In vitro chitin synthesis in an insect: formation and structure of microfibrils

Chitin microfibril synthesis in Tribolium castaneum chitin synthetase (CS) assays results in the formation of a considerable precipitate. Such precipitate does not appear in the absence of the CS substrate (uridine diphospho N-acetyl-D-glucosamine) or in the presence of polyoxin-D, a potent CS inhibitor. Diflubenzuron which blocks in vivo chitin synthesis in insects has no effect on the precipitate formation at a relatively high concentration (3 x 10(-4) M). Electron microscopic examination of the CS product reveals a network of long, parallel oriented microfibrils. The microfibrils vary in thickness from 10 to 80 nm. Thinner elements merge to form larger and thicker microfibrils which are packed in distinct bundles. Particles associated with the microfibrils are largely spheroidal and range from about 50 to 250 nm in diameter. Coiled fibroid structures enclosed inside particles were observed. Similar coiled structures or extended fibroids attached to the outside (shell) of particles were also found. The nature of the particles and their possible role in chitin synthesis and fibrillogenesis in insects are discussed.