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The Journal of Neuroscience, August 1, 2000, 20(15):5782-5791
Depletion of a Microtubule-Associated Motor Protein Induces the
Loss of Dendritic Identity
Wenqian
Yu1,
Crist
Cook1,
Carley
Sauter1,
Ryoko
Kuriyama2,
Paul L.
Kaplan3, and
Peter W.
Baas1
1 Department of Anatomy, The University of Wisconsin
Medical School, Madison, Wisconsin 53706, 2 Department of
Genetics, Cell Biology and Development, The University of Minnesota
Medical School, Minneapolis, Minnesota 55455, and
3 Creative BioMolecules, Hopkinton, Massachusetts 01748
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ABSTRACT |
Dendrites are short stout tapering processes that are rich in
ribosomes and Golgi elements, whereas axons are long thin processes of
uniform diameter that are deficient in these organelles. It has been
hypothesized that the unique morphological and compositional features
of axons and dendrites result from their distinct patterns of
microtubule polarity orientation. The microtubules within axons are
uniformly oriented with their plus ends distal to the cell body,
whereas microtubules within dendrites are nonuniformly oriented. The
minus-end-distal microtubules are thought to arise via their specific
transport into dendrites by the motor protein known as CHO1/MKLP1. According to this model, CHO1/MKLP1 transports
microtubules with their minus ends leading into dendrites by generating
forces against the plus-end-distal microtubules, thus creating drag on the plus-end-distal microtubules. Here we show that depletion of
CHO1/MKLP1 from cultured neurons causes a rapid redistribution of
microtubules within dendrites such that minus-end-distal microtubules are chased back to the cell body while plus-end-distal microtubules are
redistributed forward. The dendrite grows significantly longer and
thinner, loses its taper, and acquires a progressively more axon-like
organelle composition. These results suggest that the forces generated
by CHO1/MKLP1 are necessary for maintaining the minus-end-distal
microtubules in the dendrite, for antagonizing the anterograde
transport of the plus-end-distal microtubules, and for sustaining a
pattern of microtubule organization necessary for the maintenance of
dendritic morphology and composition. Thus, we would conclude that
dendritic identity is dependent on forces generated by CHO1/MKLP1.
Key words:
dendrite; axon; neuron; microtubule; CHO1/MKLP1; motor
protein
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INTRODUCTION |
Typical vertebrate neurons
extend a single axon and several dendrites. Dendrites are different
from the axon both morphologically and compositionally. The axon is a
long slender process of uniform diameter that is effectively unlimited
in its growth potential. In contrast, dendrites are stout tapering
processes that remain quite short compared to axons. Axons are
deficient in ribosomes and Golgi elements, whereas dendrites are rich
in these organelles. The means by which neurons generate and maintain
these differences is a central unanswered question in cellular
neuroscience. We have proposed that most or all of the features that
distinguish dendrites from the axon might be attributable, either
directly or indirectly, to differences in the organization of
microtubules within these processes (Black and Baas, 1989 ). Unlike the
uniformly plus-end-distal polarity pattern of axonal microtubules,
dendritic microtubules have a nonuniform polarity pattern (Baas et al., 1988). During neuronal development, plus-end-distal microtubules arise
first within immature processes and are then followed by the gradual
addition of minus-end-distal microtubules to those processes that
become dendrites (Baas et al., 1989). The appearance of the
minus-end-distal microtubules corresponds with the acquisition of many
distinctive features of dendritic morphology and composition, suggesting a mechanistic link between microtubule organization and the
distinct structural and compositional features of these processes. This
conclusion is also suggested by experimental studies in which key
features of axonal and dendritic morphology were shown to be mimicked
by non-neuronal cells experimentally induced to extend processes with
either uniform or nonuniform microtubule polarity patterns,
respectively (Sharp et al., 1996, 1997a).
These observations have led to a model whereby the key event in
dendritic differentiation is the specific transport of minus-end-distal microtubules into the dendrite (Sharp et al., 1995; Baas and Yu, 1996;
Baas, 1999). Studies from our laboratory strongly suggest that the
motor protein known as CHO1/MKLP1 is essential for the appearance of minus-end-distal microtubules within developing neurites
and for their differentiation into dendrites (Yu et al., 1997; Sharp et
al., 1997b; Ferhat et al., 1998). Studies in vitro have
shown that CHO1/MKLP1 transports microtubules with their minus ends
leading toward the plus ends of other microtubules and does so by
generating forces against the oppositely oriented microtubules (Nislow
et al., 1992). We have proposed that plus-end-distal microtubules are
transported into axons and dendrites by another motor, cytoplasmic
dynein, and that CHO1/MKLP1 generates its forces by pushing against
these microtubules (Baas, 1999). According to our model, this would
create drag on the anterograde movement of the plus-end-distal
microtubules, thus slowing the growth of the dendrite compared to the
axon. In addition, profound changes in membrane traffic would result
from the establishment of a nonuniform microtubule polarity pattern.
These changes would contribute to establishing the unique features that
distinguish dendrites from the axon. For example, ribosomes and Golgi
elements are thought to move toward minus ends of microtubules, and
hence would have an appropriate track to move into dendrites but not
the axon (Black and Baas, 1989).
If this reasoning is correct, the identity of the dendrite is dependent
on the forces generated by CHO1/MKLP1 on the microtubule array. To test
this, we have now performed studies in which we experimentally depleted
this motor protein from neurons with well developed dendrites. As a
result, we observe a rapid redistribution of microtubules of each
orientation and concomitant changes in both the morphology and
composition of the dendrite.
