into short pieces, but this phenomenon is suppressed by the coexpression of tau. Protection against severing is also afforded by
microtubule-associated protein 2 (MAP2), which has a tau-like microtubule-binding domain, but not by MAP1b, which has a different
microtubule-binding domain. The microtubule-binding domain of tau is required for the protection, but within itself, provides less
ure its underlying microtubule scaffolding. Recent studies indi-
cate that the capacity of a microtubule to move in a rapid and
concerted manner is directly related to its length (Dent et al.,
transport (Wang and Brown, 2002). Therefore, it follows that
reconfiguration and transport of microtubules require that
longer microtubules are severed into shorter pieces. Neurons
are rich in a protein called katanin, which is an enzyme that
hydrolyzes ATP to break the lattice of the microtubule. Ka-
tanin has been shown to be essential for severing microtubules
from the centrosome of neurons (Ahmad et al., 1999) and to
play key roles in generating short microtubules throughout all
compartments of the neuron (Karabay et al., 2004; Yu et al.,
Katanin consists of two subunits, one of which, termed P60-
katanin, is the enzyme that severs microtubules. The other sub-
unit, termed P80-katanin, targets a portion of the katanin to the
katanin compared with other cell types, and these high levels
would theoretically sever the microtubules completely to sub-
Nally and Vale, 1993). A potential clue as to how katanin is reg-
ulated derives from the observation that its severing activity is
higher activity is somehow related to phosphorylation (McNally
et al., 2002). Interestingly, however, katanin itself appears not to
be phosphorylated, suggesting that the phosphorylation of other
proteins regulates katanin.
These observations support a model whereby katanin-
induced microtubule severing is regulated by microtubule-
them to dissociate from the microtubules (Baas and Qiang,
2005). This idea was originally prompted by the observation that
the frog homolog of MAP4 diminishes the microtubule severing
that occurs in response to katanin in vitro (McNally et al., 2002).
expression in cultured neurons cause widespread severing and
loss of microtubules from cell bodies, minor processes, and den-
drites but not from the axon, where the microtubules appear to
posited that axons may be enriched with a MAP that provides
strong protection of microtubules against severing by katanin.
to speculate that tau may play this protective role in the axon,
given its tight regulation by phosphorylation (Johnson and
Stoothoff, 2004), enrichment in the axon (Mandell and Banker,
1996), and the central roles it plays in neuropathies affecting
axonal microtubules (Cairns et al., 2005; Stoothoff and Johnson,
2005). Here, we sought to determine whether tau is the chief
protector of axonal microtubules from severing by katanin.
3120 • TheJournalofNeuroscience,March22,2006 • 26(12):3120–3129
Antibodies (Abs) included the following: rabbit anti-P60-katanin poly-
clonal Ab (1:1000 for immunostaining) (Karabay et al., 2004); rabbit
anti-green fluorescent protein (anti-GFP) polyclonal Ab (1:500 for im-
munostaining; Abcam, Cambridge, UK); chicken polyclonal anti-myc
mouse monoclonal anti-?-tubulin Ab (1:200 for immunostaining; Sig-
ma); cyanine 3 (Cy3)-conjugated mouse monoclonal anti-?-tubulin Ab
(1:300 for immunostaining; Sigma); mouse monoclonal anti-MAP2 Ab
(1:200 for immunostaining and 1:1000 for Western blot; Chemicon, Te-
mecula, CA); goat polyclonal anti-MAP1b Ab (1:200 for immunostain-
ing; Santa Cruz Biotechnology, Santa Cruz, CA); rabbit polyclonal anti-
MAP1b Ab (1:500,000 for Western blot; provided by I. Fisher, Drexel
University, Philadelphia, PA); rabbit polyclonal anti-MAP4 Ab (1:100
bia University, New York, NY); rabbit polyclonal anti-tau Ab1 (1:2000
for immunostaining in experiments of RFL6 cells; also called CR; pro-
vided by G. Lee, University of Iowa, Iowa City, IA); rabbit polyclonal
anti-tau Ab2 (1:500 for immunostaining in experiments of rat neurons
monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (anti-
GAPDH; 1:500 for Western blot; Ambion, Austin, TX); and a series of
appropriate fluorescent and HRP-conjugated secondary antibodies
(Jackson ImmunoResearch, West Grove, PA).
