Microtubules that form the stationary lattice of muscle fibers are dynamic and nucleated at Golgi elements

Abstract

Skeletal muscle microtubules (MTs) form a nonclassic grid-like network, which has so far been documented in static images only. We have now observed and analyzed dynamics of GFP constructs of MT and Golgi markers in single live fibers and in the whole mouse muscle in vivo. Using confocal, intravital, and superresolution microscopy, we find that muscle MTs are dynamic, growing at the typical speed of ∼9 µm/min, and forming small bundles that build a durable network. We also show that static Golgi elements, associated with the MT-organizing center proteins γ-tubulin and pericentrin, are major sites of muscle MT nucleation, in addition to the previously identified sites (i.e., nuclear membranes). These data give us a framework for understanding how muscle MTs organize and how they contribute to the pathology of muscle diseases such as Duchenne muscular dystrophy.

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J. Cell Biol. Vol. 203 No. 2 205–213
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Correspondence to Evelyn Ralston: evelyn.ralston@nih.gov
Abbreviations used in this paper: AU, Airy units; BfA, brefeldin A; DMD,
Duchenne muscular dystrophy; ERES, ER exit sites; GalT, galactosyl-transferase;
GM, growth media; FDB, flexor digitorum brevis; MAP, MT-associated protein;
MT, microtubule; MTOC, MT-organizing center; NZ, nocodazole; SR, sarco-
plasmic reticulum.
Introduction
Microtubules (MTs) have recently been implicated in the
pathology of Duchenne muscular dystrophy (DMD; Khairallah
et al., 2012), the most common genetic disease of skeletal
muscle. This puts a spotlight on muscle MTs, whose organization
is poorly understood. During myogenesis, coordinated waves of
subcellular remodeling affect MTs as well as MT-organizing
centers (MTOCs), ER exit sites (ERES), and the Golgi complex
(Tassin et al., 1985a,b; Lu et al., 2001; Musa et al., 2003; Bugnard
et al., 2005; Srsen et al., 2009; Zaal et al., 2011). The resulting
organization has little resemblance to that of proliferating
cells. Muscle cultures can be used to study the rst phase of MT
reorganization that takes place during differentiation of myo-
blasts into multinucleated myotubes. But cultured myotubes do
not mature into the bers of which muscle is made. Therefore
most of our knowledge of MTs in adult muscle comes from
immunouorescence images of single bers, hand-teased from
rodent muscles (Ralston, 1993; Ralston et al., 1999, 2001).
Muscle bers are shaped like attened cylinders; their cytoplasm
is mostly lled with actomyosin laments. Between laments and
the plasmalemma there is a thin cytoplasmic layer that contains
nuclei, other organelles, and what we refer to as surface MTs.
Tubulin immunouorescence shows that these MTs form a
grid-like network with very few clear starting or ending points
(Fig. S1 A). There are no clues as to their organization. Also
lacking from these MTs are the asters that typically represent
MT nucleation sites in images of proliferating cells (we call
aster a ower-shaped gure formed by several MTs, which each
have one end anchored to a central point and the other end free
[Fig. S1 A, insets]). In addition, Golgi elements (the small but
numerous Golgi complexes of muscle bers) are positioned at
the vertices of the MT lattice in a unique and unexplained
organization. It became clear that we could only understand
this organization by looking at live cells.
To do so, we have introduced GFP- and mCherry-tagged
MT and Golgi markers into the mouse exor digitorum brevis
(FDB) muscle (Schertzer et al., 2006; Schertzer and Lynch, 2008;
DiFranco et al., 2009; Fig. S1 G). We have characterized their dy-
namics ex vivo, in single bers obtained by enzymatic digestion
of the muscle (Bekoff and Betz, 1977; Rosenblatt et al., 1995),
and in vivo, in muscles of live animals. We show that muscle MTs
are highly dynamic and grow from static Golgi elements. Thus,
Skeletal muscle microtubules (MTs) form a non-
classic grid-like network, which has so far been
documented in static images only. We have now ob-
served and analyzed dynamics of GFP constructs of MT
and Golgi markers in single live fibers and in the whole
mouse muscle in vivo. Using confocal, intravital, and su-
perresolution microscopy, we find that muscle MTs are dy-
namic, growing at the typical speed of 9 µm/min, and
forming small bundles that build a durable network. We
also show that static Golgi elements, associated with the
MT-organizing center proteins -tubulin and pericentrin,
are major sites of muscle MT nucleation, in addition to the
previously identified sites (i.e., nuclear membranes). These
data give us a framework for understanding how muscle
MTs organize and how they contribute to the pathology of
muscle diseases such as Duchenne muscular dystrophy.