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MATERIALS AND METHODS |
Cell culture. Cultures of rat sympathetic neurons
were generated from the superior cervical ganglia of newborn rats
essentially as previously described (Baas and Ahmad, 1993), except that
OP1 was added to the cultures 1 week after plating to assist in the rapid differentiation of robust dendritic arbors in low-density cultures (Sharp et al., 1997b).
Oligonucleotide treatments. As in our previous studies (Yu
et al., 1997; Sharp et al., 1997b), translation of CHO1/MKLP1 was suppressed by treatment of cultures with phosphorothioate-substituted DNA oligonucleotides (Research Genetics, Huntsville, AL). The antisense
oligonucleotide consisted of the sequence 5'-AGCTTTCGCTGGTTTCATG-3', which is the inverse complement of the coding sequence  1 to +18 of
hamster CHO1/MKLP1 transcript (Kuriyama et al., 1994).
Oligonucleotides were stored in serum-free medium, aliquoted, and
frozen at 80°C. Two weeks after plating, by which time robust
dendrites had formed, a portion of the plating medium was removed, and
new medium containing the oligonucleotides was added at a final
concentration of 1 µM. This medium was changed with fresh
oligonucleotide-containing medium every 12 hr for the duration of the
experiment. As a negative "sense" control, oligonucleotides with
the inverse complement of the antisense sequence were added to dishes
in an identical manner as the antisense oligonucleotides.
Immunofluorescence microscopy. Neuronal cultures were
immunostained in single-label analyses with one of four different
primary antibodies. A mouse monoclonal antibody called RMDO20
(which recognizes a poorly phosphorylated neurofilament epitope) was
purchased from Zymed (San Francisco, CA). The mouse monoclonal antibody
called CHO1 (which recognizes CHO1/MKLP1) was generated as previously described (Sellitto and Kuriyama, 1988). A mouse monoclonal
antibody termed tau-1 (which recognizes a phosphorylated variant of
tau) was provided by Lester Binder. A mouse monoclonal antibody that recognizes the full-length variant of microtubule-associated protein-2 (MAP-2) was provided by Itzhak Fischer. In the case of
CHO1, this antibody was used at a concentration of 1:1000, and an
appropriate Cy3-conjugated secondary antibody (purchased from Jackson
ImmunoResearch, West Grove, PA) was used at 1:1500. In the case of the
RMDO20, the antibody was used at 1:500. The tau-1 and MAP-2 antibodies were used at 1:100. In all cases other than CHO1, the secondary antibody (Cy3-conjugated and purchased from Jackson ImmunoResearch) was
used at 1:1000. For CHO1 staining, cultures were rinsed briefly in PBS,
fixed for 6 min in cold methanol (20°C), rehydrated three times for
5 min each in PBS, exposed to a blocking solution (containing 5%
normal goat serum) for 30 min, exposed to the primary antibody overnight at 4°C, rinsed extensively, exposed to the secondary antibody for 1 hr at 37°C, rinsed extensively, and then mounted in a
medium that reduces photobleaching. For neurofilament staining, the
same procedure was used, except that cultures were fixed with a 15 min
exposure to 4% formaldehyde, and then post-extracted with 0.5% Triton
X-100 for 5 min. For tau-1 or MAP-2 staining, the same procedure was
used, except that the cultures were fixed with a 20 min exposure to a
solution containing 4% paraformaldehyde and 0.1% glutaraldehyde,
post-extracted in graded ethanols, and then rehydrated. Photographs of
the fluorescent cells were obtained using the LSM 410 confocal
microscope (Zeiss, Thornwood, NY) with the pinhole wide open. In the
case of the CHO1-stains, all images were taken at identical brightness
and contrast settings so that fluorescence quantification could be
performed. Levels of fluorescence within individual cells were
quantified as previously described (Yu et al., 1997; Sharp et al.,
1997b).
Lucifer yellow dye injections. For injection of Lucifer
yellow dye into neurons, we used the protocol of Higgins et al.
(1991). A quantity of ~4 pl of a solution of 10 mg/ml Lucifer
yellow (Molecular Probes, Eugene, OR) was microinjected into a small
number of neurons in each culture. The neurons were spaced sufficiently
far apart to ensure little or no overlap in their neuritic arbors.
After permitting the dye to diffuse throughout the neuron for 1 hr, the
cultures were fixed in PBS containing 4% paraformaldehyde. Photographs
of the fluorescent cells were obtained using the LSM 410 confocal
microscope with the pinhole wide open.
Electron microscopy. For standard electron microscopy, we
used previously reported procedures (Yu and Baas, 1994, 1995). Neuron cultures were fixed for 20 min at 37°C in 2% glutaraldehyde,
post-fixed with 1% OsO4, rinsed twice for 2 min
in NaCl, rinsed twice for 2 min in water, contrasted for 30 min in 5%
aqueous uranyl acetate, dehydrated in ethanols, and embedded in LX-112
(Ladd, Burlington, VT). After curing overnight at 60°C, the glass
coverslips were dissolved by exposure to hydrofluoric acid. Cells of
interest were circled using a diamond-marker objective, and their
phase-contrast images were recorded using a video printer (Sony, Tokyo,
Japan). Thin sections of a uniform thickness of 100 nm were obtained
with an Ultracut S Ultramicrotome (Reichert-Jeng, Vienna, Austria), stained with uranyl acetate and lead citrate, and observed and photographed with a transmission electron microscope. The precise points along the length of the dendrite corresponding to each electron
micrograph were recorded on the video prints.
