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J. Cell Biol. Vol. 200 No. 1 109–123
Correspondence to Leonid V. Chernomordik: firstname.lastname@example.org
Abbreviations used in this paper: Anx, annexin; DM, differentiation medium; DNM,
dynamin; LPC, lysophosphatidylcholine; MHC, myosin heavy chain; MiTMAB,
myristyl trimethyl ammonium bromide; PS, phosphatidylserine; PtdIns(4,5)
P2, phosphatidylinositol(4,5)bisphosphate; rA1, recombinant Anx A1; WT,
Cell-to-cell fusion is a key step in many developmental pro-
cesses including fertilization and the formation of bone, pla-
centa, and muscles (Chen et al., 2007; Sapir et al., 2008). In
mature organisms, cell fusion is required for muscle repair
and for the formation of multinucleated giant cells during in-
flammatory reactions. In each case, initial local merger of the
membranes is followed by a transformation of the adhesive
junction between the fusing cells into an expanding cytoplas-
mic bridge. A key challenge in studying the fusion stage of
syncytium formation is to isolate the actual fusion event from
processes that prepare the cells for fusion. For example, fusion
of myoblasts—one of the very important examples of cell-
to-cell fusion—is preceded by myoblast differentiation, ac-
quisition of fusion competence, and recognition and adhesion
between myoblasts. Many proteins, including actin machin-
ery, ferlins, and certain guanine nucleotide exchange fac-
tors, are required for formation of multinucleated myotubes
(Doherty et al., 2005; Kim et al., 2007; Onel and Renkawitz-
Pohl, 2009; Rochlin et al., 2010; Sens et al., 2010; Abmayr
and Pavlath, 2012; Gruenbaum-Cohen et al., 2012). However,
these proteins are thought to mediate different pre and post-
fusion stages. The proteins that are involved in the cell fusion
event itself remain unidentified.
The dependence of myotube formation on extracellular
Ca2+ (Shainberg et al., 1969; Wakelam, 1983) and a transient
exposure of phosphatidylserine (PS) in the outer leaflet of the
plasma membrane of fusion-committed myoblasts (Sessions
and Horwitz, 1983; van den Eijnde et al., 2001; Kaspar and
Dvorák, 2008) at cell–cell contact sites (Jeong and Conboy,
2011) suggest involvement of annexins (Anxs) in myoblast
fusion. Anxs are a large family of structurally related proteins
whose common property is Ca2+-dependent binding to anionic
phospholipids such as PS (Moss and Morgan, 2004; Gerke
et al., 2005; van Genderen et al., 2008). Anxs are ubiquitous and
abundant proteins and are found in both intra- and extracellular
milieux. Anxs share a conserved C-terminal domain containing
Ca2+ binding sites but have a variable N-terminal domain (Gerke
and Moss, 2002). It has been suggested that Anxs patch membrane
microinjuries (Bouter et al., 2011), serve as membrane–membrane
rine myoblasts at the ready-to-fuse stage by blocking
formation of early fusion intermediates with lysophos-
phatidylcholine. Lifting the block allowed us to explore
a largely synchronized fusion. We found that initial merger
of two cell membranes detected as lipid mixing involved
extracellular annexins A1 and A5 acting in a functionally
yoblast fusion into multinucleated myotubes is
a crucial step in skeletal muscle development
and regeneration. Here, we accumulated mu-
redundant manner. Subsequent stages of myoblast fusion
depended on dynamin activity, phosphatidylinositol(4,5)
bisphosphate content, and cell metabolism. Uncoupling
fusion from preceding stages of myogenesis will help in
the analysis of the interplay between protein machines
that initiate and complete cell unification and in the iden-
tification of additional protein players controlling differ-
ent fusion stages.
Extracellular annexins and dynamin are important
for sequential steps in myoblast fusion
Evgenia Leikina,1 Kamran Melikov,1 Sarmistha Sanyal,1 Santosh K. Verma,1 Bokkee Eun,2 Claudia Gebert,2
Karl Pfeifer,2 Vladimir A. Lizunov,3 Michael M. Kozlov,4 and Leonid V. Chernomordik1
1Section on Membrane Biology, Program of Physical Biology; 2Section on Genome Imprinting, Program on Genomics of Differentiation; and 3Laboratory of Cellular and
Molecular Biophysics, Program of Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health,
Bethesda, MD 20892
4Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, 69978 Tel Aviv, Israel
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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 200 • NUMBER 1 • 2013 110
myoblast fusion—with an assay in which we coincubated the
cells labeled with green cell tracker and cells labeled with either
fluorescent lipid DiI or orange cell tracker to detect either lipid
mixing between the cells or an opening of a cytoplasmic con-
nection as the appearance of colabeled cells.
To detect fusion intermediates preceding syncytium for-
mation, we used primary myoblasts after the 12th passage. We
found these cells to fuse less effectively than the cells between
the 6th and 10th passages used in all other experiments (7% vs.
20–50% after 24 h in DM). We placed the myoblasts of the 12th
passage labeled with green cell tracker and myoblasts labeled
with DiI into DM. 24 h later most of the colabeled cells were
multinucleated (Fig. 1 B, arrows). However, we also observed
a considerable percentage of colabeled mononucleated cells,
where the cells labeled with aqueous probe (green cell tracker)
acquired membrane probe (Fig. 1 A, bar 1). We observed less of
multinucleated cells and more of mononucleated colabeled cells
(Fig. 1 C, arrows) after 16 h in DM than after 24 h in DM. Note
that by the time we scored fusion, fluorescent lipids incorpo-
rated into the plasma membrane because of their internalization
had been mostly labeling intracellular membranes.
In parallel experiments, we incubated primary myoblasts
labeled with green cell tracker together with myoblasts labeled
with orange cell tracker and found almost no mononucleated
cells colabeled with both aqueous probes (Fig. 1 A, bar 2; cola-
beling was observed only in 3 out of 588 cell pairs and in 1 out
of 642 after 16 and 24 h of incubation in DM, respectively).
These findings indicated that myoblast fusion starts with hemi-
fusion (merger of only outer membrane leaflets [Chernomordik
and Kozlov, 2005]) and, in contrast to cell fusion mediated by
viral fusogens (Chernomordik and Kozlov, 2005), very rarely
stalls at a stage of a nonexpanding fusion pore. Note that our
experimental approach would not distinguish between bona
fide hemifusion and fusion pores too small or too short-lived to
allow passage of the cell tracker–labeled proteins.
Further evidence that myoblast fusion proceeds through
hemifusion intermediates came from experiments with LPC. In
the case of C2C12 cells, myotubes were first observed 2 d after
the cells were placed in the DM. At this time, we coplated the
cells labeled with different fluorescent probes. The next 16 h
were the period of efficient cell fusion, detected as a rise in the
percentage of cell nuclei in multinucleated cells (a measure of
syncytium formation) and in the number of cells colabeled with
both probes (a measure of lipid mixing). Both syncytium for-
mation and lipid mixing were strongly inhibited by the LPC-
supplemented DM applied at the start of this 16-h interval.
Fusion rapidly ensued when the LPC was washed out at the end
of the 16-h incubation (Fig. 1 D, time 0). Within 30 min after
replacement of the LPC-supplemented DM with an LPC-free
one, the extent of lipid mixing and syncytium formation for
C2C12 cells was approaching the levels observed at this time in
the control experiment performed without LPC application.
LPC has also reversibly blocked fusion of primary myo-
blasts. As with C2C12 cells, LPC removal resulted in a largely
synchronous fusion process (Video 1) with a much higher rate
of fusion events than the rates observed in cells that had not
been exposed to LPC (Video 2). This robust fusion and analysis
linkers, bend and fuse membranes (Gerke and Moss, 2002; van
Genderen et al., 2008), and anchor other proteins to the mem-
branes (Gerke and Moss, 2002). Different Anxs have been im-
plicated in many intra- and extracellular processes, including
exocytosis, plasma membrane repair, blood coagulation, apop-
tosis, adhesion, and inflammation (McNeil et al., 2006; White
et al., 2006; van Genderen et al., 2008; Blume et al., 2009;
Bouter et al., 2011; Draeger et al., 2011). Intriguingly, Anx A1
and A5 are up-regulated during myotube formation in vitro
(Arcuri et al., 2002; Tannu et al., 2004; Kislinger et al., 2005;
Gonnet et al., 2008; Casadei et al., 2009; Makarov et al., 2009;
Bizzarro et al., 2010) and during muscle regeneration in vivo
(see the Public Expression Profiling Resource at http://pepr
.cnmcresearch.org/). Furthermore, Anx A1 has been implicated
in myogenic differentiation and myotube formation (Bizzarro
et al., 2010).