Microtubules that form the stationary lattice
of muscle fibers are dynamic and nucleated at
Golgi elements
Sarah Oddoux, Kristien J. Zaal, Victoria Tate, Aster Kenea, Shuktika A. Nandkeolyar, Ericka Reid, Wenhua Liu,
and Evelyn Ralston
Light Imaging Section, Office of Science and Technology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda,
MD 20892
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THE JOURNAL OF CELL BIOLOGY

JCB • VOLUME 203 • NUMBER 2 • 2013 206
EB3-GFP labeling the plus tips of MTs growing on parallel and
antiparallel tracks. Puncta can be followed on average for 4.3 ± 0.1 s,
i.e., 0.6 µm. Occasional spots (Fig. 1, A, C, and D, arrowheads)
release EB3-GFP again and again, suggesting cytoplasmic MT
nucleation sites. Projections show these spots to be at MT inter-
sections. The mean growth rate of EB3-GFP in muscle bers is
8.6 ± 0.1 µm/min (Table 1), similar to that in Drosophila mela-
nogaster neuronal dendrites (Ori-McKenney et al., 2012).
To validate plated bers as a model for muscle in vivo, we
built a setup for intravital microscopy of the FDB and surround-
ing muscles (Fig. 1 C; Videos 2 and 5; and Fig. S2, A–C). EB3-
GFP dynamics, the presence of antiparallel tracks, and the
existence of cytoplasmic nucleation centers validate all ex vivo
observations. The mean MT growth rate indicated by EB3-GFP
in vivo is 5.0 ± 0.1 µm/min.
GFP-tubulin highlights a stationary,
durable frame
To visualize the entire MTs we then examined GFP-tubulin.
Compared with EB3-GFP, GFP-tubulin in plated bers rst appears
like some other cells (Emov et al., 2007; Rivero et al., 2009;
Ori-McKenney et al., 2012), muscle bers use the Golgi complex
as a MTOC, while forming a MT network unlike any other.
Results and discussion
EB3-GFP reveals a dynamic network of
MTs in live muscle, both ex vivo and in vivo
The MT plus tip end-binding protein EB3 is arguably the best
marker of growing MTs (Stepanova et al., 2003) but its distribu-
tion in muscle bers is unusual because it is found not only at
the tips but anywhere along MTs (Fig. S1 B). Our rst goal was
therefore to clarify EB3 distribution by observing EB3-GFP in
live bers (see Materials and methods and Fig. S1 D2).
In plated bers (Fig. 1, A1 and B1), each of the EB3-GFP
puncta moves (Video 1) mostly longitudinally (parallel to the ber
axis) or transversely (perpendicular). To analyze EB3-GFP dynam-
ics we generated color-coded time-lapse projections (Fig. 1 A2)
and kymograph plots (Fig. 1 B). Puncta move indifferently left or
right (56 vs. 44%; Fig. 1 A2) and up or down, and at similar speeds
in all directions (Fig. 1 B, arrows). These results are consistent with
Figure 1. EB3-GFP shows steady MT dynamics ex vivo and in vivo, whereas GFP-tubulin highlights a durable MT frame. A single image, focused near the
surface of a plated fiber expressing EB3-GFP (A1), shows typical puncta. The dynamics can be appreciated in the corresponding time-lapse series (Video 1)
and in its projection (A2). Color coding of the projection helps to visualize movement: the first image of the series is colored blue and the last one magenta,
as in the bar. EB3-GFP puncta mostly move longitudinally and transversely, as if along parallel and antiparallel tracks (A2, arrows). Some MT intersections
(arrowheads) seem to behave as MT nucleation sites. An image from another fiber (B1) and the kymograph of the line between the arrowheads (B2; see
Materials and methods) indicate that puncta move at the same speed in either direction on the longitudinal axis (oblique lines; red arrows). EB3-GFP dynam-
ics in vivo (C1 and C2; Videos 2 and 5 and Fig. S2) validate plated fibers in all respects (arrowheads point to nucleation sites). In contrast, GFP-tubulin in
plated fibers (D–G) appears static: the color-coded projections (D2, F1, and F2), with the same number of images and frame rate as A2, are practically
white. An aster (arrowhead) indicates an MT nucleation site. The kymograph (E2) of the line between the arrowheads (E1) shows stationary MTs, with
occasional local movement (arrows). Muscle MTs show dynamic instability (G); the asterisk shows the plus end of a MT growing and shrinking over 80 s.
See Tables 1 and S1 for data quantitation and technical parameters. Bars: (A–E) 10 µm; (insets) 2 µm; (kymograph vertical time axes) 60 s.

207
Skeletal muscle microtubule dynamics • Oddoux et al.
These observations imply that muscle MTs are bundled.
However, diffraction-limited microscopy, with a resolution of
250 nm at best, cannot resolve closely spaced MTs, which have
outer diameters of 25 nm. Using G-STED superresolution micro-
scopy (Vicidomini et al., 2013), we reached an improved resolu-
tion of 65 nm and resolved MT tracks into two to four strands,
often torsading around each other (Fig. 3, B and C, red arrow-
heads). With a few exceptions (Fig. 3 C, black arrowhead) the
strands are of even intensity, suggesting that they represent single
MTs. Muscle MTs thus form small bundles with space between
strands for MT-associated proteins (MAPs; Chen et al., 1992).
