Multiple interactions between insect lipoproteins and fat body cells: extracellular trapping and endocytic trafficking.

The binding and internalization of a circulating insect lipoprotein, high density lipophorin (HDLp), by insect fat body cells was studied at the electron-microscopic level using ultrasmall gold-labeled HDLp and DiI-labeled HDLp, which were visualized by silver enhancement and diaminobenzidine photoconversion, respectively. Internalization of HDLp seems to conflict with the selective process by which the lipids are transported between HDLp and fat body cells. The pathway followed by the internalized lipoproteins was investigated. In addition, the localizations of HDLp in fat body cells of young and older adult locusts were compared because of the previously reported age-related differences in distribution of cell-associated and internalized HDLp. In the present study, internalized labeled HDLp was observed in early endosomes, late endosomes, and putative lysosomes. In older adults, these labeled structures were much less abundant than in young adults. Moreover, in these animals, the labeled endosomal/lysosomal vesicles were located close to the plasma membranes. A more intense labeling was observed in the extracellular matrix in older adults compared to young adults. In both developmental stages, an apparent accumulation of labeled HDLp was found in extracellular spaces. We propose that this entrapment of HDLp may be essential for selective lipid transport between HDLp and fat body cells.

Electron microscopic visualization of receptor-mediated endocytosis of Dil-labeled lipoproteins by diaminobenzidine photoconversion.

We present a modified diaminobenzidine (DAB) photoconversion method that enables staining of internalized Dil-labeled lipoproteins without the apparent punctate background staining that was observed with the original DAB photoconversion method. This is illustrated by the localization of Dil-labeled insect lipoproteins in natural recipient cells that internalize these lipoproteins by receptor-mediated endocytosis. Exposure to Dil-excitation light of cells that had been incubated with Dil-labeled lipoproteins yielded a light- and electron-dense DAB reaction product. In addition to the expected staining, an apparent punctate background staining of vesicular structures hindered proper identification of Dil-containing vesicles because these background-stained vesicles were indistinguishable from putative late endosomal and lysosomal structures at the electron microscopic level. This background staining was completely abrogated by inhibition of peroxisomal catalase with aminotriazole. The conversion of DAB by the emitted light of Dil was not affected by aminotriazole. We conclude that specific staining of Dil-labeled intracellular structures can be achieved with the modified DAB photoconversion method reported here.

Developmental down-regulation of receptor-mediated endocytosis of an insect lipoprotein.

Fat body cells of insects exhibit a high-affinity lipoprotein binding site at their cell surfaces. In the present study, the lipoprotein binding site was identified as an endocytotic receptor involved in receptor-mediated uptake of its lipoprotein ligand, high density lipophorin. After an initial period of high endocytotic uptake of high density lipophorin in the adult stage, this process strongly diminished. In the same period, a dramatic increase in cell surface-associated lipoproteins was observed. When animals were starved, however, internalization of lipoproteins was maintained. The pathway followed by the internalized lipoproteins appears to be different from the endosomal/lysosomal pathway, as the vast majority of apolipoproteins seemed to escape from lysosomal hydrolysis. In addition, no substantial intracellular accumulation of apolipoproteins was observed, suggesting that internalized lipoproteins were resecreted. It is unlikely that internalization is required for transport of the two major lipid components of insect lipoproteins, diacylglycerol and cholesterol, as inhibition of endocytosis neither affected the exchange of these lipids between lipoproteins and fat body cells nor influenced the loading of diacylglycerol onto lipoproteins in response to adipokinetic hormone. We postulate that the endosomal environment may facilitate transport of components which, unlike diacylglycerol and cholesterol, cannot be transported by simple aqueous diffusion.

Molecular cloning of three distinct cDNAs, each encoding a different adipokinetic hormone precursor, of the migratory locust, Locusta migratoria. Differential expression of the distinct adipokinetic hormone precursor genes during flight activity.

Three distinct cDNAs encoding the preproadipokinetic hormones I, II, and III (prepro-AKH I, II, and III), respectively, of Locusta migratoria have been isolated and sequenced. The three L. migratoria AKH precursors have an overall architecture similar to that of other precursors of the AKH/red pigment-concentrating hormone (RPCH) family identified so far. The AKH I and II precursors of L. migratoria are highly homologous to the Schistocerca gregaria and Schistocerca nitans AKH precursors. Although the L. migratoria AKH III precursor appears to be the least homologous to the Manduca sexta, Drosophila melanogaster, and Carcinus maenas AKH/RPCH precursors, we favor the opinion that the L. migratoria AKH III precursor is evolutionary more related to the M. sexta, D. melanogaster, and C. maenas AKH/RPCH precursors than to the AKH I and II precursors of S. gregaria, S. nitans, or L. migratoria. In situ hybridization showed signals for the different AKH mRNAs to be co-localized in cell bodies of the glandular lobes of the corpora cardiaca. Northern blot analysis revealed the presence of single mRNA species encoding the AKH I precursor (approximately 570 bases), AKH II precursor (approximately 600 bases), and AKH III precursor (approximately 670 bases), respectively. Interestingly, flight activity increased steady-state levels of the AKH I and II mRNAs (approximately 2.0 times each) and the AKH III mRNA (approximately 4.2 times) in the corpora cardiaca.

Organization of talin and vinculin in adhesion plaques of wet-cleaved chicken embryo fibroblasts.

We have studied the fine structure of adhesion plaques in chicken embryo fibroblasts (CEF) and visualized the localization of vinculin and talin using immunoelectron microscopy on CEF opened by 'wet-cleaving'. This procedure, performed with nitrocellulose on cells grown on electron microscope grids, cleaved the CEF close to the inner face of the ventral membrane or at a slightly higher level through the cytoplasm. In the resulting preparations, adhesion plaques were identified by their localization at the end of microfilament bundles and by their density of vinculin and talin. The plaques showed a substructure of moderately electron-dense parallel bands that were interconnected. Both the parallel bands as well as the interconnecting threads showed a high density of vinculin and talin labels, whereas neither the surrounding membrane cytoskeleton nor the overlaying bundled microfilaments were labeled. In stereomicrographs, we observed no difference between the distances from vinculin or talin label, respectively, to the plasma membrane. In early spreading cells, vinculin and talin were found to be deposited simultaneously in fine radiating streaks that covered rather large parts of the ventral membrane at areas of close contact with the substratum. These streaks, which were initially overlayed by an isotropic cytoskeletal network without filament bundles, were the apparent precursors of later formed adhesion plaques. These observations suggest that there are no separate layers of talin and vinculin, but rather that adhesion plaques consist of a dense network of talin and vinculin. The observations strongly support the model proposed by Bendori et al. (1989), J. Cell Biol. 108, 2383-2393, that was based on the location of vinculin- and talin-binding sites in the vinculin molecule.