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Keywords:

  • myogenesis;
  • F-actin;
  • Arp2/3;
  • podosome;
  • immunological synapse

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. AN OVERVIEW OF MYOBLAST FUSION
  5. TWO-TEMPORAL-PHASES OF FUSION AND THE TWO-PHASE MYOBLAST FUSION MODEL
  6. ULTRASTRUCTURAL FEATURES OF MYOBLAST FUSION
  7. KEY MOLECULAR PLAYERS AS COMPONENTS OF THE FUSION-RESTRICTED MYOGENIC-ADHESIVE STRUCTURE (FURMAS)
  8. REGULATION OF ACTIN POLYMERIZATION DURING MYOBLAST FUSION
  9. THE FURMAS MODEL FOR MYOBLAST FUSION: KEY MOLECULAR PLAYERS, THEIR LOCALIZATION AND ULTRASTRUCTURAL FEATURES
  10. COMPARISION OF TRANSIENT ADHESIVE STRUCTURES: THE FURMAS, IMMUNOLOGICAL SYNAPSES, PODOSOMES AND INVADADOPODIA
  11. FUNCTIONAL CONSERVATION BETWEEN ODROSOPHILA AND VERTEBRATE MYOBLAST FUSION
  12. PERSPECTIVES
  13. Acknowledgements
  14. REFERENCES

In Drosophila, as in mammals, myoblast fusion is fundamental for development. This fusion process has two distinct phases that share common ultrastructural features and at least some molecular players between Drosophila and vertebrates. Here, we integrate the latest data on the key molecular players and ultrastructural features found during myoblast fusion into a new working model to explain this fundamental cellular process. At cell–cell contact sites, a protein complex (FuRMAS) serves as a signalling centre and might restrict the area of membrane fusion. The FuRMAS consists of a ring of cell adhesion molecules, signalling proteins, and F-actin. Regulated F-actin branching plays a pivotal role in myoblast fusion with regard to vesicle transport, fusion pore formation, and expansion as well as the integration of the fusion-competent myoblast into the growing myotube. Interestingly, local F-actin accumulation is a typical feature of other transient adhesive structures such as the immunological synapse, podosomes, and invadopodia. Developmental Dynamics 238:1513–1525, 2009. © 2009 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. AN OVERVIEW OF MYOBLAST FUSION
  5. TWO-TEMPORAL-PHASES OF FUSION AND THE TWO-PHASE MYOBLAST FUSION MODEL
  6. ULTRASTRUCTURAL FEATURES OF MYOBLAST FUSION
  7. KEY MOLECULAR PLAYERS AS COMPONENTS OF THE FUSION-RESTRICTED MYOGENIC-ADHESIVE STRUCTURE (FURMAS)
  8. REGULATION OF ACTIN POLYMERIZATION DURING MYOBLAST FUSION
  9. THE FURMAS MODEL FOR MYOBLAST FUSION: KEY MOLECULAR PLAYERS, THEIR LOCALIZATION AND ULTRASTRUCTURAL FEATURES
  10. COMPARISION OF TRANSIENT ADHESIVE STRUCTURES: THE FURMAS, IMMUNOLOGICAL SYNAPSES, PODOSOMES AND INVADADOPODIA
  11. FUNCTIONAL CONSERVATION BETWEEN ODROSOPHILA AND VERTEBRATE MYOBLAST FUSION
  12. PERSPECTIVES
  13. Acknowledgements
  14. REFERENCES

Cell–cell fusion is a fascinating and fundamental process that only occurs among a limited number of cell types. These cell types are either neighbouring cells or cells that have migrated, recognized, and adhered correctly with each other (Oren-Suissa and Podbilewicz,2007). As a result, the lipid bilayers of these cell types fuse and a syncytium is formed; a process that, in addition to myogenesis, is also fundamental to zygote formation, hypodermis formation, osteoclast and placenta formation.

Myoblast fusion is a typical and essential feature of myogenesis in higher organisms and is required for growth and repair of muscles in vertebrates. In holometabolic insects, myogenesis occurs during embryogenesis as well as during metamorphosis (reviewed by Maqbool and Jagla,2007). In this review, we focus on myoblast fusion in the somatic mesoderm during the formation of the larval body wall musculature in Drosophila. Larval muscles form a stereotypic pattern per segment (Fig. 1A). Each larval muscle corresponds to one myotube containing 4 to 24 nuclei (Baylies et al.,1998). Research on Drosophila embryos has provided the greatest insights into the myoblast fusion process to date. The molecules that have been identified to regulate myoblast fusion in Drosophila are summarized in Table 1. Intriguingly, first homologues of these key molecular players have already been identified and functionally characterized in vertebrates.

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Figure 1. Schematic representation of muscle formation, attachment, and sacromere assembly. Following myoblast specification, mononucleated myoblasts fuse to form multinucleated myotubes. A: Muscles form a reiterated pattern from segment to segment. The muscles are visualized by anti-β3-Tubulin. Anterior is left and dorsal at the top. B: The cell adhesion molecule Duf/Kirre is distributed in a ring-shaped manner at the contact site of a growing muscle and an fcm. B1, B2, B3: Optical sections through one contact site. Fusion proceeds in two phases. C-1: During the first fusion phase, an fc fuses with fcms and a 2–3 nucleated precursor cell forms. C-2: This precursor cell behaves like an fc and undergoes subsequent fusion events until the final size of the muscle is reached. In parallel to these fusion events, the growing myotube migrates, as indicated by the arrows, towards its epidermal muscle attachment cells, the tendon cells. C-3: Attachment sites have been established. C-4: Sarcomere assembly completes the myogenesis process.

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Table 1. List of Proteins That Are Known to Be Required for Drosophila Myoblast Fusion
ProteinsProtein class and functionLocalizationSource
Transmembrane proteins   
Duf/KineIgSF, recognition and adhesionFCRuiz-Gomez et al. (2000)
HbsIgSF, recognition and adhesionFCMArtero et al. (2001); Dowark et al. (2001)
Rst/Irre CIgSF, recognition and adhesionFC and FCMStrünkelnberg et al (2001)
Singles barProposed function: vesicle traffickingFCMEstrada et al. (2007)
SnsIgSF, recognition and adhesionFC and FCMBour et al. (2000)
Signaling molecules   
Arf6GTPase, actin regulation or vesicle traffickingUncertainChen et al. (2003)
BlowF-actin regulationFCMDoberstain et al. (1997), Artero et al. (2003); Schrôter et al., (2006)
Cdc42GTPase, F-actin regulationUncertainSchäfer et al. (2007)
CrkAdaptor proteinUncertainErickson et al. (1997); Balagopalan et al. (2006)
MbcNucleotide exchange factor for RacUncertainRushton et al. (1995); Erickson et al. (1997)
Mind bomb 2E3 ubiquitin ligaseFCNugyen et al. (2007); Carrasco-Rando and Ruiz-Gomez (2008)
Rac1, Rac2GTPases, F-actin regulationUncertainHadeka-Suzuki et al. (2002)
RolsStabilization of adhesion, signallingFCChen et al. (2001); Menon et al. (2001); Rau et al. (2001)
Schizo/LonerNucleotide exchange factor for Arf6FC and FCMChen et al. (2003); Richardson et al. (2007)
Actin regulators   
ArpC1Subunit of the Arp2/3 complexUncertainMassarwa et al. (2007)
Arp3Subunit of the Arp2/3 complexUncertainBerger et al. (2008)
Kette/Nap-1Component of the Wave complexFC and FCMSchröter et al. (2004)
SCAR/WAVEArp2/3 activatorUncertainRichardson et al. (2007); Berger et al. (2008)
Vrp1WASP-interacting partnerFCMKim et al. (2007); Massarwa et al. (2007)
WASPArp2/3 activatorFCMMassarwa et al. (2007); Schäfer et al. (2007)

In Drosophila, heterologous cell adhesion between a founder cell (fc) and a fusion-competent myoblast (fcm) is the precondition for myoblast fusion followed by local F-actin accumulation and branching (Kesper et al.,2007; Kim et al.,2007; Richardson et al.,2007). It has been proposed that a signalling pathway initiated by successful cell adhesion between fcs and fcms relays fusion signals from the cell membrane to the actin cytoskeleton (reviewed by Chen and Olson,2004). Recent data show that cell adhesion leads to the assembly of a multiprotein complex at the site of fusion (Kesper et al.,2007; Kim et al.,2007; Richardson et al.,2007), which was named the FuRMAS, a specific Fusion-Restricted Myogenic-Adhesive Structure, that contains F-actin at the sites of cell contact as plugs/foci (Kesper et al.,2007).