construct; BD Biosciences, Boston, MA); pEGFP-C1-p60 (rat P60-
katanin) (Karabay et al., 2004); pCMV/myc3b-P60 (rat P60-katanin);
pMAP2c (human full-length MAP2c; provided by S. Halpain, The
(mouse full-length; provided by P. R. Gordon-Weeks, King’s College
London, London, UK) (Gordon-Weeks and Fischer, 2000); pEGFP-C3-
1992); pEGFP-C1-4R-tau (human four-repeat tau; provided by K. S.
pRc/CMVflag123c (tauMTBD; C terminus of human tau with the
microtubule-binding domain but without the projection domain; pro-
vided by G. Lee) (Leger et al., 1994); pRc/CMVflagn (tauMTNBD; N
terminus of human tau without the microtubule-binding domain; pro-
vided by G. Lee) (Hall et al., 1997); pRc/CMVflagFFT/6d (tauFFT/6d, a
2004); pEGFP-C1-MAP4 (human MAP4; provided by J. C. Bulinski)
(Chang et al., 2001).
Image acquisition and analysis
Images were acquired with an AxioVert 200M microscope (Carl Zeiss,
Oberkochen, Germany) coupled with an Orca-ER Digital CCD
(Hamamatsu, Shizouka, Japan) and a 40? Plan-Neurofluar/1.3 or a
100? Plan-Neorofluar/1.3 numerical aperture objective. Images to be
compared were taken at identical settings of exposure time, brightness,
and contrast and analyzed with Axiovision 4.0 software. All measure-
ments were taken as total fluorescence intensity per cell. Statistics were
done using Student’s t test.
Experiments on RFL6 fibroblasts
constructs and P60-katanin constructs using a Nucleofector (Amaxa,
Gaithersburg, MD) with the manufacturer’s program G-13. There were
always four different groups in the studies with each MAP construct: (1)
P60-katanin construct was used); (2) cells overexpressing P60-katanin
and flag/GFP (depending on which MAP construct was used); (3) Cells
overexpressing one of the MAPs and P60-katanin; (4) control group
(cells overexpressing GFP/myc and/or flag/GFP). Fifteen micrograms of
plated at a density of 8000 cells per well on glass coverslips mounted in
the bottom of 35-mm-diameter Petri dishes with holes drilled in the
bottom. Taxol was used at 5 ?M. All of the cells were cultured for 12 h
after transfection in the CO2incubator before being fixed.
Immunostain analyses. For immunostaining of the expressed MAPs
and/or P60-katanin, cultures were prepared as described previously
(Karabay et al., 2004). To evaluate the protein level within each MAP
overexpressed cell, the cells were fixed without previous extraction. The
antibody to mouse MAP1b was used to compare the expressed MAP
levels in RFL6 fibroblasts with the native levels of the MAP in rat hip-
pocampal neurons by measuring the average fluorescence intensity in
about equally, this calculation was used as a baseline to achieve a level of
expression approximately twice that in the neuron. Then, by comparing
expression levels of the various MAPs that shared the same tag, we ad-
justed our transfection regime and selected fibroblasts that expressed
generally similar levels of the other MAPs so that comparisons could be
made among the different MAPs. Rabbit polyclonal anti-GFP Ab was
polyclonal anti-tau Ab1 was then used to compare tau3R, tauFFT/6d,
tau4R, and tauMTNBD because this Ab recognizes all four. Flag Ab was
recognize tauMTBD. To quantify microtubule mass, RFL6 fibroblasts
were simultaneously fixed and extracted to remove free tubulin as de-
were used: pCMV/myc3b-P60 and pEGFP-C1-P60. pCMV/myc3b-P60
was used when the cells were transfected with pEGFP-C3-3R-tau,
pEGFP-C1-4R-tau, pEGFP-C1-MAP4. pEGFP-C1-P60 was used when
the cells were transfected with pMAP2c, pMAP1b, pRc/CMVflag123c,
GFP Ab followed by FITC-conjugated donkey anti-rabbit IgG was used
to reveal the various MAPs; mouse monoclonal anti-?-tubulin Ab fol-
lowed by Cy5-conjugated donkey anti-mouse IgG was used to reveal
microtubules. For the latter, rabbit polyclonal anti-GFP followed by
mouse monoclonal anti-MAP2 Ab followed by Cy5-conjugated donkey
anti-mouse IgG was used to reveal MAP2c; goat polyclonal anti-MAP1b
Ab followed by Cy5-conjugated donkey anti-goat IgG was used to reveal
donkey anti-mouse IgG was used to reveal the flag-tagged MAPs. To
immunostain microtubules in this latter group, we sequentially applied
Cy3 directly conjugated anti-?-tubulin Ab. (So that samples could be
compared against one another, controls were always stained with both
the Cy3 and the Cy5 methods, and microscope settings were adjusted to
equilibrate the signal.)
