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Annual Review of Genetics
Drosophila Myoblast Fusion:
Invasion and Resistance
for the Ultimate Union
Donghoon M. Lee1and Elizabeth H. Chen1,2
1Department of Molecular Biology,University of Texas Southwestern Medical Center,
Dallas, Texas 75390, USA; email: Elizabeth.Chen@UTSouthwestern.edu
2Department of Cell Biology,University of Texas Southwestern Medical Center, Dallas,
Texas 75390, USA
Annu. Rev. Genet. 2019. 53:2.1–2.25
The Annual Review of Genetics is online at
genet.annualreviews.org
https://doi.org/10.1146/annurev-genet-120116-
024603
Copyright © 2019 by Annual Reviews.
All rights reserved
Keywords
myoblast fusion, cell–cell fusion, Drosophila genetics, asymmetric fusogenic
synapse, actin-propelled membrane protrusions, mechanosensory response,
mechanical tension
Abstract
Cell–cell fusion is indispensable for creating life and building syncytial tis-
sues and organs. Ever since the discovery of cell–cell fusion, how cells join
together to form zygotes and multinucleated syncytia has remained a funda-
mental question in cell and developmental biology.In the past two decades,
Drosophila myoblast fusion has been used as a powerful genetic model to
unravel mechanisms underlying cell–cell fusion in vivo. Many evolutionar-
ily conserved fusion-promoting factors have been identied and so has a
surprising and conserved cellular mechanism. In this review, we revisit key
ndings in Drosophila myoblast fusion and highlight the critical roles of cel-
lular invasion and resistance in driving cell membrane fusion.
.
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INTRODUCTION
Cell–cell fusion is a fascinating process underlying fertilization, skeletal muscle development and
regeneration, bone remodeling, immune response, and placenta formation (2, 28, 126). Failure
in cell–cell fusion leads to defects such as infertility,congenital myopathy, osteopetrosis,immune
deciency, and pre-eclampsia. Despite the diversity of cell types that undergo fusion, all cell–cell
fusion events commence from the recognition and adhesion of two fusion partners and end with
the merging of their plasma membranes and union of their cytoplasm.
As with any membrane fusion event, the rate-limiting step of cell–cell fusion is bringing the
two membranes destined for fusion into close proximity of one another,thus allowing lipid mixing
and fusion pore formation (68). Cell adhesion molecules (CAMs) are obvious facilitators for cell–
cell fusion, given their function as velcro between cell membranes. However, numerous cell–cell
junctions exist in multicellular organisms between cells that do not fuse, suggesting that cell fusion
is a tightly regulated process beyond cell adhesion and that additional cellular machineries must
be involved to promote membrane juxtaposition and merger.
For the past two decades, Drosophila myoblast fusion has been used as a powerful genetic model
to study cell–cell fusion in vivo (1, 33, 77,90, 109). Unbiased genetic screens have led to the iden-
tication of CAMs, adaptor proteins, actin cytoskeletal regulators, and vesicle trafcking proteins
with roles in myoblast fusion (Table 1). Although CAMs are expected components in myoblast
fusion, the requirement for the intracellular actin cytoskeleton in promoting cell membrane fu-
sion of myoblasts was initially puzzling. Because the actin cytoskeleton is involved in many cel-
lular processes, such as cell migration, division, adhesion, contraction, protrusion formation, and
shape change (95), it was unclear at the time if the actin cytoskeleton had a general function in
maintaining the cellular homeostasis of fusion partners or if it played a specic role at sites of
fusion.
The discovery of actin-enriched structures at sites of fusion opened up a new chapter in study-
ing the cell biology of myoblast fusion (76, 79, 100). Surprisingly, these actin-enriched structures
are asymmetric and invasive, drilling ngerlike protrusions from one fusion partner into another
(114). The receiving fusion partners, conversely, build stiffer cortices to resist the invasive forces
(78). The mechanical interactions between the two fusion partners push the two cell membranes
closer than the distance achieved by CAMs alone to promote cell membrane fusion (77). Inter-
estingly, similar actin-propelled invasive protrusions have been found at sites of mammalian cell
fusion (98, 119). Indeed, many of the molecular components identied in Drosophila myoblast
fusion are also found in mammals (1, 33, 77), demonstrating conserved cellular and molecular
mechanisms underlying cell–cell fusion in higher eukaryotes. Here, we review the key ndings
in Drosophila myoblast fusion over the past two decades and highlight the critical function of the
actin cytoskeleton in cell membrane fusion.
EMBRYONIC MYOBLAST FUSION
The musculature of Drosophila is generated de novo twice during its life span. The larval mus-
culature forms during embryogenesis and is then disintegrated during metamorphosis when the
adult musculature is generated (112). Both larval and adult musculatures are composed of mult-
inucleated muscle bers created by the fusion of mononucleated muscle cells. To date, much of
the mechanistic understanding of Drosophila myoblast fusion has come from studies of the forma-
tion of larval body wall muscles during embryogenesis. We therefore focus our review largely on
embryonic myoblast fusion.
. Lee •Chen
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Tabl e 1 Molecular components of myoblast fusion
Fusion
regulator Protein type Cell type
Localization at
fusogenic synapse Function in myoblast fusion Reference(s)
Duf/Kirre Ig domain–containing CAM FC Ring-like structure Promotes FC–FCM adhesion and binds to Sns
and Hbs in trans to establish fusogenic synapse
104, 121
Rst Ig domain–containing CAM FC ND Promotes FC–FCM adhesion and binds to Sns
and Hbs in trans to establish fusogenic synapse
121
Sns Ig domain–containing CAM FCM Ring-like structure Promotes FC–FCM adhesion and binds to Duf
and Rst in trans to establish fusogenic synapse
18, 106
Hbs Ig domain–containing CAM FCM ND Promotes FC–FCM adhesion and binds to Duf
and Rst in trans to establish fusogenic synapse
5, 48
Sing Multipass transmembrane protein ND ND Potentially involved in vesicle trafcking 51
Rols7/Ants Ankyrin repeat-, tetratricopeptide
repeat-, and coiled-coil
domain–containing protein
FC Ring-like structure Replenishes Duf at the fusogenic synapse by
vesicle trafcking
27, 85, 99
Dock SH2 and SH3 domain–containing
adaptor protein
ND ND Links CAMs and actin cytoskeletal regulators 74
Drk SH2 and SH3 domain–containing
adaptor protein
ND ND Links CAMs and actin cytoskeletal regulators 74
Crk SH2 and SH3 domain–containing
adaptor protein
ND ND Links CAMs and actin cytoskeletal regulators 7, 73, 79
Sltr/WIP WASP-binding protein FCM Actin focus Recruits WASP to the fusogenic synapse 79, 84
WASP Actin NPF FCM Actin focus Promotes branched actin polymerization;
required for actin foci formation and
membrane protrusion generation
12, 59, 79,
84, 108,
114
Blow PH domain–containing protein FCM Actin focus Competes with WASP for WIP binding to
destabilize the WASP-WIP complex
38, 73
Mbc Bipartite Rac GEF FCM Actin focus Activates Rac proteins together with Elmo 50, 66, 106
Elmo Bipartite Rac GEF FCM Actin focus Activates Rac proteins together with Mbc 58
Rac1 Small G protein FCM Actin focus Activates Scar complex and group I Pak together
with Rac2
63, 82
Rac2 Small G protein FCM Actin focus Activates Scar complex and group I Pak together
with Rac1
63, 82
Scar/WAVE Actin NPF FCM, FC Actin focus Promotes branched actin polymerization;
required for actin foci formation in FCMs and
actin sheath formation in FCs
12, 59, 100,
114
(Continued)
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Tabl e 1 (Continued)
Fusion
regulator Protein type Cell type
Localization at
fusogenic synapse Function in myoblast fusion Reference(s)
Kette Component of the SCAR complex FCM, FC Actin focus Stabilizes the Scar complex; not required for
membrane protrusion generation
65, 73, 111,
114
Arp3 Component of the Arp2/3 actin
nucleator
FCM, FC Actin focus Promotes nucleation of branched actin lament 12, 100
ArpC1 Component of the Arp2/3 actin
nucleator
FCM, FC Actin focus Promotes nucleation of branched actin lament 84
DPak3 Serine/threonine kinase FCM Actin focus Promotes invasive protrusions with DPak1 44
DPak1 Serine/threonine kinase FCM Actin focus Promotes invasive protrusions with DPak3 44
Loner/Schizo Arf GEF FCM, FC Actin focus Activates Arf proteins 22, 29
Arf1 Small G protein ND ND Regulates N-Cad 42
Arf6 Small G protein ND ND Regulates Rac localization 29, 42
Rho1 Small G protein FC Actin sheath Activates Rok 78
Rok Serine/threonine kinase FC Actin sheath Phosphoactivates and activates MyoII 78
Nonmuscle
MyoII
Actin motor FC Actin sheath Mechanosensor; increases cortical tension via
actomyosin contraction
78
βH-Spectrin Spectrin cytoskeleton subunit FC Actin sheath Mechanoresponsive as a heterotetramer with
α-spectrin; restricts Duf at the fusogenic
synapse; constricts FCM protrusions
45
α-Spectrin Spectrin cytoskeleton subunit FC Actin sheath Mechanoresponsive as a heterotetramer with
βH-spectrin; restricts Duf at the fusogenic
synapse; constricts FCM protrusions
45
PIP2 Phospholipid FCM, FC Enriched on
membrane
Controls localization of actin regulators at the
fusogenic synapse
17
Dia Actin NPF FCM? Actin focus ND 34
D-Titin Giant lamentous protein FCM? Actin focus ND 83, 85, 131
WHAMY Actin NPF ND ND ND 20
Rab11 Small G protein ND ND ND 13
Abbreviations: βH-spectrin, βHeavy-spectrin; Ants, Antisocial; Arf1, ADP-ribosylation factor 1; Arf6, ADP-ribosylation factor 6; Arp3, Actin-related protein 3; ArpC1, Actin-related protein C1;
Blow, Blown fuse; CAM, cell adhesion molecule; Crk, Crk oncogene; Dia, Diaphanous; Dock, Dreadlocks; DPak1, Drosophila p21-activated kinase 1; DPak3, Drosophila p21-activated kinase 3;
Drk, Downstream of receptor kinase; Duf, Dumbfounded; Elmo, Engulfment and cell motility protein; FC, founder cell; FCM, fusion-competent myoblast; GEF, guanine nucleotide exchange
factor; Hbs, Hibris; Ig, immunoglobulin; Mbc, Myoblast city; MyoII, myosin II; ND, not determined; NPF, nucleation-promoting factor; PH, pleckstrin homology; PIP2, phosphatidylinositol
4,5-bisphosphate; Rok, Rho kinase; Rols7, Rolling pebbles 7; Rst, Roughest; Scar, Suppresser of cAMP receptor; Sing, Singles bar; Sltr, Solitary; Sns, Sticks and stones; WASP, Wiskott–Aldrich
syndrome protein; WAVE, WASP family verprolin homologs; WIP, WASP-interacting protein.
. Lee •Chen
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RENDEZVOUS OF FUSION PARTNERS: THE INITIAL ATTRACTION
AND ENGAGEMENT
Two Types of Embryonic Muscle Cells in Drosophila: Origin and Specication
The embryonic muscle cells in Drosophila arise from the somatic mesoderm (40, 123). Clusters
of mesodermal cells become promuscle groups by expressing the transcription factor Lethal of
scute (23). Within each group,one cell with higher Ras and Delta expression is specied to be the
muscle progenitor cell via Notch-mediated lateral inhibition (24). Activated Notch inhibits muscle
progenitor cell formation, whereas activated Ras promotes the progenitor cell fate (4). The muscle
progenitor cells then undergo cell division and give rise to either two muscle founder cells or a
founder cell and an adult muscle progenitor.The remaining cells in the promuscle cluster become
fusion-competent myoblasts (FCMs) (25, 103).