In this review, we suggest that FuRMASs have signalling centre properties and act as a platform to recruit fusion-relevant proteins. We first give an overview of the principle of myoblast fusion, describe the typical features, and then provide a comprehensive review of the key molecular players and signalling cascades involved. We then present the FuRMAS model that integrates current data from molecular and structural studies. We emphasize that FuRMAS share numerous features with other transient membrane-associated structures such as immunological synapse, podosomes, and invadopodia. They are all characterized by a belt of adhesion molecules in connection with local F-actin accumulation. Finally, we compare myoblast fusion in Drosophila and vertebrate and discuss the putative role of conserved signalling molecules and cellular processes that strongly suggest that the FuRMAS might be a structural feature common among species.

AN OVERVIEW OF MYOBLAST FUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. AN OVERVIEW OF MYOBLAST FUSION
  5. TWO-TEMPORAL-PHASES OF FUSION AND THE TWO-PHASE MYOBLAST FUSION MODEL
  6. ULTRASTRUCTURAL FEATURES OF MYOBLAST FUSION
  7. KEY MOLECULAR PLAYERS AS COMPONENTS OF THE FUSION-RESTRICTED MYOGENIC-ADHESIVE STRUCTURE (FURMAS)
  8. REGULATION OF ACTIN POLYMERIZATION DURING MYOBLAST FUSION
  9. THE FURMAS MODEL FOR MYOBLAST FUSION: KEY MOLECULAR PLAYERS, THEIR LOCALIZATION AND ULTRASTRUCTURAL FEATURES
  10. COMPARISION OF TRANSIENT ADHESIVE STRUCTURES: THE FURMAS, IMMUNOLOGICAL SYNAPSES, PODOSOMES AND INVADADOPODIA
  11. FUNCTIONAL CONSERVATION BETWEEN ODROSOPHILA AND VERTEBRATE MYOBLAST FUSION
  12. PERSPECTIVES
  13. Acknowledgements
  14. REFERENCES

In Drosophila, a master myoblast, also known as a founder cell (fc), dictates the identity of a muscle in regards to, e.g., its size, individual position, and specific attachment to the epidermis. The unique identity of an fc is determined by the differential expression of several transcription factors, for example, Krüppel, Eve, Slouch, and Ladybird (for reviews see Baylies et al.,1998; Frasch,1999). In contrast, only one identity conferring transcription factor, namely the Gli-like factor Lame Duck/Myoblast incompetent/Gleeful, is known for fcms (Duan et al.,2001; Ruiz-Gomez et al.,2002). After fc-fcm fusion, the genome of the fcm becomes reprogrammed and adopts the fate of the fcs. This reprogramming requires the degradation of proteins specifically expressed in the fcm. Mind bomb 2 (Mib2) has been suggested to be involved in the degradation of the fcm identity factor Lame Duck (Carrasco-Rando and Ruiz- Gomez,2008) and is further required for muscle integrity and stability (Nguyen et al.,2007).

In Drosophila, myoblast fusion proceeds in two temporal phases and is complete within 5.5 hr with individual fusion events taking only a matter of minutes (Beckett and Baylies,2007). In the first temporal phase of fusion, each fc fuses with a fusion-competent myoblast (fcms) to form a binucleated or trinucleated cell (Fig. 1C-1), known as a precursor (Bate,1990; Bate and Rushton,1993). In the second temporal phase of fusion, this precursor cell expands until the final size of the muscle is reached. In an alternative model, it has been suggested that besides temporal differences, genetic differences exist between these two phases of fusion, which means that some genes are needed to progress from the precursor stage to the final size of the myotubes (see Two-Temporal-Phases of Fusion and the Two-Phase Myoblast Fusion Model section).

During the first phase, fcs and fcms differ only slightly in size, and the diameter of the cells roughly corresponds to the area of contact and fusion (Fig. 1C-1). In the second phase, however, often several fcs attach to a growing myotube (Fig. 1C-2). At this step, though, the individual fcm is small in comparison to the growing myotube (Fig. 2A). Therefore, a mechanism to restrict the area of fusion might be more important in the second than in the first phase of fusion. During the second phase of fusion, the final number of nuclei of a specific muscle is determined by the lateral fusion of fcms to the growing myotube. In parallel, the ends of the growing myotube form filopodia that identify and contact specific epidermal attachment sites (Figs. 1C, 2, arrows). These contacts are then stabilized through the anchoring of the muscle and tendon cells into the extracellular matrix (ECM) (Fig. 1C-3). Finally, sarcomere assembly is initiated and leads to myotube maturation (Fig. 1C-4).

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Figure 2. Ultrastructural features during myoblast fusion in wild-type and mutant embryos. A: Schematic drawing depicting the ultrastructural features during the first and the second fusion phase. After the recognition and adhesion of fcs and fcms, a precursor cell forms. For the first phase of myoblast fusion, no ultrastructural features have been reported so far. One mutant that stops fusion during the first fusion phase is mbc, the only one analysed at the ultrastructural level. During the second fusion phase, electron-dense vesicles and plaques can be observed. Membrane vesiculation is a further characteristic feature of fusion. Embryos expressing activated RacV12 and wasp and vrp1 mutants stop fusion at this point. After all fusion events have been completed, a mature myotube is formed. B: The first fusion phase in a wild-type embryo. Electron-dense vesicles (arrowheads) (C) and electron-dense plaques (arrow) (D) are shown on ultrathin sections of wild-type embryos. E: A wasp3D3-035 mutant embryo with vesiculating membranes (arrowheads) and electron-dense vesicles (arrow). The membrane remnants become removed and a fusion pore forms. Subsequently, the fcm is integrated into the growing myotube. Magnifications: (B) 3,600×; (C) 12,000×; (D) 21,000×; (F) 21,000×. N, nuclei; G, Golgi apparatus, encircled by a dashed line.

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To date, a number of genes have been identified that encode fusion-relevant proteins and include cell adhesion molecules, members of signalling cascades, and regulators of the actin cytoskeleton (Table 1). fc-specific chemoattractive signals are known to direct fcms towards the fcs (Ruiz-Gomez et al.,2000; Strünkelnberg et al.,2001), and subsequent recognition and adhesion triggers a multi-step signal transduction cascade (Fig. 4) that results in the activation of the F-actin cytoskeleton machinery and ultimately fusion pore formation (Fig. 5). In addition, fusion-relevant proteins also participate in a number of other developmental processes such as muscle attachment (Schröter et al.,2004; Schäfer et al.,2007), sarcomere assembly (e.g., Kreisköther et al.,2006) and visceral fusion (Klapper et al.,2001,2002; San Martin and Bate,2001), Malpighian tubule organization (Denholm et al.,2003; Pütz et al.,2005), garland cell development (Weavers et al.,2009), as well as eye development (e.g., Carthew,2007; Fischbach et al.,2009). Thus, because of the multifunctional roles of fusion-relevant proteins, care should be taken when interpreting biochemical protein interaction assays, and the findings need to be correlated to the particular biological function.

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Figure 4. The Arp2/3 complex is activated by different F-actin regulators during the first and second fusion phase. A: The adhesion molecules in fcs and fcms trigger a signalling cascade that leads to the activation of the SCAR/WAVE complex and Vrp 1 during the first fusion phase. Intracellular components that might be involved in this signalling cascade include the nucleotide exchange factors Mbc and Schizo/Loner, the Rac-GTPases Rac1 and Rac2, as well as the Arf6-GTPase (in redundancy with another Arf-GTPase). Mutations in these genes disrupt fusion before precursor cell formation. In blow mutants, the first phase of fusion is inefficient (Beckett and Baylies,2007). B: The signalling cascades during the second fusion phase additionally involved the multidomain protein Rols7 in precursor cells and the Blow protein in fcms. In addition to the SCAR/WAVE complex, the WASP/Vrp 1 complex in fcms is activated during the second fusion phase. Whether Mbc and Schizo/Loner play a role during the second fusion phase remains to be determined. All actin regulators are shown in red.

