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First published online 14 November 2007
doi: 10.1242/dev.010678
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1 Program in Developmental Biology, Sloan Kettering Institute, New York, NY
10021, USA.
2 Weill Graduate School at Cornell Medical School, New York, NY 10021,
USA.
* Author for correspondence (e-mail: m-baylies{at}ski.mskcc.org)
Accepted 14 September 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Cell-cell fusion, Myoblast fusion, Muscle, Actin, Kette (Hem, Nap1), SCAR (WAVE), Arp2/3
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In
addition, intercellular fusion has been confirmed as a primary mechanism of
tissue repair used by stem cells
(Alvarez-Dolado et al., 2003;
Wang et al., 2003;
Weimann et al., 2003).
Although much is known about the molecules and mechanisms underlying
intracellular fusion of membrane compartments, the molecular mechanisms
underlying cell-cell fusion are not well understood. Increased knowledge of
cell-cell fusion would have significant advantages for tissue engineering and
repair.
During the development and repair of muscle, mononucleated myoblasts fuse
to form multinucleated muscle fibers
(Abmayr et al., 2003;
Chen and Olson, 2004;
Horsley and Pavlath, 2004;
Patel et al., 2002). Fusion in
Drosophila requires two cell types: founder cells (FCs), which seed
specific muscles, and fusion-competent myoblasts (FCMs), which fuse to an FC
and adopt that FC muscle program (Baylies
et al., 1998; Carmena and
Baylies, 2006; Frasch,
1999). As a result of fusion, a muscle of particular size, shape
and orientation forms. There are 30 individual muscles per hemisegment of the
Drosophila embryo; depending on the particular muscle, body wall
muscles in Drosophila embryos fuse between two and 25 times
(Bate, 1990).
A number of mutations have been identified in Drosophila that
disrupt fusion (Abmayr et al.,
2003; Chen and Olson,
2004; Taylor,
2003). The genes revealed by these mutations have been organized
into a model based on genetics, biochemistry and predicted function. The sum
of the activities of these genes lead to undefined rearrangements in the
cytoskeleton that are necessary for fusion
(Chen and Olson, 2004).
Recognition and adhesion between an FC and FCMs are mediated by four,
single-pass transmembrane proteins belonging to the immunoglobulin (IG)-domain
containing family of adhesion molecules. Two are required in FCs, Dumbfounded
[Duf; also known as Kirre - FlyBase
(Ruiz-Gomez et al., 2000)] and
Roughest [Rst; also known as Irregular Chiasm-C; IrreC
(Strunkelnberg et al., 2001)],
and two are required in FCMs, Sticks and stones [Sns
(Bour et al., 2000)] and
Hibris [Hbs (Artero et al.,
2001; Dworak et al.,
2001)]. Downstream of these adhesion proteins in the FC, signal
transduction bifurcates, with one branch of the pathway mediated by the
scaffold protein Rolling pebbles [Rols; also known as Antisocial; Ants
(Chen and Olson, 2001;
Menon and Chia, 2001;
Rau et al., 2001)], and the
second branch mediated by Loner [also known as Schizo - FlyBase
(Chen et al., 2003)], a GEF
(guanine nucleotide exchange factor) protein. Rols relays adhesion to
components of the cytoskeleton (Menon and
Chia, 2001; Zhang et al.,
2000). Rols has been shown to physically interact with Duf and
Myoblast city (Mbc), the Drosophila Dock180 homolog
(Chen and Olson, 2001;
Erickson et al., 1997;
Rushton et al., 1995). Based
on work in other systems, Mbc regulates Rac activation
(Hasegawa et al., 1996;
Kiyokawa et al., 1998;
Nolan et al., 1998). Removal
of two of the three Drosophila Rac homologs, Rac1 and Rac2, leads to
a fusion block (Hakeda-Suzuki et al.,
2002; Luo et al.,
1994).
Loner, a GEF that interacts with Duf, regulates the small GTPase, ARF6
(also known as Arf51f - FlyBase) (Chen et
al., 2003). ARF6 is required for cell shape changes and enhances
the activity of Rac to form membrane ruffles
(Donaldson, 2003;
Radhakrishna et al., 1999;
Zhang et al., 1999). In
loner mutants, Rac localization is aberrant
(Chen et al., 2003). Hence,
Loner, through its regulation of ARF6 and Rac, leads to alterations in the
cytoskeleton required for myoblast fusion.