A typical abdominal hemisegment of a Drosophila embryo contains 30 muscle founder cells.
These founder cells are further divided into different subsets based on their expression of distinct
transcription factors (40), such as the homeodomain proteins S59 (41), Even-skipped (122), and
Ladybird (71); the LIM domain protein Apterous (19); the basic helix-loop-helix proteins Vestigial
(10) and Nautilus (8); the COE family protein Collier (31); and the zinc-nger protein Krüppel
(105). In addition, the chromatin remodeling factor Sin3A is involved in founder cell fate deter-
mination (39). Each founder cell resides at a specic location of a hemisegment, and together they
pregure the stereotypical pattern of the multinucleated muscle bers.
In contrast to founder cells, all the FCMs are specied by a single transcription factor, Lame
duck (Lmd) (43). Lmd is a member of the Gli superfamily of zinc-nger-containing transcription
factors and activates the expression of FCM-specic genes. In lmd mutant embryos, although
founder cells are properly specied, all FCMs are absent, leading to an absence of multinucleated
muscle bers (43). Another zinc-nger transcription factor, Tramtrack 69, is activated by Lmd
in FCMs to repress the expression of founder cell–specic genes and to stabilize the FCM cell
fate (30).
Fusion Occurs Between Muscle Founder Cells and
Fusion-Competent Myoblasts
Fusion between embryonic muscle cells in Drosophila was rst suggested by transmission electron
microscopy (TEM) analysis that displayed disintegrated plasma membranes between muscle
cells (115), and it was later visualized by immunohistochemical studies (9) (Figure 1a). The
rst genetic evidence for the presence of founder cells in Drosophila embryos came from the
study of the myoblast city (mbc) mutant embryos, in which a subset of mononucleated muscle cells
elongate to form miniature muscle bers in the place of the wild-type multinucleated muscle
bers (106) (Figure 1b). These elongated miniature muscle cells were named muscle founder
cells because they act as founding seeds to attract the surrounding FCMs and determine the
position, orientation, size, and pattern of neuronal innervation of future muscle bers. FCMs
function as building blocks by fusing with neighboring founder cells to generate multinucleated
muscle bers. Upon the completion of each fusion event, the nucleus of the fused FCM adopts
the transcription prole of the founder cell, such that the multinucleated myotube behaves as a
larger founder cell and proceeds with additional rounds of fusion.
Cell Adhesion Molecules: Mediators of Muscle Cell Recognition and Adhesion
Once the muscle cell fates are specied, the distinct transcription factors in founder cells and
FCMs begin to activate different sets of target genes to regulate myoblast fusion. Both types
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1990 2000 2015
1995
(Rushton et al. 1995)
2005 2010
(Sens et al. 2010)
(Kim et al. 2015b)
Multinucleated muscle cells
visualized in Drosophila embryo
a
bdf
ce
Founder cells identied Discovery of actin-enriched structure
(actin focus) at the site of myoblast fusion
Discovery of the role of mechanical
tension in promoting myoblast fusion
Cell adhesion molecules
discovered
Discovery of the asymmetric
fusogenic synapse
(Bate 1990)
(Bour et al. 2000;
Ruiz-Gómez et al. 2000)
(Kesper et al. 2007;
Kim et al. 2007;
Richardson et al. 2007)
Figure 1
Milestones in the study of Drosophila myoblast fusion. (a) Multinucleated muscle cells visualized in Drosophila embryo (9). (b) Founder
cells identied (106). (c) Cell adhesion molecules discovered (18, 104). (d) Discovery of actin-enriched structure (actin focus) at the site
of myoblast fusion (76, 79, 100). (e) Discovery of the asymmetric fusogenic synapse (114). (f) Discovery of the role of mechanical
tension in promoting myoblast fusion (78).
of cells express immunoglobulin (Ig) domain–containing CAMs to mediate the recognition and
adhesion of founder cells and FCMs (Figure 2a).
Duf and Rst: founder cell–specic adhesion molecules. Dumbfounded (Duf), also known as
Kirre, was the rst CAM identied in myoblast fusion via a founder cell–specic enhancer trap,
rP298-lacZ, in which lacZ was inserted in the duf promoter (104) (Figure 1c). Overexpressing
Duf in epithelial cells attracts FCMs to ectopic locations, demonstrating Duf’s role as a myoblast
attractant (104). Loss of Duf together with its paralog Roughest (Rst) causes a complete disruption
of myoblast fusion, whereas either single mutant has wild-type musculature, demonstrating the
redundant functions of Duf and Rst in the fusion process (104, 121). Indeed,expressing either Duf
or Rst in embryonic muscle cells completely rescues the fusion defect in duf,rst double mutant
embryos (104, 121).
Sns and Hbs: fusion-competent myoblast–specic adhesion molecules. Sticks and stones
(Sns) was identied based on its fusion-defective mutant phenotype (18) (Figure 1d). Sns and its
paralog Hibris (Hbs) are specically expressed in FCMs (5, 18). Although the hbs mutant exhibits
little myoblast fusion defect (5, 48), the sns,hbs double mutant showed a complete lack-of-fusion
phenotype that is more severe than the sns single mutant, suggesting that Sns and Hbs are par-
tially redundant in the fusion process with Sns playing a major role (116). Indeed, expressing Sns
in FCMs fully rescues the sns,hbs double mutant phenotype, whereas Hbs expression only partially
rescues the fusion defect (116). Interestingly, Hbs appears to have a dominant-negative effect on
Sns, since overexpression of Hbs in wild-type embryos resulted in a myoblast fusion defect (5),
likely due to increased Sns/Hbs heterodimerization (116).
Transinteractions between immunoglobin domain–containing cell adhesion molecules.
Two lines of genetic evidence suggest potential transinteractions between the CAMs in founder
cells and FCMs. First, ectopic expression of Duf or Rst in epithelial cells attracts FCMs to these
locations (104, 121). Second, in duf,rst double mutant embryos, Sns is no longer enriched at the
founder cell and FCM contact sites, but instead becomes evenly distributed at the cell cortex (56).
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FCM
Actin focus
Actin focus
Actin sheath
FCMFounder cell
Resistance
Actin bundle
Actin lament
Membrane
Blow
Loner
Arf1
DPak1
WASP
Elmo
Mbc
Sltr
ArpC1
Arp3
Kette
Scar
α/βH-spectrin network
under shear stress
Kette
Scar
ArpC1
Arp3
Rho1
Sns
Rok
MyoII accumulation to
dilation deformation
spectrin
spectrin
Rols7
Rols7
Duf
Duf
Duf
Sns
Sns
PIP2
Duf
Hbs
Duf
Duf
Rst
Sns
Sns
Sns
Arf1
Dia
Rac2
DPak3
Crk
Crk
Dock
Drk
Rac1
Recruitment
500 nm
500 nm
Activation
Maintenance
Predicted promotion
Arf6
Arf6
500 nm
FCM
bc
a
Actin
polymerization
Actin
polymerization
spectrin
Loner
Rols7
Mechanical force
Sing
Sing
Myoll
Myoll
Myoll
PIP2
Mechanical force
(Caption appears on following page)
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Figure 2 (Figure appears on preceding page)
The asymmetric fusogenic synapse. (a) Molecular components and signaling pathways at the fusogenic synapse. Scar, Arp2/3 complex,
Loner, Arf, Sing, and PIP2 are shown once in the two founder cell boxes for the sake of simplicity. (b) Invasive protrusions at the
fusogenic synapse visualized by transmission electron microscopy.A mononucleated FCM (pink) is projecting membrane protrusions
into the binucleated myotube. Note the exclusion of ribosomes and intracellular organelles in the protrusive area, which is lled with
actin laments. Panel badapted from Reference 114 with permission. (c) The spectrin network constricts the invasive protrusions,
visualized by superresolution microscopy. Actin-propelled protrusions (green) from the FCM penetrate through microdomains free of
accumulated βH-spectrin (red) in the apposing founder cell. Panel cadapted from Reference 45 with permission. Abbreviations:
Arf, ADP-ribosylation factor; Arp2/3, actin-related protein 2/3; βH,βHeavy; Blow, Blown fuse; Crk, Crk oncogene; Dia, Diaphanous;
Dock, Dreadlocks; DPak, Drosophila p21-activated kinase; Drk, Downstream of receptor kinase; Duf, Dumbfounded; Elmo,
Engulfment and cell motility protein; FCM, fusion-competent myoblast; Hbs, Hibris; Mbc, Myoblast city; MyoII, Myosin II; PIP2,
phosphatidylinositol 4,5-bisphosphate; Rok, Rho kinase; Rols, Rolling pebbles; Rst, Roughest; Scar, Suppresser of cAMP
receptor/WAVE; Sing, Singles bar; Sns, Sticks and stones; Sltr, Solitary/WIP; WASP, Wiskott–Aldrich syndrome protein; WAVE,
WASP family verprolin homologs; WIP, WASP-interacting protein.
The specic afnities between different CAMs in trans have been revealed by S2 cell aggregation
assays, in which two groups of normally nonadherent S2 cells expressing distinct CAMs are tested
for intergroup aggregation. Duf and Rst show both homophilic interactions with themselves and
heterophilic interactions with Sns (48, 56, 116) and Hbs (48, 116), whereas no homophilic in-
teractions are detected for Sns and Hbs (56). The transheterophilic interaction between Duf and
Sns is conrmed by coimmunoprecipitation of these proteins (56). Despite their transheterophilic
interactions, overexpressing these founder cell– and FCM-specic CAMs in two populations of
S2 cells, respectively, does not induce cell fusion (29, 118). Consistent with this observation, crys-
tallographic studies of Caenorhabditis elegans orthologs of Duf/Rst (SYG-1) and Sns/Hbs (SYG-2)
showed that SYG-1 and SYG-2 interact through their most N-terminal Ig domains and form an
L-shaped rigid structure that props the cell membranes of two adherent cells apart at a distance
of approximately 45 nm (91). This distance is too large for membrane fusion to occur because
that would require much closer proximity of 1–2 nm. Thus, CAMs can bring two cells to a cer-
tain proximity, and the rigid CAM structures formed in trans may then block further membrane
juxtaposition and fusion, consistent with the nonfusogenic nature of most cell–cell junctions in
multicellular organisms.
Adaptor Proteins: Relaying the Fusion Signal
The engagement of cell type–specic CAMs establishes future sites of fusion and initiates a cascade
of signaling events in both founder cells and FCMs. Adaptor proteins play a critical role in relaying
signals from the cell membrane to intracellular components (Figure 2a).
Rols7/Ants: a founder cell–specic adaptor protein. Three independent studies uncovered
a role for rolling pebbles 7 (rols7), also known as antisocial (ants), in myoblast fusion (27, 85, 99).
Rols7 encodes a founder cell–specic protein containing ankyrin repeats, tetratricopeptide repeats,
and a RING nger (27, 85, 99). Rols7 is localized in a Duf- and Rst-dependent manner at the
site of fusion (27, 85). In the duf,rst double mutant embryos, Rols7 becomes evenly distributed
throughout the founder cell cytoplasm (27, 85, 86). Ectopic expression of Duf in salivary gland
epithelia cells or cultured S2 cells recruits Rols7 to the cell cortex (86). The recruitment of Rols7
by Duf is likely to occur through biochemical interactions of these two proteins (27) mediated by
the ankyrin repeats of Rols7 (86). Reciprocally, Rols7 maintains Duf localization at the founder
cell membrane (86). In the rols7 mutant, Duf is not detected at the fusion sites and is present at a
lower level in the founder cell cytoplasm (86). It is proposed that Rols7 promotes the enrichment
of Duf and self-enrichment at the fusion sites by cotranslocation with Duf in exocytic vesicles to
the plasma membrane.