Experiments on primary neurons
and Baas (1994)] were transfected with small interfering RNA (siRNA)
for MAP2, tau, or MAP1b using a Nucleofector (Amaxa) with the man-
ufacturer’s program G-13. For MAP2 siRNA, we used a sequence previ-
ously reported to be effective (Krichevsky and Kosik, 2002). For tau and
MAP1b, we used a mixture of four different siRNA duplexes designed
against different regions of each molecule using Dharmacon (Lafayette,
CO) custom SMARTpool siRNA service. The Dharmacon accession
for rat tau is NM_017212. The nonspecific duplex III (Dharmacon) was
used as control. siRNA was dissolved to 200 ?M, aliquoted, and stored at
?20°C. siRNA concentration at transfection was 18 ?M. The siRNA
treated neurons were cultured for 48 h in plastic dishes coated with
poly-L-lysine before being replated. All of the siRNA treated neurons
of different MAP levels.
Two days later, in each group, some of neurons were replated at a
density of 8000 cells per well on glass coverslips mounted in the bottom
tained in the plastic dishes. The transfections of pEGFP-C1-P60 were
performed 2 d after the replating. For transfections of P60-katanin, we
used Lipofectamine 2000 (11668-027; Invitrogen, San Diego, CA). Two
Qiangetal.•TauProtectsAxonalMicrotubulesfromSeveringJ.Neurosci.,March22,2006 • 26(12):3120–3129 • 3121
to four micrograms of DNA constructs and 5–10 ?g of Lipofectamine
2000 were used per 35 mm dish. The ratio of DNA and Lipofectamine
2000 was 1:2.5. The cells were incubated in the DNA/Lipofectamine-
containing medium for 5 h. After transfection, neurons were transferred
plemented with 2% B27, 0.3% glucose, 1 mM glutamine, and 5% FBS).
Twelve hours later, the cells replated on 35 mm dishes were fixed for
immunostaining, and the cells replated on plastic dishes were collected
for Western blots for the three MAPs. The levels of MAPs were detected
Immunostain analyses. Mouse monoclonal anti-MAP2 Ab, rabbit
detect the three MAPs, respectively, in the siRNA-treated hippocampal
neurons. GFP antibodies were applied to find the P60-katanin-
overexpressing neurons. To quantify microtubule mass, neurons were
simultaneously fixed and extracted to remove free tubulin as described
previously (Yu et al., 2005). To immunostain microtubules, we applied
Cy3 directly conjugated anti-?-tubulin Ab.
to established procedures (Sambrook et al., 1989). Neurons cultured in
the plastic dishes were suspended with cold 1? PBS, lysed in the sample
buffer with SDS, and then the proteins were resolved in 10 or 7.5%
of MAP2c and Tau, whereas 7.5% gel was used in the Western blots to
lulose membranes (300 mA; overnight at 4°C). Transfers were probed
with MAP2 antibody against both MAP2a/b and MAP2c, with MAP1b
antibody (provided by I. Fisher) and with Tau antibody (DD8), respec-
tively. The corresponding secondary HRP-conjugated antibody was
used. After reaction with chemiluminescent peroxidase substrate (Super
Signal; Pierce, Rockford, IL), each blot was covered with x-ray film. For
imaged using an Epson (Long Beach, CA) Perfection 1280 scanner. Pro-
tein markers were used to indicate the appropriate molecular weights.
GAPDH was used as the internal control in each group to show that the
same amount of protein was loaded in each group.
