The Rockefeller University Press $30.00
J. Cell Biol. Vol. 186 No. 3 363–369
Correspondence to James M. Ervasti: firstname.lastname@example.org
Abbreviations used in this paper: CH, calponin homology; MAP, MT-associated
protein; MOM, mouse on mouse; MT, microtubule; wt, wild type.
The plakins are a class of giant cytolinker proteins that can link
transmembrane protein complexes to the actin, intermediate fila
ment, and microtubule (MT) cytoskeletons in various combi
nations (Fuchs and Karakesisoglou, 2001). Plakins can bind
actin filaments via tandem calponin homology (CH) domains,
intermediate filaments via plakin repeat domains, and MTs
through either a Gas2related domain or a glycine/serine/
arginine domain (for review see Leung et al., 2002). The ability
to crosslink multiple components of the cellular cytoskeleton
allows cytolinkers to stabilize cells from mechanically induced
damage. Mouse knockout studies exemplify the stabilizing
effects of cytolinkers, as loss of the cytolinker plectin resulted
in skin blistering and a form of muscular dystrophy (Andra
et al., 1997), and ablation of BPAG1, another cytolinker, caused
skin blistering upon mechanical stimulation (Guo et al., 1995).
Dystrophin, the protein absent in patients with Duchenne
muscular dystrophy (Hoffman et al., 1987), shows structural and
functional similarities to cytolinkers, which suggests the hypoth
esis that dystrophin performs a cytolinker role in muscle. Dystro
phin’s large molecular mass of 427 kD, spectrinlike repeats, and
ability to bind actin filaments via a tandem CH domain (Way
et al., 1992) highlight three similarities with cytolinkers. Although
dystrophin lacks a plakin repeat domain, dystrophin–intermediate
filament interactions have been documented (Stone et al., 2005;
Bhosle et al., 2006). Thus, the ability to link the actin and inter
mediate filament cytoskeletons to the transmembrane dystro
glycan complex (Suzuki et al., 1992) illustrates how dystrophin
functions similarly to other cytolinkers. Finally, the muscle
membrane fragility associated with the loss of dystrophin (Petrof
et al., 1993) parallels the structural deficiencies observed in other
cytolinkerdeficient tissues, further demonstrating a close rela
tionship between dystrophin and other cytolinkers. Collectively,
these data support the hypothesis that dystrophin may function
as a cytolinker in skeletal muscle.
Although dystrophin exhibits many characteristics of a
cytolinker, a direct dystrophin–MT interaction has not been doc
umented. Dystrophin lacks either a Gas2related or a glycine/
serine/arginine domain, but recent studies indicated that dystro
phin at least indirectly influences MT organization or stability
(Percival et al., 2007; Ayalon et al., 2008). For instance, the
dystrophindeficient mdx mouse exhibited MT disorganization
in skeletal muscle with the costameric MTs most severely affected
(Percival et al., 2007). Dystrophin’s enrichment at costameres
plexes. Dystrophin is functionally similar to cytolinkers, as
it links the multiple components of the cellular cytoskeleton
to the transmembrane dystroglycan complex. Although no
direct link between dystrophin and MTs has been docu-
mented, costamere-associated MTs are disrupted when
dystrophin is absent. Using tissue-based cosedimenta-
tion assays on mice expressing endogenous dystrophin
or truncated transgene products, we find that constructs
ytolinkers are giant proteins that can stabilize cells
by linking actin filaments, intermediate filaments,
and microtubules (MTs) to transmembrane com-
harboring spectrinlike repeat 24 through the first third of
the WW domain cosediment with MTs. Purified Dp260, a
truncated isoform of dystrophin, bound MTs with a Kd
of 0.66 µM, a stoichiometry of 1 Dp260/1.4 tubulin
heterodimer at saturation, and stabilizes MTs from
cold-induced depolymerization. Finally, - and -tubulin
expression is increased 2.5-fold in mdx skeletal muscle
without altering the tubulin–MT equilibrium. Collectively,
these data suggest dystrophin directly organizes and/or
stabilizes costameric MTs and classifies dystrophin as a
cytolinker in skeletal muscle.