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Regulation of vesicle trafcking during myoblast fusion remains unclear. To date, two vesicle
trafcking-related proteins have been implicated in myoblast fusion, Singles bar (Sing) (51) and
Rab11 (13). Sing is a multipass transmembrane protein that belongs to the family of MARVEL
domain–containing proteins, which have been implicated in the biogenesis of exocytic vesicles
(107). Sing is required for myoblast fusion and is expressed in both founder cells and FCMs (51).
Although there is an accumulation of intracellular vesicles in sing mutant embryos, the transloca-
tion of Duf and Rols7 is not affected, leaving open the question whether Sing is involved in vesicle
trafcking during myoblast fusion (51). Rab11 is another vesicle trafcking-related protein impli-
cated in myoblast fusion. Overexpression of the constitutively active or dominant-negative form
of Rab11 caused a mild myoblast fusion defect (13). However, it is unclear whether endogenous
Rab11 and/or other Rab GTPases contribute to myoblast fusion.
SH2 and SH3 domain–containing adaptor proteins: Dock, Drk, and Crk. A potential role for
the Src Homology 2 (SH2) and Src Homology 3 (SH3) domain–containing proteins dreadlocks
(Dock), downstream of receptor kinase (Drk), and Crk oncogene (Crk) in myoblast fusion was rst
implicated by biochemical studies. Both Crk and Dock have been shown to bind the FCM-specic
CAMs Sns and Hbs in addition to downstream actin cytoskeletal regulators (7, 73, 74,79), which
suggests that these proteins function as adaptors linking the CAMs and the actin cytoskeleton. In
addition, Dock also binds Duf in founder cells (74). dock and drk single mutants and the dock,drk
double mutant do not show any myoblast fusion defect, likely due to maternal contributions and
potential functional redundancy with crk (74). The localization of crk on the small fourth chromo-
some, of which few genetic tools are available, has hampered the generation of a crk single mutant
and a dock,drk;crk triple mutant. Nevertheless, the dock mutant shows genetic interactions with
mutations in sns,hbs, and genes regulating the actin cytoskeleton, which indicates its functional
signicance in myoblast fusion (74).
INTERACTIONS BETWEEN FUSION PARTNERS: INCREASED
INTIMACY DRIVEN BY ASYMMETRIC ACTIN POLYMERIZATION
AND ACTOMYOSIN DYNAMICS
Although the Ig domain–containing CAMs are essential for myoblast recognition and adhesion,
they are not sufcient to bring cell membranes into close enough contact for fusion. Genetic
analyses have led to the identication of many actin polymerization and actomyosin contractility
regulators essential for myoblast fusion. Subsequent cell biological and ultrastructural analyses
have revealed that a major function of the actin cytoskeleton is pushing cell membranes into closer
proximity to one another at the site of fusion.
Actin Polymerization–Mediated Mechanical Force Generation by
Fusion-Competent Myoblasts
Of the two muscle cell types, FCMs are the more aggressive fusion partners. They utilize the actin
polymerization machinery to propel invasive protrusions into the apposing founder cells. In this
section, we review how actin polymerization regulators were discovered and how they function
together to control the invasive behavior of the FCMs (Figure 2a).
Actin polymerization regulators: essential roles revealed by genetic analyses. Drosophila ge-
netics has been instrumental in identifying actin polymerization regulators in myoblast fusion.
These discoveries have led to exciting directions of investigation and a deep understanding of the
cellular mechanisms underlying cell–cell fusion.
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Mbc, Elmo, and Rac. myoblast city (mbc) was the very rst myoblast fusion mutant identied
in Drosophila (106). Its human homolog DOCK180 was shown to alter cell morphology at the
time of discovery (67), suggesting a link between myoblast fusion and the actin cytoskeleton
(50). DOCK180 forms a complex with Engulfment and cell motility protein (ELMO), and the
DOCK180–ELMO complex functions as a bipartite guanine nucleotide exchange factor (GEF)
for the small GTPase Rac (21). Indeed, the Drosophila elmo maternal/zygotic mutant also exhibits
a severe myoblast fusion defect, and Elmo can enhance Mbc-dependent activation of Rac pro-
teins (58). The rst evidence linking Rac with myoblast fusion came from genetic experiments in
Drosophila expressing constitutively active Rac1V12 or dominant-negative Rac1N17 in embryonic
muscle cells, both of which resulted in severe myoblast fusion defects (82). Subsequent loss-of-
function analyses revealed normal somatic musculature in the rac1 and rac2 single mutants, but
severe fusion defects in the rac1,rac 2 double mutant (63), demonstrating that Rac1 and Rac2 func-
tion redundantly in myoblast fusion.
Kette, Scar, and Arp2/3. Rac regulates the actin cytoskeleton by activating the suppresser of
cAMP receptor (Scar)/WASP family verprolin homologs (WAVE) complex, which is a ve-subunit
protein complex consisting of Scar/WAVE, Sra1/PIR, Kette/Nap1/Hem, Abi, and HSPC300 (96).
Scar is a member of the WASP (Wiskott–Aldrich syndrome protein) family of actin nucleation-
promoting factors (NPFs) for the actin-related protein (Arp)2/3 complex, a seven-subunit protein
complex that nucleates actin monomers to form branched actin laments (120). A role for kette in
myoblast fusion was originally identied by an unbiased screen of ethyl methanesulfonate (EMS)-
induced mutant alleles (111). The kette mutant exhibits a strong myoblast fusion defect despite
undergoing a low level of fusion (111), further implicating the actin cytoskeleton in myoblast fu-
sion. Later, scar was also identied as a molecular component in myoblast fusion. Although the
scar zygotic mutant shows a mild fusion defect, embryos with reduced maternal and zygotic scar
contributions exhibit a severe fusion defect (100). Consistent with the requirement for the Scar
complex in myoblast fusion, mutants of arp3 and arpC1, which encode two components of the
Arp2/3 complex, are also defective in myoblast fusion (12, 84,100).
WA S P an d W I P. WASP is another NPF for the Arp2/3 complex (96). WASP is stabilized in a
tight complex by WASP-interacting protein (WIP) (57). A role for WASP in myoblast fusion
was uncovered through both forward and reverse genetic approaches (79, 84, 108). While the
zygotic wasp null mutant has largely normal musculature, the maternal/zygotic wasp mutant shows
severe defects in myoblast fusion, suggesting that the maternally contributed WASP masks the
effects of zygotic mutations (79, 84). Indeed, a zygotic wasp mutant carrying a truncated form
of the protein that misses the Arp2/3-binding domain functions as a dominant-negative form by
interfering with the maternally contributed WASP (84, 108). A role for Drosophila WIP (D-WIP),
also known as Solitary (Sltr), in myoblast fusion was revealed by an EMS mutagenesis screen (79)
and a reverse genetic analysis on the basis of its interaction with WASP (84). sltr mutant displays
a severe myoblast fusion defect despite a low level of fusion (79, 84). Interestingly, Sltr expression
is restricted to FCMs prior to fusion, because no Sltr expression is detected in the lmd mutant
embryos (79, 84). This is the rst actin cytoskeletal regulator found to be expressed in only one
of the two muscle cell types.
F-actin focus: zooming in to the site of fusion. The identication of many actin cytoskeletal
regulators through genetic analyses highlights the essential function of the actin cytoskeleton in
myoblast fusion. Subsequently, striking actin cytoskeletal rearrangements at sites of fusion were
revealed by confocal microscopy (76, 79, 100). Prior to each fusion event, there is a burst of actin
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polymerization at the founder cell and FCM contact site, resulting in the formation of a dense
oval-shaped F-actin focus (76, 79, 100) (Figure 1d). Time-lapse imaging conrmed that these
actin foci indeed correspond to sites of fusion and that they assemble and dissolve within a 5.7–
29.5-min time frame with an average life span of approximately 11.9 min (100). Many of the
actin regulators implicated in myoblast fusion colocalize with the actin foci, such as WASP (108),
Sltr (79), Mbc, and Kette (100). These actin foci appear to be surrounded by ring-like structures
formed by CAMs and adaptor proteins, such as Duf, Sns, and Rols7 (76, 114). Initially, however,
the cellular origin of the actin foci was unclear. One study suggested that a larger portion of each
actin focus resided in the FCM (79), whereas other studies concluded that each actin focus was
evenly divided between the adherent founder cell and the FCM (76, 100).
Breaking symmetry: discovery of the invasive podosome-like structure and the asymmetric
fusogenic synapse. Confusion over the cellular origin of the actin foci was resolved by cell type–
specic expression of green uorescent protein (GFP)-tagged actin (114). While GFP-actin ex-
pressed in FCMs colocalizes with the oval-shaped dense actin foci, GFP-actin expressed in founder
cells does not accumulate at the sites of fusion, demonstrating that the actin foci are generated ex-
clusively in FCMs (114) (Figure 1e). Although founder cells do not accumulate dense actin foci,
there is a thin sheath of actin underlying the founder cell membranes, which is barely visible in
wild-type embryos (114). The FCM-specic actin focus (with an average size of 1.7 μm2)dynam-
ically changes its shape and protrudes toward the founder cell, causing an inward curvature on
the founder cell membrane (114). Electron microscopy (EM) analyses revealed multiple actin-
propelled ngerlike protrusions, each with an average diameter of 250 nm projecting as long as
1.9 μm into the founder cell (114) (Figure 2b). Each actin focus contains an average of 4.3 n-
gerlike protrusions (114). When viewed along the protrusive axis, the dense actin focus is seen
surrounded by a ring of CAMs (76, 114). The characteristic organization of the actin focus sur-
rounded by adhesion molecules, together with its dynamic and protrusive nature, resembles that
of a podosome, which has been extensively studied in the migration and adhesion of cultured cell
(26). This actin-enriched protrusive structure in the FCM is therefore termed a podosome-like
structure (PLS) (114). Such an actin-propelled invasive structure has also been observed in cul-
tured Drosophila primary muscle cells (66). The closely juxtaposed cell membrane contact zone
mediated by invasive protrusions encircled by CAMs at the site of fusion has since been referred
to as the fusogenic synapse (114).
WASP and Scar complexes: distinct requirement,recruitment, and function. Although both
WASP and Scar are members of the WASP family and activate the actin nucleation activity of
Arp2/3, they have distinct functions in myoblast fusion due to their differential expression and
modes of recruitment.
Differential requirement in founder cells and fusion-competent myoblasts. The WASP and Scar
complexes are differentially required in the two types of muscle cells. Several lines of evidence
support an FCM-specic role for the WASP–Sltr complex. First, Sltr is specically expressed in
FCMs (79, 84). Second, no fusion defect is induced when dominant-negative forms of WASP
or Sltr are expressed in founder cells (84). And third, expressing WASP in founder cells alone
does not rescue the fusion defect in wasp mutant embryos as does WASP expression in all muscle
cells (108). Cell type–specic rescue of sltr with an UAS–Sltr transgene is technically challenging,
since the UAS–Sltr transgene has leaky expression in all muscle cells even without a GAL4 driver
(P. Jin & E. Chen, unpublished material). Conversely, the Scar complex is required in both founder
cells and FCMs, because expressing Scar (or Kette) in all muscle cells—but not in either founder
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cells or FCMs alone—fully rescues the scar (or kette) mutant phenotype (65, 114). Despite the
requirement for the Scar complex in both muscle cell types, its upstream regulators, Mbc and
Rac, have been shown to function specically in FCMs (66). FCM-specic expression of Mbc (or
Rac1) is sufcient to fully rescue the mbc (or rac1,rac 2) mutant phenotype (66). This raises the
intriguing question of how the Scar complex is regulated in founder cells.