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Figure 5. A model for FuRMAS expansion. Cell adhesion and FuRMAS formation. A: Lateral view: Duf/Kirre and SNS form a ring-like adhesion belt that is stabilized during the second fusion phase by Rols7. Please note that to date, Hibris and Rst have not been analysed in respect to the FuRMAS. In the centre of this belt, an F-actin plug (red) is found on each site of the contacting membrane and fusion-relevant proteins become localized to the contact area in fcms. B: Branched F-actin leads to the expansion of the FuRMAS: In a lateral view, opposing membranes and branched F-actin filaments are shown in detail. Cross-sectional view of the FuRMAS at the level of the opposing membranes with respect to F-actin; a view of a plug and electron-dense vesicles. The adhesion belt that defines the FuRMAS is highlighted as circles of the membrane spanning Duf/Kirre and SNS molecules. The F-actin plug expands during the fusion process and drives the expansion of the FuRMAS (schematically indicated by the arrows). As a consequence, the F-actin plug is dissolved. Electron-dense vesicles accumulate at the opposing membranes (compare to Fig. 3B for a three-dimensional model). C: Pulling of the fcm into the growing myotube. As a consequence of FuRMAS expansion, the fusing fcms become integrated into the growing myotube as indicated by the arrow. By this time, F-actin has disintegrated.

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Figure 3. Spatial distribution of paired vesicles at the opposing membranes between a growing myotub and a fcm. A: Serial section (A*– F*) reproduced from Doberstein et al. (1997, fig. 3) with permission of the publisher and tilted here to better visualize the spatial arrangement. The green lines represent landmarks showing corresponding positions in the sections. Prefusion complexes are visible at the contact zone between four cells (membranes are visualized in blue). Vesicles of the central prefusion complex existing between a growing myotube and a fcm are labelled in red. B: Schematic diagram of the arrangement of the red vesicles in A (vesicles: red in fcm, orange in growing myotube). A* to F* in B correspond to the cross-sections A* to F* in A. N, nucleus.

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TWO-TEMPORAL-PHASES OF FUSION AND THE TWO-PHASE MYOBLAST FUSION MODEL

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. AN OVERVIEW OF MYOBLAST FUSION
  5. TWO-TEMPORAL-PHASES OF FUSION AND THE TWO-PHASE MYOBLAST FUSION MODEL
  6. ULTRASTRUCTURAL FEATURES OF MYOBLAST FUSION
  7. KEY MOLECULAR PLAYERS AS COMPONENTS OF THE FUSION-RESTRICTED MYOGENIC-ADHESIVE STRUCTURE (FURMAS)
  8. REGULATION OF ACTIN POLYMERIZATION DURING MYOBLAST FUSION
  9. THE FURMAS MODEL FOR MYOBLAST FUSION: KEY MOLECULAR PLAYERS, THEIR LOCALIZATION AND ULTRASTRUCTURAL FEATURES
  10. COMPARISION OF TRANSIENT ADHESIVE STRUCTURES: THE FURMAS, IMMUNOLOGICAL SYNAPSES, PODOSOMES AND INVADADOPODIA
  11. FUNCTIONAL CONSERVATION BETWEEN ODROSOPHILA AND VERTEBRATE MYOBLAST FUSION
  12. PERSPECTIVES
  13. Acknowledgements
  14. REFERENCES

Before discussing the two-temporal phases versus the two-phase model, we want to emphasize the important morphological differences between the temporal phases of fusion. As depicted in the overview (Fig. 1C), the first and second fusion phases are different from the morphological point of view. The very first fusion leads to a bi- or trinucleated precursor. During this initial fusion step, each cell is approximately 4 μm in width (Bate,1990 [light microscope]; Schröter et al.,2006 [TEM]) and they form contact over about 1.7–1.9 μm (Fig. 2B). Within this contact area, the membranes dissolve and a syncytial cell with two nuclei and continuity of cytoplasm is formed. During the second fusion phase, fcms remain small as in the first fusion phase (Fig. 2A), whereas the precursor cell/growing myotube enlarges with every successful fusion. Therefore, in absolute terms, the contact site remains similar in size but this contact area is relatively small compared to the large precursor cell/growing myotube (Fig. 2A). As Bate (1990) showed using dye injections, precursor cells/growing myotubes migrate towards their attachment sites, which is why fusions occur laterally during the second fusion phase (Fig. 2A).

Genetic (Rau et al.,2001; Massarwa et al.,2007; Berger et al.,2008) as well as ultrastructural (Doberstein et al.,1997; Schröter et al.,2006) analyses led to the proposal that several distinct genetic players act during the two temporal fusion phases. This led to a two-phase model of myoblast fusion having common and different fusion-relevant proteins and signalling factors that are essential during the two-phase model. By contrast, Beckett and Baylies (2007) endorsed a two-temporal-phase model of myoblast fusion and proposed that there are no genetic differences. However, it should be noted that the data leading to the proposal of the two-temporal-phase (Beckett and Baylies,2007) and two-phase (Rau et al.,2001; Schröter et al.,2004; Menon et al.,2005) models are not contradictory, but differences arise due to interpretation. For example, Beckett and Baylies (2007) carefully counted the number of nuclei found in distinct muscle precursors in many known fusion mutants. Among these, only in myoblast city (mbc) mutants (Rushton et al.,1995; Erickson et al.,1997; Galletta et al.,1999) no fusion occurs at all as has been reported previously (Doberstein et al.,1997; Schröter et al.,2004,2006; Menon et al.,2005). Interestingly, in agreement with previously published data, Beckett and Baylies (2007) found different numbers of nuclei in blow, kette, schizo/loner, mbc, and rols mutants as well as states of fusion between the segments of the individual mutants and concluded that there are no genetic differences between the two-phases of myoblast fusion, but rather only a delay in the fusion process itself. Transplantation assays coupled with a fusion assay were used to test the fusion capability of rolling pebbles (rols) mutant myoblasts (Rau et al.,2001). These experiments indicate that rols mutant fcs lead to small syncytia also in a wild-type background and that these syncytia remain small even in the third instar larvae. These observations are hard to explain when taking only time into consideration. However, the depletion of the pool of fcms by “faster acting” wild-type fcs well before the third instar cannot be excluded. It is possible that some fusion-relevant proteins like Rols are needed for a better efficiency of fusion in the second phase of fusion (see An Overview of Myoblast Fusion section). In addition, several fusion-relevant proteins are maternally contributed, and with respect to fusion efficiency, all zygotic loss-of-function mutants with a strong maternal contribution of the corresponding mRNA are hard to interpret. For example, kette mutants often form small syncytia (Schröter et al.,2004; Beckett and Baylies,2007), indicating that maternally provided Kette might be central to successful initial fusion events. Finally, recent studies on differential regulation of F-actin reorganization during the first and second fusion phases (Massarwa et al.,2007; Berger et al.,2008) add weight to the model of genetic differences in addition to temporal differences (see Key Molecular Players as Components of the Fusion-Restricted Myogenic-Adhesive Structure [FuRMAS] section).

On the basis of the described data, we suggest that there are a number of fusion-relevant proteins such as Rols that play a more important role in the second compared to the first fusion phase/step in terms of restricting the area of fusion to precursor cells, which have a larger surface area compared to the small fcms. Others such as Vrp1/Wip are only expressed in fcms during the first phase of fusion, and later present in both fcm and growing myotube. We have integrated the two-temporal-phase and the two-phase models as well as incorporated the latest research on ultrastuctural features of myoblast fusion (Fig. 2).