Microtubule polarity analyses. To determine the polarity
orientation of microtubules within control and experimental dendrites, we used the standard "hooking" protocol. In this procedure, the cells are lysed in the presence of exogenous brain tubulin in a special
microtubule assembly buffer that promotes the formation of lateral
protofilament sheets on the existing microtubules. When viewed in cross
section with the electron microscope, these sheets appear as hooked
appendages on the microtubules. The curvature of the "hooks"
reveals the orientation of the microtubule. A clockwise hook as viewed
from the distal end of the process indicates that the microtubule is
oriented with its plus end distal to the cell body, and a
counterclockwise hook indicates the opposite. The procedure was
performed, and the data were interpreted as previously described (Sharp
et al., 1995, 1997b; Yu et al., 1997).
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RESULTS |
We have documented in a previous study that treatment with
antisense oligonucleotides is an effective means to specifically and
rapidly reduce levels of CHO1/MKLP1 protein within cultured rat
sympathetic neurons (Sharp et al., 1997b). In these earlier studies,
cultures were exposed to oligonucleotides before dendritic differentiation. We performed dose-response studies using two different antisense oligonucleotide sequences and their corresponding sense controls, and found both of the antisense sequences to be highly
effective in diminishing protein levels in a dose-dependent and
time-dependent fashion. There were no observed ill-effects on the
health of the neurons, other than the fact that dendrites did not
develop. No diminution in any other cytoskeletal proteins was observed
with the antisense treatments. No diminution in CHO1/MKLP1 or any other
proteins were observed with the sense sequences, nor were any
morphological changes observed. After rinsing out the antisense
oligonucleotides, CHO1/MKLP1 expression resumed, as did the normal
program of dendritic differentiation. Having already reported this set
of experiments, we felt comfortable using one of the two antisense
sequences for the present study, along with its corresponding sense
sequence as a control. To assess the results of depleting CHO1/MKLP1
after dendrites had developed, cultures were exposed to the
oligonucleotides on the fourteenth day after plating, by which time the
vast majority of neurons had developed robust dendritic arbors. A
concentration of 1 µM was used because this concentration
was shown in our earlier studies to be the minimum required to
effectively suppress CHO1/MKLP1 expression, thus leading to a rapid
depletion of the protein.
Morphological changes in cultured neurons during
antisense treatment
We first wished to assess morphological changes that occur in the
presence of the antisense oligonucleotides. Morphology was assessed
either using phase-contrast optics or using fluorescence microscopy
after immunostaining the cultures for a poorly phosphorylated epitope
of neurofilament protein that is enriched in dendrites versus axons. We
found that immunostaining with this antibody revealed both axons and
dendrites, but that the dendrites were illuminated particularly well.
Cultures that had not been treated with oligonucleotides and cultures
treated with the sense oligonucleotides showed indistinguishable
morphological characteristics, whereas cultures exposed to the
antisense oligonucleotides were markedly different. In untreated
cultures and cultures treated with sense oligonucleotides for 1, 2, or
3 d, approximately half the neurons showed a very robust dendritic
arbor (Figs. 1a-c,
2a). These cells showed
at least three thick tapering curvaceous dendrites that were each
~30- to 50-µm-long and at least 3-µm-wide at their base. Fewer
than 10% of the neurons showed no thick tapering dendrites whatsoever,
but instead consisted only of axon-like processes that only tapered
over a few micrometers near the cell body if at all (Fig.
1d). The remaining neurons consisted of at least one thick
tapering dendrite, but in general displayed a less robust dendritic
morphology than the majority of the neurons. These morphological differences among individual cells in the control cultures may result
from heterogeneity of neuronal types in the superior cervical ganglia
or may represent the failure of a small number of the neurons to
differentiate as robustly as they might have under these culture
conditions. In the antisense cultures, at days 1, 2, and 3, there were
no neurons that displayed the full robust dendritic arbor displayed by
most of the neurons in the untreated and sense-treated cultures (Figs.
2b-h). The majority of the neurons displayed no large thick
tapering processes whatsoever and were similar in appearance to the
small number of neurons in untreated and sense-treated cultures that
displayed no robust dendrites (Fig. 2h). At 1 d
of antisense treatment, ~70% of the cells showed this
"dendrite-less" morphology. This proportion progressively increased
to ~75 and 80% at 2 and 3 d in antisense, respectively. Prolonged exposure to antisense (up to 12 d) resulted in further loss of dendrites, but even after the longer exposure times, a small
number of the cells (<10%) continued to display processes with
dendritic morphology (data not shown).
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Figure 1.
Morphology of cultured rat sympathetic neurons
revealed by neurofilament immunostaining. Shown here are examples of
the morphology of rat sympathetic neurons that were cultured in the
presence of OP-1 to promote robust dendritic differentiation. After 2 weeks in culture, the vast majority of the neurons showed thick,
tapering dendrites. Cultures were immunostained with an antibody to a
poorly phosphorylated neurofilament epitope that is highly enriched in
dendrites compared to axons. Most of the neurons showed three or more
robust thick, tapering curvaceous dendrites that were at least 30-50
µm in length. Some neurons showed a somewhat less robust dendritic
arbor, but clearly showed unmistakable dendrites. Fewer than 10% of
the neurons showed no robust dendrites (d). Scale
bar, 30 µm.
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Figure 2.
Morphology of cultured rat sympathetic
neurons exposed to CHO1/MKLP1-antisense for 1 d revealed by
neurofilament immunostaining. Shown here are neurons treated with sense
or antisense oligonucleotides specific for CHO1/MKLP1. The neurons
shown here were immunostained with the same neurofilament antibody as
in Figure 1. a shows a sense control with a robust
dendritic arbor similar to that observed in typical control neurons.