The requirement for both WASP and Scar complexes in FCMs and for Scar complex alone
in founder cells correlates with the distinct actin polymerization patterns in these two cells—the
formation of oval-shaped dense actin foci in FCMs and thin actin sheaths in founder cells. In
single mutants of either NPF (or their interacting proteins), the FCM-generated actin remains
enriched at the asymmetric fusogenic synapse due to the presence of the remaining NPF (59, 66,
79, 100, 114). Only when the activities of both WASP and Scar complexes are eliminated in double
mutants, such as sltr,scar and sltr;kette, do the actin foci no longer form at the fusogenic synapse
(34, 114), which leads to a complete block of myoblast fusion (12, 114). Thus, WASP and Scar
complexes have redundant functions in actin foci formation.
Different modes of recruitment to the fusogenic synapse. All of the above-mentioned actin
cytoskeletal regulators colocalize with the actin foci at the fusogenic synapse (59, 66, 79, 100,
108, 114). The FCM-specic CAM Sns recruits Sltr, likely through the SH2 and SH3 domain–
containing adaptor protein Crk, and Sltr in turn recruits WASP to the fusogenic synapse via
biochemical interactions (79, 84). Sns may also recruit Mbc though Crk and other unknown
factors (7). The small GTPase Rac, downstream of Mbc, recruits the Scar complex to the
fusogenic synapse (59). The recruitment of the two NPF complexes is independent of each other.
For example, the Sltr enrichment at the fusogenic synapse is unaffected in the mbc mutant and
the rac1,rac2 double mutant (79), and the Scar enrichment is undisrupted in sltr mutants (59).
Distinct cellular functions. Although both WASP and Scar have similar biochemical functions in
activating Arp2/3, their cellular functions are not interchangeable. Expression of Scar in the sltr
mutant does not rescue the fusion defect caused by the absence of Sltr (114). Moreover, WASP
is required for the invasiveness of the PLS, because in sltr mutant embryos actin-lled ngers
are either wide and tubby or folded upon each other and unable to project into the apposing
founder cell (114). In contrast, kette mutant embryos show invasive protrusions similar to those
in the wild type embryos, suggesting that the Scar complex is not required for the invasiveness
of these protrusions (65, 114). Normal PLS invasion in the kette mutant raises the question of
how the loss of the Scar complex disrupts myoblast fusion. Since Scar is also required in founder
cells to generate the thin sheath of actin, it is conceivable that the loss of Scar may weaken the
response of founder cells to the invasive protrusions from the FCMs. Indeed, expressing WASP
or Sltr in founder cells to activate Arp2/3 in the kette mutant partially rescues the fusion defect
(65), likely by replenishing the actin sheath. An additional function of Kette has been proposed in
which Kette may be involved in dissolving the fusion-inhibitory cellular junctions formed by the
CAM N-cadherin (N-Cad), although the mechanism underlying the Kette and N-Cad interaction
is unknown (65).
Regulation of podosome-like structure dynamics and organization: Blow and Pak. When
the actin foci were initially characterized, it was thought that the size of the foci was a major de-
termining factor for their function (100). Subsequent analyses revealed that actin polymerization
dynamics and actin lament organization within the actin foci are the key determinants for PLS
function.
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Blown fuse. blown fuse (blow) was rst identied as an axon guidance mutant (125), but later stud-
ies found that the primary defect in the blow mutant embryos was in myoblast fusion (38). Blow
is another FCM-specic protein colocalizing with the actin foci, like WASP and Sltr (73). Its re-
cruitment to the fusogenic synapse is mediated by Sns and Crk but is independent of Mbc, Kette,
and Sltr (73). Biochemically, Blow competes with WASP for Sltr binding, thus modulating the sta-
bility of the WASP–Sltr complex (73).Because the WASP–Sltr complex binds to the barbed ends
of actin laments, Blow-mediated WASP–Sltr destabilization facilitates the dissociation of WASP
from the actin laments, which results in lament end capping and initiation of new branched
actin laments. Indeed, uorescent recovery after photobleaching analyses demonstrate rapid and
full recovery of GFP-actin at the fusogenic synapse in wild-type embryos but slow and partial
recovery in the blow mutant (73). EM studies reveal compromised protrusions in blow mutant,
suggesting that the dynamic branched actin polymerization is required for generating short and
mechanically stiff actin laments suitable for propelling invasive protrusions (73).
Group I p21-activated kinases. ADrosophila group I p21-activated kinase (Pak), DPak3, was
identied from a deciency screen as a myoblast fusion-promoting protein (44). Paks are ser-
ine/threonine kinases known to regulate actin cytoskeletal organization (3, 16). Two Drosophila
group I Paks, DPak3 and DPak1, have partially redundant functions in myoblast fusion, with
DPak3 playing a major role (44). DPak3 is specically required in FCMs, because FCM-specic
DPak3 expression fully rescues the dpak3 mutant phenotype (44). DPak3 is enriched at the fuso-
genic synapse, colocalizing with the F-actin focus within the PLS, and this enrichment is depen-
dent on Rac but independent of Sltr or Kette (44).EM analysis of dpak3 mutant embryos showed
compromised invasive protrusions that contain ribosomes and intracellular organelles, demon-
strating a role for DPak3 in organizing the actin laments within the PLS into a densely packed
network, which in turn generates sufcient mechanical force to promote PLS invasion and fusion
pore formation (44).
Other fusion-promoting factors with unclear functions in myoblast fusion. Besides the
actin polymerization regulators described above, several other proteins have actin cytoskeleton-
associated functions during myoblast fusion. However, their precise roles in the fusion process
have not been clearly demonstrated.
Loner. loner, also known as schizo, was identied in an EMS mutagenesis screen for myoblast
fusion mutants (29). Loner is a member of the BRAG family of ADP-ribosylation factor (Arf)
GEFs, which are known to activate the Arf GTPases at plasma membranes and endosomes (32).
A function of Loner in founder cells is supported by its expanded expression in Notch mutant
embryos that contain more founder cells, its localization at the vicinity of the founder cell nuclear
marker rP298-lacZ, and its recruitment by Duf to cell contact sites in cultured Drosophila cells (29).
However, because the FCM-specic transcription factor Lmd, the PLS at the fusogenic synapse,
and muscle type–specic GAL4 drivers were neither identied nor available at the time of the
initial characterization of Loner, its potential role in FCMs could not be assessed. Later studies
demonstrated a role for Loner in both founder cells and FCMs, based on its expression in both cell
populations and the partial rescue of the loner mutant phenotype by either founder cell– or FCM-
specic expression of the gene (42, 100). The function of Loner in the fusion process, however,
remains unclear. One study showed that Loner controls the localization of Rac (29), whereas an-
other showed that Loner does not play a role in actin foci formation at the fusogenic synapse (100).
Protein interaction studies revealed that Loner interacts with two CAMs, Duf (22) and N-Cad
(42). Although N-Cad itself is not required for myoblast fusion, it exhibits genetic interactions
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with loner. Removing N-cad suppresses the fusion defect of the loner mutant (42). A similar genetic
interaction was observed between N-cad and kette, suggesting that both Loner and Kette may be
involved in dissolving N-Cad-containing cellular junctions that may inhibit myoblast fusion (65).
However, it remains unclear how these N-Cad junctions are related to the fusogenic synapse.
Arf. Structure–function analysis of Loner suggests that its GEF activity is essential for myoblast
fusion (29). In vitro GDP release assays showed that the GEF domain of Loner has specic activity
toward Arf6 but not Arf1. Founder cell–specic expression of a dominant-negative form of Arf6
results in a mild fusion defect (29). In addition, a role for Arf6 in the fusion of cultured mammalian
muscle cells has been demonstrated (6). However, arf6 null mutant is homozygous viable (49, 70)
without exhibiting any myoblast fusion defects (42), suggesting that Arf6 either is not required for
myoblast fusion in vivo or has a redundant function with other Arfs in the fusion process. Similar
redundant functions among the Rac GTPases have been demonstrated in myoblast fusion (63).
For example, expression of dominant-negative Rac1 in muscle cells causes a severe myoblast fusion
defect, whereas the rac1 single mutant has normal musculature due to the redundant function of
rac2 (63, 82). A later study showed that Loner binds a dominant-negative form of Arf1 (Arf1DN)
with higher afnity than that of Arf6 (42). Moreover, the expression of Arf1DN in all muscle cells
results in a mild fusion defect in some embryos and the expression of constitutively active Arf1 in
all muscle cells partially suppresses the fusion defects in loner mutant embryos (42). Taken together,
these results suggest that Arf1 and Arf6 may have redundant functions in myoblast fusion, with
Arf1 playing a major role. Further investigations are required to resolve the Arf conundrum.
D-Titin. D-Titin is a giant lamentous protein best known for its role in sarcomere assembly
and function (124). A role for D-Titin in myoblast fusion has been identied based on a partial
loss-of-fusion phenotype in D-Titin mutant embryos (83, 131). D-Titin is expressed in the so-
matic mesoderm and accumulates at the myoblast–myotube contact sites (85, 131). Furthermore,
D-Titin colocalizes with Sltr at the fusogenic synapse (79), suggesting that it may be involved
in regulating actin lament organization within the PLS. Despite these observations, the precise
function of D-Titin in the fusion process requires further investigation.
Diaphanous. Diaphanous (Dia) is one of the Drosophila formins that promote linear actin nucle-
ation (61). Dia colocalizes with the actin foci at the fusogenic synapse in FCMs, and its recruitment
is dependent on the FCM-specic CAM Sns but independent of all the upstream regulators of
Arp2/3 (34). dia mutant embryos and embryos expressing a dominant-negative form of Dia in all
muscle cells exhibit thinner muscle bers with reduced number of nuclei, indicating a potential
block of myoblast fusion. However, it is unclear whether this effect is caused by a specic defect
in the fusion process or an earlier defect in FCM maturation, because most of the mononucle-
ated cells marked by diaDN-GFP do not appear to express the muscle structural protein myosin
heavy chain (MHC), indicating that they are not fully differentiated (34). In addition, it is unclear
whether Dia polymerizes actin at the fusogenic synapse because Dia remains enriched at these
sites in sltr;kette mutant embryos, where F-actin foci fail to form (34). Further experiments are
necessary to clarify the endogenous function of Dia in myoblast fusion.
Whamy. WHAMY is another member of the WASP family of Arp2/3 NPFs (101). The whamy
single mutant has normal musculature, but together with a mutation in wasp,thewhamy,wasp dou-
ble mutant exhibits a mild fusion defect (20). Overexpressed WHAMY in somatic mesoderm does
not localize to the fusogenic synapse but rather to muscle attachment sites (20), raising the ques-
tion of how WHAMY coordinates with WASP to regulate myoblast fusion.
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Mechanosensitive Response-Mediated Tension Generation by Founder Cells
The invasive protrusions from the FCM at the fusogenic synapse create a hand-in-glove type of in-
teraction between the two fusion partners (Figure 2a). Such an interaction signicantly increases
the cell surface contact area between the two cell membranes and allows intimate apposition of
the two lipid bilayers beyond that brought about by CAMs. Recent studies revealed that founder
cells are not passive fusion partners. Instead, they mount mechanosensitive responses to the inva-
sive protrusions from FCMs to push back the FCM (78) (Figure 1f), restrict the boundary of the
fusogenic synapse (45), and sculpt the invasive protrusions to facilitate cell membrane fusion (45)
(Figure 2a).
