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

Suzannah Rutherford,Sarada Bulchand,William Chia,Sree Devi Menon,Simi Elizabeth George

doi:10.1371/journal.pone.0009374

Suzannah Rutherford, Sarada Bulchand + Show 3 more

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https://doi.org/10.1371/journal.pone.0009374

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Abstract

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.

Highlights

Skeletal muscles perform various roles, which include coordinating movement and stabilising joints in many organisms
The transmembrane domain of Duf was replaced with that of DE Cadherin (DE Cadh)/Shotgun (Shg) that has been shown to play an important role in cell adhesion during Drosophila epithelial morphogenesis [42] (Duf TM DE-Cadh-flag) and Semaphorin 1a (Sema-1a) that is involved in axon guidance [43] (Duf TM Sema-1a-flag)
As reported previously [25], we find that full length wild type Duf (Duf flag) is enriched at the point of cell-cell contact and Rolling pebbles (Rols) and Loner translocate to these points of cell contact in a Duf dependent manner (Fig. 2A and G)

Summary

Introduction

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. 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]

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