Whether the formation of multinucleated cells by fusion evolved independently in different cell types or if there exist common underlying mechanisms is unknown. In this study we have investigated the mammalian homologues of Loner
and Myoblast city,
which have previously been found to be essential in the development of the embryonic musculature in Drosophila
(Erickson et al., 1997
; Chen et al., 2003
). Our data show that the functions of Brag2 and Dock180 are evolutionarily conserved and involve control of differentiation and cell shape in addition to fusion, via different mechanisms directed by their respective GTPases. Moreover, this study identifies Dock180 as a common molecular component that controls cell fusion in different cell types, providing the first evidence of shared fusion machinery.
Two assays to quantitate myoblast fusion, one by observation and the other biochemical, complemented each other by permitting the detection of morphological differences and by removing observer bias and error, respectively. In light of the failure of Brag2- and Dock180-deficient myoblasts to fuse to existing muscle fibers in vivo, the low level of fusion observed in vitro leads us to conclude that whereas Brag2 and Dock180 are required for myotube maturation, early fusion in mammalian myotubes is not dependent on Brag2 or Dock180. However, it cannot be ruled out that incomplete knockdown, a ubiquitous caveat of RNAi experiments, may have contributed to the low level of fusion observed.
Whereas Dock180 and Brag2 have not previously been linked to muscle cell differentiation aside from fusion, our analysis of individual cells at early stages of differentiation indicated that myoblasts deficient in either Brag2 or Dock180 failed to efficiently exit the cell cycle. These cells were likewise impaired in their ability to induce the transcription factor myogenin or the contractile protein MHC. Suppression of myogenin promoter activity has been previously observed with dominant-negative mutants of Rac (Takano et al., 1998
). However, the role of Rac in myoblast differentiation is complex and remains poorly understood, as other recent studies suggest that a drop in Rac activity is essential for cell cycle withdrawal (Heller et al., 2001
; Charrasse et al., 2007
). This implies that inappropriate cycling of cells deficient in Dock180 may occur though Rac-independent mechanisms, possibly involving interactions of Dock180 with Crk and other signaling components involved in cell cycle regulation.
In further support of Rac independent control of differentiation, Brag2 deficiency did not affect Rac activation, yet its effects on cell cycle withdrawal and differentiation were more severe when compared with Dock180. Cells deficient in both Dock180 and Brag2 more closely mimicked the differentiation and morphological defects observed in Brag2-deficient cells. Although ARF6 has not previously been linked to cell cycle, it has been recently shown that Brag2 can localize to the nucleus (Dunphy et al., 2007
), where its functions in cell cycle regulation have not been characterized to date. Their similar effects on cell cycle withdrawal and initiation of differentiation suggest that Brag2 and Dock180 function in a common pathway possibly through ARF6.
The similar defects early in differentiation notwithstanding, disparity between Brag2 and Dock180 are highlighted by the striking morphological differences between the two during fusion, demonstrating unique roles of each in myotube maturation. Defects in skeletal muscle maturation have been previously reported in myoferlin null mice, which formed myotubes with significantly reduced myofiber cross-sectional area and muscle mass (Doherty et al., 2005
). Our data indicate that Brag2 and Dock180 are dispensable for myoblasts proliferation but that they play critical roles in the orchestration of skeletal muscle formation, especially in the later stages of myotube growth. It is possible that the different morphological observations relate to the distinct roles that each GEF carries out in the regulation of its respective GTPase. We have shown that Brag2 deficiency, and to a lesser extent Dock180 deficiency, leads to ARF6 inactivity whereas Dock180 deficiency exclusively leads to a drop in Rac activity. Furthermore, with respect to Dock180, these results are in agreement with recent results in which Trio, another GEF with dual specificity for Rac and Rho, severely impairs myoblast fusion when its levels are decreased by RNAi (Charrasse et al., 2007
). Unlike Trio, Dock180 has no cross-specificity for Rho, thus it may be the combination of both Rac and Rho inactivation, which results in the severe fusion defect of Trio deficiency. Time-lapse analysis showed that in contrast to the Dock180-deficient cells, absence of Brag2 rendered nascent myotubes unable to extend and adhere to the surface. This unique Brag2 phenotype combined with previous results, which indicate that overexpression of an ARF6 GAP PAG3 decreases the recruitment of paxillin to focal adhesions (Kondo et al., 2000
), led us to analyze the localization of paxillin. During differentiation and fusion, distribution of paxillin was severely disrupted in myogenin-positive Brag2Si and DKD cells. A failure to redistribute paxillin upon differentiation accounts for the adhesion defect observed in bragballs, where a newly formed multinucleated cell is incapable of creating or maintaining functional focal adhesions, causing collapse into the bragball morphology.