In this study, we analyze in vitro myotube formation by
C2C12 and primary mouse myoblasts. We use treatment with
lysophosphatidylchloine (LPC) to uncouple the cell-to-cell
fusion stage from the earlier stages of myogenesis that prepare
the cells for fusion. LPC reversibly blocks the merger of the
contacting leaflets of the fusing membranes at the onset of di-
verse membrane fusion processes (Chernomordik and Kozlov,
2005) so that an LPC block allowed us to accumulate ready-
to-fuse cells and to observe a relatively synchronized fusion
upon LPC removal. We show that antibodies to Anx A1 and
A5 and also peptides derived from the N-terminal domain of
these Anx (A1- and A5-peptides) inhibit synchronized myo-
blast fusion and myotube formation in the experiments with-
out LPC application. Myotube formation was also inhibited by
siRNA suppression of Anx A1 and A5 expression. Similarly,
primary myoblasts isolated from either Anx A1 mutant or Anx
A5 mutant mice are deficient for in vitro myotube formation.
Reducing both Anx A1 and A5 together inhibits myoblast
fusion more effectively than lowering expression of either one
of these Anxs alone. Fusion inhibition accomplished by low-
ering the concentration of one of these Anxs can be rescued
by application of a recombinant version of either Anx A1 or
A5 (rA1 and rA5). Finally, using our LPC block synchroni-
zation system, we also examined events downstream of the
Anx-dependent early fusion stages. Syncytium formation
was inhibited by ATP depletion and was dependent on dy-
namin 2 (DNM2) and phosphatidylinositol 4,5-bisphosphate
(PtdIns(4,5)P2). These findings can bring valuable insights
into mechanisms by which mutations in DNM2 and myo-
tubularin, a protein involved in turnover of phosphoinositides,
cause centronuclear myopathies (Spiro et al., 1966; Bitoun
et al., 2005; Hnia et al., 2012).
Experimental system and fusion pathway
C2C12 cells and primary myoblasts isolated, if not stated other-
wise, from wild-type (WT) mouse were committed to myogen-
esis by placing them into differentiation medium (DM). To
distinguish early fusion intermediates, we complemented syn-
cytium formation assay—the conventional way of quantifying
111Annexins and dynamin in myoblast fusion • Leikina et al.
role for Anx expression, we incubated differentiating cells with
antibodies to Anx A1 and A5. Each antibody inhibited both
lipid mixing and syncytium formation in C2C12 cells and in
primary myoblasts (Fig. 2, D and E). Because antibody binding
to surface-associated proteins can inhibit fusion by steric hin-
drance even if the antigens are not involved in fusion, we tested
an alternative approach to block Anx A1 and A5 activities
by the peptides comprising N-terminal regions of these Anxs.
A1-peptide has been demonstrated to inhibit several Anx A1–
dependent processes (McNeil et al., 2006; Wang et al., 2011).
Here we show that this peptide and an A5-peptide inhibited
lipid mixing and syncytium formation by primary myoblasts
(Fig. 2, G and F).
We confirmed a functional requirement for Anx A1 and
A5 in myotube formation by using siRNA (Fig. 3, A and B).
Inefficient lipid mixing and syncytium formation associated
with Anx A1 suppression were rescued not only by rA1 but also
by rA5. Similarly reduced fusion efficiency in cells transfected
with A5 siRNA was rescued by rA1 as well as by rA5. Thus
both Anxs are involved in myotube formation but shortage of
of expression of myogenic markers myogenin and myosin
heavy chain (MHC; Fig. S1) indicated that 16 h in the presence
of fusion-inhibiting concentration of LPC did not block myo-
genic differentiation of the cells.
The finding that LPC reversibly blocks myoblast fusion
both supported the fusion-through-hemifusion pathway and
provided us with a way to effectively separate the fusion stage
from the upstream processes of myogenesis and to concentrate
the fusion events that would normally develop within a 16-h
span within 30 to 60 min. Combining syncytium formation as-
say and lipid mixing assay will be used here for distinguishing
conditions that affect early and late stages of myoblast fusion.
Myotube formation involves extracellular
Anx A1 and A5
We found myogenic differentiation of primary myoblasts to
be associated with a sharp boost in Anx A1 and A5 presence
on the cell surface (Fig. 2, A and B). Elevated amounts of both
Anxs were also present at the outer surface of C2C12 cells at
the time of myotube formation (Fig. 2 C). To test for a functional
Figure 1. Myoblast fusion proceeds via hemifusion intermediates and is reversibly blocked by hemifusion-inhibiting lipid LPC. (A–C) Fusion phenotypes
observed for primary myoblasts of 12th passage after 24 h (A) and 24 or 16 h (B) in DM. (A) Percentage of mononucleated cells labeled with green cell
tracker that had also acquired red fluorescence by lipid probe exchange with DiI-labeled cells (1) or in parallel experiment (2) by cell tracker exchange
with orange cell tracker–labeled myoblasts that signify cytoplasmic connection. (3) Syncytium formation quantified combining the data from the experiments
shown in 1 and 2. (B and C) Fluorescence microscopy images illustrating hemifusion phenotype. Left, phase contrast with nuclear staining; right, green cell
tracker and DiI (red). (B) Arrows mark the colabeled multinucleated cells. Bar, 50 µm. (C) An enlargement of the marked region in B (bottom) with white
arrows pointing to the mononucleated cells colabeled with membrane probe DiI (red) and green cell tracker, a hallmark of the hemifusion phenotype.
Bar, 25 µm. (D) LPC inhibited C2C12 cell fusion and concentrated the fusion events that would normally develop within 16 h to develop mostly within
30 min after LPC removal. Curves show time courses of increase in the extents of lipid mixing (green circles) and syncytium formation (red triangles) after
LPC removal at t = 0 normalized to those observed in the control experiments in which both application of LPC at t = 16 h and its removal at t = 0 were
omitted. All results are shown as means ± SEM (n ≥ 3).
JCB • VOLUME 200 • NUMBER 1 • 2013 112
individual myoblasts depends on the Anx A1 and A5 expressed
by these cells rather than on Anx expressed by other cells. These
findings suggest that most of the Anx A1 and A5 released by the
Anx-expressing cells remain associated with the surfaces of
these cells. Note that the amounts of Anx released into the ex-
tracellular medium are much lower than the amounts of rA1 and
rA5 added exogenously in Fig. 3 A.
To explore the effects of a complete lack of either Anx
A1 or A5, we compared the time courses of syncytium forma-
tion in primary myoblasts isolated from Anx A1–deficient
mice (Hannon et al., 2003), from Anx A5–deficient mouse
(Brachvogel et al., 2003), and from the WT parental strain
mice. Although primary myoblasts from Anx A1/ and from
Anx A5/ mice incubated in DM for 24 h expressed myo-
genic differentiation markers myogenin and MHC similarly to
WT myoblasts (Fig. S1), the lack of either of the two Anxs
considerably impaired lipid mixing and syncytium formation
one can be compensated for by the other, suggesting functional
redundancy. Note that not only do rA1 and rA5 rescue fusion,
they also promote fusion in C2C12 cells and WT primary myo-
blasts (Fig. S2).
The presence of nontransfected cells limits the effects of
A1 and A5 siRNAs on total levels of expression of these pro-
teins and on myotube formation. To focus on the transfected
myoblasts, we labeled them by cotransfection with GFP vector
and scored the efficiency of syncytium formation for only GFP-
labeled cells. Lowering the de novo expression of Anx A1 and
A5 in the cells transfected with the corresponding siRNAs
strongly inhibited their ability to fuse (Fig. 3, C and D). Inter-
estingly, we observed no changes in the fusion efficiency for
cells that were not transfected (defined as the cells that did not
express GFP) in the same tissue culture plate (Fig. 3 C, right) in
spite of a considerable decrease of the Anx A1 and A5 contents
in the total cell lysates (Fig. 3 B). Thus fusion competence of
Figure 2. Myotube formation involves extracellular Anx A1 and A5. (A and B) Myogenic differentiation of primary myoblasts boosts surface concentration
of Anx A1 and A5. Anx A1 (1 and 2) and A5 (3 and 4) were detected by immunofluorescence microscopy in nonpermeabilized proliferating cells (1 and 3)
and the cells that were incubated in DM for 24 h (2 and 4). Bars, 10 µm. (B) Cell surface fluorescence was quantified for 16 cells for each condition and
presented as mean ± SEM. (C) The time course of syncytium formation by C2C12 cells correlates with a rise in the surface concentration of Anx A1 and A5.
