Amontillado is required forDrosophilaSlit processing and for tendon-mediated muscle patterning

Abstract

Slit 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.

SlitC-PlexinA1 mediates iterative inhibition for orderly passage of spinal commissural axons through the floor plate

“…Thus, unlike in drosophila midline guidance for which Slit processing was reported to be dispensable (44), FL-Slits likely play limited guidance role for commissural axons. This is also consistent with previous studies of the process of muscle anchorage to tendons in drosophila, that reported the importance of Slit processing for subsequent distribution of the fragments and proper functional outcome (37). Our data suggest that the pro-protein convertase PC2, the vertebrate homolog of Amontillado, that we found able to cleave Slit2-FL and expressed in the floor plate, contributes to the generation of Slit2C and SlitN fragments distribution.…”

“…This confirmed that the cleavage is determinant for proper localization of Slit2 fragments to the axon navigation path. While the protease responsible for Slit2 cleavage is still unknown in vertebrates, the pheromone convertase Amontillado has been shown to cleave Slit during muscles and tendons development in Drosophila ( Ordan and Volk, 2016 ). Amontillado homolog in vertebrates is PC2, a proprotein convertase involved in the activation of endocrine peptides ( Smeekens et al, 1991 ).…”

Iterative inhibition of commissural growth cone exploration, not post-crossing barrier, ensures forward midline navigation through SlitC-PlxnA1 signaling

“…Thus, this confirmed that the cleavage is determinant for proper localization of Slit2 fragments to the axon navigation path. While the protease responsible for Slit2 cleavage is still unknown in vertebrates, the pheromone convertase Amontillado has been shown to cleave Slit during drosophila muscles and tendons development (Ordan and Volk 2016). Amontillado homolog in vertebrates is PC2, a proprotein convertase (PC) involved in the activation of endocrine peptides (Smeekens and Steiner, 1991).…”

Proteolytic cleavage of Slit by the Tolkin protease converts an axon repulsion cue to an axon growth cue in vivo

“…This is not surprising given that Slit cleavage is tightly regulated, and previous reports showed that 92% of Slit remains in full-length form during key axon guidance stages 14-17 ( Furrer et al, 2007 ). We were unable to replicate the lack of Slit cleavage in amontillado ( amon ) mutants, a proprotein convertase that was previously identified as a potential Slit protease ( Ordan and Volk, 2016 ). We found that two independent mutant alleles of amon cleaved Slit normally ( …”