Assembly of the Drosophila larval exoskeleton requires controlled secretion and shaping of the apical plasma membrane

Abstract

The apical plasma membrane of epithelial cells plays a central role in producing and shaping the apical extracellular matrix (aECM) that eventually adopts a stereotypic architecture required for the physical and physiological needs of the epithelium. To assess the implication of the apical plasma membrane on aECM differentiation, we have studied the function of the apical plasma membrane t-SNARE Syntaxin 1A in the embryo of the fruit fly Drosophila melanogaster during differentiation of the stratified exoskeleton, the cuticle, which is composed of proteins and the polysaccharide chitin. The cuticle layers of syntaxin1A deficient larvae are rudimentary. Consistently, Syntaxin 1A is required for the secretion of O-glycosylated proteins and components involved in pigmentation and protein cross-linking. By contrast, localization of chitin synthesis and organising proteins to the apical plasma membrane or to the extracellular space does not depend on Syntaxin 1A activity. However, chitin microfibrils have a random orientation instead of being arranged in parallel. This correlates with the lack of corrugations at the apical plasma membrane of epidermal cells, the apical undulae that have been proposed to be crucial for chitin microfibril orientation. Hence, Syntaxin 1A contributes to cuticle differentiation by controlling correct apical plasma membrane topology as well as mediating secretion of a subset of extracellular proteins required for layer organisation. Our data also indicate that yet another unidentified t-SNARE is needed in parallel to Syntaxin 1A to deliver extracellular material for complete cuticle assembly. Evidently, coordination of apical membrane modelling and two secretion routes are essential for stereotypic aECM organisation.