MT tracks may contain dynamic MTs growing alongside
stable MTs, as suggested by the stationary character of the GFP-
tubulin frame (Fig. 1 D). However, few muscle MTs have the
curly shape and posttranslational tubulin modications typical
of stable MTs (Schulze et al., 1987). Muscle MTs could be stabi-
lized by the muscle-specic isoform of MAP4 (Nguyen et al.,
1997), which lines each muscle MT (Fig. S1 C) and whose
function is not clear (Mangan and Olmsted, 1996; Casey et al.,
2003). However, it is most likely that the stationary MT frame
is entirely composed of dynamic MTs fasciculating along each
other. Although MTs can bear compressive forces and support
a cell structurally (Brangwynne et al., 2006), a dynamic frame
of short MTs may be better suited to contracting bers; indeed,
excessive MT stabilization has been implicated in cardiac
hypertrophy (Sato et al., 1997; Takahashi et al., 2003) and in
DMD (Khairallah et al., 2012).
Muscle MTs nucleate from static
Golgi elements
During muscle differentiation the MTOC redistributes from
centrosomes to nuclear membranes (Tassin et al., 1985a; Lu
et al., 2001; Bugnard et al., 2005; Zaal et al., 2011). EB3-GFP and
immobile, highlighting a frame that remains unchanged for min-
utes (Fig. 1 D1 and Video 3); the color-coded projections are
mostly white (Fig. 1 D2). However, there are pockets of grow-
ing and shrinking MTs (Fig. 1, D2 [inset] and E–G), reecting
dynamic instability (Mitchison and Kirschner, 1984). The mean
growth rate is 5.6 ± 0.2 µm/min. GFP-tubulin, like EB3-GFP,
grows indifferently left or right (45 ± 4 vs. 55 ± 4%), up or down.
When two MTs are on the same stretch of the frame they grow as
frequently in parallel as in antiparallel directions (48 vs. 52%).
In vivo recordings of GFP-tubulin also validated the
ex vivo data (Fig. S2, D and E; and Video 6). GFP-tubulin–
labeled MTs in vivo grow at 3.6 ± 0.2 µm/min. We ascribe the
apparently slower growth rate of GFP-tubulin compared with
that of EB3-GFP to the different methods of analysis (manual
vs. PlusTipTracker; see Materials and methods) and to intrinsic
differences between the two markers. Tubulin is the building
block of MTs, whereas EB3 is in dynamic association with MTs
from which it dissociates when MTs pause or shrink (explaining
why EB3 puncta can only be followed on average for 0.6 µm).
To learn more about the MT frame, we followed FRAP of
regions of interest containing one to three MT tracks (Fig. 2).
Because tubulin subunits are not in dynamic exchange with
MTs, we did not expect true recovery, i.e., a uniform progres-
sive return of the bleached track. Recovery was possible if new
GFP-tubulin–labeled MTs grew along the bleached track, as
occurred, but in only 7 out of 45 MT tracks. However, in half the
regions of interest a new MT entered the box near the bleached
track (Fig. 2 A, box 3 and arrow). Displacement of the bleached
MT portion, which would indicate motor-induced MT trans-
port, was not observed. The FRAP results are consistent with a
frame of stable, immobile MTs and/or of anchored, growing
MTs. Many of the observed MTs show dynamic instability,
which results in repeated loss and gain of the EB3–tip complex.
This can explain the apparent discrepancy between the low
number of GFP-tubulin recoveries and the larger number of
EB3-GFP puncta that would move in comparable ber areas.
The durable MT frame serves as a track
for growing MTs
To clarify the apparent differences between EB3 and tubulin
dynamics, we coexpressed EB3-GFP and mCherry-tubulin
(Fig. 3 A). Each of the markers behaves as if alone, indicating that
one does not interfere with the other and that neither affects
the network as a whole (also see immunoblotting controls in
Fig. S1, E and F). EB3-GFP moves along mCherry-tubulin–
labeled tracks (Fig. 3 A1 [arrows] and Video 4). In addition, multiple
EB3 dots move in the same or in opposite directions on a single
track. The kymograph (Fig. 3 A2) emphasizes both the contrast in
motility and the association of EB3-GFP with mCherry-tubulin.
Figure 2. FRAP showing only growth (or transport) of MTs restores fluor-
escence to the bleached area. (A) GFP-tubulin in a plated FDB fiber before
(prebleach), just after (bleach), and 28 s after photobleaching of two re-
gions of interest surrounded by orange boxes. For quantitation of recovery
(B), the lower box was divided into parts 2 and 3. In seven independent
photobleachings, more than half of the bleached boxes recover some fluor-
escence, as is the case in box 3, by growth or transport of a MT distinct
from the original one (A, arrows). Bar, 2 µm.
Table 1. Measurements of MT growth rate
MT marker Growth rate ± SEM
Plated fibers Intravital
µm/min µm/min
EB3-GFP 8.6 ± 0.1 (n = 463) 5.0 ± 0.1 (n = 220)
GFP-tubulin 5.6 ± 0.2 (n = 49) 3.6 ± 0.2 (n = 31)

JCB • VOLUME 203 • NUMBER 2 • 2013 208
could play a role in GLUT4 translocation (Semiz et al., 2003)
and lysosomal positioning (Fukuda et al., 2006; Korolchuk
et al., 2011).