Blown fuse (Blow), a PH-domain containing protein
(Doberstein et al., 1997), and
Kette (also known as Hem and Nap1)
(Schroter et al., 2004), are
also required for fusion. Kette functions in a conserved complex with Sra-1
(also known as Pir121 and CYFIP), Abi and HSPC300 to regulate the activity of
SCAR (also known as WAVE). SCAR, in turn, activates Arp2/3-dependent actin
polymerization (Ibarra et al.,
2005; Machesky and Insall,
1998; Smith and Li,
2004; Vartiainen and Machesky,
2004). How the Kette complex regulates SCAR is a subject of
debate, as both positive and negative interactions have been suggested
(Bogdan and Klambt, 2003;
Eden et al., 2002;
Ibarra et al., 2006;
Kunda et al., 2003;
Rogers et al., 2003). Recent
studies have identified mutations in WASP (also known as
WASp - FlyBase) and its regulator solitary [sltr;
also known as WASP-interacting protein (WIP) and
Verprolin 1 (Vrp1)] that disrupt myoblast fusion
(Kim et al., 2007;
Massarwa et al., 2007;
Schafer et al., 2007).
Similarly to SCAR, WASP is an activator of Arp2/3-dependent actin
polymerization, underscoring the importance of this pathway in fusion. Sltr is
recruited to sites of myoblast adhesion and is proposed to regulate actin
polymerization at these sites in FCMs (Kim
et al., 2007; Massarwa et al.,
2007).
Two questions raised by the genetic analyses are: (1) what is the nature of the cytoskeletal rearrangements at the site of fusion? and (2) what are the contributions of the identified proteins to this cytoskeleton remodeling at the fusion site? Here we apply novel methods in Drosophila to investigate the mechanisms underlying cell-cell fusion. We find a specific actin rearrangement at the fusion site, an actin focus, whose formation and dissolution precedes a fusion event. Analysis of fusion mutants has identified separable classes of genes required for the formation and dissolution of these fusion-specific actin structures. Likewise, the recruitment of the known proteins involved in myoblast fusion is altered in certain classes of mutants. By investigating the most actin-proximal of the known fusion mutants, kette, we find that Kette is required for the dissolution of actin foci. Mechanistically, we determined that the abnormally large foci result from the loss of positive regulation by Kette on SCAR: kette mutants show defects in SCAR localization and stability in vivo. Like kette, SCAR and Arp2/3 mutants show defects in myoblast fusion and actin foci dissolution, suggesting a model in which Kette-SCAR-Arp2/3-mediated actin polymerization leads to a reorganization of the actin focus that is required for the progression of cell-cell fusion. Taken together, these data provide new perspectives on the genetic, molecular and cellular requirements of myoblast fusion.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), twist-CD2
(Dunin-Borkowski and Brown,
1995), apME-GFP (this study), apME-NLS::eGFP
(this study), apME-NLS::dsRed (this study), Df(1)w67k30
[deficiency removing duf and rst
(Lefevre, and Green, 1972;
Ruiz-Gomez et al., 2000)],
ketteJ4-48 (Hummel et
al., 2000), rolsT627
(Chen and Olson, 2001),
lonerT1032 (Chen et
al., 2003), mbcC1
(Rushton et al., 1995),
snsXB3 (Bour et al.,
2000), blow1
(Doberstein et al., 1997),
Rac1J11Rac2
mtl
(Hakeda-Suzuki et al., 2002),
SCAR37 (Zallen
et al., 2002), SCARk13811
[(Spradling et al., 1999)
Berkeley Drosophila Genome Project], Arp3EP3640
(Rorth, 1996), and
D-WIPD30 (Massarwa et
al., 2007). Mutants were balanced and identified using CyO
P[w+wgen11lacZ], TM3 Sb1
Dfd-lacZ or TTG [TM3, twi-GAL4, UAS-2xeGFP
(Halfon et al., 2002)].
Germline clones (Chou and Perrimon,
1996) were generated by heat shock of hs-FLP; ovoD,
FRT40A/SCARk13811, FRT40A larvae. Germline clone females were
mated to SCARk13811/CyO at 20-22°C to create
SCARk13811 maternal/zygotic embryos
(Zallen et al., 2002).
Germline transformation and constructs
apME-GFP, apME-NLS::eGFP (gifts from Z. Kambris and M. Capovilla,
Centre National de la Recherche Scientific, Strasbourg, France) and
apME-NLS::dsRed (this study) DNA were constructed by cloning the
apterous mesodermal enhancer 680 into pGreenH-Pelican, pH-Stinger and
pRedH-Stinger [(Barolo et al.,
2000; Barolo et al.,
2004; Capovilla et al.,
2001) Berkeley Drosophila Genome Project], which,
respectively, contain cytoplasmic eGFP, eGFP and dsRed.T4 downstream of a
nuclear localization signal. Constructs were injected using established
protocols (Beckett and Baylies,
2006).
