Detergent-resistant domains in Spodoptera frugiperda midgut microvillar membranes and their relation to microapocrine secretion.

The midgut from lepidopteran insects has a particular way to release proteins to the lumen, named microapocrine secretion that could be an adaptation to release secretory contents into the lumen at water absorbing regions. In this process small vesicles (microapocrine vesicles) bud from the midgut microvilli as double membrane vesicles, where the inner membrane comes from the secretion vesicle and the outer one from the microvillar membrane. The molecular machinery associated with this process may be recruited by specific midgut microvilli membrane domains. To address to this, Spodoptera frugiperda midgut microvillar membranes, prepared by magnesium treatment and free from cytoskeleton with the hyperosmotic Tris procedure, were submitted to detergent extraction and fractionated by density gradient ultracentrifugation. Detergent-resistant membrane domains (DRM) were recovered and their proteins identified by proteomics. Microapocrine vesicles were isolated by washing the luminal surface of the midgut epithelium, followed by freezing and thawing plus centrifugation to recover only membranes. Proteins from purified microvillar membranes and microapocrine vesicle membranes were identified by proteomics. Comparison of the two populations suggests that the budding of microapocrine vesicles surrounded by microvillar membrane is not a random process, because only around 50% of the microvillar membrane proteins are in the microapocrine vesicles. From the 16 proteins from DRM, 14 were enriched in the microapocrine membrane vesicles. These results suggest that on budding, the microapocrine vesicle membrane is enclosed by DRM and a surrounding area of the microvillar membrane. It is proposed that the DRMs somehow recruit the proteins composing the secretory machinery.

Detergent-resistant domains in Spodoptera frugiperda midgut microvillar membranes and their relation to microapocrine secretion

The midgut from lepidopteran insects has a particular way to release proteins to the lumen, named microapocrine secretion that could be an adaptation to release secretory contents into the lumen at water absorbing regions. In this process small vesicles (microapocrine vesicles) bud from the midgut microvilli as double membrane vesicles, where the inner membrane comes from the secretion vesicle and the outer one from the microvillar membrane. The molecular machinery associated with this process may be recruited by specific midgut microvilli membrane domains. To address to this, Spodoptera frugiperda midgut microvillar membranes, prepared by magnesium treatment and free from cytoskeleton with the hyperosmotic Tris procedure, were submitted to detergent extraction and fractionated by density gradient ultracentrifugation. Detergent-resistant membrane domains (DRM) were recovered and their proteins identified by proteomics. Microapocrine vesicles were isolated by washing the luminal surface of the midgut epithelium, followed by freezing and thawing plus centrifugation to recover only membranes. Proteins from purified microvillar membranes and microapocrine vesicle membranes were identified by proteomics. Comparison of the two populations suggests that the budding of microapocrine vesicles surrounded by microvillar membrane is not a random process, because only around 50% of the microvillar membrane proteins are in the microapocrine vesicles. From the 16 proteins from DRM, 14 were enriched in the microapocrine membrane vesicles. These results suggest that on budding, the microapocrine vesicle membrane is enclosed by DRM and a surrounding area of the microvillar membrane. It is proposed that the DRMs somehow recruit the proteins composing the secretory machinery.

Gelsolin role in microapocrine secretion.

A role of gelsolin in opening the way along the microvilli for secretory vesicles during microapocrine secretion is proposed here. Data obtained with different techniques showed that many digestive enzymes are released by microapocrine secretion in insects. Proteins that might be involved in the machinery of microapocrine secretion were selected from our transcriptomes and literature searches. The proteins were annexin, Complex actin-related proteins 2 and 3 (ARP 2/3) cofilin, fimbrin, gelsolin 1, gelsolin 2, moesin, myosin 1, myosin 6, protein disulphide isomerase 1 (PDI 1), PDI 2 and profilin. The cDNAs coding for annexin, fimbrin, gelsolin 1, myosin 1, PDI 1 and PDI 2 were cloned and their sequences deposited in GenBank. Only gelsolin 1 and myosin 1 are expressed exclusively in the midgut (semiquantitative reverse transcriptase PCR). As myosin 1 may have a structural role in microvilli, gelsolin 1 is the best guess to be involved in the secretory machinery. A truncated recombinant gelsolin 1 was used to generate antibodies with which it was shown labelling inside and around midgut cell microvilli shown in an electron microscope, reinforcing a microvillar role for gelsolin 1. Suppression of gelsolin 1 synthesis by RNA interference prevents secretory vesicles from advancing inside the microvilli, in agreement with its putative role in severing the actin filaments to free the way for the vesicles.