Dystrophin is a microtubule-associated protein
Kurt W. Prins,1 Jill L. Humston,2 Amisha Mehta,3 Victoria Tate,3 Evelyn Ralston,3 and James M. Ervasti1,2
1Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455
2Molecular and Cellular Pharmacology Training Program, University of Wisconsin, Madison, WI 53706
3Light Imaging Section, Office of Science and Technology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda,
© 2009 Prins et al. This article is distributed under the terms of an Attribution–
Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publica-
tion date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a
Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license,
as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
T H E J O U R N A L O F C E L L B I O L O G Y
JCB • VOLUME 186 • NUMBER 3 • 2009 364
and the restoration of costameric MT organization through
virally mediated expression of a microdystrophin (Percival et al.,
2007) indicates that dystrophin is necessary for proper costameric
MT organization in skeletal muscle. Moreover, acute knockdown
of ankyrinB, a protein necessary for delivery of dystrophin to the
sarcolemma and neuromuscular junction, caused the loss of costa
meric MTs and aberrant MT organization in a subset of MTs
underlying the neuromuscular junction (Ayalon et al., 2008).
In this study, we investigated the hypothesis that dystro
phin directly interacts with costameric MTs. We confirmed that
costameric MTs were disrupted in dystrophindeficient skeletal
muscle and showed endogenous dystrophin cosedimented with
MTs in tissue homogenates. Using purified proteins, we found
that the carboxylterminal two thirds of dystrophin bound MTs
with a Kd of 0.66 µM and stabilized MTs from coldinduced de
polymerization. Finally, we documented a 2.5fold increased
expression of and tubulin without alteration in the tubulin–
MT equilibrium in mdx skeletal muscle. These results demon
strate that dystrophin is a MTassociated protein (MAP) that
stabilizes costameric MTs and functions as a costameric cyto
linker in skeletal muscle.
Results and discussion
To determine whether dystrophin and MTs localize to similar
structures in skeletal muscle, we conducted immunofluorescence
analysis on teased extensor digitorum longus muscle fibers co
labeled with antidystrophin and anti–tubulin antibodies (Fig. 1,
A and B). Dystrophin forms a subsarcolemmal network with
transverse components along the I bands and the M line and with
longitudinal components (Williams and Bloch, 1999), whereas
MTs form a subsarcolemmal lattice, which in fast fibers, has
transverse and longitudinal components plus an accumulation of
MTs around myonuclei (Ralston et al., 1999). We found that the
transverse MTs (Fig. 1 A, arrowheads) weave their course along
the I band dystrophin staining for long distances. MTs were also
associated with longitudinal lines of dystrophin (Fig. 1 A, ar
rows). These data identify domains of the subsarcolemmal cyto
skeleton where dystrophin and MTs may interact either directly
or indirectly. Next, we examined MT organization in mouse
models lacking dystrophin (mdx), dystrophin’s autosomal homo
logue utrophin (utrn/), or both dystrophin and utrophin
(mdx/utrn/). Consistent with previous results (Percival et al.,
2007), loss of dystrophin resulted in MT disorganization with the
costameric MTs appearing to be most severely affected (Fig. 1 C)
when compared with wild type (wt; Fig. 1 C). Ablation of utro
phin had no effect on MT organization (Fig. 1 C), which is likely
a result of its very low expression (Rybakova et al., 2002) and
restriction to the neuromuscular junction (Ohlendieck et al.,
1991). Finally, mdx/utrn/ skeletal muscle exhibited MT dis
organization comparable with that of mdx (Fig. 1 C). MT organi
zation in 24dold prenecrotic mdx skeletal muscle fibers was also
disorganized, whereas agematched wt mice displayed a MT lat
tice nearly identical to mature wt mice (Fig. 1 D). Collectively,
these results confirm a role for dystrophin in the stabilization and
proper organization of costameric MTs independent of muscle
necrosis and regeneration.
Figure 1. Dystrophin guides MTs at the surface of the muscle fibers and is
necessary for proper MT organization. (A) Isolated muscle fibers from the
extensor digitorum longus of 7-wk-old wt mice were costained for dystro-
phin (left) and -tubulin (middle). The right panel shows that MTs (red) follow
dystrophin (green) bands for long distances both transversely (arrowheads)
and longitudinally (arrows). (B) At a higher magnification, dystrophin stain-
ing is granular; MTs are studded with dystrophin “dots.” Arrows indicate
longitudinal MTs that follow dystrophin. (C) Muscle fibers from the extensor
digitorum longus of 7-wk-old wt, mdx, utrn/, and mdx/utrn/ mice were
stained with DM1A anti–-tubulin and Hoechst dye. Both wt and utrn/
fibers show the lattice of transverse and longitudinal MTs characteristic of
fast fibers (arrowheads). In mdx and mdx/utrn/ fibers, the regularity of
the lattice is lost, and mostly oblique MTs originate from cytoplasmic nucle-
ation points (arrows). (D) Peripherally nucleated prenecrotic muscle fibers
from 24-d-old mdx mice also displayed MT disorganization, indicating that
MT derangement occurred before muscle cell necrosis and regeneration.