There are two sets of studies presented here, one on RFL6 fibro-
RFL6 fibroblasts seek to test, in a relatively simple non-neuronal
tect microtubules from severing by overexpressed P60-katanin.
These studies also include experiments using Taxol, which stabi-
from MAPs. In the second set of studies, we take the inverse
axon become any less resistant to severing by katanin. The RFL6
cells and primary neurons are both from rat.
In a first set of studies on the RFL6 fibroblasts, we investigated
whether stabilization and bundling of microtubules by Taxol al-
ters their response to P60-katanin overexpression. We chose
ing GFP–P60-katanin but treated with Taxol. A portion of the microtubules appears as thick
Microtubules are not protected from P60-katanin-induced severing by Taxol. A
MAP2 or MAP1b in red. B, D, F, H, Immunostains for microtubules. A, B, Cells that are not
shown in E and F, MAP2c expression causes the formation of dense bundles of microtu-
tion of severing by overexpression of P60-katanin, and the microtubule mass is not re-
MAP2c protects microtubules from being severed by P60-katanin, but MAP1b
3122 • J.Neurosci.,March22,2006 • 26(12):3120–3129Qiangetal.•TauProtectsAxonalMicrotubulesfromSevering
RFL6 fibroblasts for these studies because they are very flat, and
hence excellent for imaging microtubules using immunofluores-
cence, and because MAP4 is the only traditional fibrous MAP
endogenously expressed. The levels of MAP4 in these cells, re-
vealed by immunostaining, are generally similar to the levels in
other fibroblastic cells used for microtubule-related studies over
the years (data not shown). No other traditional fibrous MAP is
used pEGFP-C1 (control construct) or pEGFP-C1-P60 (rat P60-
katanin) (Karabay et al., 2004). When P60-katanin was overex-
appeared as a dense array of long polymers extending from cell
center to cell periphery, but rather appeared as a scattering of
short polymers, generally only a few micrometers in length (Fig.
1B). There was also a marked diminution in polymer levels (to
Ab against rat P60-katanin indicated that the high expressers
generally expressed no more than 39% higher levels of P60-
katanin than control RFL6 fibroblasts. When the cells were
treated with Taxol (without the expression of P60-katanin) (Fig.
the microtubules formed dense bundles (Fig. 1D). Taxol treat-
0.05). However, the expression of P60-katanin (Fig. 1E) never-
theless caused the microtubules to break into short pieces (Fig.
1F), indicating that the stabilization and bundling of microtu-
bules per se does not protect them from katanin-induced sever-
ing. Polymer levels were down by 65.0% ( p ? 0.01) when P60-
katanin was overexpressed in Taxol-treated cells. Figure 4 shows
of studies in vitro showing that katanin effectively severs Taxol-
stabilized microtubules (Vale, 1991).
To test the capacity of MAPs to protect the microtubules against
severing, we first overexpressed MAP2c, MAP1b, or MAP4. The
DNA constructs used for these studies were: pEGFP-C1 (control
construct); pEGFP-C1-P60 (rat P60-katanin); pCMV/myc3b-
P60 (rat P60-katanin); pMAP2c (human MAP2c) (Gamblin et
al., 1996), pMAP1b (mouse MAP1b) (Gordon-Weeks and
Fischer, 2000); pEGFP-C1-MAP4 (human MAP4) (Chang et al.,
2001). To compare the results of the different MAPs against one
another, we achieved approximately equivalent levels of expres-
sion of all of the MAPs. As a baseline, we used MAP1b, because
the construct is from the mouse sequence and hence could be
compared more accurately than the other constructs against en-
dogenous levels in cultured rodent neurons. The transfection
endogenous levels in the cell body of cultured hippocampal neu-
achieved approximately the same expression levels of the other
MAP constructs by optimizing the conditions of the transfection
level achieved for MAP1b. With regard to P60-katanin, we se-
lected the most highly expressing cells for our analyses, because
they gave the most consistent results.
expression, shown in Figure 2, A and B, did not cause microtu-
bules to bundle, which was expected on the basis of previous
studies (Takemura et al., 1992). No significant changes were de-
tected in polymer levels in response to MAP1b ( p ? 0.05). As
shown in Figure 2, C and D, the microtubules were severed into
quantitative data. A potential concern with these studies was the
degree to which the ectopically expressed MAP1b actually asso-
ciates with microtubules, given the diffuse appearance of the flu-
orescence signal, and the lack of bundling induced by this MAP.
