Genetic and molecular study of chitin synthesis and assembly in the embryonic cuticle of Drosophila melanogaster
Final Report Abstract
The insect cuticle is an apical extracellular matrix (ECM) that is produced by the underlying epidermal cells. A central component of the cuticle is chitin that associates with proteins and adopts a stereotypic organisation. Chitin organisation in the embryo of the fruit fly Drosophila melanogaster requires the factors Knickkopf (Knk) and Retroactive (Rtv) that are both inserted into the apical plasma membrane. The central aim of the completed DFG project was to characterise the function of Knk and Rtv in a combined histological and molecular approach applying electron microscopy and in vitro binding assays, cell culture and transgenic flies studies, respectively. Our data suggest that the GPI-anchored Knk protein defines a cholesterol-rich membrane domain (without being necessary for apical undulae formation), where it is influencing chitin synthesis or stability acting through its Domon domain, the plastocyanin domain being idle. Our attempts to test whether the Domon domain may as predicted bind to haem or sugars were not successful. The notion that Knk is needed for correct amounts of chitin is consistent with our published finding that application of moderate concentrations of the chitin synthesis inhibiting insecticides lufenuron and diflubenzuron causes a knk-like phenotype. In another subproject, we show that the small snake-toxin-like Rtv protein requires six conserved aromatic amino acids for full function. Rtv is not needed for apical undulae formation, but is able to induce filopodialike structures in HEK293 cells suggesting that it may interact with the cytoskeleton. The molecular function of Rtv remains elusive since our chitin binding assays neither support nor dismiss the hypothesis that Rtv is a chitin binding protein. In collaboration with a research group at the Kansas State University, we demonstrate that upon reduction of Knk or Rtv by RNAi the serosal cuticle in the beetle Tribolium castaneum displays a knkor rtv-like phenotype, respectively, suggesting that their function and beyond that chitin organisation is conserved in insects. Besides the work on Knk and Rtv, we were also interested in understanding regulatory mechanisms driving epidermal and cuticle differentiation by especially investigating the functions of Wollknäuel (Wol), which is involved in protein glycosylation, and the role of ecdysone. In a recent publication, we demonstrate that Wol is needed for balanced differentiation of the epidermis and the cuticle. In collaboration with the research group of Anne Uv in Göteborg/Sweden, we also published that ecdysone signalling synchronises and controls progression of organogenesis in a tissue-autonomous manner. Based on this finding, we are now able to address the specific function of ecdysone in epidermal and cuticle differentiation in appropriate experiments. With the results of this DFG project, we have gained some insight into the molecular and cellular properties of Knk and Rtv, which will be useful for further characterisation, for instance in a chitin producing cell culture system. Moreover, with the study of Wollknäuel and ecdysone, we have continued investigating cellular mechanisms that govern epidermal and cuticle differentiation, especially strengthening our understanding of secretion control and plasma membrane dynamics. This progress primes us to extend our studies on the question of how secretion, plasma membrane dynamics, cell polarity and chitin synthesis and organisation are coordinated during cuticle differentiation.
Publications
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Effects of Benzoylphenylurea on chitin synthesis and orientation in the cuticle of the Drosophila Larva. European Journal of Cell Biology. 2009 March;88(3):167-180
Gangishetti U, Breitenbach S, Zander M, Shaik Saheb K, Müller U, Schwarz H & Moussian B
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The apical plasma membrane of Drosophila embryonic epithelia. European Journal of Cell Biology. 2010 Feb-March;89(2-3): 208-211
Uv A & Moussian B
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Tissue-autonomous EcR functions are required for concurrent organ morphogenesis in the Drosophila embryo. Mechanisms of Development. 2010 May-June;127(5-6): 308-319
Chavoshi T, Moussian B & Uv A
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The Alg5 ortholog Wollknäuel is essential for correct epidermal differentiation during Drosophila late embryogenesis. Glycobiology. 2011 Jan 2
Shaik KS, Pabst M, Schwarz H, Altmann F, Moussian B