Immunohistochemistry
Embryos were collected at 25°C on apple juice agar plates and were
fixed as described previously (Beckett and
Baylies, 2006) except that embryos were fixed in 4% EM grade
paraformaldehyde (Electron Microscopy Sciences) in 0.2 M sodium phosphate for
Kette and SCAR staining. Embryos were mounted in Prolong Gold (Molecular
Probes) for fluorescent stainings or Araldite otherwise. Antibodies were
preabsorbed (PA) where noted and used at the indicated final dilutions: mouse
anti-β-galactosidase (1:1000, Promega), chicken anti-β-galactosidase
(1:1000, Cappel), rabbit anti-Lame duck (LPA, 1:250)
(Duan et al., 2001), rabbit
anti-Kette (1:1000) (Hummel et al.,
2000), guinea pig anti-SCAR (1:500)
(Zallen et al., 2002), mouse
anti-GFP (1:400, Clontech), mouse anti-Rols7 (1:4000)
(Menon and Chia, 2001), rat
anti-Loner (PA, 1:300) (Chen et al.,
2003), mouse anti-Rac1 (1:200, BD Biosciences), rat anti-Mbc (PA,
1: 100) (Erickson et al.,
1997), rat anti-Sticks and stones (Sns, 1:100)
(Bour et al., 2000), rabbit
anti-Blow (PA, 1:500) (Doberstein et al.,
1997), rabbit anti-Slouch (PA, 1:200)
(Beckett and Baylies, 2007) and
rabbit anti-myosin heavy chain (Mhc; 1:10000; a gift from D. Kiehart, Duke
University, Durham, NC). Biotinylated secondary antibodies (Vector
Laboratories and Jackson ImmunoResearch) and the Vectastain ABC kit (Vector
Laboratories) were applied for non-fluorescent Mhc stainings. Additionally,
TSA amplification (PerkinElmer Life Sciences) was applied for Kette, Scar,
Loner, Sns and Rac1. We used Alexa Fluor 488-, Alexa Fluor 555- and Alexa
Fluor 647-conjugated fluorescent secondary antibodies and Alexa Fluor 546- and
Alexa Fluor 647-conjugated phalloidin (Invitrogen). Fluorescent images were
acquired on a Zeiss LSM 510 confocal scanning system mounted on an Axiovert
100M microscope with a 63x 1.2 NA C-Apochromat water objective. For
confocal microscopy, pinholes were set to capture an optical slice of 1.1
µm. Non-fluorescent images were acquired on a Zeiss Axiophot microscope.
Images were processed using Adobe Photoshop. 3D reconstruction was created
using Improvision Volocity software.
Live imaging
Embryos were collected and dechorionated in 50% bleach for 3 minutes.
Appropriately staged embryos were selected and mounted on glass-bottom Petri
dishes (MatTek Cultureware) using Technau glue (Scotch tape dissolved in
heptane) and covered with halocarbon 700 oil (Halocarbon Products). GFP was
excited at 488 nm and dsRed was excited at 543 nm. All pinholes were set to
capture an optical slice of 1.5 µm. All timelapse sequences were taken as a
series of z-stacks over time (4D imaging), with optical sections
captured every 1.5 µm. Fusion events were only considered valid if optical
slices were available above and below the plane of fusion, to rule out
mistaking a cell migration event for myoblast fusion. Optical projections were
created using the projection function of the Zeiss LSM software. Images were
processed with Adobe Photoshop, and movies were created from image sequences
using Apple Quicktime.
Foci size and duration measurements
Area was measured using the overlay function of the Zeiss LSM software (see
Fig. S2 in the supplementary material). Foci were measured in the optical
slice where they had the greatest radius and where FC-FCM adhesion was
verified with specific cell labeling. The edges of foci were determined by
using the range indicator function of the software and setting the edge where
there is a clear change from signal to background. Measurements were acquired
in the linear range of intensity and no relevant pixels were saturated.
Duration was calculated as the time between when a focus appeared in a
sequence and when it disappeared from detection. Foci were only included for
duration measurements if there were optical slices above and below throughout
the sequence to ensure actual dissolution and rule out cell/foci movement.
Statistical analysis was performed with Microsoft Excel.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Beckett and Baylies, 2007)],
fixed wild-type embryos were stained with phalloidin to label filamentous
actin (F-actin) and with specific reagents that distinguish FCs/myotubes and
FCMs. rp298-lacZ expresses β-galactosidase in the nuclei of FCs
(Nose et al., 1998), whereas
Lame duck (Lmd) is an FCM-specific transcription factor that has both nuclear
and cytoplasmic expression (Duan et al.,
2001; Nose et al.,
1998) (Fig. 1A,B,
and see Fig. S1A,B in the supplementary material). Hence, the relevant
myoblast cell types, their arrangements with respect to one another and
changes in actin cytoskeleton could be identified during fusion.