Bars: (A and B) 10 µm; (C and D) 20 µm.
DYSTROPHIN BINDS MICROTUBULES • Prins et al.
rod domain absent from Dp260. A small amount of Dp260 pel
leted in the absence of MTs, but substantially more Dp260 shifted
to the pellet fraction when MTs were present (Fig. 3 A). After sub
tracting selfpelleting Dp260, Dp260 displayed a concentration
dependent and saturable cosedimentation with a Dp260/
tubulin heterodimer stoichiometry of 1:1.4 and a Kd of 0.66 µM
(Fig. 3 C). As predicted, DysNTermR10 did not cosediment with
MTs up to concentrations approaching 10 µM (Fig. 3, B and C).
Next, we assessed how the presence of 1 µM Dp260 affected
the tubulin–MT equilibrium in vitro. Dp260 had no significant
effect on the fraction of tubulin in the MT fraction when incu
bated at room temperature (67.3 ± 0.72% vs. 68.6 ± 1.3%).
However, the presence of Dp260 significantly increased the
fraction of tubulin retained in the MT pellet (33.6 ± 2.9% vs.
42.2 ± 2.0%) when MTs were induced to depolymerize by incu
bating at 4°C. (Fig. 3, D and E). Collectively, these results dem
onstrate that dystrophin directly binds and stabilizes MTs from
Because misregulation of other MAPs can alter tubulin
expression and MT stability (Harada et al., 1994; Takahashi
et al., 2003), we investigated how the loss of dystrophin affects
the regulation of tubulin expression and the tubulin–MT equi
librium in skeletal muscle fibers. Tubulin levels in wt and mdx
skeletal muscle extracts were examined by quantitative West
ern blot analysis. With mAb B512, we observed no difference
Next, we performed a tissuebased MT cosedimentation
assay (Fig. 2 A) to determine whether dystrophin cosedimented
with MTs. Under conditions that induced MT depolymeriza
tion, virtually no muscle protein pelleted (Fig. 2 B). However,
numerous proteins pelleted under MTstabilizing conditions,
and this fraction of proteins represents MTs and the MAPs of
skeletal muscle (Fig. 2 B). We Western blotted each fraction
obtained from the tissue cosedimentation assay performed on
wt mice expressing fulllength dystrophin or transgenic mdx
mice expressing Dp260, microdystrophin (R423), or Dp71
(Fig. 2 C, right). Fulllength dystrophin, Dp260, and R423
all pelleted with MTs, whereas Dp 71 did not (Fig. 2 C, left).
By comparing the dystrophin domains present or absent in each
construct (Fig. 2 C, right) along with each construct’s ability
to cosediment with MTs, we suggest that spectrinlike repeat
24 through the first third of the WW domain encodes a novel
To test for a direct interaction between dystrophin and MTs,
we performed MT cosedimentation using two purified recombi
nant dystrophin constructs and purified tubulin. The two recombi
nant constructs used were Dp260, which encodes from spectrinlike
repeat 10 through the carboxy terminus of dystrophin, including
the proposed MTbinding domain, and DysNTermR10 (Rybakova
et al., 2006), which encodes the aminoterminal, tandem CH
actinbinding domain and spectrinlike repeats 1–10 of the middle
Figure 2. Dystrophin cosediments with MTs
in skeletal muscle extracts. (A) Flowchart of
tissue MT cosedimentation assay. (B) Coomassie
blue–stained SDS-PAGE showing supernatant
(S) and pellet (P) fractions in conditions that
favored MT depolymerization or polymeriza-
tion. The pellet fraction in the presence of MTs
represents the MAP fraction of skeletal muscle.