Anx concentrations are normalized to those at day 0 (at the time of placing the cells in DM). The data shown are from a single representative experiment
out of three repeats. For the experiment shown, each point and bar is based on analysis of 10 randomly chosen fields of view. (D and E) Antibodies to A1
and A5 (2 and 3, respectively) inhibit lipid mixing and syncytium formation (D) for C2C12 myoblasts at 67 h in DM and syncytium formation for primary
myoblasts at 24 h in DM (E). Control experiments with no antibodies applied (1) or with nonspecific IgG (4) are shown. (F and G) A1- and A5-peptides
inhibit syncytium formation by primary myoblasts at 24 h in DM. (F) Phase contrast with nuclear staining (blue) images of the cells incubated or not with
either A1- or A5-peptides. Bar, 50 µm. Arrows mark the multinucleated cells. (D, E, and G) Lipid mixing and syncytium formation extents are normalized to
those in the control experiments (1). All results are means ± SEM (n ≥ 3). Levels of significance relative to controls (1): **, P < 0.01; *, P < 0.05.
113 Annexins and dynamin in myoblast fusion • Leikina et al.
the time of LPC removal. As with myotube formation in the
experiments without LPC application, synchronized fusion of
C2C12 cells was inhibited by antibodies to Anx A1 and A5
(Fig. 5, A and B) and by A1- and A5-peptides (Fig. 5 C). In
contrast, neither control IgG nor the scrambled versions of the
peptides influenced fusion.
Myotube formation requires the presence of Ca2+ in the
extracellular environment (Shainberg et al., 1969). EGTA ap-
plication 30 min before LPC removal inhibited fusion between
C2C12 myoblasts (Fig. 5 D), indicating that Ca2+ is required for
the fusion stage of myogenesis. Because subsequent application
of Ca2+-containing medium did not reverse the inhibition by the
time we scored fusion, and because chelators were reported to
dissociate Anx from cell membranes (Rao et al., 1992; Fan
et al., 2004), we hypothesized that Ca2+-free medium washed
out functionally important membrane-associated Anxs. Indeed,
myotube formation was partially restored when treatment of the
cells with EGTA was followed by application of the Ca2+-
containing medium supplemented with rA1 or rA5. Fusion was not
restored if rA1 and rA5 were applied in the Ca2+-free medium.
A decrease in concentrations of Anx A1 and A5 at the surface of
(Fig. 4, A and B). Application of rA1 or rA5 rescued these
defects (Fig. 4 C).
Can myotube formation in the absence of either of the two
Anxs be explained by redundancy of their functions? We trans-
fected Anx A1/ myoblasts with A5 siRNA constructs and
transfected Anx A5/ myoblasts with A1 siRNA constructs.
The transfected myoblasts, identified by GFP vector cotrans-
fection, only very rarely joined myotubes (Fig. 4, D and E). As
expected, siRNAs targeting Anx A1 in A1/ cells or Anx A5
in A5/ cells had no effect on myotube formation. Thus, in
the absence of one of these two Anx, lowered expression of the
remaining one almost completely abolishes myotube formation,
which is consistent with their being functionally redundant.
To summarize, the dependence of myotube formation on
the concentrations and activities of Anx A1 and A5 indicates
that these Anxs play an important role in myogenesis.
Anx A1 and A5 at the fusion stage of
To focus on fusion stage we accumulated ready-to-fuse myo-
blasts using LPC block and applied Anx-targeting reagents at
Figure 3. siRNAs targeting expression of Anx A1 or A5 inhibit fusion of primary myoblasts. (A) Inhibition of lipid mixing (green) and syncytium formation
(red) at 24 h in DM by A1 and A5 siRNAs is reversed by application of rA1 or rA5. 1, cells transfected with negative control siRNA; 2–4, cells transfected
with A1 siRNA; 5–7, cells transfected with A5 siRNA. rA1 (3 and 6) and rA5 (4 and 7) were applied 3 h before scoring fusion. (B) The cells transfected
with control siRNA or A1 or A5 siRNAs were lysed after 24 h in DM and analyzed by Western blot to evaluate levels of expression of Anx A1 and A5
and tubulin (as a loading control). (C) After cotransfection of primary myoblasts with siRNA and GFP vector, we separately assayed syncytium formation
for transfected (left) and not-transfected (right) cells. 1, transfection with GFP vector alone; 2, cotransfecion with GFP vector and negative control siRNA
(taken as 100%); 3 and 4, cotransfection with GFP vector and siRNA to either Anx A1 (3) or A5 (4). (D) Phase-contrast images with nuclear staining (blue)
and GFP fluorescence (green) showing myoblasts cotransfected with GFP and control (left), Anx A1 (middle), or Anx A5 (right) siRNA. Arrows mark the
GFP-labeled multinucleated cells. Bar, 50 µm. (A and C) All results are shown as means ± SEM (n ≥ 3). Levels of significance relative to controls (1 in A
and 2 in C) are shown: **, P < 0.01; *, P < 0.05.
JCB • VOLUME 200 • NUMBER 1 • 2013 114
for Anx-deficient myoblasts in these experiments than fusion in-
hibition observed for the Anx-deficient myoblasts in the experi-
ments without LPC block (Fig. 4, A and B) confirmed that the
lack of Anxs inhibited fusion stage of myogenesis. Application of
rA1 and rA5 at the time of LPC removal restored the fusogenic
potential of the myoblasts (Fig. 6, D and E). These findings indi-
cate that Anx A1 and A5 play a vital role in myoblast fusion.
Transition from lipid mixing to
syncytium formation depends on
cell metabolism, DNM2 activity,
and PtdIns(4,5)P2 concentration
Although Anx-targeting reagents similarly affected lipid mix-
ing and syncytium formation, the ATP-depleting reagents NaN3
myoblasts after EGTA application and the recovery of mem-
brane-associated Anx after the application of Ca2+ and recom-
binant Anx have been confirmed by immunofluorescence
microscopy (Fig. S3). These findings demonstrate that the loss
of Anx A1 and A5 at the surface of myoblasts after EGTA ap-
plication correlates with the loss of fusion activity, and reap-
pearance of the Anxs at the surfaces of the cells after application
of rA1 and rA5 rescues the ability of these cells to fuse.
Anx antibodies and peptides also inhibited synchronized
fusion between primary myoblasts (Fig. 6, A and B). Moreover,
primary myoblasts from Anx A1– and Anx A5–deficient mice
demonstrated much less efficient fusion (lipid mixing and syncy-
tium formation) 30 min after LPC removal than myoblasts from
WT mouse (Fig. 6, C and D). A much stronger inhibition of fusion
Figure 4. Inhibition of myotube formation for primary myoblasts isolated from either Anx A1/ or Anx A5/ mice. (A and B) The lack of either of the
two Anxs substantially inhibited lipid mixing (A) and syncytium formation (B) assayed at different times after placement of the cells into DM (B). Curves
show myoblasts isolated from WT mice (1), Anx A1/ mice (2), and Anx A5/ mice (3). (C) Application of rA1 or rA5 rescues the fusogenic potential
of the Anx-deficient myoblasts. Lipid mixing (green) and syncytium formation (red) at 12 h after placing WT myoblasts (1) and Anx A1/ myoblasts (2)
into DM. After 10 h of incubation of Anx A1/ myoblasts in DM, we applied rA1 (3). Fusion was assayed 2 h later. Fusion in 2 and 3 was normalized to
fusion in 1. Cell fusion assayed at 14 h after placement of myoblasts from WT mouse (4) and Anx A5/ myoblasts (5) into DM. After 12 h of incubation
of Anx A5/ myoblasts in DM, we applied rA5 (6). Fusion was assayed 2 h later. Fusion in 5 and 6 was normalized to fusion in 4. (D) Myoblasts from
Anx A1/ (2–5) or Anx A5/ (6–9) were cotransfected with GFP vector and with either control siRNA (3 and 7) or siRNA to Anx A1 (4 and 8) or Anx A5
(5 and 9). (2 and 6) Cells transfected only with GFP vector. For each condition, we quantified the efficiency of formation of GFP-labeled syncytia after
24 h in DM and normalized the results to the efficiency of syncytium formation in WT myoblasts (1). (E) Phase-contrast images with nuclear staining (blue) and
GFP fluorescence (green) showing WT myoblasts cotransfected with control siRNA and GFP (left), Anx A1/ myoblasts cotransfected with Anx A5 siRNA
and GFP (middle), or Anx A5/ myoblasts cotransfected with Anx A1 siRNA and GFP (right). All cells are after 24 h in DM. Arrows mark the GFP-labeled
multinucleated cells. Bar, 50 µm. (C and D) All results are shown as means ± SEM (n ≥ 3). Levels of significance are shown: **, P < 0.01; *, P < 0.05.