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). As recognition occurred between an FC/myotube and FCM, the
FCMs assumed a tear-drop shape, and this change in cell shape was reflected in
the actin cytoskeleton (see Fig. S1B in the supplementary material). At the
sites of adhesion between FCs/myotubes and FCMs, we observed a striking
accumulation of F-actin in foci (Fig.
1B and see Movie 1 in the supplementary material)
(Kim et al., 2007;
Kesper et al., 2007). These
foci were most often spherical and located across both cell types
(Fig. 1C). To better
characterize actin foci, we measured their size (see Fig. S2 in the
supplementary material). In wild-type embryos, these foci ranged in size from
0.7-4.5 µm2, averaging 1.9 µm2 (n=100
foci; Table 1). The range
observed in these fixed preparations most likely reflects the dynamic nature
of the actin foci (see below). Actin foci were not observed in non-mesodermal
tissues nor were they detected in mesodermal cells before or after the stages
when fusion takes place (see Fig. S3A,C in the supplementary material).
Furthermore, actin foci are present prior to pore formation in membranes
between myotubes and FCMs, as cytoplasmic GFP expressed specifically in
myotubes does not leak across foci into the cytoplasm of adherent FCMs
(Fig. 1D). Thus, using these
labeling techniques, a series of actin cytoskeletal behaviors that occur
during muscle and myotube formation have been identified: actin-rich
protrusive structures, cell shape changes and an accumulation of F-actin at
the adhesion site between FCs/myotubes and FCMs.
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).
Phalloidin staining of twip-GFP-actin embryos confirmed that
both methods revealed the same range of actin-based rearrangements described
above (see Fig. S1C-E, Fig. S4 in the supplementary material). In particular,
GFP-actin foci were observed at FC/myotube and FCM adhesion sites to be of
similar size and shape to the phalloidin-stained F-actin foci (mean: 1.8
µm2, range: 0.7-4.7 µm2, n=25,
Fig. 1E, and see Fig. S4 in the
supplementary material). Live imaging revealed that an actin focus at the
adhesion site builds into the spherical structure observed in fixed embryos
(Fig. 1B). Actin foci are
dynamic structures: the lifetime of actin foci ranged from 5.7 to 29.5 minutes
with the average actin focus present for 11.9 minutes at all stages in which
myoblast fusion takes place (n=50). Actin foci build to their maximum
size in less than 2 minutes and, following their duration, completely dissolve
in less than 1 minute.
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; Capovilla et al.,
2001; Frasch,
1999). We therefore generated transgenic flies carrying both
twip-GFP-actin, which is expressed in all FCs and FCMs, and
an apterousME-NLS-dsRed construct, which is expressed in a specific
subset of FCs, their growing myotubes and resultant muscles. In this way, we
monitored single fusion events with 4D live imaging (a 3D z-stack imaged over
time) by following myoblast arrangements in time and space and by the
accompanying incorporation of an additional dsRed nucleus into the
apterousME-NLS-dsRed myotube. In all fusion events observed (>50),
an ordered sequence of events takes place. First, an actin focus forms at the
adhesion site between a fusing myotube, labeled with
apterousME-NLS-dsRed, and an FCM. Next, the actin focus dissolves,
followed by myoblast fusion and detection of dsRed in the newly added nucleus
(
5 minutes; Fig. 1E and see
Movie 2 in the supplementary material). Identical data were obtained using
apterousME-NLS-eGFP (see Fig. S5, Movie 3 in the supplementary
material). In no case was fusion observed in the absence of actin focus
formation and dissolution. Based on these studies, we concluded that the actin
focus marks the site of myoblast fusion and that dissolution of the actin
focus directly precedes a fusion event. In addition, these data revealed the
dynamics of actin foci formation and dissolution and myoblast fusion in
vivo.
Mutations in known fusion genes affect foci differently
Since we identified a specific actin rearrangement that is directly linked
to the fusion site, we next addressed whether mutations in the genes linked to
myoblast fusion led to defects in actin foci number or morphology. Actin foci
in mutant embryos were first examined in fixed preparations using phalloidin
staining at stage 14, the period when the maximum number of fusion events
takes place (Beckett and Baylies,
2007). The mutants fell into three classes based on actin foci
number and size: (1) decreased number, normal size, (2) increased number,
normal size, and (3) increased number, increased size
(Table 1,
Fig. 2 and see Fig. S2 in the
supplementary material; data not shown).
