approximately 35% of
all of the squiggles (n=64) moved, mainly anterograde, at rates
ranging from 0.08 to 1.14 µm/second (averaging 0.40±0.25
µm/second; Table 1).
Retrograde rates ranged from 0.08 to 1.22 µm/second (averaging
0.38±0.28 µm/second; Table
1). Furthermore, a calculation of their pause rates showed that
they moved 68% of the observation period
(Table 1).
As mentioned above, differentiated PC12 cells grown in the presence of NGF
for 24-72 hours exhibit dense networks of peripherin IF throughout their cell
bodies and neurites. In these cells, particle and squiggle motility was only
evident in the peripheral regions of the cell body and growth cones (see
Fig. 4A-E,G,H). In the cell
body, 67% (n=50) of the particles moved with rates similar to
those seen at earlier time points in DM
(Table 1). The net
translocation of >55% of these particles was anterograde, even though half
of the particles reversed directions at least once during the observation
period (Table 1). Particles
moved at rates between 0.08-1.11 µm/second (averaging 0.31±0.20
µm/second; Table 1) in the
anterograde direction, and in the retrograde direction between 0.08-1.14
µm/second (averaging 0.30±0.22 µm/second;
Table 1). Analyses of the
movements of peripherin squiggles in cell bodies revealed that they moved
63% of the time (Table 1).
Of these movements,
62% were directed towards the cell surface at rates
ranging from 0.08-1.09 µm/second (averaging 0.41±0.24 µm/second;
Table 1), and
38% of the
movements were in the retrograde direction at rates of 0.08-1.25 µm/second
(averaging 0.34±0.23 µm/second;
Table 1). Many squiggles were
also observed to change directions (Fig.
4G; Table 1). It
should be noted that the majority of neurite elongation or outgrowth takes
place within 12 hours of plating in DM (see Materials and Methods). At this
time the average length of neurites is
48 µm, and after 72 hours it is
62 µm (n=100, data not shown). Therefore, at these time
points the rates of neurite outgrowth were minimal and had no significant
impact on our measurements of particle and squiggle motility.
In the case of the growth cones of differentiated cells in DM for 24-72
hours (see above), observations were limited to their central domains
(Mueller, 1999) where rapid
changes in shape do not occur. In this area, particles and squiggles were
observed to move
53% (n=50;
Table 1) and
58%
(n=52; Table 1) of the
time, respectively. About half of the particle (
53%;
Table 1) and squiggle
(
51%; see Table 1)
movements were anterograde. The range and average velocities of these
peripherin structures were not significantly different from those described
above (P<0.005, using Student's t-test; see
Table 1). Similarly,
50%
of the particles and squiggles reversed direction during movement
(Fig. 4H;
Table 1). Therefore, the motile
properties of peripherin particles and squiggles in two major domains of PC12
neurons, cell bodies and growth cones, are similar. In addition, these
properties are also very similar to those found for vimentin in BHK-21
fibroblasts (Table 1).
It was also of interest to determine the motile properties of peripherin IF
in the neurites of extensively differentiated PC12 cells. For this purpose,
fluorescence recovery after photobleaching (FRAP) analyses were initially
carried out on 8-10 µm2 regions along neurites in
GFP-transfected cells grown in DM for 48 hours. The time for the bleach zones
to completely recover their fluorescence was
10-14 minutes with an
average t1/2 of
5.5 minutes (n=8; data not shown).
Interestingly, during fluorescence recovery we frequently found that particles
and squiggles would rapidly traverse the bleach zones (see
Fig. 5E-I, Movie 3, available
at
jcs.biologists.org/supplemental).