115 Annexins and dynamin in myoblast fusion • Leikina et al.
Note that DNM2 siRNA experiments were performed without
application of LPC.
Myotube formation was also inhibited by two treatments
targeting PtdIns(4,5)P2, an important regulator of many intra-
cellular processes including DNM function. The cell-permeant
polyphosphoinositide-binding peptide PBP10 binds to PtdIns
(4,5)P2 in the inner leaflet of the plasma membrane and dis-
places cytosolic PtdIns(4,5)P2-binding proteins (Cunningham
et al., 2001). PBP10 applied to myoblasts accumulated at the
LPC-arrested stage at the time of LPC removal strongly in-
hibited syncytium formation but not lipid mixing (Fig. 9,
A and B). We observed similar effects after treating the cells
with the primary alcohol 1-butanol (Fig. 9 C). This alcohol (but
not its isomers t-butanol and 2-butanol) lowers the concentra-
tion of phosphatidic acid and thus inhibits the activity of phos-
phoinositide 5-kinase, which generates PtdIns(4,5)P2 (Boucrot
et al., 2006). Note that phosphatidic acid is an important signal-
ing lipid and thus we cannot exclude 1-butanol effects that are
independent of PtdIns(4,5)P2.
In brief, late fusion stages that generate multinucleated
myotubes depend on cell metabolism, DNM2 GTPase activity,
and PtdIns(4,5)P2 content. Because the ATP-depleting reagents,
and deoxy-d-glucose added to the C2C12 cells (Fig. 7, A and B)
or the primary myoblasts (Fig. 7 C) accumulated at the ready-
to-fuse LPC-arrested stage 5 min before LPC removal inhibited
syncytium formation, but had no effect on lipid mixing.
DNM GTPase is one of many proteins whose function is
affected by ATP depletion (Schwoebel et al., 2002). We found
that the transition from early to late stages of myoblast fusion
for primary myoblasts (Fig. 8, A and B) and C2C12 cells
(Fig. S4 A) is blocked by the inhibitors of DNM GTPase dyna-
sore and myristyl trimethyl ammonium bromide (MiTMAB),
applied at the time of LPC removal. Interestingly, in the experi-
ments on C2C12 cells labeled with either green or orange cell
trackers, we observed no redistribution of the cell tracker–
labeled proteins between the cells treated with DNM inhibitors
(Fig. S4 B), suggesting that these inhibitors block fusion at a
stage that follows hemifusion (detected as lipid mixing) but pre-
cedes formation of fusion pores large enough (a few nanome-
ters in diameter) to pass the labeled proteins.
The importance of DNM2, a DNM isoform expressed in
muscles, in late stages of myoblast fusion was confirmed by inhi-
bition of syncytium formation but not lipid mixing observed for
primary myoblasts transfected with DNM2 siRNA (Fig. 8, C–E).
Figure 5. Synchronized fusion of C2C12 cells is influenced by reagents targeting extracellular Anx A1 and A5. (A) Phase-contrast images with nuclear
staining (blue) showing C2C12 cells that were incubated in DM for 51 h and then in LPC-supplemented DM for 16 h, and, finally, placed into LPC-free DM
(left) or LPC-free DM with antibodies to Anx A1 or A5 (middle and right). The images were taken 30 min after LPC removal. Arrows mark the multinucleated
cells. Bar, 50 µm. (B–D) Antibodies (B), A1- and A5-peptides (C), and EGTA (D) were applied to ready-to-fuse myoblasts at the time of LPC removal (B and C)
or 30 min before LPC removal (D). Fusion was scored 30 (B and C) or 60 (D) min after LPC removal and normalized to fusion in the control experiments
shown (B–D, 1). (B) Lipid mixing (green) and syncytium formation (red) were inhibited by antibodies to Anx A1 (2) and A5 (3) but not by nonspecific IgG
(4). 1, untreated cells released from LPC block. (C) Fusion was inhibited by the peptides to Anx A1 (2) and A5 (4) but not by their scrambled versions
(3 and 5). (D) At the time of LPC removal, the cells were placed into Ca2+- and Mg2+-free LPC-free PBS supplemented with 10 mM EGTA (2–7). (3–5)
30 min after LPC removal, we washed the cells with EGTA-free Ca2+- and Mg2+-containing PBS (3) or with the same buffer supplemented with rA1 (4) or
rA5 (5). (6 and 7) As in 4 and 5, but rA1 (6) and rA5 (7) were applied in Ca2+- and Mg2+-free PBS. (1) At the time of LPC removal, the cells were placed
into Ca2+- and Mg2+-containing PBS. All results are means ± SEM (n ≥ 3). (B–D) Levels of significance relative to 1 in B and C and to 3 in D are shown:
**, P < 0.01; *, P < 0.05.
JCB • VOLUME 200 • NUMBER 1 • 2013 116
this early hemifusion stage is not affected by several reagents
targeting cell metabolism, DNM activity, and lipid regula-
tors of cell function: PtdIns(4,5)P2 and phosphatidic acid. In
contrast, later fusion stages (a merger of the inner leaflets of
the membranes to form a fusion pore or expansion of this
pore) are blocked for ATP-depleted cells and involve DNM
Our findings suggest that myoblast fusion starts with hemi-
fusion. We blocked this stage by supplementing the outer leaf-
lets of the membranes with LPC known to block mammalian
myoblast fusion (Reporter and Raveed, 1973). The finding that
the extents of myoblast fusion within 30–60 min after LPC re-
moval approach the levels observed in the experiments per-
formed without LPC application confirmed the reversibility of
the LPC inhibition. Because all reagents and treatments that we
found to affect the myoblast fusion after lifting LPC block also
affected myotube formation in the experiments without LPC
DNM inhibitors, and PtdIns(4,5)P2-targeting reagents that we
used here are known to act very fast, the finding that lipid mix-
ing is not affected by any of these treatments suggests that by
the time of fusion, the protein machinery that mediates early
fusion stages is located at the cell surface and is not very sensi-
tive to changes in intracellular conditions.
In this work, we uncoupled the fusion stage of myotube for-
mation from the preceding stages of myogenesis that prepare
the muscle precursor cells for fusion. We dissected the path-
way of myoblast fusion into two stages controlled by differ-
ent protein machineries (Fig. 9 D). Extracellular Anx A1 and
A5, and/or possibly their protein partners, merge two mem-
branes with a lipid connection, allowing the mixing of the mem-
brane lipids of the outer leaflets of the membranes. Intriguingly,
Figure 6. Synchronized fusion of primary myoblasts is inhibited by antibodies, A1- and A5-peptides, and the lack of either of these Anxs. (A) Antibodies
to Anx A1 (2) or A5 (3) or nonspecific IgG (4) were applied at the time of LPC removal. (1) Cells released from LPC block with no immunoglobulins added.
(B) A1- and A5-peptides (2 and 3) were applied at the time of LPC removal. (A and B) Lipid mixing (green) and syncytium formation (red) were scored
30 min after LPC removal and normalized to those in the control experiments (1). (C) The extents of lipid mixing (left) and syncytium formation (right) observed
at different times after LPC removal at t = 0 for WT, Anx A1/, and Anx A5/ (curves 1,2, 3, respectively) myoblasts. (D) Phase-contrast images with
nuclear staining (blue) showing WT, Anx A1/, and Anx A5/ myoblasts 30 min after LPC removal. (bottom) Anx A1/ myoblasts with rA1 (left) and
Anx A5/ myoblasts with rA5 (right). Anxs were applied at the time of LPC removal. Arrows mark the multinucleated cells. Bar, 50 µm. (E) rA1 and rA5
restore fusogenic potential of Anx-deficient myoblasts. Lipid mixing (green) and syncytium formation (red) in Anx A1/ (2 and 3) or Anx A5/ (5 and 6)
myoblasts were scored 30 min after LPC removal in the absence (2 and 5) or in the presence of rA1 (3) or rA5 (6). Fusion extents were normalized to
those observed for Anx A1/ (1) and Anx A5/ (4) myoblasts 24 h after placement of the cells into DM in the experiments in which myogenesis was not
interrupted by LPC. All results are shown as means ± SEM (n ≥ 3). Levels of significance relative to corresponding controls (1 in A and B and 1 or 4 in E)
are shown: **, P < 0.01; *, P < 0.05.
Annexins and dynamin in myoblast fusion • Leikina et al.
et al., 2011). Although the specific mechanisms by which DNM
and PtdIns(4,5)P2 control syncytium formation remain to be
identified, our work uncovers the existence of a controlling
mechanism that determines whether early fusion connections
expand to generate a multinucleated myotube.
