Retroactive Maintains Cuticle Integrity by Promoting the Trafficking of Knickkopf into the Procuticle of Tribolium castaneum

Abstract

Molting, or the replacement of the old exoskeleton with a new cuticle, is a complex developmental process that all insects must undergo to allow unhindered growth and development. Prior to each molt, the developing new cuticle must resist the actions of potent chitinolytic enzymes that degrade the overlying old cuticle. We recently disproved the classical dogma that a physical barrier prevents chitinases from accessing the new cuticle and showed that the chitin-binding protein Knickkopf (Knk) protects the new cuticle from degradation. Here we demonstrate that, in Tribolium castaneum, the protein Retroactive (TcRtv) is an essential mediator of this protective effect of Knk. TcRtv localizes within epidermal cells and specifically confers protection to the new cuticle against chitinases by facilitating the trafficking of TcKnk into the procuticle. Down-regulation of TcRtv resulted in entrapment of TcKnk within the epidermal cells and caused molting defects and lethality in all stages of insect growth, consistent with the loss of TcKnk function. Given the ubiquity of Rtv and Knk orthologs in arthropods, we propose that this mechanism of new cuticle protection is conserved throughout the phylum.

Obstructor A Organizes Matrix Assembly at the Apical Cell Surface to Promote Enzymatic Cuticle Maturation in Drosophila

“…8). All insects establish compact and structured exoskeletal cuticles at their outermost body parts (4,29,32,33,36,37). Our data argue that structural similarities of the body wall cuticle and molecular conservation of involved proteins (8, 9, 15-19, 38 -40) point toward a highly conserved mechanism of procuticle formation among chitinous invertebrates.…”

“…This suggests that chitin matrix protection must be affected. Our findings may not exclude other potential protective proteins (32), and yet uncharacterized Drosophila Knk-like proteins (33) could depend on Obst-A. However, the wrinkled cuticle specifically observed in obst-A null and transheterozygous obst-A;knk mutants and the localization of Obst-A and Knk proteins in the outer cuticle led to the speculative hypothesis that they act in cuticle stability of the newly synthesized and packaged chitin matrix.…”

“…Molting, wounding, and other types of cuticle disruption require complex actions of enzymes and scaffold proteins that form new and remodel existing cuticles throughout development (4,32,33). To some extent, chitin synthases and chitinolytic enzymes have been studied in the past (1-7), but little is known about underlying mechanisms that control the assembly, stability, and integrity of newly synthesized cuticles.…”

Chitinase from Thermomyces lanuginosus SSBP and its biotechnological applications

“…These are primarily endochitinases that randomly cleave chitin present in the cuticle to chitooligosaccharides that are further hydrolyzed by exoenzymes to N-acetyl-glucosamine monomer, which is reused to synthesize a new cuticle (Chaudhari et al 2013). Chitinases from Bombyx mori, Manduca sexta have been extensively studied.…”

Mutation of a Cuticular Protein,BmorCPR2, Alters Larval Body Shape and Adaptability in Silkworm,Bombyx mori

“…Naked chitin fibers need to interact with chitinbinding protein to stabilize the helicoidal layers of cuticle (Rebers and Riddiford 1988;Rebers and Willis 2001;Merzendorfer and Zimoch 2003;Moussian 2010). The largest member of these chitin-binding proteins is the CPR family, which may contribute to coordinating the interactions between chitin and the proteinaceous matrix (Rebers and Riddiford 1988;Rebers and Willis 2001;Togawa et al 2004;Magkrioti et al 2004;Willis 2010;Andersen 2011), and RR-type chitin-binding protein, chitin, and chitinase are colocalized in the procuticle (Moussian et al 2006;Chaudhari et al 2011Chaudhari et al , 2013Arakane et al 2012). The recent study on Tribolium castaneum indicated that the TcCPR4 (a RR1-type cuticular protein) distribute the region nearby chitin laminae and can affect the chitin content (Arakane 2013).…”