Class one mutant embryos had fewer foci. Disruption of genes involved in FC-FCM recognition and adhesion, such as duf, rst and sns (Fig. 2B and data not shown), led to embryos with no actin foci. These data indicated that adhesion between FCs/myotubes and FCMs is critically required to initiate actin nucleation and formation of the F-actin focus. Additionally, disruption of rols led to a decrease in the number of actin foci, although those that did form were of normal size (Fig. 2C, Table 1 and data not shown). Antibodies to Sns and Rols colocalized with the actin foci (Fig. 3A,B), consistent with the actin foci marking the fusion site.
One mutant had increased numbers of wild-type size actin foci (class 2):
loner (Fig. 2D;
Table 1 and data not shown).
The increase in the number of foci was consistent with a block in myoblast
fusion in this mutant background. Despite the reported FC-specific expression
of Loner (Chen et al., 2003),
it was found to be consistently expressed within myotubes and FCMs, localized
near foci but never overlapping (Fig.
3C and data not shown). These data, together with live imaging
data (see below), suggested that, although Loner activity is required for the
progression of fusion, it regulates fusion independently of actin foci.
A third class - Rac (Rac1, Rac2, Mtl triple mutants),
kette, blow and mbc - showed enlarged foci, as well as
increased numbers of actin foci (Fig.
2E-H and data not shown). Rac localization was punctate throughout
the cell, with partially overlapping F-actin foci
(Fig. 3D). The protein products
encoded by the other genes were enriched at the sites of actin foci formation
both in the myotubes and in adhering FCMs
(Fig. 3E-G). Expression of
these proteins in both myotubes and FCMs is consistent with published data
(Erickson et al., 1997;
Schroter et al., 2006;
Schroter et al., 2004).
Enlarged foci were seen in these mutants from the earliest stages of fusion
(data not shown), and foci persisted after fusion was complete in wild-type
embryos (see Fig. S3F in the supplementary material). FCMs often clustered
together at the side of adhesion to FCs in these mutants. However, distinct
actin foci were often still discernable for an individual FC-FCM combination.
In kette mutants, the average size of an actin focus was 3.4
µm2, with a range of 1.2-8.3 µm2 (n=100;
Table 1). Likewise blow,
mbc and Rac mutants had more and larger F-actin foci than wild
type (Table 1, and data not
shown).
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Live imaging of mutants
To further understand the regulation of the actin foci at fusion sites, we
performed live imaging analysis on representative mutants from the three
classes of fusion mutants. Live imaging analysis of rols mutants,
which are capable of some fusion, indicated that fusion always follows actin
focus formation and dissolution, as in wild type (data not shown). Live
imaging of loner mutants indicated that whereas actin foci form
normally, they never dissolve, correlating with the increased numbers of foci
observed and a complete fusion block
(Beckett and Baylies, 2007)
(data not shown). These data, taken together with the wild-type size of actin
foci in loner mutants, suggested that the Loner-ARF6 pathway
regulates fusion independently of actin foci.
|
). Kette
is a member of an evolutionarily conserved complex, which regulates the
activity of SCAR. SCAR, in turn, activates Arp2/3-dependent actin
polymerization (Ibarra et al.,
2005; Smith and Li,
2004; Vartiainen and Machesky,
2004). Live imaging of kette mutant embryos revealed that
GFP-actin foci were larger, as seen in the fixed preparations, and persisted
significantly longer than in wild-type embryos (average 36.3 minutes compared
to 11.9 minutes in wild-type, n=25;
Fig. 4A,B and see Movie 4 in
the supplementary material). This measure of actin focus duration in the
kette mutant embryos is most likely an underestimate, as actin foci
were always still present at the end of an imaging sequence (see Fig. S3F in
the supplementary material). Despite the changes seen in actin foci size and
number in kette mutant embryos, the foci still formed at sites of
FC/myotube-FCM adhesion. These results indicated that the fusion defect in
kette mutant embryos (and we would suggest, in the other members of
this class; data not shown) is correlated with a failure in the dissolution of
actin foci at sites of fusion.
Localization of fusion proteins in the different foci classes
We next examined the localization of members of the known fusion proteins
in the three classes of fusion mutants
(Fig. 5). In fusion mutants
that have no actin foci (i.e. sns), the fusion proteins that normally
localize to the myoblast fusion site lost their polarized localization.