The molecular mass standards (given in kilo-
daltons) are indicated on the left. (C, left)
Western blot analysis of tissue cosedimenta-
tion assay from skeletal muscle extracts of
wt mice expressing dystrophin or mdx mice
transgenically expressing Dp260, R4-R23, and
Dp71. (right) Diagrammatic representation
of constructs analyzed in tissue cosedimenta-
tion. ABD, actin-binding domain; H, hinge
region; W, WW domain; CR, cysteine-rich
domain; CT, carboxy-terminal domain; MT BD,
JCB • VOLUME 186 • NUMBER 3 • 2009 366
in tubulin expression in mdx skeletal muscle (Fig. 4, A and B).
Because levels of and tubulin are coregulated (Gonzalez
Garay and Cabral, 1995), we investigated tubulin levels to
determine whether tubulin is upregulated in mdx skeletal
muscle. Tubulin expression was elevated 2.5fold in mdx
skeletal muscle (Fig. 4, A and B), suggesting that expression of
both and tubulin is increased in mdx skeletal muscle.
Thus, we conclude that mAb DM1A is able to recognize a pop
ulation of tubulin not detected by mAb B512. To examine
MT stability in mdx skeletal muscle, we analyzed levels of
tyrosinated tubulin, a marker of dynamic MTs (Gundersen
et al., 1984, 1987), and acetylated tubulin, a marker of long
lived MTs (Bulinski and Gundersen, 1991). The levels of tyro
sinated tubulin were increased 2.5fold in mdx extracts
(Fig. 4, A and B), whereas the levels of acetylated tubulin
were not (Fig. 4, A and B). The loss of dystrophin’s MTstabilizing
ability may explain why acetylated tubulin was not more
abundant in mdx skeletal muscle extracts, but alterations in the
tubulin–MT equilibrium could also explain the lack of more
stable MTs. Therefore, we examined the tubulin–MT equilib
rium in wt and mdx skeletal muscles and found that the loss of
dystrophin did not affect the equilibrium (Fig. 4, C and D).
Collectively, these results show that tubulins are misregulated
in dystrophindeficient skeletal muscle without affecting the
tubulin–MT equilibrium. The loss of dystrophin’s MTstabilizing
ability likely explains why there are not more stabilized MTs even
in the presence of more tubulin dimer in dystrophindeficient
An indirect link between dystrophin and MTs mediated
by ankyrinB was recently shown to be important for proper
trafficking of dystrophin and dystroglycan to the sarcolemma
(Ayalon et al., 2008). However, costameric MTs are disorga
nized in mdx skeletal muscle even in the presence of properly
localized ankyrinB (Ayalon et al., 2008). Because the MT and
ankyrinB–binding domains of dystrophin do not overlap (Fig. 5),
our results and previous results suggest that dystrophin interacts
with MTs in vivo through two distinct mechanisms. We propose
that ankyrinB delivers dystrophin to the sarcolemma depen
dent on MTs and that dystrophin and ankyrinB collaborate to
stabilize and organize MTs in skeletal muscle.
As with other cytolinkers, the ability to bind multiple com
ponents of the filamentous cytoskeleton likely allows dystrophin
to protect the sarcolemma from mechanically induced damage.
One highly truncated microdystrophin construct (R423) is
very effective in restoring function in the dystrophindeficient
mdx mouse (Harper et al., 2002). Interestingly, the R423 micro
dystrophin contains all sequences required for interaction with
the three cytoskeletal filament systems: the aminoterminal tan
dem CH domain, which binds actin (Way et al., 1992) and cyto
keratin filaments (Stone et al., 2005), the spectrinlike repeat
3 and the cysteinerich regions, which are necessary for synemin
intermediate filament binding (Bhosle et al., 2006), and the
MTbinding domain. In contrast, Dp260 lacks the cytokeratin
filament–binding domain and portions of the synemin and actin
binding domains, which likely alters the binding affinities to both
actin and synemin filaments and may explain why transgenic
overexpression of Dp260 only partially alleviates the mdx
in tubulin expression between wt and mdx skeletal muscle
extracts (Fig. 4, A and B), which was consistent with what we
(Prins et al., 2008) and others (Barton et al., 2002) reported
previously. However, mAb DM1A showed an 2.5fold increase
Figure 3. Dp260 binds and stabilizes MTs in vitro. (A) Coomassie blue–
stained SDS-PAGE showing the supernatant (S) and pellet (P) fractions of
purified Dp260 in the presence and absence of MTs. Dp260 shifted to the
pellet fraction when MTs were present, indicating that Dp260 binds MTs.