-Tubulin and pericentrin are associated
with Golgi elements
The MTOC protein -tubulin is required for both centro-
somal and noncentrosomal MT nucleation (Erhardt et al., 2002;
Bugnard et al., 2005; Emov et al., 2007; Rivero et al., 2009;
Ori-McKenney et al., 2012; Zhu and Kaverina, 2013), but it is
anchored by different proteins. In nonmuscle cells, Golgi -
tubulin is linked either to the trans-Golgi by GCC185 and
CLASP2 (Emov et al., 2007) or to the cis-Golgi by GM130
and AKAP450 (Rivero et al., 2009).
By immunouorescence we detected -tubulin on GM130-
labeled Golgi elements of muscle bers (Fig. 4 C and Table 2).
CLASP2 could not be detected at all, and AKAP450 was only
seen in the perinuclear area (Fig. 4 E). However, AKAP450
resembles another MTOC protein, pericentrin. AKAP450 and
pericentrin are large coiled-coil proteins that share a centrosome-
binding sequence (Gillingham and Munro, 2000). We found in
silico that they also share sequences in the GM130-binding re-
gion of AKAP450 (Hurtado et al., 2011). Moreover, AKAP450
and kendrin, a pericentrin isoform, collaborate to bind -tubulin to
centrosomes (Takahashi et al., 2002). Pericentrin could there-
fore replace or supplement AKAP450 in muscle bers. Peri-
centrin was detected in bers and found colocalized with Golgi
elements (Fig. 4 D and Table 3). Although these results are
consistent with cis-Golgi MT nucleation, we have not been
able to conrm the involvement of GM130 because knocking
it down by shRNA was inconclusive. It is possible that both
cis- and trans-Golgi nucleations coexist or that other cis-Golgi
proteins are involved.
Finally, we assessed the motility of Golgi elements. We
coexpressed galactosyl-transferase-mCherry (GalT-mCherry),
GFP-tubulin, however, grow from additional nucleation centers
(Fig. 1 and Videos 1–3), which are at MT intersections, where
we also nd Golgi elements (Fig. 4 A1). To assess whether Golgi
elements serve as nucleation centers, we treated muscle bers
with nocodazole (NZ) to depolymerize MTs, washed NZ away,
and analyzed MT recovery in relation to Golgi elements. After
NZ treatment (Fig. 4 A2), only curly, NZ-resistant MTs are left
(arrowheads), many of which surround nuclei. These MTs are
detyrosylated, i.e., stable (Fig. 4 F). During the rst minutes of
recovery they elongate (Fig. 4 A3, arrowheads), whereas MT
seeds appear around Golgi elements (arrows) and nuclei. These
seeds rapidly grow into well-formed asters, strikingly centered
on Golgi elements (Fig. 4 A4, arrows). MTs from different
nucleation centers become interconnected (Fig. 4 A5) and an
integrated network reforms (Fig. 4 A6). The percentage of Golgi
elements associated with asters progressively decreases (Fig. 4 B),
indicating that the asters become part of the reformed MT net-
work. We conclude that Golgi elements (in addition to the nu-
clear membranes already described in myotubes) nucleate MTs
in muscle bers.
In proliferating cells, the involvement of the Golgi com-
plex in MT nucleation has been conrmed by treatment with
brefeldin A (BfA), which redistributes Golgi components in-
cluding Golgi-nucleated MTs to ERES (Rivero et al., 2009).
In muscle bers we found no convincing effect of BfA, likely
because the Golgi elements are dispersed and colocalized
with ERES even in the absence of BfA (Ralston et al., 1999;
2001; Lu et al., 2001).
Muscle bers thus have at least two categories of MT
nucleation sites, nuclei and Golgi elements. We do not know
whether there are any biochemical or functional differences
between MTs nucleated from these distinct sites, but one can
hypothesize that perinuclear MTs are involved in the position-
ing of nuclei (Elhanany-Tamir et al., 2012; Metzger et al., 2012;
Wilson and Holzbaur, 2012), whereas Golgi-nucleated MTs
Figure 3. Dynamic MTs grow along MTs and
are bundled. EB3-GFP and mCherry-tubulin
were coexpressed and simultaneously im-
aged in plated fibers (A1; Video 4). EB3-GFP
(green) moves along static mCherry-tubulin
tracks (red; A2 kymograph). The asterisk indi-
cates a nucleation spot and the arrows point to
EB3-GFP–labeled MTs growing toward each
other. G-STED superresolution microscopy of
FDB fibers stained for -tubulin resolves MTs
into two or more components (B and C; panels
with blue arrowheads show confocal images;
panels with red arrowheads show the cor-
responding G-STED image at a resolution of
65 nm). A black arrowhead points to a proba-
bly unresolved MT. Bars: (A) 10 µm; (B and C)
2.5 µm; (A2, vertical time axis) 60 s.

209Skeletal muscle microtubule dynamics • Oddoux et al.
and its absence in the mdx mouse prevents MTs from form-
ing an orthogonal grid (Percival et al., 2007; Prins et al., 2009).