Instead, Blow and Mbc became cortically distributed, while Kette was punctate
in the cytoplasm (Fig. 5A and
data not shown). This suggested that adhesion is required for proper
localization of this subset of fusion proteins that co-localize with the actin
foci. In fusion mutants with normally sized actin foci (loner), the
localization of this subset of fusion proteins is indistinguishable from
wild-type (Fig. 5B and data not
shown). Finally, in fusion mutants with enlarged actin foci (kette, mbc,
blow, Rac), this subset of the known fusion proteins continued to
colocalize with F-actin and were present at high levels throughout the
abnormally large actin foci at sites of myoblast adhesion
(Fig. 5C and data not shown).
Taken together, these data indicated that the formation of an actin focus
correlates with the proper localization of a specific subset of the known
fusion proteins. Enlarged actin foci were associated with an accumulation of
this subset of known fusion proteins that normally localize at the site of
myoblast fusion. Interestingly, the localization of Loner, which does not
colocalize with actin foci in wild-type embryos, was not altered in any class
of mutants at the time of foci formation (data not shown).
Sltr (D-WIP) is reported to regulate the formation of actin foci
specifically in FCMs (Kim et al.,
2007). Therefore, we tested if foci were asymmetrically disrupted
in fusion mutants with enlarged actin foci. Use of an antibody against the
FC/myotube-specific protein Rols indicated that the enlarged actin foci
localize across both cell types in mbc, blow and kette
mutant embryos (Fig. 5D-F).
These data suggested that, unlike Sltr (D-WIP), these gene products are not
required specifically in one cell type.
SCAR loss of function leads to a fusion block and prevents actin focus dissolution
To further understand the mechanism underlying the foci dissolution defect
in kette mutants, we examined the function of SCAR in myoblast
fusion. The regulation of SCAR by Kette has been studied in a variety of
systems. Depending on the context, the Kette complex is thought to negatively
or positively regulate the activity of SCAR
(Bogdan and Klambt, 2003;
Eden et al., 2002;
Ibarra et al., 2006;
Kunda et al., 2003;
Rogers et al., 2003).
Therefore, we examined the final pattern of the embryonic musculature in
SCAR loss-of-function mutants. Embryos homozygous for a null mutation
of SCAR exhibited a moderate, but completely penetrant, myoblast
fusion defect (Fig. 6B).
Embryos with reduced maternal and zygotic SCAR contributions,
however, had a more severe myoblast fusion defect than removal of zygotic
SCAR alone, with increased numbers of free myoblasts and thinner
muscles (Fig. 6C).
Krüppel-expressing myotubes were most often mononucleate in maternal and
zygotic SCAR mutants, with an occasional binucleate cell, suggesting
a severe fusion block [1.24±0.43 nuclei/myotube (mean±s.d.)
n=22 hemisegments; data not shown]. Altogether, these data indicated
that SCAR is crucial for myoblast fusion.
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Arp2/3 is required for fusion
The requirement of SCAR for proper myoblast fusion and actin focus
dissolution prompted us to examine the role of the Arp2/3 complex in these
processes. Examination of a zygotic loss-of-function allele of Arp3/Arp66B, an
essential component of the Arp2/3 complex, revealed a moderate myoblast fusion
defect, similar to the defect seen in a SCAR zygotic loss-of-function
(Fig. 6D). Likewise foci size
was similar to that observed in SCAR zygotic mutants
(Fig. 6G,
Table 1). As with SCAR, this
relatively mild defect could be due to the presence of maternally contributed
Arp3. However, further analysis of the Arp2/3 complex is complicated by an
earlier requirement, as germline clones with available reagents do not develop
to the stages of muscle formation (Zallen
et al., 2002). Nevertheless these data suggested that Arp2/3, like
Kette and Scar, is required for actin focus dissolution.
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Although a number of these genes have been implicated in
cytoskeletal rearrangements, neither the nature of these rearrangements nor
contributions of the individual genes to these rearrangements were known. We
have applied novel imaging methods in Drosophila to investigate the
cellular and molecular mechanisms underlying cell-cell fusion. Critical to a
mechanistic understanding of myoblast fusion is the identification of the
specific site of fusion. We have identified that site, and find that the
formation and subsequent dissolution of an F-actin focus at that site directly
precedes a fusion event. Our live-imaging approaches have also revealed both
the dynamics of these foci and timing of cell-cell fusion, indicating that
Drosophila myoblast fusion is a rapid process. With these new assays,
we reassessed the functions of the known fusion genes, added new genes and
provided a new framework for understanding the identity and sequence of
cellular events required for myoblast fusion.