The mechanosensitive actomyosin network: building up the resistance. A function for the
small GTPase Rho1 was uncovered in myoblast fusion when expressing a dominant-negative form
of Rho1 in muscle cells caused a fusion defect (78). It is well known that Rho1 activates Rho kinase
(Rok), which in turn phosphorylates the regulatory light chain (RLC) of the nonmuscle myosin II
(MyoII) to activate MyoII (15). Rho1, Rok, and MyoII are all enriched at the fusogenic synapse,
and, strikingly, only on the side of the founder cell (78). Despite the normal musculature in the rho1
and rok single mutant embryos due to maternal contribution,the rok;rho1 double mutant embryos
exhibit a myoblast fusion defect (78). Founder cell–specic expression of phosphomimetic, but not
nonphosphorylatable, MyoII RLC partially rescues the rok;rho1 mutant phenotype, demonstrating
that the function of Rho1–Rok is to activate MyoII in founder cells (78).
The enrichment of MyoII at the fusogenic synapse can be mechanically triggered, because
MyoII still accumulates in the absence of Duf-mediated Rho1–Rok accumulation in Drosophila
embryos (78). In support of this, mechanosensitive accumulation of MyoII has been demonstrated
by two complementary biophysical approaches, micropipette aspiration (MPA) and atomic force
microscopy (AFM), which apply pulling and pushing forces to cells, respectively. Under both con-
ditions, MyoII accumulates in response to applied mechanical force prior to Rho1–Rok accumu-
lation in a motor domain–dependent manner (78). These experiments highlight a role of MyoII
as a mechanosensor during myoblast fusion.
The accumulation of MyoII at the fusogenic synapse increases cortical stiffness and provides
resistance to the invasive protrusions. This has been demonstrated by the presence of abnormally
long invasive protrusions from FCMs in embryos where MyoII activity is decreased in founder
cells (78). Moreover, when MyoII is knocked down in the receiving fusion partner in a cell-fusion
culture system (118), cortical stiffness decreases as measured by MPA and AFM accompanied by a
defect in cell–cell fusion (78). However, overexpression of the actin crosslinker Fimbrin in MyoII
knockdown cells restores the cortical tension and signicantly rescues the cell fusion defect (78).
Thus, MyoII elevates cortical stiffness in the receiving fusion partner to resist the PLS invasion
and keep the apposing cell membranes at a close distance for fusion.
The mechanosensitive spectrin network: restricting the boundary of the fusogenic synapse
and sculpting the invasive protrusions. Spectrin was identied by a deciency screen for my-
oblast fusion mutants (45). Prior to the functional studies of spectrin in myoblast fusion, it was best
known as a membrane skeleton scaffold protein critical for maintaining cell shape and providing
mechanical support for the plasma membrane (11, 129). The basic unit of spectrin is a exible,
chain-like α/βheterotetramer with actin-binding domains localized at the two ends. Of the two
β-spectrins in Drosophila,βHeavy-spectrin (βH-spectrin), also known as Karst (Kst), and β-spectrin,
only the former plays a role in myoblast fusion (45). Cell type–specic rescue experiments demon-
strate a founder cell–specic role for the α/βH-spectrin heterotetramers in myoblast fusion (45).
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Correspondingly, α/βH-spectrin are enriched at the fusogenic synapse in the founder cell,closely
abutting the actin focus in the FCM (45).
In contrast to its well-established scaffolding function, βH-spectrin is dynamically recruited to
the fusogenic synapse and dissolves together with the actin focus upon fusion pore formation (45).
Recruitment of βH-spectrin to the fusogenic synapse still occurs in the absence of Duf-mediated
chemical signaling, suggesting that βH-spectrin may accumulate in response to mechanical
stimuli. Indeed, biophysical analyses using MPA and AFM demonstrate a mechanosensitive
accumulation of βH-spectrin, which requires its actin-binding and tetramerization activities (45).
Moreover, MPA experiments and mathematical modeling demonstrate that βH-spectrin responds
to shear deformation, which corresponds to the base areas of invasive protrusions (45). Accumu-
lated α/βH-spectrin, in turn, forms a physical barrier for future protrusions from the FCM, such
that new protrusions can penetrate only through spectrin-free domains and trigger additional
spectrin accumulation in these areas. Eventually, an uneven α/βH-spectrin network forms with
a few small spectrin-free microdomains that allow the penetrance of narrow invasive protrusions
(45). Such a spectrin network at the fusogenic synapse has at least two functions. First,it serves as
a cellular fence to restrict CAMs to the fusogenic synapse via biochemical interactions and steric
hindrance. Second, it serves as a cellular sieve to constrict the diameter of the invasive protrusions
(45) (Figure 2c). The increased mechanical tension generated by the narrow protrusions helps
overcome energy barriers for membrane apposition and drives cell membrane fusion.
LIPID BILAYER MERGER: FORMATION OF FUSION PORES
The ultimate goal of CAM engagement and actin cytoskeletal rearrangement/contraction at the
fusogenic synapse is to bring the two lipid bilayers into close proximity and prime them for fusion.
To date, fusogenic proteins directly involved in fusion pore formation in Drosophila muscle cells
have not be identied. However, EM studies have revealed the morphology of fusion pores be-
tween fusing muscle cells, and the functions of membrane lipids in the fusion process have begun
to be uncovered.
Ultrastructural analyses of the fusogenic synapse. The rst comprehensive EM study of em-
bryonic myoblast fusion identied several characteristic structures along the muscle cell contact
zone: paired electron-dense vesicles (termed prefusion complexes); relatively rare electron-dense
plaques; and multiple membrane discontinuities (MMDs) with diameters between 50 and 250 nm
(38). It was proposed that the vesicles release electron-dense materials to form the plaques, which
induce the formation of multiple fusion pores along the muscle cell contact zone (38). Subse-
quent studies over the next ten years reproduced these structures using the same conventional
room temperature chemical xation method, making this a prevailing model describing the dis-
tinct steps of myoblast fusion. However, additional EM studies using the same method revealed
similar MMDs between muscle cells in fusion mutants, as well as between cells that do not nor-
mally fuse (114), raising the question whether these MMDs on the plasma membrane are indeed
fusion pores. Because the conventional xation method may not allow ultrafast penetration of x-
atives into tissues underneath the epithelial cell layer,it is prone to generating artifacts. Thus, the
high-pressure freezing and freeze substitution (HPF/FS) method was applied to Drosophila em-
bryos in order to achieve optimal preservation, especially of plasma membranes in mesodermal
cells.
With the HPF/FS method, MMDs are no longer observed between muscle cells in wild-type
and fusion mutant embryos (114), indicating that these may be artifacts generated by the con-
ventional method due to insufcient xation and the subsequent extraction of the membranous
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materials by osmium treatment. HPF/FS EM analyses revealed fusion pores as single-channel
openings connecting the two fusing cells (114). Because of the ultrafast expansion of fusion pores
under 200 nm demonstrated by biophysical analysis (75, 93, 94), the smallest fusion pores observed
by EM so far have diameters of approximately 300 nm (114).
The FCM-projected ngerlike protrusions at the fusogenic synapse were rst revealed by
HPF/FS EM analyses and conrmed by conventional EM studies (114). The invasive nger-
like protrusions provide a clear morphological marker for the fusogenic synapse at the ultra-
structural level. No electron-dense vesicles or plaques are associated with the invasive protru-
sions at the fusogenic synapses, suggesting that these structures either may correspond to early
events in the fusion process prior to PLS formation or are irrelevant to myoblast fusion. The
former possibility is supported by the presence of electron-dense vesicles associated with micro-
tubules pointing toward muscle cell contact sites without actin enrichment, suggesting that these
vesicles may undergo exocytosis and may be involved in CAM trafcking (79). The function of
electron-dense plaques is completely unknown. A recent study found an N-Cad-dependent in-
crease of electron-dense plaques in the kette mutant embryos, suggesting that the plaques may
block myoblast fusion (65). However, it is unclear how these plaques are related to the fusogenic
synapse.
Phosphatidylinositol 4,5-bisphosphate: involvement in myoblast fusion. Fusion pore forma-
tion requires the destabilization of the two lipid bilayers, leading to the formation of a membra-
nous opening that connects the two cells. Thus, investigating the function of lipids is important
for understanding myoblast fusion. To date, only one lipid has been implicated in Drosophila my-
oblast fusion, which is phosphatidylinositol 4,5-bisphosphate (PIP2) (17). PIP2 is enriched at the
fusogenic synapse, and its enrichment is dependent on CAMs but not actin regulators (17). It has
been shown that overexpressing PIP2-binding pleckstrin homology (PH) domain of phospholi-
pase C gamma (PLCγ) in muscle cells severely blocks myoblast fusion presumably by sequestering
PIP2 (17). In addition, Rac, Scar, and WASP are no longer enriched at the fusogenic synapse in
PLCγPH-overexpressing embryos, a nding that supports a potential contribution of PIP2 sig-
naling in actin foci formation (17). However, most of the unfused cells in these fusion-defective
embryos were labeled by PLCγPH-GFP expression and were MHC negative (17), suggesting
that these mononucleated cells may not have fully differentiated. Therefore, the specic function
of PIP2 at the fusogenic synapse needs to be assessed in embryos with decreased PIP2 levels in
properly differentiated muscle cells.
PUPAL MYOBLAST FUSION
Drosophila adult musculature accommodates diverse types of movements, including ying, walk-
ing, mating, and feeding. Adult muscles form by myoblast fusion during pupal development either
by building on a larval muscle scaffold or through a de novo process (62). Specically, the larval
oblique muscles escape histolysis and become the template for the dorsal longitudinal muscles
(DLMs) (52, 54). On the other hand, the dorsoventral muscles (DVMs) are generated de novo
without a pre-existing template (54). DLMs and DVMs constitute the indirect ight muscles
(IFM).
Relatively little is known about the mechanisms underlying pupal myoblast fusion, partly be-
cause most loss-of-function alleles of genes promoting pupal myoblast fusion have early lethal
phases (112). The application of RNA interference (RNAi) (37, 89) has made pupal muscu-
lature a genetically amenable system to study the molecular mechanisms of myoblast fusion
(88, 110).
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A Repeated Fate Determination of Myoblasts—Similar Fusion Partners
and Adhesion Molecules
The adult muscle progenitors are rst set aside during embryogenesis and then proliferate to
generate myoblast precursors during metamorphosis (55). Similar to embryonic myoblast fusion,
adult myoblast precursors are also specied into two populations, founder cells and FCMs (40).
Most of the precursors differentiate into FCMs, whereas a small population takes on the founder
cell fate. Interestingly, adult founder cell specication does not require Notch-mediated lateral
inhibition (46) but requires broblast growth factor signaling (47). The founder cells, together
with the persisting larval muscle scaffold for the DLMs, specically express Duf (46, 60) and seed
future adult muscle bers (102). Most studies of pupal myoblast fusion have been performed with
the DLMs, within which the larval muscle templates can be clearly identied.
A unique aspect of pupal myoblast fusion is the requirement for long-range cell migration
prior to the fusion process. As myoblasts reach the vicinity of founder cells or the larval muscle
scaffold, their Notch signaling is switched off such that they can differentiate into mature FCMs
expressing Sns and Hbs (60). Subsequent local migration of FCMs is mediated by CAMs (60). Ex
vivo culture of isolated IFMs identied the presence of long lopodia emanating from the template
myotubes, which facilitate heterotypic adhesion between myotubes and FCMs (113). Formation of
the lopodia requires a processive actin polymerase, Enabled, and a BAR-domain protein, IRSp53
(113). Founder cell– and FCM-specic CAMs accumulate at the sites of contact between myotube
lopodia and FCMs (113). Sns and Hbs function more equally in the pupal FCMs than in embryos,
and double knockdown of Sns and Hbs, but not single knockdowns, results in pupal myoblast
fusion defects (60). EM analysis of sns and hbs double knockdown muscles showed looser adhesion
between FCMs and template myotubes, where most of the contact sites are more than 50 nm
apart, compared to <22 nm apart in wild type (35), consistent with a function of Sns and Hbs in
mediating muscle cell adhesion.