Our immunoprecipitation and immunofluorescence data identify an association between paxillin and ARF6. Furthermore, there is a strong correlation between a deficiency in ARF6 activity and improper localization of paxillin during fusion. The intermediate drop in ARF6 activity observed in Dock180Si cells, according to our model (), accounts for the moderate paxillin distribution defect observed during differentiation and the minor paxillin clustering observed during fusion. It is possible that the observed increase in total β1-integrin protein levels in Dock180-deficient cells combined with normal cell motility during differentiation (Video 3) could account for the ability of Dock180Si myotubes to elongate. Further analysis will determine if other structural components of focal adhesions such as vinculin and focal adhesion kinase are specifically transported by ARF6 activation after differentiation, and how integrin levels affect morphology in Dock180-deficient cells.
Figure 10. Model for the roles of Brag2 and Dock180 in cell–cell fusion. Upon receipt of signals from fusion receptors on the cell surface Brag2 activates ARF6, which in turn transports paxillin to sites of focal adhesion, where it complexes with integrins (more ...)
In contrast to skeletal muscle, there is little known about the intracellular mechanisms of cell fusion in nonmuscle cell types (Vignery, 2005
). Knockdown in primary macrophages, which are differentiated cells not amenable to passage in culture, required introduction of shRNA constructs in dividing hematopoietic stem cells (HSCs). In the case of Brag2, an unexpected role for this molecule in HSC biology was revealed by the repeated inability to achieve bone marrow reconstitution using HSCs infected with the Brag2 shRNA. Further experiments will determine the function of Brag2 in HSC homing, but defects in adhesion and niche recognition are two potential explanations. Generation of shRNA-expressing intraperitoneal macrophages was possible in the case of Dock180. Deficiency in Dock180 prevented the formation of MNGCs and the rare fusion events that did occur resulted in cells that had low nuclear number, and aberrant morphology that lacked the characteristic centrally located nuclear cluster common in the control MNGCs. From our data, it is apparent that the loss of Dock180 leads to a significant fusion defect in both myoblasts and macrophages. These findings are the first to identify a role for a single molecule in fusion in different lineages, and the results suggest that muscle and macrophages use redundant intracellular machinery during the fusion process.
It is becoming apparent that the dynamic morphological state of a cell reflects the particular balance of GEFs present at a given time. For example, a recent study (Yeh et al., 2007
) found that the introduction of single, engineered GEFs and their domains could induce morphological changes including the formation of filopodia or lamellipodia. This study and others (Lee et al., 2001
; Dunphy et al., 2006
; Francis et al., 2006
; Otani et al., 2006
; Charrasse et al., 2007
) suggest that cell morphology is subject to manipulation by the repression or expression of particular GEF activities. We show that GEFs control the differentiation-specific morphology of skeletal muscle cells and MNGCs. The effects of individual GEFs are unique, an example being the markedly different impact of Dock180 and Brag2 on muscle morphology. In addition, their functions are cell type specific as indicated by analyses of a variety of nonfusion-related functions of Dock180 and Brag2 in mammalian cells (Hasegawa et al., 1996
; Dunphy et al., 2006
; Hiroi et al., 2006
; Handa et al., 2007
). Conversely, our experiments demonstrate that there are fundamental GEF-controlled fusion processes that are conserved across lineages, underscored by the similar role of Dock180 in fusion of different cell types. This paper furthers our understanding of the generation of multinucleated cells and the GEF-mediated intracellular functions responsible for their formation and maintenance.