ULTRASTRUCTURAL FEATURES OF MYOBLAST FUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. AN OVERVIEW OF MYOBLAST FUSION
  5. TWO-TEMPORAL-PHASES OF FUSION AND THE TWO-PHASE MYOBLAST FUSION MODEL
  6. ULTRASTRUCTURAL FEATURES OF MYOBLAST FUSION
  7. KEY MOLECULAR PLAYERS AS COMPONENTS OF THE FUSION-RESTRICTED MYOGENIC-ADHESIVE STRUCTURE (FURMAS)
  8. REGULATION OF ACTIN POLYMERIZATION DURING MYOBLAST FUSION
  9. THE FURMAS MODEL FOR MYOBLAST FUSION: KEY MOLECULAR PLAYERS, THEIR LOCALIZATION AND ULTRASTRUCTURAL FEATURES
  10. COMPARISION OF TRANSIENT ADHESIVE STRUCTURES: THE FURMAS, IMMUNOLOGICAL SYNAPSES, PODOSOMES AND INVADADOPODIA
  11. FUNCTIONAL CONSERVATION BETWEEN ODROSOPHILA AND VERTEBRATE MYOBLAST FUSION
  12. PERSPECTIVES
  13. Acknowledgements
  14. REFERENCES

Interestingly, during the second temporal phase, distinct membrane-associated electron-dense structures, namely, a prefusion complex and electron-dense plaques as well as membrane vesiculation, were observed using TEM (Doberstein et al.1997). However, during the first temporal phase, the prefusion complex and electron-dense plaques were not observed (Fig. 2B), but membrane vesiculation was (Schröter et al.,2006). The failure of finding a prefusion complex and electron-dense plaques in the first fusion phase could mean that these structures are restricted to the second fusion phase. But it cannot be excluded that they are more fragile, short-lived, or rare, and thus more difficult to identify in the first fusion phase.

The Prefusion Complex

At the ultrastructural level, electron-dense vesicles can be observed during the second phase (Fig. 2C). Doberstein et al. (1997) suggested that these vesicles form a prefusion complex that can consist of up to 15 vesicles (40 nm in diameter) per contact site. From the Golgi to the membrane, they might be transported by F-actin, which disintegrates after prefusion complex formation (Kim et al.,2007). A systematic analysis of these vesicles in wild-type embryos suggests that this complex consists of an average of 1.4 vesicles per complex in an individual section (Estrada et al.,2007). Kim et al. (2007) also described variable numbers of vesicles, which were also not all paired, a finding that is supported by Doberstein et al. (1997). These observations raise two key questions: (1) Why are the vesicle numbers so different? (2) Why are the vesicles not always paired? We think that this depends to some extent on the sections (Fig. 3) themselves since the sections in part show the vesicles on their way from the Golgi to the membrane before they can be visualized as paired vesicles at the opposing membranes. A further question concerns the number and spatial distribution of paired vesicles at the opposed membranes. Here, the serial sections of Doberstein et al. (1997) are highly informative since a total of 45 pairs of vesicles were found in six serial sections (each 100 nm), and these vesicles are distributed among four cells, one probably being a fc/growing myotube, and the three others probably being fcms (Fig. 3A). Approximately 15 pairs of vesicles were identified at each individual contact site and were distributed over a large area (1 μm2) at opposing membranes (paired vesicles at one contact site are marked in red and membranes in blue; Fig. 3A). Figure 3B depicts this process for a contact site between a growing muscle and a single fcm. For the FuRMAS model, discussed later in the review (see The FuRMAS Model for Myoblast Fusion: Key Molecular Players, Their Localization and Ultrastructural Features section), it is important to keep in mind that vesicles spread over this area.

Electron-Dense Plaques

Doberstein et al. (1997) proposed that the vesicles of the prefusion complex dissolve and form electron-dense plaques that are up to 500 nm in length with 10-nm-thick material along the opposed membranes (see Fig. 2D). Interestingly, membrane breakdown is visible at one site in the vicinity of these plaques (Doberstein et al.,1997). These plaques resemble desmosomes between epithelial cells (for desmosomes see Getsios et al.,2004; Gumbiner,2005). However, these plaques are rarely seen in wild-type, whereas they are frequently seen in certain mutants (see Membrane Vesiculation section), suggesting that they are transient structures. So far, electron-dense plaques and vesicles have never been observed together. This finding suggests that they might not occur at the same time. Alternatively, they might not be present in the same section due to different spatial localization.

Membrane Vesiculation

Finally, the membranes start to vesiculate along the zone of contact forming sacs of membranes (Fig. 2E) that are eventually removed during membrane breakdown (Doberstein et al.,1997). However, since serial sections taken during membrane vesiculation have not been studied so far, we are currently unable to compare the area of paired vesicles with that of membrane breakdown.

Ultrastructural analysis of known fusion mutants have provided new insights into the role of key fusion-relevant proteins for the progression of fusion relative to the described ultrastructural features (Fig. 2A). For example, blown fuse (blow) (Doberstein et al.,1997) and singles bar (sing) (Estrada et al.,2007) mutants do not progress beyond the prefusion-complex stage, thereby indicating a role for blow and sing in mediating fusion of electron-dense vesicles to the plasma membrane as already proposed for blow by Doberstein et al. (1997). For the first fusion phase, only myblast city (mbc) mutants have been analysed at the ultrastructural level and no vesicles nor electron-dense plaques were detected (Doberstein et al.,1997; Schröter et al.,2006). Both sns15 (previously named rost15; Paululat et al.,1995) and kette loss-of-function mutants accumulate aberrant electron-dense plaques that are considerably longer and thinner than in wild-type and these two mutants do not progress to the final membrane vesiculation stage (Doberstein et al.,1997; Schröter et al.,2004). Note that it is still not known whether sns15 is a null mutant or not. Finally, mutations in actin regulators (see Fig. 2E for wasp3D3-035 and Key Molecular Players as Components of the Fusion-Restricted Myogenic-Adhesive Structure [FuRMAS] section) and expression of constitutively active DRacG12V (Doberstein et al.,1997) led to the vesiculation of opposing membranes, but not to an open fusion pore.

KEY MOLECULAR PLAYERS AS COMPONENTS OF THE FUSION-RESTRICTED MYOGENIC-ADHESIVE STRUCTURE (FURMAS)

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. AN OVERVIEW OF MYOBLAST FUSION
  5. TWO-TEMPORAL-PHASES OF FUSION AND THE TWO-PHASE MYOBLAST FUSION MODEL
  6. ULTRASTRUCTURAL FEATURES OF MYOBLAST FUSION
  7. KEY MOLECULAR PLAYERS AS COMPONENTS OF THE FUSION-RESTRICTED MYOGENIC-ADHESIVE STRUCTURE (FURMAS)
  8. REGULATION OF ACTIN POLYMERIZATION DURING MYOBLAST FUSION
  9. THE FURMAS MODEL FOR MYOBLAST FUSION: KEY MOLECULAR PLAYERS, THEIR LOCALIZATION AND ULTRASTRUCTURAL FEATURES
  10. COMPARISION OF TRANSIENT ADHESIVE STRUCTURES: THE FURMAS, IMMUNOLOGICAL SYNAPSES, PODOSOMES AND INVADADOPODIA
  11. FUNCTIONAL CONSERVATION BETWEEN ODROSOPHILA AND VERTEBRATE MYOBLAST FUSION
  12. PERSPECTIVES
  13. Acknowledgements
  14. REFERENCES

Establishment and Known Components of the FuRMAS

A number of fusion-relevant proteins have been identified (Table 1), which we discuss here with respect to their role in fusion and their localization to the FuRMAS. The FuRMAS was defined as a ring-like arrangement of cell adhesion molecules (and interacting molecules) with local F-actin accumulation in plugs at the site of myoblast fusion (Kesper et al.,2007; see Fig. 1B1–B3 for the ring-like expression of Duf in fcs). F-actin and D-Titin are known to accumulate in fc/growing myotubes and fcms, while Blow is concentrated in fcms (Kesper et al.,2007; Richardson et al.,2007). The F-actin plugs at the site of fusion have also been termed actin foci (Kim et al.,2007; Richardson et al.,2007). However, before cell adhesion rings (Kesper et al.,2007) and actin plugs/foci (Richardson et al.,2007) can be established, successful cell adhesion must take place. Here, we describe the establishment of the FuRMAS by heterophilic cell adhesion (Figs. 4, 5).