However, previous studies indicate that ectopically expressed
against disassembly (as assessed by nocodazole sensitivity) and
Qiangetal.•TauProtectsAxonalMicrotubulesfromSevering J.Neurosci.,March22,2006 • 26(12):3120–3129 • 3123
MAP1b associates with microtubules. We found this also to be
be particularly careful about this result, so we overexpressed the
MAP1b construct at levels several times the endogenous levels
found in neurons. Even at these very high levels, no evidence for
protection against P60-katanin-induced severed was observed.
Figure 2E–H shows the results with MAP2c. We chose to use
MAP2c rather than the larger adult MAP2 because the former is
present in developing axons (whereas the latter is more relevant
al., 1993). Figure 2, E and F, shows marked bundling of microtu-
bules by MAP2c, although there were also numerous unbundled
microtubules as well. Polymer levels were increased by 28.0%
( p ? 0.05) in the presence of MAP2c. Overexpression of P60-
katanin did not alter the length of the microtubules, their bun-
Unbundled microtubules remained long, indicating that it was
not the bundling per se that impeded severing. There was no
significant loss of microtubule mass from either group ( p ?
0.05). Figure 4 shows the quantitative data.
very different microtubule-binding domains. However, the
microtubule-binding domain of MAP2c is very similar to that of
tau and MAP4 (Seitz et al., 2002), suggesting that tau and MAP4
may be similar to MAP2c with regard to their capacity to protect
the microtubule against severing. Also shown in Figure 4 are the
quantitative data for results obtained with MAP4; no significant
els ( p ? 0.05). When P60-katanin was overexpressed with
As with the MAP2c studies, unbundled microtubules were pro-
tected against severing as well as bundled microtubules. Thus,
MAP4 provides protection against P60-katanin, but not to the
same degree as MAP2c.
tau constructs and examined their capacity to alter the response
of the fibroblasts to P60-katanin overexpression. For these stud-
ies, we used pEGFP-C1 (control construct); pEGFP-C1-P60 (rat
3R-tau (human three-repeat tau) (Lee and Rook, 1992); pEGFP-
C1-4R-tau (human four-repeat tau) (Lu and Kosik, 2001); pRc/
CMVflag123c (tauMTBD; C terminus of human tau with the
microtubule-binding domain but without the projection do-
nus of human tau without the microtubule-binding domain)
(Hall et al., 1997); and pRc/CMVflagFFT/6d (tauFFT/6d; a nat-
ural human tau isoform that cannot bind to microtubules) (Luo
et al., 2004).
is Tau4R. Figure 3, B and b, is Tau-MTBD. Figure 3, C and c, is
microtubule mass by 20.8% above control levels ( p ? 0.05);
coexpression with P60-katanin produced no diminution in mi-
crotubule mass ( p ? 0.05). Tau3R increased microtubule mass
by 5.1% above control levels ( p ? 0.05); coexpression with P60-
katanin reduced the microtubule mass by 25.5% ( p ? 0.05),
indicating that Tau3R protects the microtubules against P60-
katanin, but not as well as MAP2c or Tau4R. Tau-MTBD in-
creased microtubule mass by 9.2% above control levels ( p ?
0.05); coexpression with P60-katanin reduced the microtubule
mass by 37.2% ( p ? 0.05), indicating that the microtubule-
binding domain alone protects the microtubules against P60-
katanin, but not as well as MAP2c or Tau4R and not even as well
as Tau3R. The two tau isoforms that lacked the microtubule-
binding domain resulted in no increase in microtubule mass;
expression of P60-katanin diminished the microtubule mass by
58.6 and 66.6%, which were statistically indistinguishable from
the diminution obtained with cells overexpressing P60-katanin
but not expressing any tau constructs ( p ? 0.05). Thus, the
microtubule-binding domain is essential for the protection but
does not, within itself, provide as much protection as the intact
molecule. As with the MAP2c and MAP4 studies, unbundled
microtubules were protected against severing as well as bundled
microtubules. Quantitative data are shown in Figure 4.