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) has
suggested that there are distinct events in the fusion process: after
recognition and adhesion, the membranes between an FC and an FCM align, paired
vesicles carrying electron dense material are then recruited to these sites
(termed the `prefusion complex'), these vesicles then release the electron
dense material, leading to plaque formation. Subsequently, the plasma membrane
breaks down, leading to cytoplasmic continuity between the cells. TEM studies
indicated that, in kette mutant embryos, myoblast fusion is blocked
at plaque formation (Schroter et al.,
2004). Similarly our data indicate that in kette mutant
embryos, fusion is blocked due to enlarged actin foci at sites of fusion. It
is thus tempting to equate the actin foci with the enlarged plaques seen by
TEM. However, there is a strong argument against this conclusion. We see
enlarged actin foci in a number of fusion mutants, including mbc and
blow. Whereas all show enlarged foci, TEM analysis reveals that only
kette mutant embryos show a block at plaque formation. mbc
mutant embryos show a block prior to the recruitment of the electron dense
vesicles, whereas embryos mutant for blow show a block at the
prefusion complex, with no plaque formation detected
(Doberstein et al., 1997).
Hence actin foci cannot be equated with the electron dense plaques.
It is consistent, however, with both data sets that the subcellular events
observed by TEM, namely membrane alignment, formation of the prefusion complex
and plaque formation, happen concurrently with the actin focus formation that
we report here. We see that the aligned membranes are still intact when a
focus is detected (Fig. 1).
Recent TEM work suggests that actin is important for the targeting of vesicles
(Kim et al., 2007). How the
diffuse actin observed in these studies relates to the dynamic but
concentrated accumulation of F-actin into foci at the plasma membrane in our
study remains in question. Lastly, these observations highlight the larger
question of how the events we detected with confocal microscopy relate to the
events distinguished by TEM.
Kette positively regulates SCAR-Arp2/3 to allow focus dissolution
Important to the interpretation of the mechanisms contributing to enlarged
foci is our finding that SCAR and Arp2/3 are also required for myoblast
fusion. Reduction of maternal and zygotic contributions of SCAR leads to a
block in myoblast fusion, like that seen in kette mutant embryos.
Reduction of zygotic Arp3 also leads to a block in fusion, although analysis
of complete loss of Arp2/3 activity is precluded by the reagents currently
available. Together these proteins provide an important direct link to an
actin polymerization event at the site of fusion.
There has been some controversy as to how the Kette complex regulates SCAR.
In biochemical assays, the Kette complex negatively regulates SCAR, holding it
in an inactive state until the complex is activated by Rac
(Eden et al., 2002). In
support of this view, reducing the dose of SCAR can partially rescue
the kette phenotype in the central nervous system of the
Drosophila embryo (Bogdan and
Klambt, 2003). By contrast, the Kette complex in
Drosophila tissue culture cells positively regulates SCAR by
correctly localizing and stabilizing SCAR
(Kunda et al., 2003;
Rogers et al., 2003). Our data
support a positive regulation of SCAR by the Kette complex in the context of
myoblast fusion. In kette mutant embryos, SCAR protein levels are
reduced significantly and the residual protein is not localized properly. It
has been suggested that the differences in SCAR regulation by the Kette
complex may be a reflection of the relative differences in the amounts of SCAR
and the components of the regulatory complex in different contexts
(Kunda et al., 2003).
The actin focus phenotype in SCAR and Arp3 mutants has
also provided mechanistic insight into the role of the Kette-SCAR-Arp2/3
pathway controlling its behavior. If SCAR or Arp2/3 was required for the
polymerization of actin leading to foci formation, we would have expected
smaller or absent actin foci in SCAR and Arp3 mutants.
Instead, we find that, similar to kette mutants, enlarged foci are
present in SCAR and Arp3 mutants. These data suggest that an
enlarged actin focus results from the loss of a Kette-SCAR-Arp2/3-dependent
actin polymerization event required for actin foci dissolution. This actin
polymerization event is presumably transient, as we have not been able to
detect a site of post-focus actin polymerization, other than cortical actin in
our imaging assays. Aberrant actin accumulation in the absence of Kette and
SCAR has been seen both in Drosophila
(Hummel et al., 2000;
Kunda et al., 2003;
Zallen et al., 2002) and in
Dictyostelium (Ibarra et al.,
2006). These observations suggest that the Arp2/3-dependent actin
polymerization machinery can function more generally in the regulation of
actin cytoskeletal organization and is capable of both forming and dissolving
visible F-actin structures. An additional activator of Arp2/3, WASP, may also
have a potential role in the regulation of actin foci. Recent studies have
indicated that WASP plays an essential role in myoblast fusion, although the
regulation of actin foci was not tested
(Massarwa et al., 2007;
Schafer et al., 2007).