Another Round of Mechanical Interactions Between Muscle Cells
Pupal and embryonic myoblast fusion appear to share similar molecular and cellular mechanisms
of bringing membranes into close proximity following cell adhesion. The rst genetic clue indi-
cating a function for the actin cytoskeleton came from myoblast-specic expression of dominant-
negative Rac1, which causes fusion defects in DLM and DVM (46, 53). Subsequently, a role for
wasp in pupal myoblast fusion was uncovered (88). In wasp hypomorphic mutants, muscle bers
are underdeveloped with differentiated but unfused myoblasts clumping around them, and so-
matic muscle expression of dominant-negative WASP results in a similar phenotype (88). RNAi
knockdown of WASP, Scar, and Arp2/3 also leads to compromised fusion, demonstrating a critical
role for Arp2/3-mediated branched actin polymerization in pupal myoblast fusion (88). Consistent
with the similar molecular requirement, actin enrichment is observed at the contact sites between
pupal myoblasts and myotubes (88), as is observed at the fusogenic synapses in embryos (76, 79,
100). Moreover, cell type–specic labeling of F-actin with Moesin-GFP showed FCM-specic
actin foci and a myotube-specic actin sheath, recapitulating the asymmetric fusogenic synapse
in embryonic myoblast fusion (88, 114). Not surprisingly, the Arp2/3 complex and its NPFs are
enriched within the actin foci (88).
Ultrastructural analysis of the pupal fusogenic synapse also revealed ngerlike protrusions (35)
similar to those in embryos (114). These protrusions are relatively rare in the wild type as com-
pared to the fusion-defective wasp mutant or the kette or sing knockdown pupae (35), consistent
with fewer actin foci in wild-type compared to fusion-mutant embryos. The distances between
adherent muscle cells were measured in the pupae, and the wild-type cells were found to have the
. Lee •Chen
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closest distance, followed by the intermediate distance between the Arp2/3 knockdown cells and
the largest distance between sns and hbs double knockdown cells (35). However, these measure-
ments were not taken in the areas of invasive protrusions. Therefore, it is unclear how these mea-
surements are related to the fusogenic synapse. Additional EM analyses showed multiple closely
abutting points of membrane along the muscle cell contact zone, as well as multiple membra-
nous openings (35). Because these EM studies used a hybrid method of both conventional room
temperature chemical xation and HPF/FS, it is unclear whether the small openings (<50 nm)
resulted from insufcient chemical xation as shown in embryos (38, 114).
COMMON MECHANISMS AND MAJOR UNANSWERED QUESTIONS
Common mechanisms of cell–cell fusion have emerged from studies of both embryonic and pupal
myoblast fusion in Drosophila. Both systems use actin-propelled invasive membrane protrusions
to bring two cell membranes into close proximity following cell adhesion (Figure 2a). Similar
protrusions have also been observed in cultured Drosophila S2R+cells induced to fuse (118), in
fusing mammalian myoblasts and osteoclasts (98, 119), and in C. elegans epithelial seam cell–hyp7
cell fusion (127). These ndings suggest that different cell–cell fusion events utilize a common
cellular strategy to promote cell–cell fusion.
Despite these exciting discoveries, several major questions remain unanswered in Drosophila
myoblast fusion. First, transmembrane fusogenic proteins have yet to be identied. Recent stud-
ies of mouse myoblast fusion uncovered a bipartite myoblast-specic fusogen, Myomaker and
Myomixer/Myomerger/Minion (14, 87, 97, 128). Although these two proteins are conserved in
vertebrate myoblast fusion (36, 80, 117, 130), neither has a Drosophila homolog, suggesting that
fusogens are likely to be species- and tissue-specic. Second, the functions of various lipids in
Drosophila myoblast fusion remain unclear.Mammalian studies implicated phosphatidylserine (PS)
(72, 81) and PS receptors BAI1, BAI3, and Stabilin-2 in myoblast fusion (64, 69, 92). The potential
functions of PS and many other lipids in Drosophila myoblast fusion remain elusive. Third, cal-
cium signaling has been implicated in myoblast fusion for decades, but its potential function at the
fusogenic synapse is completely unknown. Fourth, although there have been signicant insights
into the process of actin polymerization at the fusogenic synapse, we do not yet understand how
the actin foci are depolymerized upon fusion pore formation.
CONCLUDING REMARKS
The past two decades have witnessed signicant progress in our understanding of conserved mech-
anisms underlying myoblast fusion. The discovery of the asymmetric fusogenic synapse involving
invasive protrusions and the corresponding mechanosensitive response led to a biophysical frame-
work of cell–cell fusion. This new conceptual framework highlights the interplay of pushing and
resisting forces between the two fusion partners, which brings apposing cell membranes into close
proximity and facilitates fusogen engagement and membrane fusion (Figure 2a). These ndings
have fundamentally changed our view of how cells fuse and provide a solid foundation upon which
future studies can be built. With a sophisticated toolbox leveraging new methods from genetics,
cell biology, biochemistry, and biophysics, the next decade will undoubtedly bring about many
new discoveries in this exciting eld.
DISCLOSURE STATEMENT
The authors are not aware of any afliations, memberships, funding, or nancial holdings that
might be perceived as affecting the objectivity of this review.
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ACKNOWLEDGMENTS
This work was supported by the National Institutes of Health grants R01 AR053173 and R01
GM098816, an American Heart Association Established Investigator Award, and a Howard
Hughes Medical Institute Faculty Scholar Award to E.H.C. D.M.L. is supported by a Canadian
Institute of Health Research postdoctoral fellowship.
LITERATURE CITED
1. Abmayr SM, Pavlath GK. 2012. Myoblast fusion: lessons from ies and mice. Development 139:641–56
2. Aguilar PS, Baylies MK, Fleissner A, Helming L, Inoue N, et al. 2013. Genetic basis of cell–cell fusion
mechanisms. Trends Genet. 29:427–37
3. Arias-Romero LE, Chernoff J. 2008. A tale of two Paks. Biol. Cell 100:97–108
4. Artero R, Furlong EE, Beckett K, Scott MP, Baylies M. 2003. Notch and Ras signaling pathway effec-
tor genes expressed in fusion competent and founder cells during Drosophila myogenesis. Development
130:6257–72
5. Artero RD, Castanon I, Baylies MK. 2001. The immunoglobulin-like protein Hibris functions as a dose-
dependent regulator of myoblast fusion and is differentially controlled by Ras and Notch signaling.
Development 128:4251–64
6. Bach A-S, Enjalbert S, Comunale F, Bodin S, Vitale N, et al. 2010. ADP-ribosylation factor 6 regulates
mammalian myoblast fusion through phospholipase D1 and phosphatidylinositol 4,5-bisphosphate sig-
naling pathways. Mol. Biol. Cell 21:2412–24
7. Balagopalan L, Chen M-H, Geisbrecht ER, Abmayr SM. 2006. The CDM superfamily protein MBC di-
rects myoblast fusion through a mechanism that requires phosphatidylinositol 3,4,5-triphosphate bind-
ing but is independent of direct interaction with DCrk. Mol. Cell. Biol. 26:9442–55
8. Balagopalan L, Keller CA, Abmayr SM. 2001. Loss-of-function mutations reveal that the Drosophila
nautilus gene is not essential for embryonic myogenesis or viability. Dev. Biol. 231:374–82
9. Bate M. 1990. The embryonic development of larval muscles in Drosophila. Development 110:791–804
10. Bate M, Rushton E, Frasch M. 1993. A dual requirement for neurogenic genes in Drosophila myogenesis.
Dev. 119(Suppl.):149–61
11. Bennett V, Lorenzo DN. 2013. Spectrin- and ankyrin-based membrane domains and the evolution of
vertebrates. Curr. Top. Membr. 72:1–37
12. Berger S, Schäfer G, Kesper DA, Holz A, Eriksson T, et al. 2008. WASP and SCAR have distinct roles
in activating the Arp2/3 complex during myoblast fusion. J. Cell Sci. 121:1303–13
13. Bhuin T, Roy JK. 2009. Rab11 is required for myoblast fusion in Drosophila. Cell Tissue Res. 336:489–99
14. Bi P, Ramirez-Martinez A, Li H, Cannavino J, McAnally JR, et al. 2017. Control of muscle formation
by the fusogenic micropeptide myomixer. Science 356:323–27
15. Bishop AL, Hall A. 2000. Rho GTPases and their effector proteins.Biochem. J. 348:241–55
16. Bokoch GM. 2003. Biology of the p21-activated kinases. Annu. Rev. Biochem. 72:743–81
17. Bothe I, Deng S, Baylies M. 2014. PI(4,5)P2 regulates myoblast fusion through Arp2/3 regulator local-
ization at the fusion site. Development 141:2289–301
18. Bour BA, Chakravarti M, West JM, Abmayr SM. 2000.Drosophila SNS, a member of the immunoglobulin
superfamily that is essential for myoblast fusion. Genes Dev. 14:1498–511
19. Bourgouin C, Lundgren SE, Thomas JB. 1992. apterous is a Drosophila LIM domain gene required for
the development of a subset of embryonic muscles. Neuron 9:549–61
20. Brinkmann K, Winterhoff M, Önel S-F, Schultz J, Faix J, Bogdan S. 2016. WHAMY is a novel actin
polymerase promoting myoblast fusion, macrophage cell motility and sensory organ development in
Drosophila.J. Cell Sci. 129:604–20
21. Brugnera E, Haney L, Grimsley C, Lu M, Walk SF, et al. 2002. Unconventional Rac–GEF activity is
mediated through the Dock180–ELMO complex. Nat. Cell Biol. 4:574–82
22. Bulchand S, Menon SD, George SE, Chia W. 2010. The intracellular domain of Dumbfounded affects
myoblast fusion efciency and interacts with Rolling pebbles and Loner. PLOS ONE 5:e9374
. Lee •Chen
Review in Advance first posted on
July 5, 2019. (Changes may still
occur before final publication.)
Annu. Rev. Genet. 2019.53. Downloaded from www.annualreviews.org
Access provided by University of Texas Southwestern Medical Center on 10/01/19. For personal use only.
GE53CH02_Chen ARjats.cls June 17, 2019 14:52
23. Carmena A, Bate M, Jiménez F. 1995. lethal of scute, a proneural gene, participates in the specication of
muscle progenitors during Drosophila embryogenesis. Genes Dev. 9:2373–83
24. Carmena A, Buff E, Halfon MS, Gisselbrecht S, Jiménez F, et al. 2002. Reciprocal regulatory interac-
tions between the Notch and Ras signaling pathways in the Drosophila embryonic mesoderm. Dev. Biol.
244:226–42
25. Carmena A, Gisselbrecht S, Harrison J, Jiménez F, Michelson AM. 1998. Combinatorial signaling
codes for the progressive determination of cell fates in the Drosophila embryonic mesoderm. Genes Dev.