However, dystrophin does not line MTs as MAP4 does; instead,
dystrophin appears to dene domains along which MTs grow
preferentially (Prins et al., 2009). Dystrophin may capture MTs;
those that start at an oblique angle (Fig. 1 D2, colored MTs)
often abruptly change their orientation (Fig. 5 B) when they
encounter the transverse bands that contain dystrophin. Studies
of MT dynamics and directionality in mdx muscles will help us
to further develop this model.
Skeletal muscle MTs, like the proverbial canary in the
mine, are affected by practically all physiological and patho-
logical changes in muscle, most likely because they are sen-
sitive to patterned contractions (Ralston et al., 2001). They
reach and dynamically connect all domains of muscle bers.
a marker of the Golgi complex, with GFP-tubulin or EB3-GFP
(Fig. S3, Video 7, and Video 8). In both plated bers (Fig. S3 A)
and in vivo (Fig. S3 B) Golgi elements are static. Thus, the clas-
sic model that MTs position the Golgi complex is turned upside
down in muscle, where, instead, static Golgi elements position
MT nucleation. The lack of motility of muscle Golgi elements
is consistent with their steady positioning along Z bands (Kaisto
and Metsikkö, 2003).
Building a model of muscle MT organization
At this point, we can start building a model (Fig. 5 A). At
steady-state, MTs nucleated from Golgi elements grow along
other dynamic and/or stable MTs to form bundles. To explain
the orthogonal grid of MTs, we must involve dystrophin, the
protein missing in DMD. Dystrophin is a MAP (Prins et al., 2009)
Figure 4. MTs are nucleated on Golgi ele-
ments that concentrate -tubulin and pericen-
trin. To investigate MT nucleation, plated FDB
fibers were treated with NZ to depolymerize
MTs and fixed after different periods of recov-
ery. They were then stained with anti–-tubulin
(-tub) and anti-GM130 (gm) to label MTs
and Golgi elements. In a control fiber (A1),
Golgi elements are along MTs, especially at
crossings, and around nuclei. After NZ, before
recovery (A2), only a few curly MTs remain (ar-
rowheads). These contain both detyrosylated
and tyrosylated tubulins (detyr-tub and Y-tub; F),
indicating the presence of both stable and
dynamic MTs. In the first minute of recovery,
NZ-resistant MTs elongate (A3, arrowheads)
and MT seeds appear around Golgi elements
(A3, arrows). A few minutes later, full asters
centered on Golgi elements become prominent
(A4, arrows). MTs then progressively reform a
network (A5 and A6). During early recovery,
90% of Golgi elements are at the center
of MT asters; at steady-state, only 10% are
(B; data are from a single representative ex-
periment out of three; n = 100, from five fibers,
for each time point). Fibers at early stages of
recovery (as shown in A3) were stained with
anti-GM130 and with antibodies against -
tubulin (-tub), pericentrin, and AKAP450.
-Tubulin and pericentrin are concentrated
on Golgi elements of MT seeds (C1–C4 and
D1–D4); AKAP450 is only detected around
nuclei (E1–E3). Most MT seeds are associ-
ated with Golgi elements ± -tubulin and some
with -tubulin alone (C5; Table 2). Similar
results are obtained for quantitation with peri-
centrin (D5; Table 3; two independent ex-
periments, nine fibers, 400 MT seeds for C5,
and 322 MT seeds for D5). Bars: (A–F) 10 µm;
(C4 and D4) 2 µm.
Table 2. Association of MT seeds with GM130 and -tubulin
Associated with Fraction of MT seeds ± SEM
%
GM130 + -tubulin 20.1 ± 2.8
GM130 only 25.4 ± 2.9
-Tubulin only 14.1 ± 1.7
None 40.4 ± 4.0
Based on two experiments and 322 MT seeds.
Table 3. Association of MT seeds with GM130 and pericentrin
Associated with Fraction of MT seeds ± SEM
%
GM130 + pericentrin 45.5 ± 4.9
GM130 only 6.1 ± 0.8
Pericentrin only 13.6 ± 1.5
None 34.8 ± 4.5
Based on two experiments and 400 MT seeds.

JCB • VOLUME 203 • NUMBER 2 • 2013 210
AKAP350 from J. Goldenring (Medical College of Georgia, Augusta, GA;
Shanks et al., 2002). Other antibodies were purchased commercially:
rat anti-tyrosylated tubulin from Accurate; rabbit anti–-tubulin and mouse
anti-GAPDH from Abcam; mouse anti–-tubulin, rabbit anti-acetylated tubu-
lin, and rabbit anti–-tubulin from Sigma-Aldrich; rabbit anti-detyrosylated
tubulin from EMD Millipore; mouse anti-GM130 and mouse anti-pericentrin
from BD; rabbit anti-GFP from Cell Signaling Technology; and rabbit anti-
pericentrin from Covance. Goat anti–mouse and anti–rat conjugated with
DyLight 488, 549, and 647 were obtained from Jackson ImmunoResearch
Laboratories, Inc. and goat anti–rabbit conjugated with Alexa 488, 546,
568, and 647 were obtained from Molecular Probes. For Western blot anal-
yses we used horseradish peroxidase–conjugated goat anti–rabbit and goat
anti–mouse purchased from Bio-Rad Laboratories and Alexa 680–conjugated
goat anti–rat and anti–mouse antibodies purchased from Invitrogen.