(B) Coomassie blue–stained SDS-PAGE showing the supernatant and pellet
fractions of DysN-R10 in the presence and absence of MTs. The presence
of MTs did not cause a shift of DysN-R10 into the pellet fraction, indicating
that DysN-R10 does not bind MTs. (C) Concentration-dependent binding
of Dp260 to taxol-stabilized MTs (2 µM tubulin) from three independent
experiments. DysN-R10 did not show significant MT-binding activity.
(D) Coomassie blue–stained SDS-PAGE of the supernatant and pellet frac-
tions of MTs induced to depolymerize by incubating at 4°C in the presence
or absence of 1 µM Dp260. (E) Quantification of tubulin in the MT fraction
when induced to depolymerize by incubating at 4°C (n = 6). The presence
of 1 µM Dp260 significantly (t test; *, P ≤ 0.05) increased the amount of
tubulin in the MT fraction, indicating that Dp260 stabilizes MTs. Error bars
represent mean ± SEM. (A, B, and D) Molecular mass standards (given in
kilodaltons) are indicated on the left.
367 DYSTROPHIN BINDS MICROTUBULES • Prins et al.
pathophysiology. For example, disorganized MTs are also asso
ciated with Golgi mislocalization (Percival et al., 2007), which
in combination, would likely lead to impaired trafficking of
membranebound proteins and may explain the decreased levels
of dystroglycan and the sarcoglycans at the sarcolemma of mdx
skeletal muscle (Ohlendieck and Campbell, 1991). Because no
MT knockout mouse has been generated, the exact function of
MTs in skeletal muscle remains unknown. However, the impor
tance of MTs in skeletal muscle biology is illustrated by the muscle
weakness and increased levels of serum creatine kinase associ
ated with colchicine toxicity in human patients (Boomershine,
2002; Caglar et al., 2003; Wilbur and Makowsky, 2004; Altman
et al., 2007). Therefore, it is possible that derangement of the
MT cytoskeleton contributes to some of the phenotypes associ
ated with dystrophin deficiency.
Materials and methods
Control C57BL/6 and mdx mice were initially obtained from The Jackson
Laboratory. The utrn/ and mdx/utrn/ mice were provided by D. Lowe
(University of Minnesota, Minneapolis, MN). Mdx mice transgenically ex-
pressing Dp260 and R4-R23 were provided by J. Chamberlain (Univer-
sity of Washington, Seattle, WA), and the Dp71 line was provided by
J. Rafael-Fortney (Ohio State University, Columbus, OH). All animals were
housed and treated following guidelines set by the University of Minnesota
Institutional Animal Care and Use Committee.
The mAbs to -tubulin (B512), -tubulin (D66), tyrosinated tubulin (TUB 1-A2),
and acetylated tubulin (6-11B-1) were purchased from Sigma-Aldrich. The mAb
to -tubulin (DM1A) was purchased from Abcam. The mAb to dystrophin (Dys2)
was purchased from Novacastra. The polyclonal antibody to dystrophin (Rb2)
was described previously (Rybakova et al., 1996). Infrared dye–conjugated
anti–mouse secondary antibodies were purchased from LI-COR Biosciences.
phenotype (Warner et al., 2002). Because Dp116 harbors only the
MTbinding domain and one of two syneminbinding sequences,
an inability to bind cytokeratin filaments and actin filaments likely
explains why transgenic overexpression of Dp116 fails to rescue
the mdx phenotype (Judge et al., 2006). Finally, the mild muscle
phenotypes of cytoactin (Sonnemann et al., 2006) or keratin 19
knockout mice (Stone et al., 2007) may be explained by dystro
phin’s linkage with the remaining components of the cortical cyto
skeleton. Collectively, these results support the hypothesis that
dystrophin must bind all three components of the cellular cyto
skeleton to function properly in skeletal muscle.
Although the dystrophin–MT interaction fits well with the
structural/organization functions previously ascribed to dystro
phin, the importance of MTs in trafficking of proteins, vesicles,
organelles, and mRNAs (for review see Hirokawa and Noda,
2008; Gennerich and Vale, 2009) also suggests how MT disrup
tion in mdx skeletal muscle could contribute to the dystrophic
Figure 4. Mdx mice exhibit tubulin misregulation in the
absence of an altered tubulin–MT equilibrium. (A) Repre-
sentative Western blots of tubulin levels in skeletal muscle
extracts from wt and mdx mice. (B) Quantification of tubu-
lin levels from three wt and three mdx extracts. (C) Repre-
sentative Western blots of tubulin (tub) and MT fractions
of skeletal muscle extracts from wt and mdx mice using
mAb DM1A. (D) Quantification of tubulin–MT equilibrium
from five wt and four mdx tibialis anterior muscles. Loss
of dystrophin does not affect the tubulin–MT equilibrium.