In order to observe the movements of these structures in more detail and to
determine their contribution to fluorescence recovery, we bleached much larger
regions (35-50 µm2) along the length of neurites (see
Fig. 5A1-A3). The overall rate
of fluorescence recovery for these larger areas containing longer peripherin
IF was similar to that described above
(Fig. 5A). However, after
dividing these larger photobleach zones into smaller areas of equal size
(Fig. 5C1-C3), we frequently
detected transient spikes in fluorescence intensity that were much brighter
than the intensity recorded just prior to photobleaching
(Figs. 5B,C). These spikes were
due to the rapid movements of particles and squiggles into and out of bleach
zones (Fig. 5B-1 to B-3 and
Fig. 5C-1 to C-3). These rapid
and bi-directional movements of particles and squiggles became evident only
during the period preceding the recovery of dense peripherin networks (see
Movie 3 at
jcs.biologists.org/supplemental).
|
We also determined the rates and directions of movements of particles and
squiggles within neurites after photobleaching at 48 hours in DM and compared
their properties to those observed during the early stages of neurite
outgrowth (0.5-6 hours, see above). Although particles (n=77) were
observed to move into the bleach zone from both ends, the majority of
movements were anterograde (65%; Table
1). The rates of anterograde particle movements ranged from 0.08
to 1.45 µm/second (average of 0.33±0.24 µm/second;
Table 1) and retrograde rates
were from 0.08 to 1.54 µm/second (average of 0.30±0.20 µm/second;
Table 1). Interestingly, very
few reversals were observed (Fig.
5J). For example, of the 77 particles studied, only 8% reversed
their direction (see Table 1).
Determination of pause times revealed that in neurites, peripherin particles
moved
75% of the time.
Further analysis of motile squiggles (n=50) in neurites after 48
hours in DM revealed that 62% moved in the anterograde direction at rates
of 0.08 to 1.21 µm/second (average of 0.31±0.29 µm/second;
Table 1). Retrograde movements
ranged from 0.08-1.0 µm/second (average of 0.30±0.28 µm/second;
Table 1). Calculation of pause
times demonstrated that squiggles moved
70% of the time
(Table 1). In addition, almost
no peripherin squiggle reversals were observed
(Table 1). Overall, the results
of these studies showed that the majority of peripherin particles and
squiggles moved rapidly in the anterograde direction along neurites with fewer
pauses and reversals than detected in either cell bodies or growth cones.
Mechanisms underlying the motility of particles and squiggles
Previous studies have shown that the motility of the various structural
forms of IF proteins are dependent upon MT
(Prahlad et al., 2000;
Prahlad et al., 1998
;
Shah et al., 2000
;
Yoon et al., 1998
). Therefore,
FRAP analyses were performed on GFP-peripherin-expressing PC12 cells grown in
DM for 48 hours and treated with colchicine for 15 minutes-2 hours (see
Materials and Methods). Phase contrast images revealed that no significant
neurite retraction occurred during the observation period, and no MT could be
detected by immunofluorescence at 15 minutes after adding colchicine (data not
shown). Time-lapse observations of bleach zones demonstrated that only
23% (n=10) of the total fluorescence was recovered, even at 1
hour post-photobleaching (Fig.
5D). This recovery was much slower than that recorded for controls
(Fig. 5A-C). In addition, there
were no transient increases in fluorescence intensity
(Fig. 5D1-D3), as no particles
or squiggles could be detected traversing the bleach zones. Fixation and
staining after photobleaching demonstrated that a dense array of peripherin IF
remained in the bleach zone (data not shown; Materials and Methods). These
observations suggest that the movements of particles and squiggles into bleach
zones are required for normal fluorescence recovery.
Immunofluorescence was employed to determine the relationships between peripherin particles, squiggles and MT in the growth cones of non-transfected PC12 cells after 4 hours in DM. These studies showed that the majority of particles (78%; n=96) and squiggles (88%; n=184) present in several different growth cones were associated with MT (Fig. 6A-D).
|
The findings that MT are required for the motility of peripherin particles
and squiggles suggested that MT-associated motors provide the motive force for
their movements. Immunofluorescence observations of PC12 cells replated in DM
for 0.5-12 hours showed that the majority of peripherin particles (79%;
n=154) and squiggles (80%; n=137) were associated with
conventional kinesin (Fig. 6E). Similar results were obtained after 24-72 hours in DM in the peripheral
regions of cell bodies and growth cones (data not shown). Since retrograde
movements were also detected, the relationship between dynein, dynactin and
peripherin was determined. This involved double labeling with anti-peripherin
and dynein intermediate chain, or anti-peripherin and either p50 (dynamitin)
or p150Glued. The results were indistinguishable for each
of these antibodies (see, for example, Fig.