katanin together with various MAP constructs. A, The quantitative data graphically on the
alone); MAP2c plus katanin (the cells expressing MAP2c and P60-katanin); MAP1b (MAP1b
plus P60-katanin (cells expressing tau4R and P60-katanin); tauMTBD (tauMTBD alone);
tauMTBD plus katanin (cells expressing tauMTBD and P60-katanin); tauMTNBD (tauMTNBD
alone); tauMTNBD plus katanin (cells expressing tauMTNBD and P60-katanin); tauFFT/6d
3124 • J.Neurosci.,March22,2006 • 26(12):3120–3129 Qiangetal.•TauProtectsAxonalMicrotubulesfromSevering
Over the years, a variety of different methods have been used to
early 1990s using antisense oligonucleotides to deplete MAPs
from cultured cerebellar macro-neurons, which are quite similar
to cultured hippocampal neurons. Both types of neurons un-
dergo a series of developmental stages in which the neuron first
extends a broad lamellipodium (stage I) that coalesces into sev-
eral essentially identical minor processes (stage II), after which
one of the minor processes elongates rapidly and becomes the
axon (stage III). The studies using antisense oligonucleotides
processes (Caceres et al., 1992), whereas tau and MAP1b are im-
eres et al., 1991; Brugg et al., 1993; DiTella
et al., 1996). In our studies, we used
siRNA, which was introduced by nucleo-
fection at the time of plating (He et al.,
2005). Tau and MAP1b were depleted by
using combinations of four siRNA se-
quences, whereas MAP2 was depleted us-
ing a sequence reported previously (see
Materials and Methods). After 2 d in cul-
ture, most of the MAP protein was de-
pleted (as assessed by quantitative immu-
nostains and Western blots) (Fig. 5), at
which time the neurons were triturated
and replated so that we could observe the
capacity of the neuron to differentiation
an axon after the MAP protein had been
depleted. The protein level of MAPs re-
termined to be 95–99% depleted by the
fifth total day (3 d after replating) (Fig.
5G). Unlike previous studies using anti-
sense oligonucleotides, we observed no
major phenotypic deficiencies in these
neurons as they underwent development
after depletion of tau, MAP2, or MAP1b.
Lamellipodia, minor processes, and axons
all developed and appeared morphologi-
cally normal. However, we did note that
the timetable for axonal differentiation
was slower in neurons depleted of MAP1b
(but not neurons depleted of tau or
MAP2) compared with control neurons,
with only 51% in stage III on the third day
after replating as opposed to 77% for con-
trols. In addition, axons were somewhat
compared with controls) or MAP2 (14%
shorter compared with controls) and
shorter yet in neurons depleted of MAP1b
(24% shorter compared with controls).
There was no difference in the lengths of
the minor processes with any of the MAP
depletions. Microtubule levels in the cell
bodies were down by 13% in neurons de-
pleted of MAP1b but were unchanged in
neurons depleted of tau or MAP2. These
data are shown in detail with statistical
analyses in Figure 6 and its legend. Inter-
estingly, in the axons of about one-third of the neurons depleted
distribution (but not total levels) of microtubules; microtubules
were denser in the distal half of the axon and sparser in the prox-
imal half, compared with controls.
These results add to those obtained from a variety of studies
using different techniques to knock out, knock down, or inhibit
MAP functions in neurons, some of which show dramatic phe-
notypes and some of which do not (Tint et al., 1998; Dawson et
al., 2001; Teng et al., 2001; Gonzalez-Billault et al., 2002; Harada
but it may have to do with compensatory mechanisms that are
the 1990s are most similar to ours but produced very different
the loss of MAP2, MAP1b, and tau in siRNA treated neurons are 95, 99, and 99%, respectively. G, Western blot of whole-cell
extracts probed with the MAP2, MAP1b, and tau Ab, confirming the protein-lowering effect. GAPDH was used as the internal
Qiangetal.•TauProtectsAxonalMicrotubulesfromSeveringJ.Neurosci.,March22,2006 • 26(12):3120–3129 • 3125
confidence in the siRNA method, which
has been shown to be more specific and
less problematic than the older antisense
approach (Rossi and Eckstein, 2003).
