The Intracellular Domain of Dumbfounded Affects Myoblast Fusion Efficiency and Interacts with Rolling Pebbles and Loner

Chia W (2010) The Intracellular Domain of Dumbfounded Affects Myoblast Fusion Efficiency and Interacts with Rolling Pebbles and Loner. PLoS ONE 5(2): e9374. doi:10.1371/journal.pone.0009374 The Intracellular Domain of Dumbfounded Affects Myoblast Fusion Efficiency and Interacts with Rolling Pebbles and Loner Sarada Bulchand 0 1 2 Sree Devi Menon 0 1 2 Simi Elizabeth George 0 1 2 William Chia 0 1 2 Suzannah Rutherford, Fred Hutchinson Cancer Research Center, United States of America 0 Funding: Temasek Lifesciences Laboratory. Singapore Millenium Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript 1 Temasek Lifesciences Laboratory, National University of Singapore , Singapore, Singapore 2 Domain Analysis of Duf Drosophila body wall muscles are multinucleated syncytia formed by successive fusions between a founder myoblast and several fusion competent myoblasts. Initial fusion gives rise to a bi/trinucleate precursor followed by more fusion cycles forming a mature muscle. This process requires the functions of various molecules including the transmembrane myoblast attractants Dumbfounded (Duf) and its paralogue Roughest (Rst), a scaffold protein Rolling pebbles (Rols) and a guanine nucleotide exchange factor Loner. Fusion completely fails in a duf, rst mutant, and is blocked at the bi/trinucleate stage in rols and loner single mutants. We analysed the transmembrane and intracellular domains of Duf, by mutating conserved putative signaling sites and serially deleting the intracellular domain. These were tested for their ability to translocate and interact with Rols and Loner and to rescue the fusion defect in duf, rst mutant embryos. Studying combinations of double mutants, further tested the function of Rols, Loner and other fusion molecules. Here we show that serial truncations of the Duf intracellular domain successively compromise its function to translocate and interact with Rols and Loner in addition to affecting myoblast fusion efficiency in embryos. Putative phosphorylation sites function additively while the extreme C terminus including a PDZ binding domain is dispensable for its function. We also show that fusion is completely blocked in a rols, loner double mutant and is compromised in other double mutants. These results suggest an additive function of the intracellular domain of Duf and an early function of Rols and Loner which is independent of Duf. - Skeletal muscles perform various roles, which include coordinating movement and stabilising joints in many organisms. Understanding how they develop has been the focus of several studies [1]. Studying this process in vertebrate models is complicated by the relative inaccessibility of their muscles and long developmental times. Simple model organisms like Drosophila melanogaster have been widely used instead. Its somatic/body wall muscles (analogous to vertebrate skeletal muscles) are easily accessible and several muscle specific genes are conserved with those of vertebrates [2]. Also, some principles of muscle development are similar [3,4]. Drosophila somatic muscles develop during mid to late embryogenesis, display their contractile function during late embryogenesis and continue to function in the developing larva where they are critical for motility [5,6]. During early embryogenesis, two types of myoblasts are specified, the founder cells (FCs) and the fusion competent myoblasts (FCMs) [7,8]. The FCs express the myoblast attractants, Dumbfounded (Duf)/Kin of irregular-chiasm-C (Kirre) and its paralogue Roughest (Rst)/Irregular chiasm-C (IrreC) [9,10]. They also express muscle identity genes, like Even-skipped (Eve) [11], that are responsible for the specification of muscle size, position with respect to the body axis, points of epidermal attachment and points of innervation [8,12]. The FCMs on the other hand constitute a more homogeneous population of cells expressing the Duf/Rst ligands, Sticks and Stones (SNS) [13] and Hibris (Hbs) [14]. The FCMs contribute to muscle size by fusing with the FCs [8,15]. Fusion always occurs between FC/myotube and FCM and never between cells of the same type [16]. Recent studies have shown that myoblasts are spatially organised in the embryo. Fusion initiates between an FC and FCM that are in its vicinity. As development proceeds, FCMs appear to migrate towards the FC for further rounds of fusion [17]. Duf/Rst expressed on the FC surface and SNS/Hbs expressed on the FCM surface are thought to bring about myoblast attraction, and have been suggested to actively participate in this process [9,14,18,19]. Upon FC-FCM contact and adhesion, the plasma membranes breakdown leading to cytoplasmic continuity [4]. The presence of vesicles and electron dense plaques at the site of adhesion prior to membrane breakdown have also been observed but the nature and content of these vesicles are unknown [20]. Further studies have revealed the accumulation of an F actin focus (FuRMAS) at the site of myoblast adhesion [21] and live imaging data indicate that the Factin focus marks the site of fusion [22]. Proteins like Duf and SNS localise to this focus suggesting that this might be their site of activity during fusion [21]. Upon fusion, the nuclei of the FCMs are entrained by the FC nucleus and begin to express FC specific molecules [6]. The process of fusion is reiterative. Events are repeated in a stepwise manner first leading to the formation of a bi/trinucleate precursor, followed by more such rounds of fusion, accompanied by growth at the ends of the myotube. As embryogenesis proceeds the newly formed muscles attach to specific sites at the epidermis leading to the formation of approximately 30 muscles per hemisegment [8]. Genetic screens have identified a large number of molecules required for myoblast fusion that fall into several categories depending on their predicted functions [23,24]. Mutation of these genes, in most cases, leads to the formation of defective mini muscles with reduced nuclei, ending in embryonic lethality. Duf and Rst are Type I single pass transmembrane receptors with an N terminal extracellular domain and C terminal intracellular domain, belonging to the Immunoglobulin superfamily of proteins [10,25]. Their function is redundant in the FC. In mutant embryos that lack both duf and rst, Df(1)w67k30 (henceforth called the duf, rst mutant), there is no attraction and adhesion between FCs and FCMs leading to a complete block in fusion [9,10]. Both the extracellular and intracellular domains of Duf have been shown to be critical for the attraction of FCMs and sustenance of fusion respectively [25]. In the absence of the extracellular domain FCMs are not attracted towards the FC and fusion fails. In the absence of the intracellular domain fusion is not sustained beyond the first phase, stalling at the bi/tri nucleate precursor stage [25]. This suggests that the intracellular domai (...truncated)