6F). Approximately 73% (n=250) of the peripherin
particles and 86% (n=300) of the squiggles were closely
associated with dynein and dynactin (Fig.
6F).
Since individual GFP-peripherin particles and squiggles were observed to
move in one direction and then rapidly reverse, it was of interest to further
determine the relationships between peripherin, dynein, dynactin and kinesin.
Double label immunofluorescence analyses of 15 different growth cones in
GFP-peripherin-expressing cells revealed that 40% of the particles
(n=423) were associated with both kinesin and dynein
(Fig. 7A-F);
25% were
associated only with dynein (Fig.
7A,C,E);
21% associated only with kinesin
(Fig. 7A,B,D); and
14% did
not appear to associate with either motor
(Fig. 7F). In addition,
56% of the squiggles (n=300) were associated with both motors,
15% were associated with dynein only,
18% were associated with
kinesin only and
11% did not appear to associate with either motor.
|
To be certain that the associations observed among peripherin, kinesin and/or dynein were not random, statistical analyses were carried out as described (see Materials and Methods). Briefly, double- and triple-labeled fluorescence images of PC12 cells grown in DM for 2-4 hours were used for analyses. The total areas of randomly selected cytoplasmic regions as well as the total number and average size of peripherin, kinesin and/or dynein particles were determined. We also determined the actual number of peripherin particles that associated with kinesin and/or dynein. Using these values, we calculated that the probability of the associations observed between peripherin and kinesin, between peripherin and dynein, and among peripherin, kinesin and dynein by chance alone was less than 1 in 10,000. On the basis of these analyses, we are confident that a significant population of peripherin particles is associated with both kinesin and dynein.
At higher resolution, platinum replica immunogold electron microscopy of
cytoskeletal preparations that preserve the integrity of IF and MT
(Helfand et al., 2002)
confirmed the observation that perpherin particles were closely associated
with MT in the central domains of growth cones
(Fig. 8). In addition, in
double-labeled preparations, the clusters of gold particles seen with the
peripherin antibody frequently colocalized with anti-kinesin
(Fig. 8A-C) or anti-dynein
(Fig. 8D-F) antibodies.
|
We also determined whether kinesin, dynein and dynactin were present in PC12 IF-enriched cytoskeletal preparations at 72 hours after replating in DM (see Materials and Methods). Immunoblot analyses of these preparations revealed, in addition to the major peripherin band, the presence of kinesin heavy chain and components of the dynein and dynactin complexes including IC, LIC1 and 2, HC, dynamitin (p50), p150Glued and Arp-1 (Fig. 9; Materials and Methods).
|
Dynein, dynactin and kinesin are required for maintaining peripherin
IF organization
The relationships between peripherin, MT and their associated motors were
also studied by disrupting the activities of conventional kinesin and
cytoplasmic dynein in PC12 cells. To test whether kinesin is required to
maintain the organization of the peripherin IF network, differentiated PC12
cells (48 hours in DM) were microinjected with either kinesin antibody (0.75
mg/ml) or, as a control, with non-immune serum (see Materials and Methods)
(Prahlad et al., 1998). Cells
were processed for immunofluorescence with peripherin antibody 0.5-4 hours
after microinjection. In every cell injected with kinesin antibody
(n=35) virtually all of the peripherin was located in the
juxtanuclear region within the cell body
(Fig. 10C,D). Very few, if
any, peripherin IF, particles or squiggles could be detected in neurites. In
controls, typical peripherin networks were seen
(Fig. 10A,B). We observed no
significant retraction of PC12 processes during these time intervals after
microinjection (see Materials and Methods).
|
To determine the role of dynein in peripherin IF network organization, PC12
cells grown for 24-48 hours in DM were transfected with myc-dynamitin cDNA
[see Materials and Methods (Echeverri et
al., 1996; Helfand et al.,
2002
)]. Forty-eight hours later, the cells were fixed and
processed for double label immunofluorescence using antibodies against
peripherin and c-myc. Observations of control mock-transfected cells revealed
typical peripherin networks (Fig.