Plasmids
All plasmids expressed in this study are based on pEGFP-N1 or pEGFP-
C1 vectors (Takara Bio Inc.). p-EB3-GFP-N1 cDNA was a gift from A.
Akhmanova (Stepanova et al., 2003), p-EGFP-tubulin-C1 was obtained
from Takara Bio Inc., and p-mCherry-tubulin-C1 was constructed from p-eGFP-
tubulin-C1 and p-mCherry-C1. p-GalT-mCherry-N1 was constructed from
p-GalT-eGFP-N1 (Zaal et al., 1999) and p-mCherry-N1 (a gift from G. Patterson
[National Institute of Biomedical Imaging and Bioengineering, Bethesda, MD]).
p-EGFP-EMTB-N1 was subcloned from p-EMTB-N1 (a gift from C. Bulinski
[Columbia University, New York, NY]).
cDNA injection and electroporation into mouse muscles
All animal protocols were reviewed and approved by the National Institute
of Arthritis and Musculoskeletal and Skin Diseases Animal Care and Use
Committee. Mice were C57BL/6 (The Jackson Laboratory), 6 to 8 wk old
unless otherwise mentioned. To obtain cDNA expression we followed the
protocol of DiFranco et al. (2009) with a few modifications. Mice were
anesthetized with 4% isoflurane throughout the procedure and received a
subcutaneous injection of 0.05 mg/kg buprenorphine-HCl to avoid pain.
To loosen the extracellular matrix and allow the plasmid to reach the
FDB muscle, which extends along the sole of the foot, 10 µl of 0.5 U/µl
(0.36 mg/ml) hyaluronidase was injected through the skin at the heel.
After 1 h, 20–50 µg of endotoxin-free plasmid was injected (Genewiz) at
5 mg/ml in sterile DPBS. After 15 min, acupuncture needles (0.20 ×
25 mm, Tai Chi; Lhasa OMS) were placed under the skin at the heel and at
the base of the toes and connected to an ECM 830 BTX electroporator
(BTX Harvard Apparatus). Six pulses of 20 ms each at 1 Hz were applied
to yield an electric field of 75 V/cm. 5–7 days later the animal was killed
to collect muscles or prepared for intravital imaging. Expression of the
injected cDNA is easily verified under fluorescence illumination on the dis-
section microscope (Fig. S1 G). The efficiency of expression ranges from
20 to 80% of the fibers depending on the plasmid.
Selection of EB3-GFP and GFP-tubulin constructs to track MTs
in muscle fibers
In search of suitable MT markers we expressed GFP-tubulin (Fig. S1 D1),
EB3-GFP (Fig. S1 D2), and the MT-binding domain of the MAP ensconsin
(EMTB-GFP; Fig. S1 D3). Our criteria for accepting a construct were as fol-
lows: pattern and location indistinguishable from native MTs (Fig. S1 A),
even at moderate expression levels, normal appearance of the whole MT
network, and useful signal-to-noise ratio. EB3-GFP and GFP-tubulin satisfied
all criteria. Regrettably, EMTB (Faire et al., 1999) caused abnormalities of
MTs in all but the lowest expressing fibers. We verified by immunoblotting
(Fig. S1 F) that GFP-tubulin undergoes detyrosylation and acetylation, the
normal posttranslational modifications of muscle tubulin (Gundersen et al.,
1989), and that EB3-GFP overexpression does not affect the posttransla-
tional modifications of tubulin (Fig. S1 E). We also checked by immunofluor-
escence that the expression of the GFP constructs does not alter the
respective patterns of detyrosylated and tyrosylated tubulin. Finally we veri-
fied that mCherry constructs gave results similar to those with the corre-
sponding GFP construct.
Intravital imaging
Mice injected with cDNA and electroporated as described in a previous
paragraph were anesthetized by intraperitoneal injection of 75 mg/kg
of sodium pentobarbital. A flap of skin was removed from the plant of
the foot to expose the FDB. The mouse was placed in a tub-shaped stage
insert (custom designed at the National Institute of Arthritis and Musculo-
skeletal and Skin Diseases for the TCS SP5 confocal microscope [Leica]),
the bottom of which was made of a no. 1.5 coverglass. The exposed FDB,
MTs are moored by Golgi elements through ERES to the ER/
sarcoplasmic reticulum, which extends longitudinally in the
myobrillar core (Kaisto and Metsikkö, 2003). They interact with
the triad junctions of T-tubules and sarcoplasmic reticulum
(Fourest-Lieuvin et al., 2012), with dystrophin, and also with
muscle-specic protein networks involved in the maintenance
of sarcomeric organization (Ayalon et al., 2008, 2011; Randazzo
et al., 2013). For a long time, the relevance of muscle MTs
and the consequences of their perturbations were not clear. Now
muscle MTs are nally starting to receive long overdue attention.