The remaining panels show antisense-treated neurons with clearly
altered morphologies. The neuron shown in b displays
dendrites that are longer than typical control dendrites, less tapered
but still curvaceous. The vast majority of the experimental dendrites
lost their curvaceous appearance and appeared straight and taut.
c shows a neuron with dendrites that are somewhat
thinner and less tapered than control dendrites, and less curvaceous.
At least one of the dendrites is longer than typical controls. The
remaining panels (d-h) show neurons in more advanced
stages of their loss of dendritic morphology. The tapered regions of
the dendrites appear to wither in a distoproximal fashion, but the
actual length of the processes is difficult to assess because of
bundling with neighboring axons. Scale bar, 40 µm.
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We focused most of our attention on the cultures treated with antisense
for 1 d, because the most dramatic changes in morphology occurred
over the first 24 hr. In addition, we felt that the most informative
cells to examine were those whose processes were in a transitional
phase of losing their dendritic characteristics. As dendrites are lost
from the cultures, do they gradually decay, do they retract into the
cell body, or do they undergo transformation into processes with axonal
characteristics? The "experimental" dendrites of these cells were
typically different in appearance from any of the processes observed in
the untreated or sense-treated cultures. The neuron shown in Figure
2b displays experimental dendrites that are clearly
longer than typical control dendrites, less tapered, but still
curvaceous. Unlike the dendrites of this cell, the vast majority of
dendrites in the antisense-treated cultures lost their curvaceous
appearance and appeared much straighter and more taut. Figure
2c shows a neuron with dendrites that are somewhat
thinner and less tapered than control dendrites, and clearly less
curvaceous. At least one of the dendrites is longer than typical
controls. The remaining panels (panels d-h) show neurons
with processes in more advanced stages of their loss of dendritic characteristics.
Even with the assistance of the neurofilament staining, it was still
impossible to conclude whether the dendrites of the antisense-treated cells were shortening or whether they were becoming thinner, losing their taper, and actually elongating. In cultures of rat sympathetic neurons, axons tend to bundle with dendrites, and hence it is difficult
to discern the actual tip of a dendrite amid the neighboring axons that
surround it. To resolve the issue, we microinjected a small number of
cells in each culture with Lucifer yellow, a fluorescent dye that
rapidly diffuses throughout the neuron (Higgins et al., 1991). A total
of 50 cells under each experimental condition were examined. As shown
in Figure 3, a and
b, the dye injections were very effective at revealing the
tips of the dendrites in untreated and sense-treated neurons. Each
dendrite was 30-50 µm in length. As shown in Figure 3, c
and d, the dye injections revealed that the long slender
axon-like regions that extended from the withering tapered regions of
the experimental dendrites of the antisense-treated neurons were indeed
directly continuous with these regions. We found that these elongated
processes usually became too thin and too tortuous to identify their
tips with confidence, but nevertheless, it was clear that their lengths
typically exceeded 100 µm and often exceeded hundreds of micrometers,
even after the first day in antisense. Thus, during antisense
treatment, the thick-tapering regions of the dendrites gradually
withered toward the cell body, whereas the total length of the process underwent significant elongation.
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Figure 3.
Morphology of individual neurons revealed by
Lucifer yellow dye injections. Immunostains shown in Figures 1 and 2
suggest that during antisense treatment, the tapering regions of the
dendrites wither, but it is unclear whether the entire length of the
process increases or decreases because of the bundling of the dendrites
with axons from neighboring cells. To investigate this issue, we
injected Lucifer yellow into individual neurons to reveal their
neuritic arbor. a shows an untreated neuron,
b shows a sense-treated neuron, and c and
d show antisense-treated neurons. Treatments were for
1 d. In the untreated and sense-treated neurons, the tips of the
dendrites are clearly distinguishable. Typical dendrites were 30-50
µm in length. In the antisense-treated neurons, the dendrites are
substantially longer than controls. In many cases, the dendrites had
elongated to well over 100 µm over the first day in antisense. Scale
bar, 30 µm.
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Reduction in CHO1/MKLP1 levels during antisense treatment
As noted above, our previous study demonstrated the specificity
and efficacy of the antisense treatment in terms of diminishing CHO1/MKLP1 levels in cultured rat sympathetic neurons. Such efficacy and specificity have also been shown in studies using the same CHO1/MKLP1 antisense sequence on cultured neuroblastoma cells (Yu et
al., 1997) and cultured podocytes (Kobayashi et al., 1998). Nevertheless, we chose to perform quantitative immunofluorescence analyses on the levels of CHO1/MKLP1 remaining in neurons after treatment with the antisense to ascertain the degree to which the
levels were reduced and also to investigate whether the levels of the
protein correspond in any way to specific features of neuronal morphology. Fluorescence intensities were expressed in arbitrary fluorescence units (AFUs). Oligonucleotide treatments were for 1 d. In 13 untreated and 15 sense-treated neurons with robust dendritic
arbors, the CHO1/MKLP1 levels were all >100 AFUs (151 ± 30 AFUs
for untreated cultures and 130 ± 21 AFUs for sense-treated cultures). In seven untreated neurons and six sense-treated neurons with less robust dendrites, the CHO1/MKLP1 levels were all >50 AFHs
but <100 AFUs (83 ± 12 and 78 ± 11 AFUs, respectively). In nine untreated and nine sense-treated neurons with no dendrites, the
levels were all <60 AFUs (49 ± 9 and 44 ± 13 AFUs,
respectively). In 10 antisense-treated neurons with withering tapered
regions, the levels were between 70 and 90 AFUs (75 ± 9 AFUs). In
10 antisense-treated neurons with no tapering regions remaining, the
levels were all <50 AFUs (40 ± 11 AFUs). These results, examples
of which are shown in Figure 4, indicate
that the antisense treatment (but not the sense treatment) was
effective in reducing CHO1/MKLP1 levels (by an average of ~40%
across cells), and that the levels of this protein roughly correspond
to the robustness of the dendritic arbor, both in antisense-treated and
control neurons.