We next performed an experiment in
which P60-katanin was overexpressed for
12 h in these experimental neurons on the
2005), in which the levels of P60 were ele-
vated ?39% by the end of the 12 h time
frame of expression. We performed this
experiment in neurons treated with con-
trol siRNA (Fig. 7A,B,a,b) or siRNA for
MAP1b (Fig. 7C,D,c,d), MAP2 (data not
shown), or tau (Fig. 7E,F,e,f). (In Fig. 7,
panels with capital letters are the original
panels with small letters show pseudocol-
ored regions of axons to better illustrate
fluorescence intensity.) As reported previ-
ously, the microtubule levels were sub-
stantially reduced in the cell bodies (36%
diminution) and minor processes (65%
diminution) but not in the axons of the
pression. The same result was obtained in
the case of neurons depleted of MAP1b
and MAP2. However, in the case of neu-
rons depleted of tau, the overexpression of P60-katanin resulted
in a substantial loss of microtubule mass from the axon (63%
diminution). These data, which are shown quantitatively in Fig-
ure 7G, indicate a unique role for tau in the protection of axonal
microtubules against severing by katanin.
MAPs with axonal microtubules modulates the capacity of ka-
tanin to sever them (Baas and Qiang, 2005). According to this
model, dissociation of the MAP from the microtubule, presum-
ably by local phosphorylation of the MAP, allows katanin to ac-
cess the microtubule lattice. This model is appealing because it
can explain how the neuron maintains a population of very long
regulated severing events. For example, the local phosphoryla-
tion of the MAP within a discrete region of the axon could result
in the formation of a collateral branch by allowing microtubules
to be locally severed in that region (Baas and Qiang, 2005). Our
(Brugg et al., 1993; Meichsner et al., 1993; Mandell and Banker,
cells and some types of neurons (Chapin et al., 1995).
tion of the microtubules against severing by overexpressed P60-
katanin. All three of these MAPs share a similar microtubule-
binding domain (Chapin and Bulinski, 1991; Dehmelt and
Halpain, 2004). The level of protection relates to the binding
four-repeat form of tau protect somewhat better and have stron-
ger binding affinities to the microtubule than the three-repeat
form of tau (Seitz et al., 2002). We found that the microtubule-
binding domain of tau provides some protection against micro-
molecule. However, without the binding domain, the rest of the
molecule provides no protection whatsoever. Thus, the likely
mechanism by which MAPs regulate microtubule severing is to
physically restrict access of the severing proteins to the microtu-
bule rather than interacting directly with the severing proteins.
We speculate that the projection domain provides additional
steric hindrance of the access of katanin to the microtubule.
In contrast with the tau/MAP2/MAP4 family of MAPs, we
found that MAP1b provides no protection against katanin-
induced microtubule severing, nor does Taxol. Both of these
molecules stabilize the microtubules against disassembly. Taxol
bundles microtubules but MAP1b does not. We do not believe
tion phenomenon because Taxol-induced bundles are very sen-
sitive to severing and because the unbundled microtubules in
cells overexpressing tau, MAP2, or MAP4 were also protected
against severing. In contrast with the other expressed MAPs,
MAP1b did not cause microtubules to bundle and displayed a
more diffuse nonfilamentous appearance, suggesting that not all
of the protein associated with the microtubules. This may be
because of the phosphorylation state of the expressed protein
reason, we also examined cells that were overexpressing several-
tion against microtubule severing (although the microtubules
group and the groups depleted of MAP2 or tau. In contrast, depleting MAP1b delayed their development ( p ? 0.01). B, The
3126 • J.Neurosci.,March22,2006 • 26(12):3120–3129Qiangetal.•TauProtectsAxonalMicrotubulesfromSevering
were stabilized), providing support for the conclusion that
MAP1b is not a likely candidate for protecting axonal microtu-
bules from severing.
These observations suggest that the protection phenomenon
may be specific to the tau/MAP2/MAP4 family of related MAPs.
