Amontillado is required for Drosophila Slit processing and for tendon-mediated muscle patterning.

Elly Ordan,Talila Volk

doi:10.1242/bio.020636

Abstract

ABSTRACTSlit cleavage into N-terminal and C-terminal polypeptides is essential for restricting the range of Slit activity. Although the Slit cleavage site has been characterized previously and is evolutionally conserved, the identity of the protease that cleaves Slit remains elusive. Our previous analysis indicated that Slit cleavage is essential to immobilize the active Slit-N at the tendon cell surfaces, mediating the arrest of muscle elongation. In an attempt to identify the protease required for Slit cleavage we performed an RNAi-based assay in the ectoderm and followed the process of elongation of the lateral transverse muscles toward tendon cells. The screen led to the identification of the Drosophila homolog of pheromone convertase 2 (PC2), Amontillado (Amon), as an essential protease for Slit cleavage. Further analysis indicated that Slit mobility on SDS polyacrylamide gel electrophoresis (SDS-PAGE) is slightly up-shifted in amon mutants, and its conventional cleavage into the Slit-N and Slit-C polypeptides is attenuated. Consistent with the requirement for amon to promote Slit cleavage and membrane immobilization of Slit-N, the muscle phenotype of amon mutant embryos was rescued by co-expressing a membrane-bound form of full-length Slit lacking the cleavage site and knocked into the slit locus. The identification of a novel protease component essential for Slit processing may represent an additional regulatory step in the Slit signaling pathway.

Highlights

A functional musculoskeletal system depends on proper recognition and connection of muscles and tendons (Schweitzer et al, 2010)
In the Drosophila embryo each muscle is formed through fusion of a single founder cell with fusion-competent myoblasts, forming a syncytium that elongates towards its attachment sites, the ectodermal tendon cells (Schejter and Baylies, 2010; Schnorrer and Dickson, 2004b; Soler et al, 2016)
Our previous analysis demonstrated that Slit cleavage is essential for proper elongation of the lateral transverse muscles 1-3 (LT1-3), as well as for the extension of the dorsal acute muscle 3 (DA3) towards its corresponding tendon attachment cell. These results indicated that, upon cleavage, Slit-N remains tethered to the cell surfaces of the tendon cell, forming a short-range repulsive signal that is important for keeping the elongating LT muscles on their correct paths in the middle of the segment, and for providing a stop signal for the DA3 muscle once it has reached the tendon site (Ordan et al, 2015)

Summary

Introduction

A functional musculoskeletal system depends on proper recognition and connection of muscles and tendons (Schweitzer et al, 2010). These results indicated that, upon cleavage, Slit-N remains tethered to the cell surfaces of the tendon cell, forming a short-range repulsive signal that is important for keeping the elongating LT muscles on their correct paths in the middle of the segment, and for providing a stop signal for the DA3 muscle once it has reached the tendon site (Ordan et al, 2015).

Results

Conclusion