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Figure 4.
Immunofluorescence analyses on the levels of
CHO1/MKLP1 in control and antisense-treated neurons. After 1 d in
antisense, cultures were fixed in cold methanol, and CHO1/MKLP1 was
visualized using immunofluorescence microscopy. DIC images of the fixed
cells are shown in the left-hand column, whereas
corresponding immunofluorescence images are shown in the
right-hand column. a and
a' show an untreated neuron with robust dendrites and
strong CHO1/MKLP1 immunoreactivity in the cell body and dendrites but
not the axonal network. b and b' show
another untreated neuron with somewhat less robust dendrites. The
immunoreactivity is correspondingly somewhat less intense.
c and c' show an antisense-treated neuron
wherein the withered tapered regions of dendrites are still apparent.
Immunoreactivity is significantly lower than in control neurons.
d and d' show an
antisense-treated neuron wherein the dendrites have almost completely
reverted to an axonal morphology. The immunoreactivity is even lower
yet. Scale bar, 35 µm.
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Ultrastructural changes during antisense treatment
To assess ultrastructural changes in dendrites that result from
the antisense treatment, untreated and antisense-treated neurons were
prepared for standard transmission electron microscopy. In control
neurons (see Fig. 5a for
phase-contrast image), ribosomes were abundant throughout the length of
the dendrite and were especially rich in the most proximal third of the
process near the cell body (Fig. 5d). Microtubules were
somewhat scattered near the cell body (Fig. 5d), but were
more paraxial (but not as paraxial as in axons) farther down the length
of the dendrite (Fig. 5e). Neurofilaments were plentiful
throughout and formed dense bundles that were characteristic of
dendrites but not axons (Baas et al., 1991). In the antisense-treated cultures that showed withering tapered regions (see Fig. 5,
b and c, for phase-contrast images), ribosomes
were still plentiful near the cell body of antisense-treated dendrites
(approximately the proximal fifth; Fig. 5f), but were
dramatically diminished with distance and virtually absent from the
thinner distal regions that still fell within the original length of
the dendrite (Fig. 5h) and from the newly grown regions
(Fig. 5i,j). Neurofilament bundles remain plentiful
throughout most of the original length of the dendrite (Fig.
5f-h), but only sparse unbundled neurofilaments were
present in the newly grown regions (Fig. 5i,j). Microtubules appear at relatively lower levels within the more proximal regions of
the original dendrite (Fig. 5f), but at higher levels
more distally (Fig. 5h-j). The microtubules appear to be
more paraxial than in controls, especially in the more distal regions
of the antisense-treated dendrites. The levels of internal membranous elements (presumably much of which is Golgi; Sharp et al., 1995) also
appear to diminish with antisense treatment and with distance down the
length of the antisense-treated dendrites. The cytoplasm of processes
that had completely lost their taper was entirely similar to the
cytoplasm within the thin distal regions of the "transitional"
dendrites shown in Figure 5, i and j (data not shown). Thus, the cytoplasm of the dendrite becomes gradually more
axonal in character during antisense treatment, and the organelles that
are normally present in dendrites but not axons appear to retreat
proximally toward the cell body as the dendrites lose their thickness
and taper.
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Figure 5.
Electron microscopic analyses on dendrites of
control and CHO1/MKLP1-antisense-treated neurons. Top
panels show phase-contrast micrographs of embedded samples.
a is an untreated neuron, whereas b and
c are antisense-treated. The remaining panels show
electron micrographs from the indicated regions. MT,
Microtubule; NF, neurofilament bundles typical of
dendrites; R, ribosomes. Control neurons show abundant
ribosomes near the cell body and fewer but still abundant levels
farther down the length of the dendrite. Microtubules are scattered
near the cell body, but more paraxial (but still not as paraxial as in
axons) farther down the length of the dendrite. Neurofilament bundles
are plentiful throughout. Ribosomes are still plentiful near the cell
body of antisense-treated dendrites, but are dramatically diminished
with distance, and virtually absent from the thinner distal regions of
the original dendrite and the newly grown regions. Neurofilament
bundles remain plentiful throughout most of the original length of the
dendrite, but only sparse unbundled neurofilaments are present in the
newly grown regions. Microtubules appear at relatively lower levels
within the more proximal regions of the original dendrite, but at
higher levels more distally. The microtubules appear to be more
paraxial than in controls
especially in the more distal regions of the
antisense-treated dendrites. The levels of internal membranous elements
(presumably Golgi) also appear to diminish with antisense treatment.
Thus, the cytoplasm of the antisense-treated dendrites becomes more
axonal in character. The change in microtubule distribution is
consistent with the idea that the minus-end-distal microtubules are
moving out of the dendrite back toward the cell body, and the
plus-end-distal microtubules are translocating anterogradely during
antisense treatment. Scale bar, 0.5 µm.