Analysis of mutants in the WASP regulator, Sltr (D-WIP), indicates that actin
foci do form (Kim et al.,
2007). We find that the actin foci are approximately wild-type
size in these mutants (Table
1).
A revised molecular model of cell fusion
On a molecular level, the current model placed the intracellular signaling
events downstream of recognition into two distinct pathways that converge on
cytoskeletal rearrangements required for fusion. Our data distinguish among
the identified fusion genes with respect to actin reorganization at the site
of fusion. Moreover, our data suggests new relationships between the fusion
mutants, leading to a revision of the existing model
(Fig. 7).
Downstream of recognition and adhesion, actin foci form in the myotube and
FCM. Mbc-Rac activities and the Blow-Kette-SCAR-Arp2/3 pathway acts to promote
FCM cell shape change and target actin reorganization, leading to the
dissolution of the actin focus in both FCMs and myotubes. Moreover, Blow and
Kette have similar protein localization and actin focus phenotype, consistent
with observed genetic interactions
(Schroter et al., 2004).
Interestingly, absence of any one of these gene products does not prohibit
localization of the other members of this genetic pathway at the fusion site.
Rac has been found to regulate the Kette complex in several contexts
(Eden et al., 2002;
Steffen et al., 2004), adding
further support to this aspect of our model. Based on previous studies in a
number of systems (Ibarra et al.,
2005; Machesky and Insall,
1998; Vartiainen and Machesky,
2004), the target of SCAR activity is the Arp2/3 complex. Our data
support this view: mutants in both SCAR and Arp3 show fusion
defects. Although we do not yet know how all the biochemical activities of
these proteins are coordinated, the sum of the activities of all these
proteins is to dissolve the actin focus, through actin reorganization in both
the FCM and myotube. Our data suggests that dissolution of the focus is
required for fusion to proceed and would be coupled to membrane breakdown and
cytoplasmic mixing between the two cells.
A second pathway involving Loner and ARF6 also contributes to myoblast fusion. However, this pathway does not appear to directly regulate actin foci, despite the block in myoblast fusion. Unlike mutants in the Mbc-Rac-Kette-SCAR-Arp2/3 pathway, where actin focus size is dramatically increased, foci size remains wild-type in loner mutants. Our protein localization studies are consistent with the actin foci data: Loner does not colocalize with the actin foci, but is most often found near actin foci. Likewise, analysis of the subset of known fusion components that colocalize with the foci do not change in loner mutants. We predict that this pathway is required for additional behaviors, either upstream or downstream of the foci, necessary for fusion, such as myoblast searching or migration, microtubule rearrangements or other subcellular functions such as membrane trafficking.
Our data provide new insight concerning the function of Rols in myoblast
fusion. Rols is proposed to serve as an adapter
(Chen and Olson, 2001;
Chen and Olson, 2004) between
the recognition and adhesion protein Duf and Mbc, a GEF for Rac
(Hasegawa et al., 1996;
Kiyokawa et al., 1998;
Nolan et al., 1998), linking
adhesion to cytoskeletal rearrangements. Biochemical data from overexpression
studies in Drosophila S2 cells suggest a direct interaction between
Rols and Mbc (Chen and Olson,
2001). Our data, however, would indicate that this relationship is
not required for focus formation and dissolution. Rols appears not to be
necessary for the recruitment of Mbc to the fusion site, as average focus size
is wild-type in rols mutants whereas it is enlarged in mbc
mutants. Moreover, we find that localization of Rols and Mbc is not identical
at foci (data not shown). Our data supports the alternative model in which
Rols is required for efficient Duf recruitment to the FC membrane
(Menon et al., 2005). The
drastically reduced number of actin foci in rols mutants suggests
difficulties in myoblast recognition and/or adhesion. Those actin foci that do
appear are of wild-type size and correlate with fusion events, consistent with
a model of reduced efficiency of fusion in rols mutants.
Our model leaves the function of the actin focus unresolved. Actin
rearrangements can be linked to the active organization of membrane domains
(Liu and Fletcher, 2006),
membrane and protein trafficking (Egea et
al., 2006; Kaksonen et al.,
2006; Qualmann and Kessels,
2002; Stamnes,
2002), and structural support. Our data indicate that formation
and dissolution of actin foci are essential for the progression of fusion. We
have never observed a fusion event that has not been linked to an actin focus.
The identification of the site of fusion, a particular actin structure at this
site, new methods of analysis and key regulators of this structure open
avenues of study for the process of cell-cell fusion in invertebrate and
vertebrate biology.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/24/4357/DC1
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ACKNOWLEDGMENTS |
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