The initial merger of myoblast membranes involves extra-
cellular Anx A1 and A5. We found elevated concentrations of
these Anxs at the outer surface of murine myoblasts at the time
of fusion. Recombinant Anxs promoted myoblast fusion and
thus facilitated fusion of cells that would normally not fuse by
this time. In contrast, antibodies and A1- and A5- peptides, as
well as lowering or abolishing the expression of either of these
Anxs, inhibited myoblast fusion. All these modifications of the
Anx activity had much stronger effects on the synchronized
myoblast fusion than on myotube formation in the experiments
without LPC application, and at earlier times after placement of
the cells into DM than at the later times. These differences most
likely reflect the fact that under normal conditions the cell fu-
sion step takes a small fraction of the total time in DM required
for myotube formation.
Although our results suggest the important role of Anx
A1 and A5 in myoblast fusion and myogenesis, Anx A1–
(Hannon et al., 2003), Anx A5– (Brachvogel et al., 2003), and
both Anx A5– and A6–deficient mice (Belluoccio et al., 2010)
are viable and fertile. None of those studies reported any al-
terations in the development of the musculoskeletal system
application, it is unlikely that LPC modifies rather than merely
blocks the fusion pathway. LPC has been reported to inhibit
many disparate membrane fusion processes (Chernomordik and
Kozlov, 2005), and thus synchronization of cell fusion using re-
versible LPC block can be of help in uncoupling the fusion
stage from the stages that prepare the cells for fusion in other
The cell machinery that drives the expansion of nascent
membrane connections remains to be identified for any cell-
to-cell fusion process. Our findings indicate that the transition
from early hemifusion connections between myoblast mem-
branes to opening and expansion of fusion pores is dependent
on cell metabolism and controlled by intracellular machinery.
As in cell-to-cell fusion mediated by viral fusogens (Richard
et al., 2011), late stages of myoblast fusion involve DNM
GTPase and are inhibited by lowering of the membrane con-
centration of accessible PtdIns(4,5)P2. Our findings are consis-
tent with earlier ones that contact zones between C2C12 cells at
the time of fusion are enriched in PtdIns(4,5)P2 (Nowak et al.,
2009) and that a decrease in PtdIns(4,5)P2 content inhibits
myotube formation (Bach et al., 2010). Our work identifies the
PtdIns(4,5)P2-dependent stage of myoblast fusion as the one
downstream of a local membrane merger. PtdIns(4,5)P2 can in-
fluence myotube formation by regulating DNM activity or the
function of numerous other PtdIns(4,5)P2 binding proteins such
as GRAF1, which is implicated in myoblast fusion (Doherty
Figure 7. The transition from lipid mixing to syncytium
formation depends on cell metabolism. (A and B) A mix of NaN3
and 2-d-deoxyglucose was applied to fusion-committed C2C12
cells 5 min before LPC removal. (A) Images of the cells treated
(bottom) and untreated (top) with ATP-depleting mix were
taken 30 min after LPC removal. Left, phase contrast with
nuclear staining; right, DiI (red) and green cell tracker. Red
and white arrows mark the multinucleated cells and colabeled
mononucleated cells, respectively. Bar, 50 µm. (B and C) In
contrast to lipid mixing (green), syncytium formation (red) is
blocked by ATP depletion for both C2C12 cells (B) and pri-
mary myoblasts (C). Fusion extents for the ATP-depleted cells
(2) were normalized to those for the untreated cells released
from LPC block (1). All results are shown as means ± SEM
(n ≥ 3). Levels of significance relative to controls (1) are
shown: **, P < 0.01.
JCB • VOLUME 200 • NUMBER 1 • 2013 118
Anx-dependent myoblast fusion is likely regulated by
PS exposure, by an increase in the local concentration of
membrane-bound Anx A1 and A5, and/or by mechanisms es-
tablishing close cell contacts. Anx A1 and A5 interact with
many cell surface proteins (Moss and Morgan, 2004) and can
control myoblast fusion by influencing the activities of other,
as yet unidentified, proteins. Alternatively, Anxs can directly
mediate membrane fusion. Indeed some Anxs, including A1,
have been implicated in intracellular fusion processes (Kubista
et al., 2000; McNeil et al., 2006) and are known to aggregate
and fuse PS-containing liposomes (Francis et al., 1992; Bitto
and Cho, 1999). Because Anx A5, on its own, is inefficient
in inducing liposome aggregation and lipid mixing (Hoekstra
et al., 1993; Bitto and Cho, 1999), fusogenic activity of this
Anx can depend on membrane–membrane binding mediated
by other proteins, as reported for reovirus fusion machinery
(Salsman et al., 2008).
Some prefusion stages of myogenesis may also depend on
Anxs (Bizzarro et al., 2010) and be disrupted by an excess of
extracellular Anx (van den Eijnde et al., 2001; Kaspar and
Dvorák, 2008; Jeong and Conboy, 2011). We also found that
and specifically examined any characteristics of skeletal mus-
cle. In our experiments, the lack of Anx A1 or A5 substan-
tially inhibited synchronized myoblast fusion and slowed
down myotube formation, but did not completely block either
process, suggesting a functional redundancy between these
Anxs. Indeed, we found that a shortage of one of these Anxs
can be compensated for by application of a recombinant ver-
sion of another one. Furthermore, transfecting Anx A1/
myoblasts with A5 siRNA, or vice versa, and transfecting
Anx A5/ myoblasts with A1 siRNA resulted in a very potent
inhibition of myotube formation. Although our work has
focused on the contributions of Anx A1 and A5 in murine
myoblast fusion, other representatives of the Anx family are
also expressed in fusing myoblasts (Clemen et al., 1999;
Tannu et al., 2004; Kislinger et al., 2005) and can be involved
in fusion, especially in the absence of either A1 or A5 and for
other animals (Duan, 2008). We have systematically studied
only Anx A1 and A5 but found that siRNA knockdown of Anx
A2 did not inhibit myotube formation for Anx A1/ myo-
blasts (Fig. S5 A), suggesting that Anx A2 does not play an
important role in fusion in murine myoblasts.
Figure 8. In contrast to lipid mixing, syncy-
tium formation by primary myoblasts depends
on DNM activity. (A and B) After incubation for
16 h in LPC, cells were washed to remove LPC
block to cell fusion and then treated immedi-
ately with vehicle (DMSO) as a control or with
DNM inhibitors (100 µM dynasore or 10 µM
MitMAB). (A) Images of the cells treated with
either dynasore (middle) or MitMAB (right)
and vehicle-treated cells (left) were taken
30 min after LPC removal. (top) Phase contrast
with nuclear staining; red arrows mark the mul-
tinucleated cells. (bottom) DiI (red) and green
cell tracker; white arrows mark the colabeled
mononucleated cells. Bar, 50 µm. (B) 50 and
100 µM dynasore (2 and 3) and 2.5 and 10 µM
MitMAB (4 and 5) inhibit syncytium formation
(red) but do not inhibit lipid mixing (green).
Fusion extents were normalized to those for the
vehicle-treated cells released from LPC block
(1). (C) Syncytium formation (red) and lipid
mixing (green) for myoblasts transfected with
control siRNA (1), with 50 (2), and with 100
(3) pM of DNM2 siRNA. Fusion extents were
assayed after 40 h in DM and normalized
to those for the cells transfected with control
siRNA (1). (B and C) All results are shown as
means ± SEM (n ≥ 3). Levels of significance
relative to controls (1) are shown: **, P <
0.01; *, P < 0.05. (D) The cells transfected
with control or DNM2 siRNAs were lysed after
40 h in DM and analyzed by Western blotting
to evaluate levels of expression of DNM2 and
tubulin (a loading control). (E) Images of the
cells transfected with 100 pM of the control
siRNA (1 and 2) and DNM2 siRNA (3 and 4).
Images were taken after 40 h in DM. Images 1
and 3 show phase contrast with nuclear stain-
ing (blue) and images 2 and 4 show DiI (red)
and green cell tracker. Red arrows (1 and 3)
and white arrows (4) mark the multinucleated
cells and colabeled mononucleated cells, re-
spectively. Bar, 50 µm.
119 Annexins and dynamin in myoblast fusion • Leikina et al.
for synchronized myoblast fusion and, thus, at the fusion stage
of myogenesis. We dissected myoblast fusion into two distinct
stages with an early stage of membrane merger involving ex-
tracellular Anx A1 and A5 and subsequent syncytium forma-
tion dependent on DNM activity and PtdIns(4,5)P2 content.