Materials and methods
Antibodies and other reagents
Several antibodies were gifts: rabbit anti–mouse/human detyrosylated
tubulin from G. Cooper IV and T. Galien (Veterans Affairs Medical Center,
Charleston, SC; Sato et al., 1997); rabbit anti–mouse/human EB3 from
A. Akhmanova (Utrecht University, Utrecht, Netherlands; Stepanova et al.,
2003); rabbit anti–mouse mMAP4 from J. Olmsted (University of Rochester,
Rochester, NY; Casey et al., 2003); and rabbit anti–human AKAP450/
Figure 5. Model of MT organization in skeletal muscle fibers at steady-
state. (A) MTs nucleating from Golgi elements grow parallel or antiparal-
lel to existing MTs, thereby forming small bundles, which are guided or
restricted by the dystrophin bands positioned along Z lines, M bands, and
longitudinal stripes. MTs starting at an oblique angle reorient upon con-
tact with other MTs or dystrophin bands, as observed (B) in GFP-tubulin–
expressing fibers. The MT lattice that results is both durable and dynamic.
Bars, 2 µm.

211Skeletal muscle microtubule dynamics • Oddoux et al.
detection on the gated HyD detectors of a TCS SP8 X G-STED system
(Leica) with pulsed white light laser excitation. The conditions for confocal
imaging were identical to those for time-lapse series, i.e., pinhole opened
to 1.5 AU to increase brightness and depth of field, whereas G-STED
parameters were optimized for best resolution, i.e., pinhole between 0.5
and 1.0 AU.
Image rendering and analysis
Two techniques were used to make dynamics perceptible in single static
images: color coding and kymograph plotting. Color coding of confocal
time series was done with Photoshop CS5. Each image of the 38-s time se-
ries had its red, green, and blue levels set to obtain a progressive change
of colors, the first frame being blue (red = 0, green = 0, and blue = 255)
and the last frame magenta (red = 255, green = 0, and blue = 255). A
moving object appears rainbow colored in projection whereas a station-
ary object is white. Kymographs were done on image stacks in ImageJ
with the Reslice tool. The position of the line that is extracted from each
image and repeated in the kymograph is indicated by two arrowheads on
one of the images. An object that moves along the selected line during the
recording of the series appears as an oblique dash in the kymograph, a
stationary object on the line appears as a vertical line, and an object that
crosses the line appears as a single dot.
EB3-GFP speed was analyzed with PlusTipTracker, a MatLab-based
open source software (Applegate et al., 2011). The tracks were detected
with PlusTip GetTracks, with the following parameters, adapted depending
on the quality of the movie: gap length = 3, angle = 10–15, radius range =
1–8, fluctuation radius = 2, and maximum shrinkage factor = 1.5. Each
track was checked by hand with PlusTip SeeTracks to avoid false positives.
GFP-tubulin MT growth rate was analyzed with ImageJ. The path covered
was tracked, measured manually, and converted to a growth rate in mi-
crometers per minute.
To quantitate the association of nascent MTs with Golgi elements,
-tubulin, and/or pericentrin (Fig. 4, C5 and D5; and Tables 2 and 3),
we examined the tubulin staining of triple-stained immunofluorescence im-
ages while hiding the other channels, marked the position of presumed MT
seeds (at least three MT fragments), and then examined whether Golgi and
-tubulin or pericentrin staining were present. Because MT seeds at early
recovery times are less organized than the later asters, it is possible to count
as seeds the simple crossing of two MTs. It is likely that this explains the rel-
atively high percentage of MT seeds not linked to any of the components.
Immunoblots
Fibers were prepared as described in a previous paragraph but instead
of being plated they were rinsed three times in DPBS, lysed in 40 µl of
loading buffer (National Diagnostics), and boiled. Protein concentration
was assayed with the 2D Quant Kit (GE Healthcare). Proteins were sepa-
rated on 10% acrylamide gels and immunoblotted according to standard
procedures. Protein bands were detected either with the Odyssey infrared
imaging system (Li-Cor) or with photographic film.
Online supplemental material
Fig. S1 presents background and quality control data: immunofluorescence
of muscle fibers stained for tubulin, EB3, and MAP4, and characterization
of muscle fibers expressing GFP constructs by fluorescence and immuno-
blotting. Fig. S2 illustrates the setup for and images from intravital imaging of
EB3-GFP and GFP-tubulin. Fig. S3 illustrates simultaneous recordings of the
Golgi marker GalT-mCherry and of a MT marker, ex vivo and in vivo. Videos
provide examples of time-lapse recordings and are essential to perceive MT
dynamics. Videos 1 and 2 present EB3-GFP dynamics ex vivo and in vivo,
respectively. Video 3 shows GFP-tubulin ex vivo. Video 4 shows simultaneous
EB3-GFP and mCherry-tubulin recordings ex vivo. Video 5 shows a more ex-
tensive muscle area expressing EB3-GFP in vivo. Video 6 shows GFP-tubulin
in vivo. Video 7 shows simultaneous GFP-tubulin and GalT-mCherry record-
ings ex vivo, and Video 8 shows EB3-GFP and GalT-mCherry in vivo. On-
line supplemental material is available at http://www.jcb.org/cgi/content/
full/jcb.201304063/DC1. Additional data are available in the JCB Data-
Viewer at http://dx.doi.org/10.1083/jcb.201304063.dv.