Neurons express a variety of tau and MAP2 isoforms (Dehmelt
and Halpain, 2004; Luo et al., 2004), and it has been known for
many years that neurons compartmentalize many of these iso-
forms (Peng et al., 1986; Ferreira et al., 1987). Recent work sug-
gests that overexpression of these MAPs can attenuate the inter-
action of molecular motor proteins with the microtubule lattice
bules are so much more resistant to ka-
tanin than microtubules in any other
compartment of the neuron may relate to
the levels of the particular MAP in the
axon versus other neuronal compart-
ments, the phosphorylation state of the
some aspect of the protection not revealed
by the RFL6 studies. In addition, we
should note that neurons express a variety
of other proteins that can bind microtu-
bules, such as doublecortin (Francis et al.,
1999) and stable-tubule-only-polypeptide
(Slaughter and Black, 2003), and we have
not exhaustively determined whether
these various proteins might be able to af-
ford protection against katanin. Instead,
we decided to shift our attention to bona
crotubules against severing.
For these studies on primary neurons,
we used a contemporary siRNA approach
tured hippocampal neurons, after which
we replated the neurons to allow them to
extend processes after depleting the MAP.
ative to controls. P60-katanin was then
overexpressed for 12 h to evaluate for po-
tential changes in the sensitivity of the mi-
crotubules to severing. We were initially
one of these MAPs, were fully capable of
extending lamellipodia, minor processes,
and an axon. The time table of differentia-
tion was noticeably slower after depletion
of MAP1b, with fewer cells differentiating
axons by the third day after replating. The
time table of differentiation was unaf-
fected by depletion of tau or MAP2. The
length of the axon on day 3 was ?10%
?21% shorter after depletion of MAP1b.
The only other differences we detected,
compared with controls, was that the mi-
crotubule levels were diminished by 20%
in the cell bodies (but not axons or minor
and the microtubules were more unevenly distributed in their
axons, with a higher density more distally. These results indicate
normally in the absence of any one of these MAPs, at least to the
point of axonal differentiation.
As reported previously (Yu et al., 2005), overexpression of
from the cell body and minor processes of control neurons, but
result was obtained. However, after depletion of tau, the loss of
microtubule mass from the axon was equally dramatic as any-
where else in the neuron, indicating that tau is indeed the factor
of red, orange, and yellow indicating intermediate levels. G, The quantification of microtubule mass in cell bodies, in minor
siRNA-treated groups leads to a 33–40% diminution in microtubule mass from cell bodies ( p ? 0.01). Overexpression of
P60-katanin, leading to a 63% diminution ( p ? 0.001). The microtubules in axons of the other three groups showed no
Qiangetal.•TauProtectsAxonalMicrotubulesfromSeveringJ.Neurosci.,March22,2006 • 26(12):3120–3129 • 3127
in the axon that is responsible for rendering the microtubules
more resistant to katanin. We do not think that tau is simply
stabilizing the microtubules against disassembly after severing,
because depletion of tau does not, within itself, diminish the
microtubule mass, nor does it render axonal microtubules any
more sensitive to depolymerization by drugs such as nocodazole
(Tint et al., 1998).
The fact that 12 h of P60-katanin overexpression after tau
depletion does not entirely eliminate all microtubules from the
cell body or minor processes (or dendrites) (Yu et al., 2005) sug-
MAP2, the isoforms of which are widespread in developing neu-
rons, although not the chief protector, could presumably afford
some protection, and as noted above, there may be other
microtubule-binding proteins with the capacity to protect
the only protector in the axon is supported by the fact that there
were no obvious changes in the axonal microtubules, at least at
experimental increase in P60-katanin levels above the endoge-
depleted during siRNA treatment is sufficient to provide some
degree of protection in our experimental regimen, or other mol-
ecules contribute to the protection.
The axon is endowed with very long microtubules, which are
presumably important for the axon to obtain such great lengths.
also serve to offset potential retraction of the axon (Baas and
Ahmad, 2001). Our studies suggest that tau is important for en-
suring that katanin does not have unfettered access to the micro-
tubules and is thereby critical for the maintenance of the axonal
to spastin, another microtubule-severing protein present in the
neuron (Errico et al., 2002; Evans et al., 2005; Roll-Mecak and
Vale, 2005). These ideas are particularly exciting in light of the
the gradual dissociation of tau from the microtubules (Stoothoff
and Johnson, 2005). We propose that the sustained loss of tau
to endogenous severing proteins, thus causing the microtubule
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