Error bars represent mean ± SEM.
Figure 5. Diagrammatic representation of MT- and ankyrin-B–binding
domains of dystrophin. Numbers indicate amino acids of full-length dys-
trophin. H, hinge region; W, WW domain; CR, cysteine-rich domain;
CT, carboxy-terminal domain; MT BD, MT-binding domain; Ank-B BD,
JCB • VOLUME 186 • NUMBER 3 • 2009 368
30 min at 4°C. The supernatant and pellet fractions were prepared and
quantified as described in the previous paragraph.
Western blot analysis and quantification
Western blot analysis and quantification from three wt and three mdx skel-
etal muscle extracts were performed as described previously (Prins et al.,
2008). In brief, 25 µg of skeletal muscle extract was subjected to SDS-
PAGE and transferred to nitrocellulose membranes, which were washed/
blocked in a 5% milk solution in PBS for 1 h. The membranes were incu-
bated overnight with primary antibody at room temperature. The primary
antibodies and dilutions used were mAb Dys2 (1:50), mAb B512 (1:250),
mAb DM1A (1:250; Sigma-Aldrich), mAb D66 (1:100), and mAb 6-11B-1
(1:100). Membranes were washed two times for 10 min in 5% milk solu-
tion at room temperature, incubated with infrared dye–conjugated second-
ary antibody (1:10,000) for 30 min at room temperature, and the
membranes were washed in a 0.5% Tween solution in PBS two times for
10 min. Western blots were imaged and quantified with an infrared imag-
ing system (Odyssey; LI-COR Biosciences). The Coomassie blue–stained
posttransfer gel was analyzed densitometrically using UVP software and
served as the loading control.
In vivo tubulin–MT equilibrium assay
The tibialis anterior was dissected, immediately placed in 1 ml of MT stabi-
lization buffer (1% Triton X-100, 50% glycerol, 5% DMSO, 10 mM
Na2HPO4, 0.5 mM EGTA, and 0.5 mM MgSO4), and homogenized with
10 strokes in a homogenizer. The resulting homogenate was centrifuged at
100,000 g for 30 min at 25°C. The soluble portion (tubulin containing)
was saved for analysis, whereas the pelleted portion (MT fraction) was re-
suspended in 1 mL of 1% SDS buffer then boiled for 10 min. The pellet
fraction was centrifuged at 13,000 g for 10 min, and the soluble portion
was saved for analysis. 25 µl of the tubulin and MT fraction was analyzed
and quantified via Western blot using mAb DM1A on the infrared imaging
All data are presented as mean ± SEM. Comparison between groups was
performed using a t test with significance defined as P ≤ 0.05.
We would like to thank Dr. Kevin Sonnemann for generating the Dp260
baculovirus expression construct and Dr. Sonnemann and Thomas Cheever for
their helpful discussions.