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Changes in microtubule polarity orientation as a result of
CHO1/MKLP1 depletion
The alterations in dendritic morphology and composition are
consistent with a loss of minus-end-distal microtubules during CHO1/MKLP1 antisense treatment. To assess microtubule polarity orientation, we used the standard "hooking" procedure. In this method, cells are extracted in the presence of exogenous brain tubulin
in a special buffer that promotes the addition of the exogenous tubulin
onto the sides of pre-existing microtubules. The exogenous tubulin
forms lateral curved appendages that appear as "hooks" when viewed
in cross section electron microscopically. As viewed from the tip of
the dendrite, clockwise hooks indicate plus-end-distal microtubules,
whereas counterclockwise hooks indicate minus-end-distal microtubules.
As previously reported, in control dendrites, slightly more than half
of the microtubules were plus-end-distal in proximal and middle
regions, with progressively higher percentages in the more distal
regions (Baas et al., 1989, 1991). Data from two such control dendrites
are shown in Figure 6a. We
viewed >20 sections from dendrites that had completely thinned along
their lengths and in all cases, the percentage of clockwise hooks was >95%, indicating uniformly plus-end-distal microtubules. Shown in
Figure 6b are the data from six examples of
"transitional" dendrites that still showed withering tapered
regions. In all cases, the thinner distal regions showed predominantly
or entirely clockwise hooks. In two of the six cases, the proportion of
plus-end-distal microtubules was significantly higher than controls at
corresponding sites throughout the length of the dendrite. In four of
the six cases, this increase in the proportion of plus-end-distal
microtubules was observed throughout most of the length of the
dendrite, except in the most proximal region near the cell body, which
actually showed a reduction in the proportion of clockwise hooks
(indicating an increase in the proportion of minus-end-distal
microtubules). Examples of the electron micrographs are shown in Figure
6c-e. Taken together, these data are consistent with the
view that minus-end-distal microtubules are gradually cleared from the
antisense-treated dendrites and that microtubules of this orientation
are chased back toward the cell body.
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Figure 6.
Microtubule polarity analyses on control and
CHO1/MKLP1-antisense treated cultures. The standard "hooking"
procedure was used to assess microtubule polarity orientation.
Clockwise hooks indicate plus-end-distal microtubules, whereas
counterclockwise hooks indicate minus-end-distal microtubules.
a shows the data from two control dendrites, whereas
b shows the data from six antisense-treated dendrites.
The data are expressed as the percentages of clockwise hooks in
different sampled regions. In control dendrites, slightly more than
half of the hooks were clockwise in proximal and middle regions, with
progressively higher percentages in the more distal regions. In
dendrites that had completely thinned along their lengths, the
percentage of clockwise hooks was >95%, indicating uniformly
plus-end-distal microtubules (data not shown). The dendrites shown in
b are "transitional," in the sense that they still
showed withering tapered regions. In all cases, the thinner distal
regions showed predominantly or entirely clockwise hooks. In two of the
six cases, the proportion of plus-end-distal microtubules was
significantly higher than controls at corresponding sites throughout
the length of the dendrite. In four of the six cases, this increase in
the proportion of plus-end-distal microtubules was observed throughout
most of the length of the dendrite, except in the most proximal region
near the cell body, which actually showed a reduction in the proportion
of clockwise hooks. Examples of the electron micrographs are shown in
c-e, and the corresponding regions of the dendrite from
which the electron micrographs were taken are shown by
lettered-marked arrows in b. Scale bar:
a, b, 40 µm; c-e, 0.45 µm.
|
|
Distribution of microtubule-associated proteins during
antisense treatment
Several studies have shown that the full-length isoform of MAP-2
is concentrated within dendrites, whereas a specific phosphorylated isoform of tau (recognized by the tau-1 antibody) is concentrated within the axon (Binder et al., 1985; Matus, 1994). The mechanisms responsible for this compartmentation and its functional significance remain unclear. In our hands, dendrites of untreated cultures stained
lightly with the tau-1 antibody and were clearly surrounded by more
intensely staining axons (Fig.
7a). The axons in the culture varied in their level of intensity, perhaps as a function of their length or distance from the cell body. The withering regions of the
antisense-treated dendrites showed no apparent increase in staining for
tau-1 and were still surrounded by more intensely stained axons (Fig.
7b). Given the variability in the levels of axonal staining,
it was unclear whether the elongated distal regions of these dendrites
stained any more or less intensely for tau compared to typical axons.
The dendrites of untreated cultures stained intensely for full-length
MAP-2, whereas the axons showed little or no staining (Fig.
7c). As the dendrites thinned and elongated, there was no
immediate diminution in MAP-2 staining (Fig. 7d), although
the intense MAP-2 staining was gradually diminished as the thick
tapering regions of the dendrites withered (Fig. 7e). After
the complete transformation of the dendrites to an axonal morphology,
they were indistinguishable from the axons in the culture on the basis
of their staining for these MAPs (data not shown). These studies show
that a transformation to a more axonal MAP composition eventually
occurs in the experimental dendrites, but that the signs of this
occurring are not detectable as early as the signs of transformation to
a more axonal morphology and ultrastructure.
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Figure 7.
Tau-1 and MAP-2 immunofluorescence analyses
on control and CHO1/MKLP1 antisense-treated neurons. a
and b show an untreated and an antisense-treated neuron,
both stained with the tau-1 antibody. The dendrites of untreated
cultures stained lightly and were clearly surrounded by more
intensely-staining axons (a). The withering
regions of the antisense-treated dendrites showed no apparent increase
in staining for tau-1 and were still surrounded by more intensely
stained axons (b). a shows an
untreated neuron, whereas d and e show
antisense-treated neurons, all stained for full-length MAP-2. The
dendrites of untreated cultures stained intensely for full-length
MAP-2, whereas the axons showed little or no staining
(c). As the dendrites thinned and elongated in
response to antisense-treatment, there was no immediate diminution in
MAP-2 staining (d), although the intense MAP-2
staining was gradually diminished as the thick tapering regions of the
dendrites became progressively more withered (e).
