Submitted on January 18, 2008
Accepted on July 11, 2008
A megalin-like receptor is involved in protein endocytosis in the midgut of an insect (Bombyx mori, Lepidoptera)
Morena Casartelli1*, Gaia Cermenati1, Simona Rodighiero2, Francesco Pennacchio3, and Barbara Giordana1
1 Biology, University of Milan, Milan, Italy
2 CIMaINa, University of Milan, Milan, Italy
3 Entomologia e Zoologia Agraria, University of Naples Federico II, Naples, Italy
* To whom correspondence should be addressed. E-mail: morena.casartelli{at}unimi.it.
The mechanism responsible for FITC-albumin (fluorescein isothiocyanate-albumin) internalisation by columnar cells in culture obtained from the midgut of Bombyx mori larvae was examined by confocal laser scanning microscopy. Protein uptake changed over time and it appeared to be energy-dependent, as it was strongly reduced by both low temperatures and metabolic inhibitors. Labelled albumin uptake as a function of increasing protein concentration showed a saturation kinetics with a Km value of 2.0 ± 0.6 µM. These data are compatible with the occurrence of receptor-mediated endocytosis. RT-PCR analysis and colocalisation experiments with an anti-megalin primary antibody indicated that the receptor involved was a putative homologue of megalin, the multiligand endocytic receptor belonging to the low-density lipoprotein (LDL)-receptor family, responsible for the uptake of various molecules, albumin included, in many epithelial cells of mammals. This insect receptor, like the mammalian counterpart, required Ca2+ for albumin internalization and was inhibited by gentamicin. FITC-albumin internalisation was clathrin-mediated, since two inhibitors of this process caused a significant reduction of the uptake and clathrin and albumin colocalised in the intermicrovillar areas of the apical plasma membrane. The integrity of actin and microtubules organisation was essential for the correct functioning of the endocytic machinery.