10E,F). Cells over-expressing dynamitin displayed a dramatic
decrease in peripherin in the perinuclear area
(Fig. 10E,F). The majority of
the peripherin was concentrated in the distal regions of neurites and in some
cases near the surface of the cell body. On the basis of these observations
and those described above for kinesin, it appears that both plus-end- and
minus-end-directed MT-associated motors are required for the maintenance of
normal peripherin networks in differentiated PC12 cells.
![]() |
Discussion |
---|
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---|
In BHK-21 fibroblasts, and in the cell bodies and growth cones of PC12
cells, particles and squiggles move 50% of the time (see
Table 1). However, PC12
neurites contain particles and squiggles that spend a significantly greater
proportion of their time moving (
75%). In addition, the motility of these
structures within neurites appears to be more directed when compared to their
motility within cell bodies and growth cones. This is supported by the
findings that there are very few reversals of particle and squiggle movements
observed within neurites (Table
1). Taken together, it appears that there are specific mechanisms
within neurites that are not present within fibroblasts, neuronal growth cones
or cell bodies which enhance IF motility. Possible explanations for these
alterations in motile behavior may lie in the changes in the phosphorylation
states of IF and/or motor proteins known to take place specifically within
neurites (Jung et al., 2000a
;
Lee and Hollenbeck, 1995
;
Lee et al., 1986
;
Nixon et al., 1987
;
Oblinger et al., 1987
;
Pfister et al., 1996
; Salata,
2001; Sternberger and Sternberger,
1983
; Yabe et al.,
2000
).
Our results show that IF protein is present within all regions of growth
cones. As mentioned above, the behavior of peripherin particles and squiggles
within the central domain of growth cones, known to contain both MT and actin
(Mueller, 1999), was similar
to that described in cell bodies. In addition, this is the first study to
detect IF protein in the form of non-filamentous particles in the peripheral
domain of growth cones. This domain is defined by its lack of MT and its
enriched actin content (Mueller,
1999
). Preliminary observations of GFP-peripherin particles within
the peripheral domain reveals that the vast majority move in a retrograde
direction at much slower rates (data not shown). This suggests that these
movements may be linked to the actomyosin system. Further support for this
possibility comes from the observation that peripherin can associate with
actin through myosin Va, a processive actin-associated motor that is enriched
in growth cones (Rao et al.,
2002
; Wolff et al.,
1999
). Therefore, it is possible that different structural forms
of peripherin can also be transported by the actomyosin system.
As indicated above, the range of rapid movements recorded for the type III
IF peripherin particles and squiggles in PC12 cells reported in this study and
for the short type IV IF (NF) described in cultured sympathetic nerve cells
(Roy et al., 2000;
Wang et al., 2000
) are very
similar. In contrast, the overall distances traveled by these different types
of neural IF proteins can be explained by their dramatically different pause
times. One explanation for this difference may be related to the structure of
the triplet proteins comprising the short motile NF observed in sympathetic
neurons. Both NF-M and NF-H have unusually long highly charged C-terminal
tails that project from the core IF structure
(Hirokawa et al., 1997
;
Hisanaga and Hirokawa, 1988
).
It has been suggested that these domains, and their modification by
phosphorylation, promote filament stability and modify NF transport in axons
by regulating interactions with MT and MT-dependent motors
(Chen et al., 2000
;
Hisanaga and Hirokawa, 1988
;
Jung et al., 2000b
;
Nakagawa et al., 1995
;
Yabe et al., 2001b
;
Yabe et al., 1999
). Therefore,
it is possible that the tail domains of NF-M and NF-H could be involved,
either actively or passively, in determining pause intervals, thereby
influencing the total distances traveled by NF. In support of this
possibility, it has been shown that the initiation of NF-H expression during
postnatal development is coincident with a decrease in the overall rates of
axonal transport (Cote et al.,
1993
; Marszalek et al.,
1996
; Willard and Simon,
1983
). Furthermore, the disruption of NF-M or NF-H genes in mice
accelerates axonal transport of NF-L
(Jacomy et al., 1999
;
Zhu et al., 1998
). On the
basis of these observations, it appears that the rapid movements of peripherin
particles and squiggles may be related to the absence of the long highly
charged tail domains that are characteristic of mature NF.