Scale bar, 12 µm.
|
|
| |
DISCUSSION |
In previous studies, it was established that CHO1/MKLP1 is
enriched in dendrites but not axons and that inhibition of its expression before dendritic development prohibits dendrites from forming (Yu et al., 1997; Sharp et al., 1997b). On the basis of these
observations, we tentatively concluded that CHO1/MKLP1 is essential for
the transport of minus-end-distal microtubules into nascent dendrites
and that the presence of the minus-end-distal microtubules is requisite
for the acquisition of a dendritic morphology and organelle
composition. Of course, there are alternative explanations for the
manner by which a motor protein might be essential for dendritic
differentiation, and it is important to note that the issue of
microtubule transport has been controversial (for discussion, see Baas,
2000). In the present study, we have suppressed CHO1/MKLP1 expression
after dendrites had already formed and observed the results of a
gradual depletion of the protein. If CHO1/MKLP1 functions as we have proposed, we can make predictions about how microtubules should redistribute, how organelles should redistribute, and how the
morphology of the dendrite should change as the levels of CHO1/MKLP1
are diminished. Our model for the establishment of the neuronal
microtubule arrays, shown schematically in Figure 8a, is based on the manner by
which CHO1/MKLP1 and cytoplasmic dynein transport microtubules in
vitro and in the mitotic spindle (for discussion, see Baas, 1999).
In this model, cytoplasmic dynein transports microtubules into axons
and dendrites with their plus ends leading by pushing against the actin
cytomatrix (Ahmad et al., 1998), whereas CHO1/MKLP1 transports
microtubules with their minus ends leading only into dendrites by
pushing against the plus-end-distal microtubules (Sharp et al., 1997b).
Once CHO1/MKLP1 is sufficiently depleted, the unopposed dynein-driven
forces should redistribute the microtubules by transporting them all
with their plus ends leading. This would drive the minus-end-distal
microtubules back into the cell body, and the plus-end-distal
microtubules would now move more efficiently forward. As the
microtubule polarity pattern becomes gradually more plus-end-distal,
the morphology and cytoplasmic composition of the dendrites should
become gradually more axon-like.
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|
Figure 8.
A model for the role of CHO1/MKLP1 in the
dendrite. In our model (a), cytoplasmic dynein
transports microtubules with their plus ends leading (pushing against
the actin cytomatrix) into the axon and the dendrites, whereas
CHO1/MKLP1 transports microtubules anterogradely with their
minus-ends-leading (pushing against the plus-end-distal microtubules)
into the dendrites but not the axon. The forces generated by CHO1/MKLP1
create drag on the plus-end-distal microtubules, which impedes their
dynein-driven transport. Both the suppression of dynein-driven
microtubule transport and the nonuniform microtubule polarity pattern
itself result in the characteristic morphological and compositional
features that distinguish dendrites from the axon. This model makes
certain predictions regarding what would happen if CHO1/MKLP1 were
depleted (b). The model predicts that when
CHO1/MKLP1 is depleted, the forces generated by cytoplasmic dynein
should no longer be antagonized. The plus-end-distal microtubules
should therefore be better able to move anterogradely, and the
minus-end-distal microtubules should be transported (with their plus
ends leading) back toward the cell body. As the minus-end-distal
microtubules are cleared and the plus-end-distal microtubules move
rapidly forward, the dendrite should gradually lose its taper, thin
out, and elongate. These predictions are consistent with the
redistribution of microtubules and the alterations in dendritic
morphology observed in our depletion studies. Observed compositional
changes are also consistent with the predictions of the model (data not
shown in the schematic).
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|
The results of our studies bear out these predictions (Fig.
8b). As the levels of CHO1/MKLP1 are diminished, there is a
dramatic redistribution of microtubules. Minus-end-distal microtubules gradually vacate the dendrite until the dendrite completely reverts to
a uniformly plus-end-distal pattern of microtubule organization. During
this transition, we documented cases in which there were abnormally
high percentages of minus-end-distal microtubules in the proximal
region of the dendrite at the same time as the proportion of
plus-end-distal microtubules increased in the middle and more distal
regions. These results are not consistent with the idea that the
minus-end-distal microtubules simply vanish or depolymerize from the
dendrite, but are entirely consistent with the predictions of our
model, namely that these microtubules are chased back to the cell body
by dynein-driven transport. In addition, these observations provide a
new line of evidence favoring the view that tubulin is actively
transported within neurons in the form of polymers (Baas, 2000). As
this redistribution of microtubules occurred, there was a dramatic
alteration in the morphology of the dendrite; the region still
containing minus-end-distal microtubules continued to show taper until
most or all of the microtubules of this orientation were depleted. The
region cleared of minus-end-distal microtubules became thinner,
elongated dramatically, and took on the morphological appearance of an
axon. Eventually, the entire dendrite acquired an axonal morphology as
the microtubule polarity pattern became uniformly plus-end-distal.
We suspect that there are two reasons for the morphological changes.
The first reason relates directly to the forces generated by the motor.