Directed migration and recognition might require chemoattractive signals to be released from the fc to attract fcms that might also play a role in fc-fcm adhesion. Indeed, the transmembrane protein Dumbfounded/Kin of Irre (Duf/Kirre) is expressed only in fcs and was shown to attract fcms towards the epidermis when expressed ectopically with wingless-GAL4 (Ruiz-Gomez et al.,2000). However, it remains to be clarified whether Duf/Kirre acts as a membrane-spanning molecule or whether part of the extracellular domain is secreted. In the fcs of the embryo, Duf/Kirre and another transmembrane protein, namely Roughest/Irregular C (Rst/IrreC), have overlapping and therefore redundant roles. Like Duf/Kirre, Rst/IrreC is a member of the Immunoglobulin Superfamily (IgSF) and can also attract myoblasts (Strünkelnberg et al.,2001); however, in contrast to Duf/Kirre, Rst/IrreC is expressed in both myoblast types. In aggregation experiments with S2 cells, the fcm-specific IgSF protein Sticks and Stones (SNS) was observed to mediate heterotypic adhesion with Duf/Kirre- and Rst/Irre C–expressing cells (Bour et al.,2000; Galletta et al.,2004). Hibris (Hbs), the paralogue of SNS, is also expressed in fcms (Artero et al.,2001; Dworak et al.,2001), but interestingly, Hbs seems not to be absolutely essential for myoblast fusion since its loss of function does not disturb myoblast fusion (Artero et al.,2001; Dworak et al.,2001). Whereas Artero et al. (2001) suggested based on genetic interaction that SNS and Hbs function antagonistically, Dworak et al. (2001) proposed that both genes act in functional redundancy. This finding was confirmed by double mutant experiments from Menon et al. (2005) and Shelton et al. (2009).

For Duf/Kirre and SNS, it is known that they are distributed in a ring-shaped manner within the FuRMASs at the contact sites of the fc/growing myotube and fcms (Fig. 5). Following the recognition and adhesion between fcs and fcms, fusion-relevant proteins are thought to transfer signals from the membrane to initiate the myoblast fusion process. The cytoplasmatic tails of the adhesion molecules are most likely to be involved in this signal transduction process (Sink,2006). For example, the intracellular domain of Duf/Kirre is needed to recruit the multidomain protein Rolling Pebbles 7 (Rols7), also known as Antisocial (Ants) (Chen and Olson,2001; Menon and Chia,2001). This leads to colocalization of Rols and Duf/Kirre in a ring-like manner within the FuRMAS in the fc/growing myotube (Kesper et al.,2007).

Immunoprecipitaion (CoIPs) studies with Duf and Rols/Ants or Rols and Mbc after transfection into S2 cells revealed interaction of Rols/Ants with Duf as well as with Mbc (Chen and Olson,2001). Thus, Rols7/Ants might serve as a linker between the membrane and the actin cytoskeleton via Mbc and Rac (Fig. 4). This notion is supported by evidence for the localization of Mbc to the actin plug/foci and by the distribution of Rac in distinct spots throughout the cytoplasm with some spots overlapping with the actin plugs/foci (Richardson et al.,2007). Recently, Geisbrecht et al. (2008) performed genetic interaction studies and thereby showed that Mbc and ELMO/CED-12 co-operate to activate the small GTPases Rac1 and Rac2 in eye development during metamorphosis. Moreover, the ankyrin repeats of Rols/Ants are essential for the recruitment of Rols7 to the membrane (Menon et al.,2005), whereas the TPR repeats interact with the intracellular domain of Duf/Kirre (Kreisköther et al.,2006). Zhang et al. (2000) first showed that the cytoskeleton component D-Titin accumulates at the site of fusion. Kesper et al. (2007) confirmed this and reported that D-Titin is localized in the same pattern as F-actin in plugs/foci of the FuRMASs. This localization depends on Rols7, which recruits D-Titin to the site of fusion (Menon and Chia,2001).

Mutations in schizo/loner lead to a strong myoblast fusion phenotype. Schizo/Loner, a GEF for the Adenosin ribosylation factor (Arf) family of GTPases, is suggested to be another signalling molecule within fcs/growing myotubes and was shown to bind Arf6 in in vitro assays (Chen et al.,2003). arf6 loss-of-function mutants exhibit a wild-type muscle pattern and mutant flies are vital but male sterile (Dyer et al.,2007). This finding indicates that Arf6 is not or not the only Arf GTPase which can activate Schizo/Loner. Richardson et al. (2007) observed Schizo/Loner in both fcs/growing myotubes and fcms using confocal microscopy. Furthermore, they also found Schizo/Loner outside of the actin plugs/foci and not at the adjacent membranes between fcms and fcs. Thus, Schizo/Loner seems not to be a prominent component of the FuRMASs. However, Schizo is clearly essential for fusion, but its exact role needs further clarification.

Although a signal molecule that mediates transduction in fcms in either of the two fusion phases has not yet been identified, a detailed analysis of the rescue capacity of SNS deletion constructs showed that the cytoplasmic domain of SNS possesses multiple redundant domains (Kocherlakota et al.,2008). This finding further strengthens the notion that there are different signalling cascades at play.

Dynamics of FuRMAS

Here, we discuss the dynamics of the FuRMAS during fusion. In the wild-type, rings of different sizes ranging from 1 to 5 μm have been observed. As the size of the cell adhesion rings in mutants (blow, kette, mbc, and rols) are less than 2 μm, it is likely that the observed size differences in the wild-type reflect a change in size of individual rings. This speculation led us to postulate that the FuRMAS expands during the individual fusion process from 1 to 5 μm in diameter (Fig. 5). The ring (initially 1 μm in diameter) of SNS/Duf and Rols in FuRMAS (Kesper et al.,2007) surrounds an actin area of 0.78 μm2. This raises the question whether the actin plugs/foci also change their size. Indeed, in life-imaging experiments of wild-type embryos, the size of these actin plugs/foci is very dynamic and ranges from 0.7 to 4.5 μm2 (Richardson et al.,2007) when measured in a lateral view (see Fig. 5). In the absence of intracellular components that regulate the formation of branched F-actin such as Kette, the FuRMAS is formed but does not expand completely (Kesper et al.,2007) although the actin plug size is enlarged (Richardson et al.,2007). These actin plugs/foci are highly dynamic structures and are established within 2 min and dissolve completely in less than 1 min but can be present at the individual fusion site for up to around 12 min on average (Richardson et al.,2007).

REGULATION OF ACTIN POLYMERIZATION DURING MYOBLAST FUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. AN OVERVIEW OF MYOBLAST FUSION
  5. TWO-TEMPORAL-PHASES OF FUSION AND THE TWO-PHASE MYOBLAST FUSION MODEL
  6. ULTRASTRUCTURAL FEATURES OF MYOBLAST FUSION
  7. KEY MOLECULAR PLAYERS AS COMPONENTS OF THE FUSION-RESTRICTED MYOGENIC-ADHESIVE STRUCTURE (FURMAS)
  8. REGULATION OF ACTIN POLYMERIZATION DURING MYOBLAST FUSION
  9. THE FURMAS MODEL FOR MYOBLAST FUSION: KEY MOLECULAR PLAYERS, THEIR LOCALIZATION AND ULTRASTRUCTURAL FEATURES
  10. COMPARISION OF TRANSIENT ADHESIVE STRUCTURES: THE FURMAS, IMMUNOLOGICAL SYNAPSES, PODOSOMES AND INVADADOPODIA
  11. FUNCTIONAL CONSERVATION BETWEEN ODROSOPHILA AND VERTEBRATE MYOBLAST FUSION
  12. PERSPECTIVES
  13. Acknowledgements
  14. REFERENCES

The dynamics of F-actin plugs/foci within the FuRMAS requires regulated F-actin polymerization and depolymerization. Indeed, to date, there is an accumulating body of evidence to show that the precise regulation of actin polymerization is crucial for successful myoblast fusion (Schröter et al.,2004; Kesper et al.,2007; Kim et al.,2007; Massarwa et al.,2007; Richardson et al.,2007; Berger et al.,2008). Actin-related proteins like the Arp2/3 complex initiate the formation of new F-actin filaments by anchoring new filaments to a pre-existing actin network (reviewed by Pollard,2007). During myoblast fusion, the underlying activation of the Arp2/3 complex is differentially regulated in fcs and fcms and in the first and second fusion phase (Fig. 4; Table 1). Rho, Rac, and Cdc42 are small GTPases, which are involved in many actin-dependent processes (reviewed by Etienne-Manneville and Hall,2002). Cdc42 seems not to play a major role in myoblast fusion (Schäfer et al.,2007) in contrast to Rac (see below). The first actin regulators that were identified to be important for myoblast fusion were the small rac-GTPases rac1, rac2 (Luo et al.,1994; Doberstein et al.,1997; Hakeda-Suzuki et al.,2002), and kette, the Drosophila homologue of Nap1/Hem-2 (Schröter et al.,2004). Interestingly, kette interacts genetically with the fcm-specifically expressed gene blow (Schröter et al.,2004). Fusion mutants such as kette have distinct fusion interruption at the ultrastructural level (see Ultrastructural Features of Myoblast Fusion section).