It is also possible that particulate non-filamentous forms of NF triplet
proteins, similar to those described for vimentin IF precursors
(Prahlad et al., 1998), and
the peripherin particles described in this study, could move at fast transport
rates. In support of this possibility, rapidly moving NF particles containing
the triplet proteins have been described in squid axoplasm
(Prahlad et al., 2000
), dorsal
root ganglion neurons and neuroblastoma cells
(Yabe et al., 2001a
).
Unfortunately, none of these studies calculated the pause times required to
determine whether the NF particles are components of a rapid transport
system.
FRAP studies of the peripherin network along neurites in differentiated
PC12 cells demonstrate that the t1/2 for fluorescence recovery is
almost identical to that recorded for another member of the type III IF
family, vimentin (Yoon et al.,
1998). Interestingly, the fluorescence recovery of both peripherin
and vimentin IF slow down significantly in the absence of microtubules [see
Fig. 5D (Yoon et al., 1998
)]. However,
the recovery of GFP-peripherin fluorescence is even more sensitive to MT
inhibitors, as only
23% recovery was recorded at 1 hour
post-photobleaching (see Fig.
5D). In addition, under these conditions, no peripherin particle
or squiggle motility was observed in bleach zones made along the length of
neurites. These observations suggest that the majority of subunit exchange
required for normal fluorescence recovery along neurites may be dependent on
the MT-based transport of IF precursors such as particles and squiggles. It is
also possible that the partial recovery (
23%) detected under these
conditions may be related to an actomyosin-based transport system for
delivering IF precursors. In support of this, actomyosin-based transport has
been reported in nerve cells (Evans and
Bridgman, 1995
; Tabb et al.,
1998
).
Two theories have been proposed to describe the mechanisms of neural IF
protein transport within axons. The subunit transport theory holds that neural
IF are transported along MT as oligomeric complexes
(Hirokawa et al., 1997). The
second theory states that neural IF are transported within axons as fully
assembled polymers (Bass and Brown,
1997
). Our observations of live cells suggest that aspects of both
theories are correct as we have demonstrated that both non-filamentous
(particles) and short neural IF (squiggles) can be transported in a
MT-dependent manner within all regions of PC12 cells. We have also
demonstrated that the majority of particles and squiggles associate with both
conventional kinesin and cytoplasmic dynein (see
Fig. 7). This finding
complements other studies that have demonstrated both kinesin- and
dynein-dependent transport of type III IF proteins in fibroblasts and type IV
NF proteins in neurons (Helfand et al.,
2002
; Prahlad et al.,
2000
; Prahlad et al.,
1998
; Shah et al.,
2000
; Yabe et al.,
1999
). It is also of interest to note that although many of the
peripherin particles are associated with both kinesin and dynein, the majority
of movements are anterograde. This may reflect specific modifications that
regulate MT-associated motor components
(Lee and Hollenbeck, 1995
;
Morfini et al., 2002
;
Reese and Haimo, 2000
;
Salata et al., 2001
).
The finding that neural IF proteins are transported along MT by motor
proteins also has important implications for understanding numerous human
neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) and
Parkinson's Disease (PD). The pathological hallmarks of these diseases are
abnormal accumulations of neural IF within axons and cell bodies
(Gotow, 2000;
Julien and Mushynski, 1998
).
Our studies suggest that similar accumulations can occur following the
disruption of either kinesin or dynein function in PC12 cells. This is further
supported by recent findings demonstrating progressive neuronal degeneration
in transgenic mice that overexpress dynamitin in mature motor neurons
(LaMonte et al., 2002
). The
motor neurons in these mice display large aggregates of NF, and this is
coincident with the development of motor neuron disease
(LaMonte et al., 2002
).
In conclusion, our study demonstrates that non-filamentous, non-membrane-bound particles and short filaments containing peripherin move along neurites at rates consistent with rapid transport. It therefore appears likely that a subpopulation of cytoskeletal IF proteins can move at rapid rates along axons, providing a mechanism for the timely turnover, replacement and repair of cytoskeletal components within the most distal reaches of neurons.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
* These authors contributed equally to this work
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References |
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