Interestingly, some mutations in DNM2 and myotubularin, a
protein involved in turnover of phosphoinositides, cause cen-
tronuclear myopathies characterized by an abnormal localiza-
tion of cell nuclei and, possibly, are related to a delay in
muscle fiber maturation (Spiro et al., 1966; Bitoun et al., 2005;
Hnia et al., 2012).
Materials and methods
rA1 and A5 were either purchased from US Biological or BD, respectively,
or expressed in Escherichia coli (BL21DE3) and purified in-house. Anx A1
plasmid DNA (Blume et al., 2009) was a gift from K. Lauber (University of
Tübingen, Tübingen, Germany) and Anx A5 plasmid (plasmid pET12a-
PAPI) was purchased from Addgene. The cells grown in LB medium in the
presence of 100 mg/ml ampicillin and induced with 1 mM IPTG for protein
expression were lysed by sonication. In the case of Anx A1 we followed
the protocol described in Logue et al. (2009). The soluble fraction of the
cell extract was incubated overnight with Ni-NTA agarose (QIAGEN).
After three washes with buffer containing 25 mM imidazole, 6-His–tagged
Anx A1 was eluted with the buffer containing 100 mM imidazole. The
eluted protein was dialyzed overnight in PBS, pH 7.4, without imidazole
and then treated with biotinylated thrombin to cleave the N-terminal His
tag; the thrombin was removed by incubating the protein with streptavidin
agarose. In the case of Anx A5, we followed Elegbede et al. (2006). The
overexpressed protein was purified from the inclusion bodies. The sonica-
tion was performed in the presence of 5 mM Ca2+. Anx A5 binds to
anionic lipids of the broken cell membranes and ends up in the insoluble
fraction of the cell lysate. The cell pellet containing Anx A5 was dissolved
in a buffer containing 6 M urea and 6 mM EDTA and left to stir overnight
application of rA1 or rA5 at the time of placement of the cells
into DM rather than later, as in the experiments presented here,
inhibited rather than promoted myotube formation.
Earlier work has indicated that formation of multinu-
cleated myotubes is accompanied by changes in membrane
concentrations of some lipids (e.g., cholesterol and polyphos-
phoinositides; Wakelam, 1985), in fatty acid contents of the
membranes (Yin et al., 2009), in lipid domain organization
of the plasma membrane (Mukai et al., 2009), and in the dis-
tribution of some lipids (e.g., PS) between different leaflets
of plasma membrane (van den Eijnde et al., 2001). Our find-
ings further emphasize the importance of membrane lipids
in the cell fusion stage of myogenesis, suggesting that PS
exposure recruits extracellular proteins involved in fusion,
LPC blocks membrane merger, and PtdIns(4,5)P2 controls
late stages of fusion.
Some of the features of myoblast fusion that are essential
for Anx-involving fusion processes have also been reported for
other developmental cell–cell fusion processes. Macrophage
fusion in giant cell and osteoclast formation, sperm–egg fusion,
and cytotrophoblast fusion in placental morphogenesis all in-
volve transient exposure of PS in the outer leaflet of the plasma
membrane and depend on extracellular Ca2+ (Yanagimachi,
1978; Jin et al., 1990; Gauster and Huppertz, 2008). Although it
is widely assumed that each of these fusion processes is driven
by a specific protein fusogen, it is tempting to hypothesize that
some of these relatively slow fusions between PS-exposing
cells involve ubiquitous extracellular Anxs.
To summarize, the LPC-block approach developed in
this work can be used for identification of proteins required
Figure 9. The transition from lipid mixing to
syncytium formation by C2C12 cells depends
on PtdIns(4,5)P content. (A) Images of the
fusion-committed cells accumulated by LPC
block treated with PtdIns(4,5)P-binding PBP10
(7.5 µM) applied at the time of LPC removal
(right) or the untreated cells released from
LPC block (left). Images show DiI (red) and
green cell tracker fluorescence and were taken
30 min after LPC removal. Notched arrows
mark the multinucleated cells and smaller ar-
rows mark the colabeled mononucleated cells.
Bar, 50 µm. (B and C) Reagents lowering the
concentration of accessible PtdIns(4,5)P2 in
the plasma membrane inhibit fusion of C2C12
myoblasts assayed as lipid mixing (green) and
myotube formation (red). We applied 5 and
7.5 µM PBP10 (B, 2 and 3) as well as 1%
1-butanol and, in the control experiments, its
inactive isomers 2-butanol and t-butanol (C,
2, 3, and 4, respectively) at the time of LPC
removal. Lipid mixing and syncytium forma-
tion extents were normalized to those for the
untreated cells released from LPC block (B and
C, 1; presented as means ± SEM; n ≥ 3). Lev-
els of significance relative to controls (1) are
shown: **, P < 0.01; *, P < 0.05. (D) The pro-
posed pathway of the fusion stage of myotube
formation. Anx A1 and A5 at the PS-exposing
surface of fusion-committed myoblasts directly
or via other proteins initiate membrane merger.
Subsequent stages of syncytium formation are
controlled by DNM- and PtdIns(4,5)P2-dependent
intracellular protein machinery.
JCB • VOLUME 200 • NUMBER 1 • 2013 120
at 1,000 rpm for 5 min, and cell pellet was dissociated in 10 ml of F10 me-
dium (Invitrogen) supplemented with 10 ng/ml of basic fibroblast growth fac-
tor (PeproTech) and 10% cosmic calf serum (Hyclone; referred to as growth
medium 1 [GM1]). Finally, the cells were preplated onto a normal tissue cul-
ture dish twice for 1 h to deplete the population of fibroblasts, which generally
adhere faster than myoblasts. The preplating was repeated during the follow-
ing four passages. Starting from the fifth passage, the cells were used in the
experiments. The minimal myogenic purity of our primary myoblast cultures as
assessed by immunofluorescence using rabbit antibodies to the muscle cell
marker desmin (Abcam) exceeded 95%. We compared time courses of myo-
tube formation and the extents of inhibition of myotube formation by A1 and
A5 siRNAs for multiple (three to five) independently isolated WT cultures and
found no major differences between different isolates. Based on these results,
each of the primary myoblast cultures used in the experiments (WT, Anx
A1/, and Anx A5/) represented a single independent isolate originated
by pooling together cells from three to four pups.
Mouse-derived C2C12 myoblasts (Blau et al., 1983; American Type
Culture Collection) were cultured in DMEM (Invitrogen) containing 2 mM
l-glutamine supplemented with antibiotics (10,000 U/ml penicillin and
10 mg/ml streptomycin) and 10% heat-inactivated fetal calf serum, re-
ferred to as growth medium 2 (GM2).
To induce differentiation, primary myoblasts and C2C12 cells at
75% confluency were placed into DMEM containing antibiotics and 5%
horse serum (Invitrogen), referred to as DM.
We labeled cells with DiI, green, and orange cell tracker, as recommended
by the manufacturer. C2C12 cells were labeled 48 h after placement in
DM. Differently labeled cells were allowed to attach for 2–3 h after coplat-
ing before application of LPC or other reagents. Fusion was scored 16 h
later, i.e., 67 h after triggering of myogenic differentiation.
In the experiments on the time course of myogenesis for primary
myoblasts, we labeled proliferating cells in GM1, lifted them with versene,
and coplated differently labeled cells in DM to start the myogenesis (t = 0).
To synchronize fusion of primary myoblasts, 7 h later we placed the cells
into LPC-supplemented DM for 16 h, and then applied LPC-free DM supple-
mented or not with different reagents.
A 10-mg/ml stock solution of LPC was freshly prepared in water. To revers-
ibly block fusion and accumulate ready-to-fuse myoblasts, we incubated
the cells in DM supplemented with 300 µM LPC. Fusion ensued when LPC
was removed in three washes with LPC-free DM. The membrane-inserted
LPC that mediates fusion inhibition is rapidly internalized and metabolized,
and because we needed to keep the fusion blocked for 16 h, we used lau-
royl LPC to maintain a continuous supply of our lipid inhibitor in the me-
dium. Importantly, the relatively high solubility of lauroyl LPC in water also
facilitated its removal and thus the removal of the fusion block by washing
the cells with LPC-free medium.
