Group I chitin deacetylases are essential for higher order organization of chitin fibers in beetle cuticle

Mi Young Noh,Subbaratnam Muthukrishnan,Karl J Kramer,Yasuyuki Arakane

doi:10.1074/jbc.ra117.001454

Mi Young Noh, Subbaratnam Muthukrishnan + Show 2 more

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https://doi.org/10.1074/jbc.ra117.001454

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Abstract

Roles in the organization of the cuticle (exoskeleton) of two chitin deacetylases (CDAs) belonging to group I, TcCDA1 and TcCDA2, as well as two alternatively spliced forms of the latter, TcCDA2a and TcCDA2b, from the red flour beetle, Tribolium castaneum, were examined in different body parts using transmission EM and RNAi. Even though all TcCDAs are co-expressed in cuticle-forming cells from the hardened forewing (elytron) and ventral abdomen, as well as in the softer hindwing and dorsal abdomen, there are significant differences in the tissue specificity of expression of the alternatively spliced transcripts. Loss of either TcCDA1 or TcCDA2 protein by RNAi causes abnormalities in organization of chitinous horizontal laminae and vertical pore canals in all regions of the procuticle of both the hard and soft cuticles. Simultaneous RNAi for TcCDA1 and TcCDA2 produces the most serious abnormalities. RNAi of either TcCDA2a or TcCDA2b affects cuticle integrity to some extent. Following RNAi, there is accumulation of smaller disorganized fibers in both the horizontal laminae and pore canals, indicating that TcCDAs play a critical role in elongation/organization of smaller nanofibers into longer fibers, which is essential for structural integrity of both hard/thick and soft/thin cuticles. Immunolocalization of TcCDA1 and TcCDA2 proteins and effects of RNAi on their accumulation indicate that these two proteins function in concert exclusively in the assembly zone in a step involving the higher order organization of the procuticle.

Highlights

Roles in the organization of the cuticle of two chitin deacetylases (CDAs) belonging to group I, TcCDA1 and TcCDA2, as well as two alternatively spliced forms of the latter, TcCDA2a and TcCDA2b, from the red flour beetle, Tribolium castaneum, were examined in different body parts using transmission EM and RNAi
We previously reported that RNAi for TcCDA1 or TcCDA2 at the larval stage led to the arrest of growth at the subsequent
TcCDA1 and TcCDA2 are required at all developmental stages, the requirement for TcCDA2a and TcCDA2b indicates tissue specificity

Summary

Introduction

Roles in the organization of the cuticle (exoskeleton) of two chitin deacetylases (CDAs) belonging to group I, TcCDA1 and TcCDA2, as well as two alternatively spliced forms of the latter, TcCDA2a and TcCDA2b, from the red flour beetle, Tribolium castaneum, were examined in different body parts using transmission EM and RNAi. Loss of either TcCDA1 or TcCDA2 protein by RNAi causes abnormalities in organization of chitinous horizontal laminae and vertical pore canals in all regions of the procuticle of both the hard and soft cuticles. In Drosophila melanogaster embryos, disruption of DmCDA1 (serpentine) and DmCDA2 (vermiform) genes resulted in an abnormal chitin core in the tracheal lumen and elongated tracheal tubes [12, 13] These two proteins along with two other cuticular proteins, Obstructor A and Knickkopf, have been proposed to interact in the assembly zone of the cuticle to provide epidermal cuticle integrity of D. melanogaster larval cuticle [17, 18]. The authors reported that in LmCDA2-deficient insects, the chitinous laminae were not helicoidally arranged based on the trajectory of the pore canals, which followed a straight path through the cuticle rather than the helicoidal path observed in abdominal cuticles of control insects

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