Abstract
The process of myogenesis includes the recognition, adhesion, and fusion of committed myoblasts into multinucleate syncytia. In the larval body wall muscles of Drosophila, this elaborate process is initiated by Founder Cells and Fusion-Competent Myoblasts (FCMs), and cell adhesion molecules Kin-of-IrreC (Kirre) and Sticks-and-stones (Sns) on their respective surfaces. The FCMs appear to provide the driving force for fusion, via the assembly of protrusions associated with branched F-actin and the WASp, SCAR and Arp2/3 pathways. In the present study, we utilize the dorsal pharyngeal musculature that forms in the Drosophila embryo as a model to explore myoblast fusion and visualize the fusion process in live embryos. These muscles rely on the same cell types and genes as the body wall muscles, but are amenable to live imaging since they do not undergo extensive morphogenetic movement during formation. Time-lapse imaging with F-actin and membrane markers revealed dynamic FCM-associated actin-enriched protrusions that rapidly extend and retract into the myotube from different sites within the actin focus. Ultrastructural analysis of this actin-enriched area showed that they have two morphologically distinct structures: wider invasions and/or narrow filopodia that contain long linear filaments. Consistent with this, formin Diaphanous (Dia) and branched actin nucleator, Arp3, are found decorating the filopodia or enriched at the actin focus, respectively, indicating that linear actin is present along with branched actin at sites of fusion in the FCM. Gain-of-function Dia and loss-of-function Arp3 both lead to fusion defects, a decrease of F-actin foci and prominent filopodia from the FCMs. We also observed differential endocytosis of cell surface components at sites of fusion, with actin reorganizing factors, WASp and SCAR, and Kirre remaining on the myotube surface and Sns preferentially taken up with other membrane proteins into early endosomes and lysosomes in the myotube.
Highlights
Myoblast fusion is initiated by cell recognition and adhesion, and culminates in loss of membrane between the adjoining cells and absorption of the nucleus into the developing syncytia
The dorsal pharyngeal musculature (DPM) comprises a tightly associated array of myotubes, each generated by fusion of a single Founder Cells (FC) with multiple Fusion-Competent Myoblasts (FCMs)
Sns and Kirre colocalize at points of FCM-myotube contact. (N–R) Dorsal view of stage 15 embryos immunostained for dMef2 and tropomyosin
Summary
Myoblast fusion is initiated by cell recognition and adhesion, and culminates in loss of membrane between the adjoining cells and absorption of the nucleus into the developing syncytia. Numerous studies have revealed F-actin foci at the contact site between FCMs and either FCs or myotubes [1, 2] that disappear just prior to fusion [2] These actin foci form in the center of a ring of Sticks-and-stones (Sns) and Kin-of-IrreC (Kirre), cell surface molecules that are essential for myoblast fusion [1, 3]. Studies have shown that Antisocial (Ants)/Rolling pebbles (Rols) stabilizes Kirre at the myotube surface through interaction with its cytoplasmic domain [13] Subsequent to their heterotypic interaction at the cell surface, Kirre and Sns function as signaling molecules to mediate intracellular events [14] that lead to actin polymerization and fusion
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