Application of reagents
In the experiments on synchronized fusion, different reagents were added to
the LPC-free medium in which the cells were kept after LPC withdrawal. In the
experiments in which we did not block the fusion stage with LPC, the re-
agents were applied 16 or 3 h before scoring fusion for C2C12 cells and
primary myoblasts, respectively. We used antibodies to Anx A1 at 2.5 µg/ml;
antibodies to Anx A5 or control IgG both at 10 µg/ml; A1- and A5-
peptides and their scrambled versions at 100 µg/ml; and rA1 and rA5 at
50 µg/ml. Inhibitor of DNM GTPase dynasore was used at 80 µM for C2C12
cells and 50 or 100 µM for primary myoblasts. Another DNM inhibitor, MiT-
MAB, was used at 25 µM for C2C12 cells and 2.5 or 10 µM for primary
myoblasts. In the experiments with reagents lowering the concentration of
plasma membrane PtdIns(4,5)P2 accessible for interactions with intracellular
proteins, we used 5 or 7.5 µM PBP10 and 1% 1-butanol or its inactive iso-
mers 2-butanol and t-butanol. To explore the effects of intracellular ATP deple-
tion on the synchronized fusion stage, we applied a mix of 10 mM sodium
azide and 20 mM 2-d-deoxyglucose in DM or in PBS to fusion-committed pri-
mary myoblasts or C2C12 cells, respectively, in the presence of LPC 5 min
before LPC removal. EGTA was applied to C2C12 cells 30 min before LPC
removal as Ca2+- and Mg2+-free LPC-containing PBS supplemented with
10 mM EGTA. At the time of LPC removal, we replaced this buffer with Ca2+-
and Mg2+-free LPC-containing PBS supplemented with 10 mM EGTA.
Cell fusion assays
To quantify the efficiency of myoblast fusion, we fixed the cells in phos-
phate buffered 10% wt/vol formalin solution (Electron Microscopy Sciences).
at room temperature. The suspension was centrifuged and the soluble frac-
tion was diluted 10-fold as in a snap-dilution method in the refolding buffer
containing 5mM Ca2+. This solution was left to stir overnight at 4°C, produc-
ing a cloudy suspension of the protein. The resulting cloudy suspension was
centrifuged and the pellet was resuspended in the extraction buffer contain-
ing 10 mM EDTA and 10 mM EGTA. The pellet was stirred for 4 h at room
temperature and then centrifuged. The supernatant containing Anx A5 was
extensively dialyzed. Purified Anxs were aliquoted and stored at 80°C.
Synthetic peptide mimicking N-terminal regions of human Anx
A1 (2–26; Ac-AMVSEFLKQAWFIENEEQEYVQTVK), peptide with the
same amino acid composition but a scrambled sequence (Ac-EMQS-
NAAVQYVEIKTWLEFEVKEQF), and peptide mimicking the N-terminal re-
gion of human Anx A5 (2–20; Ac-AQVLRGTVTDFPGFDERAD) and its
scrambled version (Ac-LVATGGAVRPEDTFDRQDF) were custom synthe-
sized by AnaSpec. Neither the A1- nor the A5-peptide affected fusion of
those myoblasts that did not express the corresponding Anx (Fig. S5 B).
The siRNA against murine Anx A1 and A5, DNM2, and control siRNA, a
scrambled sequence not causing the specific degradation of any cellular
message, were purchased from Santa Cruz Biotechnology, Inc. Rabbit
polyclonal antibodies to Anx A1and A5 used in functional and immunofluor-
escence experiments were purchased from Abcam. We used primary
myoblasts from Anx A1– (Hannon et al., 2003) and Anx A5–deficient
(Brachvogel et al., 2003) mice to verify that these antibodies to Anx A1
and A5 have no cross-reactivity in immunofluorescence and in Western
blots. Non-specific rabbit polyclonal IgG and antibodies to -tubulin were
purchased from Abcam. The rabbit polyclonal anti–Anx A1 antibody used
in Western blots was purchased from Santa Cruz Biotechnology, Inc.
The fluorescent lipophilic tracers Vybrant DiI and membrane-permeant
green CMFDA and orange CMRA cell trackers were purchased from
Molecular Probes. LPC (1-lauroyl-2-hydroxy-sn-glycero-3-phosphocholine)
was purchased from Avanti Polar Lipids, Inc. 1-butanol, 2-butanol, t-butanol,
sodium azide, 2-d-deoxyglucose, and DNM GTPase inhibitors dynasore
(Macia et al., 2006) and MiTMAB (Quan et al., 2007) were purchased
from Sigma-Aldrich. The polyphosphoinositide binding peptide PBP10, a
rhodamine B-tagged 10-residue peptide derived from the PtdIns(4,5)P2
binding region in segment 2 of gelsolin (Cunningham et al., 2001), was
purchased from EMD Millipore.
National Institutes of Health and Public Health Service policy was followed
for all animal research, which was approved by the Eunice Kennedy
Shriver National Institute of Child Health and Human Development Animal
Care and Use Committee. Anx A1 knockout animals (Hannon et al., 2003)
were purchased from Charles River. These animals carry an insertion/deletion
mutation that interrupts exon 2 and deletes exons 3 and 4 of Anx A1. Anx
A5 knockout animals (Brachvogel et al., 2003) were a gift of E. Pöschl
(University of East Anglia, Norwich, UK) and B. Brachvogel (University of
Cologne, Cologne, Germany) and carry an insertion/deletion mutation
that interrupts exon 3 and deletes exon 4 of Anx A5. Both mouse strains
are in a C57BL/6 background. WT C57BL/6 mice were obtained from
National Cancer Institute, Frederick. To generate offspring for the in vitro
culture of primary mouse myoblasts, matings were set up for each geno-
type. To confirm the homozygous deletion of the Anx A1 and A5 genes in
the offspring of Anx A1/ mice and Anx A5/ mice, we performed toe
clipping at P4 and extracted genomic DNA using Direct PCR (Tail) solution
(Viagen Biotech) and Proteinase K (Roche). Anx A1/ genotyping was
performed by individual PCRs for the WT and Anx A1/ genotypes. We
used primers 5-GCCTTGCACAAAGCTATCATGG-3 and 5-GCATTG-
GTCCTCTTGGTAAGAATG-3 to generate a 700-bp product specific to the
WT allele and primers 5-TACTGTCGTCGTCCCCTCAAACTG-3 and
5-GTTGCACCACAGATGAAACGC-3 to detect a 220-bp amplicon spe-
cific to the mutant allele (Hannon et al., 2003). Standard PCR reactions
and conditions were also used to genotype the Anx A5 deletion using the
following primer pairs (suggested by E. Pöschl): WT, 5-TGGGGAGAGA-
CTTGCCAAC-3 (Anxa5-Int2) and 5-AATTAAACGTTACCCAAGCC-3
(Anxa5-Int/Ex3.rev); knockout, 5-TGGGGAGAGACTTGCCAAC-3
(Anxa5-Int2) and 5-TGCGGGCCTCTTCGCTATTACG-3 (Anxa5-LacZ.
rev). Pups were killed at postnatal day 6 to dissect neonatal muscle tissue.
We isolated primary myoblasts from the forelimbs and hindlimbs of three to
four 5-d-old pups of the same littermate as described previously (Bois and
Grosveld, 2003). The dissected/minced muscle was enzymatically disaggre-
gated in 6 ml PBS including 1.5 U/ml dispase II and 1.4 U/ml collagenase
D (Roche) and triturated with a 10-ml pipette every 5 min for 20 min at 37°C.
Next, the cells filtered through 70-µm mesh (BD) were collected by pelleting
121 Annexins and dynamin in myoblast fusion • Leikina et al.
Immunofluorescence assay for myogenin and MHC
To analyze the levels of expression of myogenin and MHC, the cells were
fixed for 10 min at 37°C in 4% formaldehyde in phosphate buffer with 5%
FBS and, after four washes with PBS with 5% FBS, permeabilized in 0.1%
Triton/PBS solution for 3 min at room temperature. Cells were incubated
with primary antibodies (1:1,000) for an hour at room temperature,
washed four times with PBS, and then incubated with secondary antibodies
(Alexa Fluor 488 goat anti–rabbit IgG; Invitrogen; 1:1,000 dilution) for an
hour and finally washed four times with PBS. Images were recorded either
on a confocal microscope (LSM 510; Carl Zeiss) using manufacturer-
supplied software package or using Micro-Manager software package on
AxiObserver.D1 (Carl Zeiss) inverted microscope equipped with iXon 885
EMCCD camera and pE-2 LED light source (CoolLed). In both cases Plan-
Apochromat 20×/0.8 (Carl Zeiss) objective lens was used. Images were
analyzed in ImageJ. To create frequency distribution of myogenin expres-
sion level within cell nuclei, we used Hoechst staining to automatically cre-
ate a region of interest for each cell nucleus and measure average pixel
intensity for anti-myogenin staining within these regions. Anti-myogenin
signal was corrected for background using ImageJ built-in rolling-ball algo-
rithm with the radius parameter equal twice the diameter of the largest cell
nucleus. Because cytosolic distribution of MHC made automated analysis
of images significantly more difficult, we do not present cell distribution
analysis for MHC labeling.