The small Rac-GTPase and the vertebrate homologue of Kette Nap-1/Nap-125 are known to be involved in activating the Arp2/3 regulator SCAR/WAVE. A protein complex including Nap-125 keeps SCAR/WAVE in an inactive state, whereas the binding of Rac activates SCAR/WAVE, which in turn stimulates the Arp2/3 complex (Takenawa and Suetsugu,2007). Mbc is required for myoblast fusion (Rushton et al.,1995; Erickson et al.,1997) and is thought to be the GEF that activates Rac (Côté and Vuori,2002; see Fig. 4). Indeed, Mbc and Rac GTPases colocalize with F-actin plugs (Richardson et al.,2007). The stimulation of the Arp2/3 complex by the nucleation-promoting factors such as SCAR/WAVE and WASP is achieved through three short evolutionary conserved motifs that are also known as the VCA module: The V motif interacts with actin monomers and the CA motif can bind the Arp2/3 complex and actin. In Drosophila, immunhistochemical studies suggest that Kette is also required for the correct localization of SCAR/WAVE (Richardson et al.,2007). Embryos that lack zygotic scar/wave activity show a weak fusion defect (Richardson et al.,2007; Berger et al.,2008), a phenotype that can be enhanced by the additional reduction of maternal scar/wave (Richardson et al.,2007). Besides SCAR/WAVE, the Wiskott-Aldrich Syndrome protein WASP is required for myoblast fusion. Analysis of a new wasp allele wasp3D3-035 that lacks the CA domain of the VCA module and acts as a dominant-negative mutation revealed that this domain is essential for correct myoblast fusion (Schäfer et al.,2007). Double mutant experiments with Arp3 and wasp mutant embryos as well as the loss of maternal and zygotic WASP activity imply that WASP is only required during the second fusion phase (Massarwa et al.,2007; Berger et al.,2008). Moreover, cell type–specific rescue experiments indicate that WASP is only required in fcms (Schäfer et al.,2007); however, this issue needs further clarification as Massarwa et al. (2007) reported contradictory results. In addition, WASP is known to form a complex with its fcm-specifically expressed interaction partner Verprolin 1 (Vrp1) (also known as D-Wip or Solitary) (Massarwa et al.,2007; Kim et al.,2007), and homozygous vrp1 null mutants display arrested fusion after precursor formation. On the ultrastructural level, Kim et al (2007) observed mislocalization of the prefusion complex and accumulation of vesicles, and thus proposed that F-actin regulation is required to target vesicles to the membrane; however, the membranes appear to remain intact. In contrast, Massarwa et al. (2007) and Berger et al. (2008) observed membrane vesiculation in vrp1 mutants and found no evidence of accumulation of vesicles. The reason for these differing findings remains to be clarified, e.g., different fixation methods (high-pressure freezing; chemical fixation) could give different results. In wasp mutants, the prefusion complex is also correctly formed and no vesicle accumulation is observed (Berger et al.,2008; Massarwa et al.,2007). Interestingly, wasp mutants exhibit a highly similar phenotype to vrp1 mutants (Berger et al.,2008; Massarwa et al.,2007), a finding that fits well with the fact that WASP and Vrp1 are interacting proteins. To clarify the role of SCAR/WAVE, WASP, and Vrp1 during myoblast fusion, scar/wave vrp1 double mutants were analysed and found to display no evidence of fusion taking place. Taken together, the evidence strongly suggests that SCAR/WAVE and Vrp1 control the first fusion phase, whereas SCAR/WAVE, WASP, and Vrp1 are all required for the second fusion phase (Berger et al.,2008). This in turn suggests that Arp2/3 is activated independently of WASP in the first fusion phase. Indeed, it was proposed that vertebrate Wip could interact with Cortactin to induce actin polymerization via the Arp2/3 complex (Kinley et al.,2003). However, whether this is also the case for myoblast fusion remains to be investigated.

The Arp2/3 complex consists of 7 proteins: 2 actin-related proteins, namely Arp2 and Arp3, as well as 5 further subunits (ArpC1–5). Mutations in the Arp3 and ArpC1 genes have been shown to disrupt fusion (Massarwa et al.,2007; Richardson et al.,2007; Berger et al.,2008). Molecular players that regulate F-actin branching during the first and second fusion phase in a cell-type-specific manner are summarized in Figure 4. Interestingly, on the ultrastructural level, myoblast fusion in wasp and vrp1 mutants is disrupted at a different step than in Arp3schwächling mutants. In detail, wasp and vrp1 mutants stop fusion during membrane vesiculation (Massarwa et al.,2007; Berger et al.,2008), whereas Arp3schwächling mutants stop fusion after membrane remnants have been removed and a fusion pore has been formed, but the fcm is not integrated into the growing myotube (Berger et al.,2008). From these data, a model has been proposed that suggests that the enlargement of the fusion pore depends on Arp2/3-mediated F-actin branching. Membrane vesiculation and breakdown, however, seem to be controlled by other factors, e.g., a so far unknown fusogene (a protein that fuses membranes). We strongly suggest the presence of co-ordinated pathways leading to fusion pore formation and expansion, and also due to the complexity of the intracellular domain of SNS, the presence of multiple pathways at least in fcms (Kocherlakota et al.,2008). Finally, to add weight to this notion, biochemical experiments have provided evidence to suggest that SNS interacts with Crk, a SH2-SH3 type of adaptor protein, and that Vrp1 mediates the formation of a Crk-Vrp1-WASP complex in fcms (Kim et al.,2007).

THE FURMAS MODEL FOR MYOBLAST FUSION: KEY MOLECULAR PLAYERS, THEIR LOCALIZATION AND ULTRASTRUCTURAL FEATURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. AN OVERVIEW OF MYOBLAST FUSION
  5. TWO-TEMPORAL-PHASES OF FUSION AND THE TWO-PHASE MYOBLAST FUSION MODEL
  6. ULTRASTRUCTURAL FEATURES OF MYOBLAST FUSION
  7. KEY MOLECULAR PLAYERS AS COMPONENTS OF THE FUSION-RESTRICTED MYOGENIC-ADHESIVE STRUCTURE (FURMAS)
  8. REGULATION OF ACTIN POLYMERIZATION DURING MYOBLAST FUSION
  9. THE FURMAS MODEL FOR MYOBLAST FUSION: KEY MOLECULAR PLAYERS, THEIR LOCALIZATION AND ULTRASTRUCTURAL FEATURES
  10. COMPARISION OF TRANSIENT ADHESIVE STRUCTURES: THE FURMAS, IMMUNOLOGICAL SYNAPSES, PODOSOMES AND INVADADOPODIA
  11. FUNCTIONAL CONSERVATION BETWEEN ODROSOPHILA AND VERTEBRATE MYOBLAST FUSION
  12. PERSPECTIVES
  13. Acknowledgements
  14. REFERENCES

Based on our previous studies (Kesper et al.,2007), we propose that during myoblast fusion, an adhesion structure called the FuRMAS keeps the membranes in close contact. Kesper et al. (2007) outlined a model that integrates the FuRMAS data obtained at the cellular and subcellular levels. We have now incorporated recent new data from several laboratories into this model and have expanded it in order to include the role of F-actin regulation during myoblast fusion (Fig. 5). Numerous actin regulators are known to colocalize with the actin plugs/foci (Fig. 5A and B; see Key Molecular Players as Components of the Fusion-Restricted Myogenic-Adhesive Structure (FuRMAS) section). We propose that F-actin has multiple functions during myoblast fusion that occur in part in parallel. First, F-actin is likely to transport the electron-dense vesicles to the site of fusion to form the prefusion complex at the opposing membranes (Kim et al.,2007), and it is likely that these vesicles accumulate over an area of 1 μm2 (Doberstein et al.,1997; see Fig. 3). Second, we further propose that F-actin might be required for adhesion belt expansion from 1 to 5 μm in diameter (Kesper et al.,2007; Fig. 5B), which is in accordance with the observed reduced size of fusion pores found in Arp3schwächling mutants (Berger et al.,2008). Third, Massarwa et al. (2007) proposed that fusion pore enlargement is dependent on Vrp1-mediated WASP activation as wasp and vrp1 mutants show multiple small fusion pores (Massarwa et al.,2007; Berger et al.,2008). Thus, Arp2/3 activation seems to be necessary to remove vesiculating membrane residuals and thus create an open fusion pore. Fourth, Arp2/3 activation seems to be essential to integrate the fcm into the growing myotube as Arp3schwächling mutant embryos do not integrate fcms into the growing myotube (Berger et al.,2008; see Fig. 5C).