12:3910–22
26. Chen EH. 2011. Invasive podosomes and myoblast fusion. Curr. Top. Membr. 68:235–58
27. Chen EH, Olson EN. 2001. Antisocial, an intracellular adaptor protein, is required for myoblast fusion
in Drosophila.Dev. Cell 1:705–15
28. Chen EH, Olson EN. 2005. Unveiling the mechanisms of cell-cell fusion. Science 308:369–73
29. Chen EH, Pryce BA, Tzeng JA, Gonzalez GA, Olson EN. 2003. Control of myoblast fusion by a guanine
nucleotide exchange factor, loner, and its effector ARF6. Cell 114:751–62
30. Ciglar L, Girardot C, Wilczy ´
nski B, Braun M, Furlong EEM, et al. 2014. Coordinated repression and
activation of two transcriptional programs stabilizes cell fate during myogenesis. Development 141:2633–
43
31. Crozatier M, Vincent A. 1999. Requirement for the Drosophila COE transcription factor Collier in for-
mation of an embryonic muscle: transcriptional response to notch signalling. Development 126:1495–504
32. D’Souza RS, Casanova JE. 2016. The BRAG/IQSec family of Arf GEFs. Small GTPases 7:257–64
33. Deng S, Azevedo M, Baylies M. 2017. Acting on identity: myoblast fusion and the formation of the
syncytial muscle ber. Semin. Cell Dev. Biol. 72:45–55
34. Deng S, Bothe I, Baylies MK. 2015. The formin Diaphanous regulates myoblast fusion through actin
polymerization and Arp2/3 regulation. PLOS Genet. 11:e1005381
35. Dhanyasi N, Segal D, Shimoni E, Shinder V, Shilo B-Z, et al. 2015. Surface apposition and multiple cell
contacts promote myoblast fusion in Drosophila ight muscles. J. Cell Biol. 211:191–203
36. Di Gioia SA, Connors S, Matsunami N, Cannavino J, Rose MF, et al. 2017. A defect in myoblast fusion
underlies Carey–Fineman–Ziter syndrome. Nat. Commun. 8:16077
37. Dietzl G, Chen D, Schnorrer F, Su K-C, Barinova Y, et al. 2007. A genome-wide transgenic RNAi
library for conditional gene inactivation in Drosophila. Nature 448:151–56
38. Doberstein SK, Fetter RD, Mehta AY, Goodman CS. 1997. Genetic analysis of myoblast fusion: blown
fuse is required for progression beyond the prefusion complex. J. Cell Biol. 136:1249–61
39. Dobi KC, Halfon MS, Baylies MK. 2014. Whole-genome analysis of muscle founder cells implicates the
chromatin regulator Sin3A in muscle identity. Cell Rep. 8:858–70
40. Dobi KC, Schulman VK,Baylies MK. 2015. Specication of the somatic musculature in Drosophila.Wiley
Interdiscip.Rev. Dev. Biol. 4:357–75
41. Dohrmann C, Azpiazu N, Frasch M. 1990. A new Drosophila homeo box gene is expressed in mesodermal
precursor cells of distinct muscles during embryogenesis. Genes Dev. 4:2098–111
42. Dottermusch-Heidel C, Groth V, Beck L, Önel S-F. 2012. The Arf-GEF Schizo/Loner regulates
N-cadherin to induce fusion competence of Drosophila myoblasts. Dev. Biol. 368:18–27
43. Duan H, Skeath JB, Nguyen HT. 2001. Drosophila Lame duck, a novel member of the Gli superfamily,
acts as a key regulator of myogenesis by controlling fusion-competent myoblast development. Develop-
ment 128:4489–500
44. Duan R, Jin P, Luo F, Zhang G, Anderson N, Chen EH. 2012. Group I PAKs function downstream of
Rac to promote podosome invasion during myoblast fusion in vivo. J. Cell Biol. 199:169–85
45. Duan R, Kim JH, Shilagardi K, Schiffhauer E, Lee D, et al. 2018. Spectrin is a mechanoresponsive
protein shaping fusogenic synapse architecture during myoblast fusion. Nat. Cell Biol. 20:688–98
46. Dutta D, Anant S, Ruiz-Gomez M, Bate M, VijayRaghavan K. 2004. Founder myoblasts and bre num-
ber during adult myogenesis in Drosophila. Development 131:3761–72
47. Dutta D, Shaw S, Maqbool T, Pandya H, VijayRaghavan K. 2005. Drosophila Heartless acts with Heart-
broken/Dof in muscle founder differentiation. PLOS Biol. 3:e337
www.annualreviews.org •Drosophila Myoblast Fusion .
Review in Advance first posted on
July 5, 2019. (Changes may still
occur before final publication.)
Annu. Rev. Genet. 2019.53. Downloaded from www.annualreviews.org
Access provided by University of Texas Southwestern Medical Center on 10/01/19. For personal use only.
GE53CH02_Chen ARjats.cls June 17, 2019 14:52
48. Dworak HA, Charles MA, Pellerano LB, Sink H. 2001. Characterization of Drosophila hibris,agene
related to human nephrin. Development 128:4265–76
49. Dyer N, Rebollo E, Domínguez P, Elkhatib N, Chavrier P, et al. 2007. Spermatocyte cytokinesis re-
quires rapid membrane addition mediated by ARF6 on central spindle recycling endosomes. Development
134:4437–47
50. Erickson MRS, Galletta BJ, Abmayr SM. 1997. Drosophila myoblast city encodes a conserved protein that
is essential for myoblast fusion, dorsal closure, and cytoskeletal organization. J. Cell Biol. 138:589–603
51. Estrada B, Maeland AD, Gisselbrecht SS, Bloor JW, Brown NH, Michelson AM. 2007. The MARVEL
domain protein, Singles Bar, is required for progression past the pre-fusion complex stage of myoblast
fusion. Dev. Biol. 307:328–39
52. Fernandes J, Bate M, Vijayraghavan K. 1991. Development of the indirect ight muscles of Drosophila.
Development 113:67–77
53. Fernandes JJ, Atreya KB, Desai KM, Hall RE, Patel MD, et al. 2005. A dominant negative form of Rac1
affects myogenesis of adult thoracic muscles in Drosophila. Dev. Biol. 285:11–27
54. Fernandes JJ, Keshishian H. 1996. Patterning thedorsal longitudinal ight muscles (DLM) of Drosophila:
insights from the ablation of larval scaffolds. Development 122:3755–63
55. Figeac N, Jagla T, Aradhya R, Da Ponte JP, Jagla K. 2010. Drosophila adult muscle precursors form a
network of interconnected cells and are specied by the rhomboid-triggered EGF pathway. Development
137:1965–73
56. Galletta BJ, Chakravarti M, Banerjee R, Abmayr SM. 2004. SNS: adhesive properties, localization re-
quirements and ectodomain dependence in S2 cells and embryonic myoblasts. Mech. Dev. 121:1455–68
57. García E, Jones GE, Machesky LM, Antón IM. 2012. WIP: WASP-interacting proteins at invadopodia
and podosomes. Eur. J. Cell Biol. 91:869–77
58. Geisbrecht ER, Haralalka S, Swanson SK, Florens L, Washburn MP, Abmayr SM. 2008. Drosophila
ELMO/CED-12 interacts with Myoblast city to direct myoblast fusion and ommatidial organization.
Dev. Biol. 314:137–49
59. Gildor B, Massarwa R, Shilo B-Z, Schejter ED. 2009. The SCAR and WASp nucleation-promoting
factors act sequentially to mediate Drosophila myoblast fusion. EMBO Rep. 10:1043–50
60. Gildor B, Schejter ED, Shilo B-Z. 2012. Bidirectional Notch activation represses fusion competence in
swarming adult Drosophila myoblasts. Development 139:4040–50
61. Goode BL, Eck MJ. 2007. Mechanism and function of formins in the control of actin assembly. Annu.
Rev. Biochem. 76:593–627
62. Gunage RD, Dhanyasi N, Reichert H, VijayRaghavan K. 2017. Drosophila adult muscle development
and regeneration. Semin. Cell Dev. Biol. 72:56–66
63. Hakeda-Suzuki S, Ng J,Tzu J, Dietzl G, Sun Y, et al. 2002. Rac function and regulation during Drosophila
development. Nature 416:438–42
64. Hamoud N, Tran V, Croteau L-P, Kania A, Côté J-F. 2014. G-protein coupled receptor BAI3 promotes
myoblast fusion in vertebrates. PNAS 111:3745–50
65. Hamp J, Löwer A, Dottermusch-Heidel C, Beck L, Moussian B, et al. 2016. Drosophila Kette coordi-
nates myoblast junction dissolution and the ratio of Scar-to-WASp during myoblast fusion. J. Cell Sci.
129:3426–36
66. Haralalka S, Shelton C, Cartwright HN, Katzfey E, Janzen E, Abmayr SM. 2011. Asymmetric Mbc,
active Rac1 and F-actin foci in the fusion-competent myoblasts during myoblast fusion in Drosophila.
Development 138:1551–62
67. Hasegawa H, Kiyokawa E, Tanaka S, Nagashima K, Gotoh N, et al. 1996. DOCK180, a major CRK-
binding protein, alters cell morphology upon translocation to the cell membrane. Mol.Cell. Biol. 16:1770–
76
68. Hernández JM, Podbilewicz B. 2017. The hallmarks of cell–cell fusion. Development 144:4481–95
69. Hochreiter-Hufford AE, Lee CS, Kinchen JM, Sokolowski JD, Arandjelovic S, et al. 2013. Phos-
phatidylserine receptor BAI1 and apoptotic cells as new promoters of myoblast fusion. Nature 497:263–
67
. Lee •Chen
Review in Advance first posted on
July 5, 2019. (Changes may still
occur before final publication.)
Annu. Rev. Genet. 2019.53. Downloaded from www.annualreviews.org
Access provided by University of Texas Southwestern Medical Center on 10/01/19. For personal use only.
GE53CH02_Chen ARjats.cls June 17, 2019 14:52
70. Huang J, Zhou W, Dong W, Watson AM, Hong Y. 2009. Directed, efcient, and versatile modications
of the Drosophila genome by genomic engineering. PNAS 106:8284–89
71. Jagla T, Bellard F, Lutz Y, Dretzen G, Bellard M, Jagla K. 1998. ladybird determines cell fate decisions
during diversication of Drosophila somatic muscles. Development 125:3699–708
72. Jeong J, Conboy IM. 2011. Phosphatidylserine directly and positively regulates fusion of myoblasts into
myotubes. Biochem. Biophys. Res. Commun. 414:9–13
73. Jin P, Duan R, Luo F, Zhang G, Hong SN, Chen EH. 2011. Competition between Blown fuse and
WASP for WIP binding regulates the dynamics of WASP-dependent actin polymerization in vivo. Dev.
Cell 20:623–38
74. Kaipa BR, Shao H, Schäfer G, Trinkewitz T, Groth V, et al. 2013. Dock mediates Scar- and WASp-
dependent actin polymerization through interaction with cell adhesion molecules in founder cells and
fusion-competent myoblasts. J. Cell Sci. 126:360–72
75. Kaplan D, Zimmerberg J, Puri A, Sarkar DP, Blumenthal R. 1991. Single cell fusion events induced
by inuenza hemagglutinin: studies with rapid-ow, quantitative uorescence microscopy. Exp. Cell Res.
195:137–44
76. Kesper DA, Stute C, Buttgereit D, Kreisköther N, Vishnu S, et al. 2007. Myoblast fusion in Drosophila
melanogaster is mediated through a fusion-restricted myogenic-adhesive structure (FuRMAS). Dev. Dyn.