This work was supported by the National Institutes of Health Training
Program in Muscle Research (AR007612), a grant from the National Institutes
of Health (AR042423), and Gregory Marzolf Muscular Dystrophy Fellow-
ships to J.L. Humston and K.W. Prins. K.W. Prins is a member of the Medical
Scientist Training Program at the University of Minnesota. E. Ralston, V. Tate,
and A. Mehta were supported by the National Institutes of Health Intramural
Submitted: 11 May 2009
Accepted: 6 July 2009
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To analyze the MT lattice in dystrophic animal models, hindlegs of wt
(3 and 8 wk), mdx (3, 5, and 8 wk), utrn/ (8–10 wk), and mdx:utrn/
(3, 5, and 8 wk) mice were skinned, cut as close as possible to the body,
and fixed at room temperature for 2 h with 4% para-formaldehyde in phos-
phate buffer. They were stored in phosphate buffer until the extensor digito-
rum longus muscle was dissected and separated with fine forceps into
mostly single fibers. These were transferred to a 24-well tissue culture plate
and incubated with mouse on mouse (MOM)–blocking buffer (Vector Labo-
ratories) for 2 h at room temperature. Blocking buffer and every subse-
quent buffer for incubation or washing contained 0.04% saponin for
permeabilization and 0.05% sodium azide. Fibers were incubated over-
night with mouse antitubulin (DM1A, 1:500; or B512, 1:4,000) in MOM
diluent, washed three times for 20 min, and stained with 1:500 dilutions
of Alexa Fluor 488 anti–mouse and Alexa Fluor 568 anti–rabbit secondary
antibodies (Invitrogen) in MOM diluent for 2 h at room temperature. After
three 20-min washes, one of which contained the nuclear stain Hoechst
33342 (Sigma-Aldrich) at 2 µg/ml, fibers were mounted onto a glass slide
in a drop of Vectashield (Vector Laboratories). Confocal images were cap-
tured with a 63× NA 1.4 oil immersion lens on a TCS SP5 confocal micro-
scope (Leica) in the Light Imaging Section of the National Institute of
Arthritis and Musculoskeletal and Skin Diseases. Gain and laser power set-
tings were adjusted to avoid saturation and use the whole linear range of
fluorescence intensity. Unless specified, the parameters were adjusted for
each new fiber imaged. The raw TIF images were transferred to a com-
puter (Macintosh G5; Apple), opened in Photoshop (CS2; Adobe), assem-
bled into montages, and adjusted for brightness when needed. The final
illustrations give a faithful representation of the collected images.
Tissue MT cosedimentation assay
Tissue-based cosedimentation was performed as described previously
(Hughes et al., 2008) with the following exception. The starting material
was 200 mg of frozen skeletal muscle that was pulverized in a mortar and
pestle cooled with liquid nitrogen then added to MT buffer (1% Triton X-100,
50 mM Hepes, 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 0.75 mM benz-
amidine, 0.1 mM PMSF, 0.6 µg/ml pepstatin A, 0.5 µg/ml aprotinin,
0.5 µg/ml leupeptin, iodoacetamide, and E64). The extracts were incu-
bated for 1 h at 4°C and centrifuged at 100,000 g for 40 min at 25°C. 1 mM
of both GTP and DTT was added to the soluble fraction of the extract and
incubated at 37°C for 5 min. The extract was split into two fractions, one
that was incubated on ice for 15 min, and 20 µM taxol was added to the
other and incubated at 37°C for 15 min. 300 µl of each fraction was lay-
ered onto a cushion buffer (MT buffer plus 40% sucrose) and centrifuged
at 100,000 g for 30 min at 25°C. The supernatant was removed, and the
pellet fraction was resuspended in a Laemmli sample buffer.
A cDNA-encoding Flag-tagged Dp260 (Warner et al., 2002) provided by
J. Chamberlain was cloned into pFASTbac1 to generate a recombinant
baculovirus expression vector using previously described methods (Rybakova
et al., 2002). Dp260 and dystrophin Nterm-R10 were expressed and purified
using the baculovirus expression system and anti-Flag M2 affinity chroma-
tography, respectively, as previously described (Rybakova et al., 2002),
except the proteins were dialyzed into MT buffer without Triton X-100. Pro-
tein concentration was determined using A280 using Nanodrop software
with an extinction coefficient of 272,495 M1 cm1 for Dp260 and
221,115 M1 cm1 for DysN-R10 as predicted by the Expert Protein Analy-
sis System proteomics server (Swiss Institute of Bioinformatics).
MT cosedimentation analysis
MT cosedimentation assay was performed as described by the manufactur-
er’s instructions (Cytoskeleton, Inc). In brief, increasing amounts of purified
protein were added to preformed MTs then centrifuged at 100,000 g for
30 min. The amount of free and bound protein was determined densito-
metrically from Coomassie blue–stained gels of the supernatant and pel-
leted fractions using imaging system software (UVP). The fraction of protein
pelleting in the absence of MTs was subtracted from each data point. The
resultant data from three independent experiments were fitted to a hyper-
bolic binding equation using nonlinear regression analysis on Prism soft-
ware (GraphPad Software, Inc.).
Cold-induced depolymerization assay
Tubulin was induced to polymerize as described by the manufacturer’s in-
structions (Cytoskeleton, Inc.) and incubated with 1 µM Dp260. The reac-
tions were incubated at 4°C for 30 min then centrifuged at 100,000 g for
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