However, what might be the role of the prefusion complex? Doberstein et al. (1997) postulated that the vesicles of the prefusion complex dissolve to form electron-dense plaques. Alternatively, these vesicles may play a role in transporting fusion-relevant molecules to the site of fusion, and such molecules might be analogous to the fusogenes Eff1 and Aff1 identified in epithelial fusion in C. elegans (Mohler et al.,2002; Podbilewicz et al.,2006; Sapir et al.,2007). Although such fusogenes have so far not been identified in Drosophila, one could hypothesize that such proteins might trigger membrane vesiculation and subsequent breakdown during myoblast fusion.

COMPARISION OF TRANSIENT ADHESIVE STRUCTURES: THE FURMAS, IMMUNOLOGICAL SYNAPSES, PODOSOMES AND INVADADOPODIA

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. AN OVERVIEW OF MYOBLAST FUSION
  5. TWO-TEMPORAL-PHASES OF FUSION AND THE TWO-PHASE MYOBLAST FUSION MODEL
  6. ULTRASTRUCTURAL FEATURES OF MYOBLAST FUSION
  7. KEY MOLECULAR PLAYERS AS COMPONENTS OF THE FUSION-RESTRICTED MYOGENIC-ADHESIVE STRUCTURE (FURMAS)
  8. REGULATION OF ACTIN POLYMERIZATION DURING MYOBLAST FUSION
  9. THE FURMAS MODEL FOR MYOBLAST FUSION: KEY MOLECULAR PLAYERS, THEIR LOCALIZATION AND ULTRASTRUCTURAL FEATURES
  10. COMPARISION OF TRANSIENT ADHESIVE STRUCTURES: THE FURMAS, IMMUNOLOGICAL SYNAPSES, PODOSOMES AND INVADADOPODIA
  11. FUNCTIONAL CONSERVATION BETWEEN ODROSOPHILA AND VERTEBRATE MYOBLAST FUSION
  12. PERSPECTIVES
  13. Acknowledgements
  14. REFERENCES

A ring of cell-adhesion molecules and local F-actin branching in adhesive structures is characteristic for several cellular structures such as the immunological synapse, podosomes and invadopodia (Table 2). The immunological synapse is a transient adhesive structure that connects an antigen-presenting cell (APC) with a T-cell (Monks et al.,1997; Shaw and Dustin,1997; Grakoui et al.,1999). In terms of transient cell adhesion and local F-actin branching, this structure is similar to the FuRMAS that is formed between fcs/growing myotubes and fcms during myoblast fusion. Furthermore, both these structures are transient and regulate the spatial and temporal communication between cells. Podosomes (reviewed by Linder and Kopp,2005; Linder,2007) and invadopodia (Vignjevic and Montagnac,2008) are known to form adhesion structures between the filopodia and the extracellular matrix (ECM) and to be involved in matrix degradation that ultimately can lead to invasion. Both of these structures have recently been compared to the immunological synapse (reviewed by Wernimont et al.,2008). Interestingly, these structures are also characterized by local F-actin accumulation with common F-actin regulators (Table 2). Finally, immunological synapses, podosomes, and invadopodia also serve to create a restricted area where lytic processes take place, e.g., matrix degradation (Table 2). However, it remains to be determined whether such lytic processes are also essential for myoblast fusion.

Table 2. Key Characteristics of the FuRMAS, Immunological Synapse, Podosome, and Invadopodia
 FuRMASImmunological synapsePodosomesaInvadopodiaa
  • a

    Taken from Linder (2007).

  • b

    Size of the area that is surrounded by Duf and Sns (Kesper et al.,2007).

  • c

    Size of the actin plug in a lateral view (Richardson et al.,2007).

  • d

    The ability of each structure to degrade the matrix is indicated by the number of + signs.

  • e

    HEM-1 is the hematopoietic form of Nap-1 and HS1 is a hematopoietic-cell-specific form of the actin regulatory protein Cortactin.

Cell typeFC/growing myotubeT-cellMonocytic cellsCarcinoma cells
 FCMAPC  
     
Adhesion moleculesDuf and RstIntegrins (LFA-1)Integrins Vinculin TalinIntegrins Talin
 SnsICAMs  
     
F-actin regulatorsNap-1HEM-1e  
WAVEWAVE
 WASPWASPWASPWASP
 WIPWIPWIPWIP
 Arp2/3Arp2/3Arp2/3Arp2/3
 Rho-family GTPasesRho-family GTPasesRho-family GTPasesRho-family GTPases
   GelsolinGelsolin
  Cofilin Cofilin
  VASP VASP
  HS1eCartactin 
     
Appearance of actinPlugPlugDot-likeDot-like
     
Size0.8 μmb?0.4 μm240 μm2
 0.7–4.5 μmc   
     
PersistenceUp to 12 minOver hours2–12 minUp to 1 hr
     
ECM degradationd??++++

FUNCTIONAL CONSERVATION BETWEEN ODROSOPHILA AND VERTEBRATE MYOBLAST FUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. AN OVERVIEW OF MYOBLAST FUSION
  5. TWO-TEMPORAL-PHASES OF FUSION AND THE TWO-PHASE MYOBLAST FUSION MODEL
  6. ULTRASTRUCTURAL FEATURES OF MYOBLAST FUSION
  7. KEY MOLECULAR PLAYERS AS COMPONENTS OF THE FUSION-RESTRICTED MYOGENIC-ADHESIVE STRUCTURE (FURMAS)
  8. REGULATION OF ACTIN POLYMERIZATION DURING MYOBLAST FUSION
  9. THE FURMAS MODEL FOR MYOBLAST FUSION: KEY MOLECULAR PLAYERS, THEIR LOCALIZATION AND ULTRASTRUCTURAL FEATURES
  10. COMPARISION OF TRANSIENT ADHESIVE STRUCTURES: THE FURMAS, IMMUNOLOGICAL SYNAPSES, PODOSOMES AND INVADADOPODIA
  11. FUNCTIONAL CONSERVATION BETWEEN ODROSOPHILA AND VERTEBRATE MYOBLAST FUSION
  12. PERSPECTIVES
  13. Acknowledgements
  14. REFERENCES

Since skeletal muscle formation in mammals is experimentally less accessible than in Drosophila, most studies of myoblast fusion have been performed using vertebrate myoblast cell lines. However, in mammals, the cellular processes underlying myoblast fusion have been studied extensively on a cytological level (Knudsen and Horwitz,1977,1978; Wakelam,1985). Interestingly, comparison of these data with those from Drosophila revealed clear similarities for the myoblast fusion process. For example, both mammals and Drosophila exhibit two-phase fusion processes (Pavlath and Horsley,2003), and like in Drosophila, mammalian myoblast fusion encompasses the same cellular events of recognition, adhesion, alignment, and membrane union. In addition, electron-dense vesicles and plaques were also observed in vertebrates as in Drosophila (Rash and Fambrough,1973; Doberstein et al.,1997). However, the fusion-relevant molecules that have been identified by in vitro assays to date seem to differ between Drosophila and mammalian myoblast fusion (recently reviewed by Richardson et al.,2008). Here, we focus on functionally conserved fusion-relevant molecules that are found across species (for molecules known so far solely for vertebrates, see Maqbool and Jagla, 2008; Richardson et al.,2008).