To immunoblot Anxs, the culture plates were incubated on ice for 15 min.
Then the cells were washed with ice-cold PBS. The cells were lysed with
RIPA buffer containing protease inhibitor cocktail (Sigma-Aldrich). The ly-
sate was transferred into an Eppendorf tube, vortexed for 60 s, and cen-
trifuged for 10 min at 15,000 rpm. The soluble fraction of the lysate was
added to the denaturing sample buffer (Invitrogen), boiled, loaded onto a
4–12% NuPAGE Bis-Tris gel (Invitrogen) or 3–8% NuPAGE Tris-acetate gel
(Invitrogen), and separated with SDS-PAGE. The separated proteins were
transferred electrophoretically to a PVDF membrane (Invitrogen). After the
transfer, the membrane was blocked with 5% nonfat dry milk dissolved in
PBS containing 0.05% Tween 20 (Sigma-Aldrich). The membrane was in-
cubated at room temperature for 1 h with the rabbit polyclonal anti-Anx A1
and rabbit polyclonal anti-Anx A5 antibodies. After incubation, the mem-
brane was washed three times with PBS containing 0.05% Tween 20, and
then incubated with alkaline phosphatase–conjugated goat anti–rabbit IgG
(Thermo Fisher Scientific). The membrane was washed three times again
with PBS containing 0.05% Tween 20, and the protein bands were visual-
ized with enhanced chemifluorescence (ECF reagent; GE Healthcare). To
immunoblot DNM2, we lysed the cells for 1 h at room temperature with
0.1 Triton X-100 and 0.2% SDS in phosphate buffer in the presence of
protease inhibitors and used 3–8% NuPAGE Tris-acetate gel.
Proliferating cells coplated in 35 × 10 collagen-covered dishes (BD) in
GM1 (primary myoblasts) or GM2 (C2C12 cells) at 80% confluency were
transfected with siRNAs (100 pM/plate) with or without GFP vector
(0.5 µg/plate) or with GFP vector only using Lipofectamine 2000 (Invitro-
gen), as recommended by the manufacturer. The cells were placed into DM
16 h later. Fusion was scored 24 or 36 h later for primary and C2C12
cells, respectively. In some experiments, we then lysed the cells and ana-
lyzed the lysates using quantitative Western blotting. We verified that A1
siRNA had no significant effect on expression of Anx A5, and vice versa.
In the experiments with DNM2 siRNA, we incubated primary myoblasts for
16 h in DM, labeled them, and then transfected coplated cells and let the
cells continue differentiation in DM. Fusion was scored 24 h later.
Analysis and presentation of experimental data
We prepared graphs and performed statistical analyses using Sigmaplot
v.11.0 (Systat Software). We compared normally distributed data using
the unpaired Student’s t test, and when the data were not normally dis-
tributed or failed the equal variance test, we used the Mann-Whitney rank
sum test instead. Data are presented as the means ± SEM with the number
of experiments stated.
Online supplemental material
Fig. S1 shows that neither deficiency of Anx A1 or A5 in primary myo-
blasts nor 16-h incubation of WT myoblasts in the presence of LPC blocks
myogenic differentiation assayed as expression of myogeninin and MHC.
Fig. S2 shows promotion of myoblast fusion by rA1 and rA5. Fig. S3
shows elevation of the amounts of Anx A1 and A5 at the outer surface of
Cell nuclei were labeled with Hoechst-33342 (Molecular Probes). Images
were taken at room temperature in PBS on Axioskop microscope (Carl
Zeiss) equipped with 20×/0.3 LD A-Plan objective lens (Carl Zeiss) and
ORCA C4742-98 charge-coupled device camera (Hamamatsu Photonics)
using MetaMorph 6.1 software (Molecular Devices). We prepared and
analyzed images using ImageJ (National Institutes of Health). For each
condition we took images of 10 randomly chosen fields of view (the total
numbers of nuclei per condition averaged 800–1,000 for C2C12 cells
and 1,000–1,800 for primary myoblasts). The efficiency of myotube for-
mation was quantified as the percentage of cell nuclei present in myotubes
normalized to the total number of cell nuclei. We also assayed fusion as
the redistribution of DiI and green cell tracker between differently labeled
cells and presented it as the percentage of nuclei in colabeled cells (includ-
ing both mono- and multinucleated cells) compared with the number of
contacts of differently labeled cells. The percentage of nuclei in multinucle-
ated cells (syncytium formation assay) and the percentage of nuclei in co-
labeled cells (lipid mixing assay) after different treatments were normalized
to those in the parallel control experiments (20–50% for both C2C12 cells
and WT primary myoblasts).
In the experiments with the primary myoblasts that were cotrans-
fected with GFP vector and siRNAs, we separately evaluated the efficiency
of cell fusion (myotube formation) for transfected cells and, within the same
plate, cells that were not transfected. For each field of view, we separately
scored fusion for GFP-labeled (transfected) and unlabeled (nontransfected)
cells. For each condition, the percentage P of cell nuclei in the mononucle-
ated cells compared with the total number of cell nuclei in the field of view
was used to estimate the efficiency of myotube formation as (100-P)%.
In each experiment on synchronized fusion, we had controls in
which we measured fusion extents 30 min after application of LPC (if not
stated otherwise) and fusion extents observed if LPC was not washed out
(FLPCon). We also measured fusion extents for the cells that were released
from LPC block without being treated with the reagents studied (Fc). FLPCon
and Fc varied from day to day in the 6–10% and 20–40% range, respec-
tively. To normalize the data for each condition, we subtracted FLPCon from
the measured fusion extents, divided the result by (Fc FLPCon), and multi-
plied by 100 to present as a percentage.
Time-lapse images were recorded every 2 min using an iXon 885 EMCCD
camera (Andor Technology) and Micro-Manager software package
(Edelstein et al., 2010) on an AxiObserver D1 inverted microscope (Carl
Zeiss) equipped with a 10×/0.45 Ph1 Plan-Apochromat objective lens
(Carl Zeiss) and 617-nm LED transmitted light source (Mightex Systems).
Temperature was maintained at 37°C using DH-35iL culture dish incubator
(Warner Instruments) under constant stream of 95% air/5% CO2 gas mix-
ture. Image sequences were converted into uncompressed avi files using
ImageJ 1.47d (National Institutes of Health) and then compressed into mp4
files using Handbrake 0.9.8 open source software.
Immunofluorescence assay for cell surface–bound Anx 1 and Anx 5
The cells were incubated with primary antibodies in DM for 2 h at 4°C,
washed four times with cold PBS supplemented with 5% FBS, and then
fixed for 10 min at 37°C in 4% formaldehyde in phosphate buffer with 5%
FBS. After four washes with PBS supplemented with 5% FBS, the cells were
incubated with secondary antibodies (Alexa Fluor 488 goat anti–rabbit
IgG; Invitrogen; 1:300 dilution) for an hour followed by four washes with
PBS. Fixed cells were imaged using an inverted fluorescence microscope
(Eclipse Ti; Nikon) equipped with a 60×, 1.49 NA objective (Nikon), TIRF-
illumination arm, custom-built laser combiner (405, 488, 561, and 640 nm;
Coherent), and Ixon 885 EMCCD camera. Microscope, lasers, and cam-
era were controlled using Micro-Manager 1.4.10. The incident angle of
the laser beam was set by a motorized TIRF unit to wide-field illumination
(90°). Fluorescence and transmitted light channels were collected using al-
ternating 488-nm laser and transmitted light sources through a quad-band
dichroic and emission filter set (405/488/561/640; Semrock). 30 frames
were acquired for each field of view and averaged together to increase
signal to noise ratio. Images were manually thresholded to separate cells
from cell-free regions (the same threshold was used for all experimental
conditions). The median fluorescence signal from the cells was corrected
for background by subtraction of the median fluorescence signal from cell-
free regions. These values were further corrected for nonspecific antibody
binding by subtraction of the background corrected signal measured in a
sample incubated with labeled secondary antibodies but not with Anx-
specific primary antibodies. Finally, these values were normalized to a sig-
nal observed for cells not induced to myoblast differentiation.
JCB • VOLUME 200 • NUMBER 1 • 2013 122
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The Anx A5 knockout mice were provided by Drs. Ernst Pöschl and Bent
Brachvogel, and Anx A1 plasmid DNA was a gift from Dr. Kirsten Lauber.
The authors thank Drs. Alexander Grinberg and Joshua Zimmerberg for help-
This research was supported by the Intramural Research Program of the
Eunice Kennedy Shriver National Institute of Child Health and Human Develop-
ment, National Institutes of Health.
Submitted: 2 July 2012
Accepted: 5 December 2012
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