Recent studies have shown that Bombyx mori larval midgut can transport proteins unaltered following the transcellular pathway by transcytosis. In insects, the steps involved in this complex process are still unknown. Recently, this topic attracts increasing research efforts because of the demand of new and efficient strategies for the oral delivery of bioinsecticides targeting haemocoelic receptors. A promising tool to investigate these aspects is represented by midgut cells in culture. We developed a culture of B. mori midgut cells following the procedure of Sadrud-Din et al. (1994; 1996). We analysed the characteristics of the culture, showing that up to 60% of the stem cells isolated from the midgut, differentiated after three weeks in culture into columnar and goblet cells, the two predominant cell types in the epithelium. These cells presented in vitro the same shape, morphology and polarity recorded in vivo, even if their dimensions were slightly reduced. Moreover, cultured cell homogenates displayed aminopeptidase N and alkaline phosphatase activity, proving that these two enzymes, involved in vivo in the intermediate and final digestion of nutrients, are expressed also in vitro. We used this cell culture to assess the specific mechanism involved in the endocytosis and the sequence of intracellular events implicated in the movement of endocytic vesicles, of fluorescein isothiocyanate (FITC)-albumin, a protein absorbed by transcytosis in the midgut of B. mori larvae. We demonstrated that FITC-albumin uptake increased over time and was energy dependent, since it was strongly reduced by both low temperature and metabolic inhibitors. Labelled albumin uptake as a function of increasing protein concentration showed a saturation kinetics and was inhibited by extracellular unlabelled albumin in a concentration dependent manner. These data are compatible with the occurrence of a receptor-mediated endocytosis. FITC-albumin internalization 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. 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 internalisation. Albumin uptake was also inhibited by gentamycin, insulin and transferrin, proteins known to be megalin ligands. We demonstrated also that the integrity of actin and microtubule organisation was essential for the correct functioning of the endocytic machinery. Once internalised, albumin colocalized with early endosomes and lysosomes, suggesting that only a part of the protein is transcytosed, since a conspicuous amount is directed to the degradative pathway. Searching for enhancers of protein transport, we evaluated the ability of Cell-Penetrating Peptides (CPPs) to cross the intestinal cell membrane delivering a fused model protein. One of the best known and more often used CPPs is Tat, a peptide formed by 11 amino acid residues, that derives from the human immunodeficiency virus type 1 (HIV-1) transactivator of transcription (Tat) peptide, Tat-(47-57). Tat-EGFP (Enhanced Green Fluorescent Protein) fusion protein, produced in the laboratory of Prof. Rosa Rao (University of Naples Federico II), was used in the experiments. Tat-EGFP was internalised by columnar cells in culture more efficiently than EGFP alone and its uptake increased over time. The internalisation process appeared to be energy-independent, since it was not reduced by either low temperature or metabolic inhibitors. The ability of Tat to translocate EGFP entering the cell across the brush border membrane was observed from experiments performed in the midgut isolated in a perfusion apparatus and incubated with the fusion protein added to the mucosal side of the epithelium. Densoviruses (DNVs) are insect parvoviruses which are lethal for several insects at larval stages, including agronomical pests and insects vector-borne diseases. During my permanence at the “Laboratoire de Biologie Intégrative et Virologie” in Montpellier (France), under the supervision of Dr. Mylene Ogliastro, we studied the interaction between Junonia coenia Densovirus (JcDNV) and the permissive host Spodoptera frugiperda. Cultures of S. frugiperda midgut cells were prepared to detect the sites of penetration of JcDNV and to analyse the internalisation mechanism. The virus was unable to infect stem cells and differentiated goblet cells. In columnar cells with well developed microvilli, whether at an early phase of differentiation or fully differentiated, the virus was visible after 10 minutes on the cell surface at both the apical and the basolateral side, and after 30 minutes within the cytoplasm, but never in the nucleus. Virus particles were apparent as spots, suggesting a distribution in intracellular compartments. Virus internalisation appeared to be energy dependent, since it was strongly reduced by low temperature.
|Titolo:||Primary cultures of insect midgut cells : a system to study membrane permeability to proteins and specific tools to enhance permeation|
|Data di pubblicazione:||11-mar-2009|
|Settore Scientifico Disciplinare:||Settore BIO/09 - Fisiologia|
|Citazione:||Primary cultures of insect midgut cells : a system to study membrane permeability to proteins and specific tools to enhance permeation ; B. Giordana, M. Casartelli. - Milano : Università degli studi di Milano. DIPARTIMENTO DI BIOLOGIA, 2009 Mar 11. ((21. ciclo, Anno Accademico 2007/2008.|
|Digital Object Identifier (DOI):||10.13130/cermenati-gaia_phd2009-03-11|
|Appare nelle tipologie:||13 - Tesi di dottorato discussa entro ottobre 2010|