Albumin uptake by lepidopteran midgut cells in culture

Protein transport across the intestinal epithelium occurs in mammals by transcytosis, a complex process in which the macromolecule is internalised at one pole of the cell plasma membrane and transported by vesicles within the cytoplasm to the plasma membrane at the opposite side. Transcytosis is an active transport mechanism, mediated by a number of endocellular steps and modulated by the cytoskeleton. In insects, the transepithelial transport of macromolecules has not yet been investigated in depth, even if it has been unequivocally demonstrated in recent years that orally-delivered proteins are able to cross the intestinal barrier in vivo, reaching the haemocel undegraded. We have recently shown that Bombyx mori isolated midgut can transport proteins across the epithelium by transcytosis. A powerful approach to explore the intracellular pathway followed by proteins during transcytosis is represented by primary cultures of insect midgut cells. We investigated the mechanism involved in the internalization of the model protein FITC-albumin and the role played by the cytoskeleton in the process in B. mori midgut columnar cells. Albumin uptake by these cells was time and temperature dependent, was reduced by metabolic inhibitors and inhibited in a concentration-dependent manner by the presence of unlabelled albumin in the external medium. These data indicate that albumin uptake in midgut cells occurs by receptor-mediated endocytosis. Immunolocalization experiments and western blot analysis showed that clathrin, but not caveolin, was present in these midgut cells. Chlorpromazine and phenylarsine oxide, two inhibitors of clathrin-mediated endocytosis, inhibited albumin internalisation, providing further evidence that this mechanism can be responsible for albumin uptake. The cytoskeleton played a critical role in this process, since its disorganisation strongly affected albumin endocytosis.