We wish to acknowledge the late George Cooper IV for generous advice
over the years. We thank all colleagues who shared reagents; Jonathan Boyd
and Geoff Daniels for essential help with G-STED imaging; Gary Melvin for
design and fabrication of hardware for intravital imaging; and Nina Raben,
Rachel Myerowitz, Dan Sackett, and Andrew Milgroom for critical reading of
the manuscript.
facing the objective lens, was held on the coverglass by a small lever arm
(Fig. S3, A and B). The mouse temperature was maintained at 37°C with a
heating lamp, and breathing of the animal was monitored visually through-
out the experiment.
FDB fiber preparation
Mice were killed by CO2 followed by cervical dislocation. FDB muscles
were dissected in sterile DPBS under an MZ FLIII dissecting microscope
(Leica), rinsed in sterile DMEM, and incubated with rotation for 3 h at 37°C
in DMEM containing 1.5 mg/ml type I collagenase from Clostridium histo-
lyticum (Sigma-Aldrich) and 1 mg/ml BSA. Fibers were then freed from the
muscle by trituration and plated on Mattek dishes or Lab-Tek chambered
coverglass that had been coated for 1 h with a 1:10 dilution of Matrigel
(BD). Fibers were plated in 0.1 ml of growth media (GM), consisting of
DMEM supplemented with 20% FBS and 0.2% chicken embryo extract.
After 2 h they were fed with GM supplemented with penicillin-streptomycin.
The fibers were used within 24 h, ahead of the reorganization of MT and
other cytoskeletal components, which occurs after denervation or when
patterned activity is modified (Ralston et al., 2001, 2006). The only dam-
age directly linked to the collagenase treatment is a degradation of the
fine structure of the neuromuscular junction. We have not searched for
neuromuscular junction–specific MTs (Schmidt et al., 2012). FDB fibers
are predominantly type IIA (intermediate type). Their MTs are organized
similarly to those in fast-twitch fibers (Ralston et al., 1999).
Drug treatments
To prevent MT polymerization and induce loss of MTs, plated fibers were
incubated for 4 h at 37°C in 4 µg/ml NZ (Sigma-Aldrich) in GM. After
washing out the drug with GM at 37°C, MTs were left to recover at 37°C
in GM for 2 min to 24 h. Fibers were then fixed for staining, either with
methanol at 20°C or with 4% PFA (Electron Microscopy Sciences). The
Golgi complex was disrupted with 5 µg/ml BfA (Sigma-Aldrich) at 37°C
for 1 h. For unrecovered NZ or BfA controls, the drug was added to PFA;
alternately, methanol was used for fixation. When combined with NZ treat-
ment, BfA was added for the last hour of the 4-h incubation with NZ and
throughout the washout and fixation process.
Immunofluorescence
Fixed fibers were blocked for 2 h at RT in PBS containing either 5% BSA,
1% normal goat serum, and 0.04% saponin or the blocking reagent from
the Mouse On Mouse basic kit (Vector Laboratories). Fibers were then incu-
bated with primary antibodies for 2 h at RT (or overnight at 4°C) and with
secondary antibodies for 2 h at RT, counterstained with Hoechst 33342,
and mounted in Vectashield (Vector Laboratories). Rinses between and
after antibody incubations were three times 5 min with PBS containing
0.04% saponin.
Microscopy
Confocal images were collected using a 63× 1.4 NA or a 40× 1.25 NA
oil immersion objective lenses on a TCS SP5 (Leica) driven by the LAS AF
2.6.1 software or on an LSM 780 confocal microscope (Carl Zeiss) driven
by Zen 2011. In both cases, images were collected in sequential scanning
to avoid cross talk. Live fibers were imaged in phenol red–free GM with
25 mM Hepes at 37°C, using a Tokai Hit heated stage insert on the SP5
and a Pecon Lab-Tek S1 stage insert on the LSM 780. We used a confocal
pinhole between 1 and 3 Airy units (AU) to increase depth of field and sig-
nal intensity while limiting potential laser damage to live fibers.
For FRAP sequences on the LSM 780, we wanted to achieve close
to complete photobleaching of target regions without damaging them. We
tested different protocols and settled on 10 bleaching iterations with a
40-mW Argon laser at 80%. We were satisfied that no damage was done
because MTs that had been photobleached continued to grow outside of
the bleached area.
Unless otherwise mentioned, the images shown are single frames.
Camera icons refer to the corresponding video, available online. Projec-
tions of images were done as “maximum” projections. Images were ex-
ported in 8-bit TIF format and linearly adjusted using Photoshop CS5, and
then cropped and resized if needed for composing montages. Some mov-
ies were processed in ImageJ (National Institutes of Health) with an image
stabilizer and Kalman filter. For black and white images, the grayscale
was inverted to facilitate viewing. Settings used for still images and videos
can be found in Table S1.
Superresolution microscopy
Fibers stained with anti-tubulin followed by Alexa 488 or 647 goat anti–
mouse IgG were mounted in Prolong Gold. They were imaged using time-gated

JCB • VOLUME 203 • NUMBER 2 • 2013 212
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