236:404–15
77. Kim JH, Jin P, Duan R, Chen EH. 2015. Mechanisms of myoblast fusion during muscle development.
Curr. Opin. Genet. Dev. 32:162–70
78. Kim JH, Ren Y, Ng WP, Li S, Son S, et al. 2015. Mechanical tension drives cell membrane fusion. Dev.
Cell 32:561–73
79. Kim S, Shilagardi K, Zhang S, Hong SN, Sens KL, et al. 2007. A critical function for the actin cytoskele-
ton in targeted exocytosis of prefusion vesicles during myoblast fusion. Dev. Cell 12:571–86
80. Landemaine A, Rescan PY, Gabillard JC. 2014. Myomaker mediates fusion of fast myocytes in zebrash
embryos. Biochem. Biophys. Res. Commun. 451:480–84
81. Leikina E, Melikov K, Sanyal S, Verma SK, Eun B, et al. 2013. Extracellular annexins and dynamin are
important for sequential steps in myoblast fusion. J. Cell Biol. 200:109–23
82. Luo L, Liao YJ, Jan LY, Jan YN. 1994. Distinct morphogenetic functions of similar small GTPases:
Drosophila Drac1 is involved in axonal outgrowth and myoblast fusion. Genes Dev. 8:1787–802
83. Machado C, Andrew DJ. 2000. D-Titin: a giant protein with dual roles in chromosomes and muscles.
J. Cell Biol. 151:639–52
84. Massarwa R, Carmon S, Shilo B-Z, Schejter ED. 2007. WIP/WASp-based actin-polymerization ma-
chinery is essential for myoblast fusion in Drosophila.Dev. Cell 12:557–69
85. Menon SD, Chia W. 2001. Drosophila Rolling pebbles: a multidomain protein required for myoblast
fusion that recruits D-Titin in response to the myoblast attractant Dumbfounded. Dev. Cell 1:691–703
86. Menon SD, Osman Z, Chenchill K, Chia W. 2005. A positive feedback loop between Dumbfounded
and Rolling pebbles leads to myotube enlargement in Drosophila.J. Cell Biol. 169:909–20
87. Millay DP, O’Rourke JR, Sutherland LB, Bezprozvannaya S, Shelton JM, et al. 2013. Myomaker is a
membrane activator of myoblast fusion and muscle formation. Nature 499:301–5
88. Mukherjee P, Gildor B, Shilo B-Z, VijayRaghavan K, Schejter ED. 2011. The actin nucleator WASp is
required for myoblast fusion during adult Drosophila myogenesis. Development 138:2347–57
89. Ni J-Q, Zhou R, Czech B, Liu L-P, Holderbaum L, et al. 2011. A genome-scale shRNA resource for
transgenic RNAi in Drosophila. Nat. Methods 8:405–7
90. Önel SF, Rust MB, Jacob R, Renkawitz-Pohl R. 2014. Tethering membrane fusion: common and differ-
ent players in myoblasts and at the synapse. J. Neurogenet. 28:302–15
91. Özkan E, Chia PH, Wang RR, Goriatcheva N, Borek D, et al. 2014. Extracellular architecture of the
SYG-1/SYG-2 adhesion complex instructs synaptogenesis. Cell 156:482–94
92. Park S-Y, Yun Y, Lim J-S, Kim M-J, Kim S-Y, et al. 2016. Stabilin-2 modulates the efciency of myoblast
fusion during myogenic differentiation and muscle regeneration. Nat. Commun. 7:10871
93. Plonsky I, Cho M-S, Oomens AGP, Blissard G, Zimmerberg J. 1999. An analysis of the role of the target
membrane on the Gp64-induced fusion pore. Virology 253:65–76
www.annualreviews.org •Drosophila Myoblast Fusion .
Review in Advance first posted on
July 5, 2019. (Changes may still
occur before final publication.)
Annu. Rev. Genet. 2019.53. Downloaded from www.annualreviews.org
Access provided by University of Texas Southwestern Medical Center on 10/01/19. For personal use only.
GE53CH02_Chen ARjats.cls June 17, 2019 14:52
94. Plonsky I, Zimmerberg J. 1996.The initial fusion pore induced by baculovirus GP64 is large and forms
quickly. J. Cell Biol. 135:1831–39
95. Pollard TD, Cooper JA. 2009. Actin, a central player in cell shape and movement. Science 326:1208–12
96. Pollitt AY, Insall RH.2009. WASP and SCAR/WAVE proteins: the drivers of actin assembly. J. Cell Sci.
122:2575–78
97. Quinn ME, Goh Q, Kurosaka M, Gamage DG, Petrany MJ, et al. 2017. Myomerger induces fusion of
non-fusogenic cells and is required for skeletal muscle development. Nat. Commun. 8:15665
98. Randrianarison-Huetz V, Papaefthymiou A, Herledan G, Noviello C, Faradova U, et al. 2018. Srf con-
trols satellite cell fusion through the maintenance of actin architecture. J. Cell Biol. 217:685–700
99. Rau A, Buttgereit D, Holz A, Fetter R, Doberstein SK, et al. 2001. rolling pebbles (rols) is required in
Drosophila muscle precursors for recruitment of myoblasts for fusion. Development 128:5061–73
100. Richardson BE, Beckett K, Nowak SJ, Baylies MK. 2007. SCAR/WAVE and Arp2/3 are crucial for
cytoskeletal remodeling at the site of myoblast fusion. Development 134:4357–67
101. Rottner K, Hänisch J, Campellone KG. 2010. WASH, WHAMM and JMY: regulation of Arp2/3 com-
plex and beyond. Trends Cell Biol. 20:650–61
102. Roy S, VijayRaghavan K. 1998.Patterning muscles using organizers: Larval muscle templates and adult
myoblasts actively interact to pattern the dorsal longitudinal ight muscles of Drosophila.J. Cell Biol.
141:1135–45
103. Ruiz-Gómez M, Bate M. 1997. Segregation of myogenic lineages in Drosophila requires Numb. Devel-
opment 124:4857–66
104. Ruiz-Gómez M, Coutts N, Price A, Taylor MV, Bate M. 2000. Drosophila dumbfounded: a myoblast
attractant essential for fusion. Cell 102:189–98
105. Ruiz-Gómez M, Romani S, Hartmann C, Jäckle H,Bate M. 1997. Specic muscle identities are regulated
by Krüppel during Drosophila embryogenesis. Development 124:3407–14
106. Rushton E, Drysdale R, Abmayr SM, Michelson AM, Bate M. 1995. Mutations in a novel gene, my-
oblast city, provide evidence in support of the founder cell hypothesis for Drosophila muscle development.
Development 121:1979–88
107. Sánchez-Pulido L, Martıín-Belmonte F, Valencia A, Alonso MA. 2002. MARVEL: a conserved domain
involved in membrane apposition events. Trends Biochem. Sci. 27:599–601
108. Schäfer G, Weber S, Holz A, Bogdan S, Schumacher S, et al. 2007. The Wiskott-Aldrich syndrome
protein (WASP) is essential for myoblast fusion in Drosophila.Dev. Biol. 304:664–74
109. Schejter ED. 2016. Myoblast fusion: experimental systems and cellular mechanisms. Semin. Cell Dev.
Biol. 60:112–20
110. Schnorrer F, Schönbauer C, Langer CCH, Dietzl G, Novatchkova M, et al. 2010. Systematic genetic
analysis of muscle morphogenesis and function in Drosophila. Nature 464:287–91
111. Schröter RH, Lier S, Holz A, Bogdan S, Klämbt C, et al. 2004. kette and blown fuse interact genetically
during the second fusion step of myogenesis in Drosophila. Development 131:4501–9
112. Schulman VK, Dobi KC, Baylies MK. 2015. Morphogenesis of the somatic musculature in Drosophila
melanogaster. Wiley Interdiscip.Rev. Dev. Biol. 4:313–34
113. Segal D, Dhanyasi N, Schejter ED, Shilo B-Z. 2016. Adhesion and fusion of muscle cells are promoted
by lopodia. Dev. Cell 38:291–304
114. Sens KL, Zhang S, Jin P, Duan R, Zhang G, et al. 2010. An invasive podosome-like structure promotes
fusion pore formation during myoblast fusion. J. Cell Biol. 191:1013–27
115. Shaq SA. 1963. Electron microscopic studies on the indirect ight muscles of Drosophila melanogaster.
II. Differentiation of myobrils. J. Cell Biol. 17:363–73
116. Shelton C, Kocherlakota KS, Zhuang S, Abmayr SM. 2009. The immunoglobulin superfamily member
Hbs functions redundantly with Sns in interactions between founder and fusion-competent myoblasts.
Development 136:1159–68
117. Shi J, Bi P, Pei J, Li H, Grishin NV, et al. 2017. Requirement of the fusogenic micropeptide myomixer
for muscle formation in zebrash. PNAS 114:11950–55
118. Shilagardi K, Li S, Luo F,Marikar F, Duan R, et al. 2013. Actin-propelled invasive membrane protrusions
promote fusogenic protein engagement during cell–cell fusion. Science 340:359–63
. Lee •Chen
Review in Advance first posted on
July 5, 2019. (Changes may still
occur before final publication.)
Annu. Rev. Genet. 2019.53. Downloaded from www.annualreviews.org
Access provided by University of Texas Southwestern Medical Center on 10/01/19. For personal use only.
GE53CH02_Chen ARjats.cls June 17, 2019 14:52
119. Shin N-Y, Choi H, Neff L, Wu Y, Saito H, et al. 2014. Dynamin and endocytosis are required for the
fusion of osteoclasts and myoblasts. J. Cell Biol. 207:73–89
120. Stradal TE, Scita G. 2006. Protein complexes regulating Arp2/3-mediated actin assembly. Curr. Opin.
Cell Biol. 18:4–10
121. Strünkelnberg M, Bonengel B, Moda LM, Hertenstein A, de Couet HG, et al. 2001. rst and its paralogue
kirre act redundantly during embryonic muscle development in Drosophila. Development 128:4229–39
122. Su M-T, Fujioka M, Goto T, Bodmer R. 1999. The Drosophila homeobox genes zfh-1 and even-skipped
are required for cardiac-specic differentiation of a numb-dependent lineage decision. Development
126:3241–51
123. Tixier V, Bataillé L, Jagla K. 2010. Diversication of muscle types: recent insights from Drosophila.Exp.
Cell Res. 316:3019–27
124. Tskhovrebova L, Trinick J. 2010. Roles of titin in the structure and elasticity of the sarcomere.J. Biomed.
Biotechnol. 2010:1–7
125. Van Vactor D, Sink H, Fambrough D, Tsoo R, Goodman CS. 1993. Genes that control neuromuscular
specicity in Drosophila. Cell 73:1137–53
126. Willkomm L, Bloch W. 2015. State of the art in cell–cell fusion. In Cell Fusion, ed. K Pfannkuche,
pp. 1–19. Methods Mol. Biol. 1313. Clifton, NJ: Humana
127. Yang Y, Zhang Y, Li WJ, Jiang Y, Zhu Z, et al. 2017. Spectraplakin induces positive feedback between
fusogens and the actin cytoskeleton to promote cell-cell fusion. Dev. Cell 41:107–20.e4
128. Zhang Q, Vashisht AA, O’Rourke J, Corbel SY, Moran R, et al. 2017. The microprotein Minion controls
cell fusion and muscle formation. Nat. Commun. 8:15664
129. Zhang R, Zhang CY, Zhao Q, Li DH. 2013. Spectrin: structure, function and disease. Sci. China Life Sci.
56:1076–85
130. Zhang W, Roy S. 2017. Myomaker is required for the fusion of fast-twitch myocytes in the zebrash
embryo. Dev. Biol. 423:24–33
131. Zhang Y, Featherstone D, Davis W, Rushton E, Broadie K. 2000. Drosophila D-Titin is required for
myoblast fusion and skeletal muscle striation. J. Cell Sci. 113:3103–15
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