The first functional evidence that myoblast fusion events are evolutionarily conserved came from studies on zebrafish embryos. During zebrafish embryogenesis, two types of skeletal muscle fibres are formed: mono-nucleated slow-twitch fibres and multinucleated fast-twitch fibres. Recently, the Drosophila homologue of Duf/Kirre named Kirrel has been identified in zebrafish and shown to localize to fusion-competent myoblasts of the fast-twitch lineage (Srinivas et al.,2007). In addition, homologues of some of the intracellular signalling molecules (see Table 1) found in Drosophila have also been identified in zebrafish, e.g., Rac and the Myoblast City homologues Dock1 and Dock5 as well as the Crk-related adaptor proteins Crk and Crk-like. Knocking down either Dock1, Dock5, Crk, or Crk-like was seen to significantly reduce myoblast fusion efficiency in zebrafish (Moore et al.,2007). However, myoblast fusion events are affected differently when overexpressing activated Rac. For example, in Drosophila, activated Rac blocks myoblast fusion (Luo et al.,1994; Doberstein et al.,1997), whereas in zebrafish, it leads to hyperfusion in fast-twitch muscles (Srinivas et al.,2007).

Moreover, the Arf6 guanine nucleotide exchange factor Schizo/Loner and the Arf6-GTPase are evolutionarily conserved in both vertebrates and Drosophila (Chen et al.,2003; Önel et al.,2004). In myogenesis, Arf6 probably has an overlapping and, therefore, redundant function with another Arf GTPase (see Ultrastructural Features of Myoblast Fusion section). A dominant-negative version of Arf6 was reported to reduce fusion efficiency in transfected C2C12 murine myoblast cell culture (Chen et al.,2003), which implies that Arf6 might also play a role during vertebrate myogenesis. Indeed, also using C2C12 murine myoblast cell culture in an RNAi approach, Pajcini et al., (2008) analysed the role of GEF Brag2 (Schizo/Loner in Drosophila) and GEF Dock180 (Mbc in Drosophila) and observed a lowered fusion index. In addition, the recently identified myoblast fusion-relevant components of the actin cytoskeleton machinery, e.g., WASP, Vrp1, and SCAR/WAVE, are evolutionarily conserved among vertebrates and Drosophila. siRNA knockdown of WASP and Vrp1 as well as Latrunculin-mediated F-actin destabilization leads to a lower fusion index in C2C12 murine myoblast cell culture (Kim et al.,2007). In murine myoblast cell culture models, F-actin seems to play a role in the alignment of cells, and fusion pores have been suggested to appear in the actin-free regions (Duan and Gallagher,2009), a suggestion also made by Kim et al. (2007) for Drosophila myoblast fusion. Chen et al. (2008) report for the viral fusogene gp64 that fusion pore expansion during syncytium formation in a cell culture assay is also restricted by an actin network, suggesting again a common mechanism. In addition, Crk, Dock180 (Dock1, Mbc homolog) and Kette/NAP1/Hem are known to be expressed in C2C12 murine myoblast cells (Kesper et al.,2007). Importantly, Dock180 null mice have severely reduced levels of all skeletal muscle tissues due to a strong decrease in myoblast fusion events (Laurin et al.,2008). Taken together, these findings suggest that myoblast fusion might be controlled by a similar mechanism in vertebrates as found in Drosophila.

PERSPECTIVES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. AN OVERVIEW OF MYOBLAST FUSION
  5. TWO-TEMPORAL-PHASES OF FUSION AND THE TWO-PHASE MYOBLAST FUSION MODEL
  6. ULTRASTRUCTURAL FEATURES OF MYOBLAST FUSION
  7. KEY MOLECULAR PLAYERS AS COMPONENTS OF THE FUSION-RESTRICTED MYOGENIC-ADHESIVE STRUCTURE (FURMAS)
  8. REGULATION OF ACTIN POLYMERIZATION DURING MYOBLAST FUSION
  9. THE FURMAS MODEL FOR MYOBLAST FUSION: KEY MOLECULAR PLAYERS, THEIR LOCALIZATION AND ULTRASTRUCTURAL FEATURES
  10. COMPARISION OF TRANSIENT ADHESIVE STRUCTURES: THE FURMAS, IMMUNOLOGICAL SYNAPSES, PODOSOMES AND INVADADOPODIA
  11. FUNCTIONAL CONSERVATION BETWEEN ODROSOPHILA AND VERTEBRATE MYOBLAST FUSION
  12. PERSPECTIVES
  13. Acknowledgements
  14. REFERENCES

Our understanding of the myoblast fusion process in Drosophila and vertebrates has substantially increased over the last few years. Work on vertebrates has provided molecular evidence for conserved components in myoblast fusion over kingdoms and serves to demonstrate that the research on Drosophila myoblast fusion can prepare the ground for health-relevant translational research in higher organisms since fusion is relevant for growth and regeneration of muscles after injuries. During these processes, satellite cells, the stem cells of the myotubes, are activated, divide, and fuse to existing myotubes. To date, studies in Drosophila have provided new key insights into cell adhesion and regulation of F-actin branching and advanced understanding of the key players underpinning myoblast fusion events. However, the most central question that remains to be answered in Drosophila myoblast fusion is: how do the fusion pores form? The fusing membranes must be brought into close proximity to each other. In other systems, e.g., organelle or virus-cell fusion, there is an intermediate stage of fusion called hemifusion that precedes membrane fusion (Sapir et al., 2008). Proteins that can bend the membrane are known to be involved in the hemifusion process. To date, no such protein has been identified in Drosophila. However, its existence is likely and the search for such a protein remains a major challenge.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. AN OVERVIEW OF MYOBLAST FUSION
  5. TWO-TEMPORAL-PHASES OF FUSION AND THE TWO-PHASE MYOBLAST FUSION MODEL
  6. ULTRASTRUCTURAL FEATURES OF MYOBLAST FUSION
  7. KEY MOLECULAR PLAYERS AS COMPONENTS OF THE FUSION-RESTRICTED MYOGENIC-ADHESIVE STRUCTURE (FURMAS)
  8. REGULATION OF ACTIN POLYMERIZATION DURING MYOBLAST FUSION
  9. THE FURMAS MODEL FOR MYOBLAST FUSION: KEY MOLECULAR PLAYERS, THEIR LOCALIZATION AND ULTRASTRUCTURAL FEATURES
  10. COMPARISION OF TRANSIENT ADHESIVE STRUCTURES: THE FURMAS, IMMUNOLOGICAL SYNAPSES, PODOSOMES AND INVADADOPODIA
  11. FUNCTIONAL CONSERVATION BETWEEN ODROSOPHILA AND VERTEBRATE MYOBLAST FUSION
  12. PERSPECTIVES
  13. Acknowledgements
  14. REFERENCES

The authors thank Detlev Buttgereit, Susanne Berger, and Bettina Bonn for stimulating discussions and critical reading. We thank Detlev Buttgereit, Roxane Schröter, Katja Geßner, and Gritt Schäfer for providing figures. The electron microscopy work was done in Prof. Dr. Beck's laboratory and we thank his technician Helga Kisselbach-Heckmann for technical assistance. Work in the R. R.-P. and S. Ö. laboratory referred to in this review has been supported by grants from the Deutsche Forschungsgemeinschaft (GRK 1216, Re628/14-3, Re628/15-2, OE 311/4-1) and the European Network of Excellence (MYORES). We apologise to those authors whose work was not cited because of space limitations.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. AN OVERVIEW OF MYOBLAST FUSION
  5. TWO-TEMPORAL-PHASES OF FUSION AND THE TWO-PHASE MYOBLAST FUSION MODEL
  6. ULTRASTRUCTURAL FEATURES OF MYOBLAST FUSION
  7. KEY MOLECULAR PLAYERS AS COMPONENTS OF THE FUSION-RESTRICTED MYOGENIC-ADHESIVE STRUCTURE (FURMAS)
  8. REGULATION OF ACTIN POLYMERIZATION DURING MYOBLAST FUSION
  9. THE FURMAS MODEL FOR MYOBLAST FUSION: KEY MOLECULAR PLAYERS, THEIR LOCALIZATION AND ULTRASTRUCTURAL FEATURES
  10. COMPARISION OF TRANSIENT ADHESIVE STRUCTURES: THE FURMAS, IMMUNOLOGICAL SYNAPSES, PODOSOMES AND INVADADOPODIA
  11. FUNCTIONAL CONSERVATION BETWEEN ODROSOPHILA AND VERTEBRATE MYOBLAST FUSION
  12. PERSPECTIVES
  13. Acknowledgements
  14. REFERENCES