Once the CHO1/MKLP1-driven forces are diminished, a "drag" is
removed on the anterograde transport of the plus-end-distal microtubules, thus permitting these microtubules to move as rapidly in
dendrites as they do in the axon. This would promote rapid growth of
the process. A shift in these forces might also explain why the
experimental dendrites become less curvaceous and more taut. We have
recently shown that tension in the axon results from myosin-driven
forces on the microfilament array and that these forces are attenuated
by the dynein-driven forces between the microtubule and the
microfilament arrays (Ahmad et al., 2000). The CHO1/MKLP1-driven forces
would be additive to the dynein-driven forces, thus further reducing
the tension in the dendrite. Less overall tension would result in a
less taut and more curvaceous process. Under conditions of depleted
CHO1/MKLP1, we would expect tension in the dendrite to increase, thus
producing a more taut and less curvaceous process. The other
explanation for the morphological changes relates to the transport of
membranous vesicles needed for the growth of the processes at their
tips. Assuming that these vesicles move toward plus ends of
microtubules, a uniformly plus-end-distal microtubule array would
provide a unidirectional vector for the vesicles to move to the distal
tip of the process. Thus, the process would be disposed to grow longer.
A nonuniformly oriented microtubule array would not provide such a
vector, and hence the vesicles would probably add to the dendrite along
its sides, which would promote a thickening over an elongation of the process.
Compositional features of the dendrite also become gradually more
axonal as CHO1/MKLP1 is depleted. Ribosomes and dendrite-enriched membranous structures (presumably Golgi elements) vacated the dendrite
in a similar distoproximal manner as the minus-end-distal microtubules.
In our model, the minus-end-distal microtubules provide a substrate for
the transport of these organelles into the dendrite (Baas et al.,
1988). In the absence of microtubules of this orientation, we would
expect these organelles to move toward the minus ends of the
plus-end-distal microtubules back toward the cell body. These
observations support our hypothesis that these organelles are
distributed on the basis of their transport toward minus-ends of
microtubules. However, it should be noted that there are still many
unresolved issues regarding exactly how such traffic is regulated. For
example, for these organelles to accumulate within dendrites, it seems
likely that there would have to be selective retention mechanisms in
addition to selective transport mechanisms (Black and Baas, 1989). The
present observations indicate that, if such mechanisms exist, they can
be overwhelmed by the motor-driven forces that would transport these
organelles retrogradely if not for the presence of the minus-end-distal microtubules.
Not all features of dendritic identity are so easy to understand on the
basis of microtubule polarity orientation. The neurofilaments offer an
interesting puzzle because they appear in both axons and dendrites, but
differ in their phosphorylation state and bundling patterns in each
type of process. Perhaps specific kinases or other regulators of
neurofilament organization are mediated by features of microtubule
organization, but how such a mechanism might be orchestrated remains
unclear. The mechanisms by which MAP-2 and tau-1 isoforms are
compartmentalized also remain mysterious. These proteins alter their
distribution as a result of CHO1/MKLP1 depletion, but this occurs
more slowly than the morphological changes that we have documented.
Interestingly, a plus-end-directed kinesin has recently been discovered
which appears to be targeted for dendrites but not axons
(Marszalek et al., 1999), and the mechanism for this targeting is also
unclear. We find it compelling to contemplate that perhaps the
acquisition of nonuniform microtubule polarity orientation could set
into motion a cascade of events that leads to all of these various
features of dendritic identity. Although this hypothesis will require
more testing, initial support for it is provided by our studies showing
that with sufficient time, depletion of CHO1/MKLP1 causes dendrites to
lose their identity and literally "become axons" by all of the
criteria that we have explored.
Our quantitative immunofluorescence studies suggest that neurons might
normally regulate dendritic morphology by modulating the levels of
CHO1/MKLP1. The control neurons with more robust dendritic arbors
consistently showed stronger immunoreactivity. Treatment with the
antisense drastically reduced the levels of the protein in most
neurons, but the degree to which the protein was diminished varied from
cell to cell. Neurons with less of the protein showed more significant
loss of dendritic characteristics, which supports the idea that the
robustness of the dendritic arbor could be regulated by the absolute
amount of this protein. The antisense results also suggest that neurons
might regulate CHO1/MKLP1 through changes in its half-life. After
1 d in antisense, the remaining CHO1/MKLP1 protein diminished more
slowly, suggesting that there may be a more long-lived pool of the
protein as well as a more short-lived pool. The fact that a small
number of cells maintained CHO1/MKLP1 immunoreactivity and fairly
robust dendrites even after 12 d in antisense suggests that the
proportion of protein with a longer half-life may increase during
neuronal maturation or vary significantly from cell to cell. Our
previous in situ hybridization analyses showed that the
levels of CHO1/MKLP1 messenger RNA are significantly lower in adult
neurons (Ferhat et al., 1998), which would be consistent with a longer
half-life of the available protein. Another possibility is that smaller
amounts of CHO1/MKLP1 are needed later in development because more
mature neurons use other proteins such as MAP-2 to retain the
minus-end-distal microtubules through structural cross-links. These and
other issues will require further study.
| |
FOOTNOTES |
Received March 15, 2000; revised April 20, 2000; accepted May 10, 2000.
This work was funded by grants from the National Institutes of Health
and the National Science Foundation to P.W.B. We thank Dennis Higgins,
David Sharp, and Itzhak Fischer for helpful discussions. We thank
Itzhak Fischer and Lester Binder for providing antibodies.
Correspondence should be addressed to Peter W. Baas, Department of
Anatomy, The University of Wisconsin Medical School, 1300 University
Avenue, Madison, WI 53706. E-mail: pwbaas{at}facstaff.wisc.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
