C Mirre’s research while affiliated with Holiste Laboratoire et Développement and other places

To understand the molecular mechanisms underlying sorting of apical and basolateral membrane components in human intestinal epithelial cells, we have cloned the human homolog of rat syntaxin 3 and looked for its subcellular localization. Endogenous human syntaxin 3 was found to be localized at the apical membrane of colon epithelial and Caco-2 cells. This apical localization was confirmed by confocal microscopy after transfection of the cDNA coding for either full length or N-terminally truncated human syntaxin 3 in Caco-2 cells. Furthermore the signal(s) and machinery targeting human syntaxin 3 to the apical membrane of epithelial cells are conserved between species since human syntaxin 3 was also localized at the apical membrane of canine MDCK cells and of epithelial cells in transgenic Drosophila melanogaster.

In this study we analyzed the relationship between detergent-resistant microdomains and caveolae in Caco-2 cells. Caveolin was not detected on Western blots or Northern blots or by immunoprecipitation in these cells, in contrast to A 431 cells. Triton X-100-resistant membranes from Caco-2 and A 431 cells showed the same morphological aspect by electron microscopy and peaked at the same isopycnic density on sucrose gradients. Detergent-resistant microdomains from Caco-2 cells were enriched in glycosyl phosphatidylinositol (GPI)-anchored proteins, in sucrase-isomaltase, an apical marker, and in most of the proteins found in caveolin-rich membranes such as src-like proteins, fimbrin, ezrin, and Gi alpha. Caveolae-like structures were present in A 431 but absent from Caco-2 cells at the electron microscopic level. Detergent-resistant microdomains from Caco-2 cells resemble caveolin-rich microdomains in their molecular composition but do not seem to derive from morphologically identified caveolae. Our results also indicate that caveolin is not necessary for sorting of GPI-linked proteins to the apical membrane of Caco-2 cells.

In this study, we have investigated the possibility that glycosyl-phosphatidylinositol (GPI)-anchored proteins form insoluble membrane complexes in Caco-2 cells and that transmembrane proteins are associated with these complexes. GPI-anchored proteins were mainly resistant to Triton X-100 (TX-100) extraction at 4 degrees C but fully soluble in n-octyl-glucoside. Resistance to Triton X-100 extraction was not observed in the endoplasmic reticulum but appeared during transport through the Golgi complex. It was not dependent upon N-glycosylation processing, or pH variation from 6.5 to 8.5, and was not affected by sterol-binding agents. Other apical or basolateral transmembrane proteins were well solubilized in TX-100, with the exception of sucrase-isomaltase, which was partly insoluble. We isolated a membrane fraction from Caco-2 cells that contained GPI-anchored proteins and sucrase-isomaltase but no antigen 525, a basolateral marker, or dipeptidylpeptidase IV, an apical one. These data suggest that GPI-anchored proteins cluster to form membrane microdomains together with an apical transmembrane protein, providing a possible apical sorting mechanism for intestinal cells in vitro that might be related to apical sorting in MDCK cells, and that other mechanisms might exist to sort proteins to the apical membrane.

By means of immunocytochemistry with a battery of specific antibodies, we describe the expression of the collagen IV chain produced by the gene DCg1 during the two phases of Drosophila neurogenesis. DgC1 was not expressed in neuronal tissues as shown by in situ hybridization, but the onset of its expression in cells of mesodermal origin was concomitant with the appearance of collagen IV on early axon pathways and peripheral nerve roots in the embryonic developing CNS. A similar situation was found during imaginal CNS development at metamorphosis, when collagen IV immunoreactivity was detected on centrifugal and centripetal nerve pathways, and specially on retinula axons that develop from the eye imaginal disc towards the lamina anlage in the brain optic lobe. Our results strongly suggest that collagen IV could be involved, together with other informative molecules of basement membranes, in a dynamic process of cell-matrix interactions during the establishment of initial axon pathways and neurite outgrowth in vivo.

We have developed and characterized a battery of specific polyclonal antibodies directed against specific portions of the alpha-chain of collagen type IV synthesized in Drosophila by the gene DCg1. Here, we describe the use of these antibodies together with in situ hybridization experiments in an attempt to study the expression and localization of collagen type IV during Drosophila oogenesis and early embryogenesis. The results clearly demonstrate that DCg1 is maternally expressed by follicle cells and that the collagen type IV chain produced is stockpiled in the growing oocyte. During the gastrulation stages, this component of Drosophila basement membranes concentrated on cells involved in the gradual invaginations leading to morphogenetic furrows. The presence of collagen type IV, which is an RGD-bearing molecule, during early stages of Drosophila development is discussed in comparison with the crucial, active role its vertebrate counterpart is supposed to play in morphogenetic processes.

We report the purification and characterization of three sequence-specific polyclonal antibodies raised against specific portions of the Drosophila αIV collagen chain produced from the gene DCg1. These antibodies were used for immunolocalization experiments on tissue sections from embryonic organogenesis stages (13–17) and first larval stages. This analysis was paralleled by in situ hybridization experiments with a labeled fragment of the gene DCg1. We demonstrated that, by late embryogenesis, the DCg1 αIV chain was synthesized by individual mesoblasts and deposited in basement membranes of skeletal and visceral muscles. These sites of αIV collagen deposition were the same, by first and second instars, but the protein was then synthesized by fat body cells. Our results were reminiscent of those obtained for vertebrate in vitro myogenesis, they suggested, moreover, a tissue-specific composition of basement membranes in Drosophila melanogaster.

We have examined directly the expression of one collagen gene (DCg1) during Drosophila melanogaster embryogenesis by means of in situ hybridization. Transcripts of this gene, which were demonstrated to encode a basement membrane type IV collagen chain, began to accumulate specifically in mesodermal derivatives at stages 12-13 of embryogenesis, and not before. Cells expressing this gene overlap, or are closely intermingled with, somatic and visceral mesoderm in stages 12-14. In stages 15-17, in addition to the strongly positive fat bodies, highly labelled cell spots are found scattered around all the parts of the gut and symmetrically on each side of the ventral nerve cord. They correspond to circulating mesodermal cells which we consider to be haemocytes or mesoblasts.

Type IV collagen, a major structural component of basement membrane, has been characterized only in vertebrates. It is unique among the collagenous proteins in that it forms specific lattice networks by end-to-end interactions. In particular, in mammals the C-terminal noncollagenous domain (NCl) of collagen IV was shown to be one of the major cross-linking sites in the network assembly. Here, we give the first direct evidence of type-IV-related collagen in invertebrates by sequence analysis of cDNA and genomic DNA clones for the 3'-end of a previously characterized Drosophila collagen gene. The data describe the C-terminal 190 amino acid residues of the triple helix and the entire noncollagenous domain (231 amino acids) of the chain encoded for by this gene. Comparison with data reported for human and mouse alpha 1(IV) chains reveals that triple-helix regions are quite different, while NC1 structures are very similar. This suggests different constraints on triple-helix and NC1 domains during evolution. Present data support the assumption that the NC1 structure originated from duplication of an ancestral sequence; the extent of both interspecies and intramolecular homologies suggests the maintenance in vertebrates and invertebrates of an ancestral specific function.

We report a direct examination of the expression of one collagen gene (DCg1) during Drosophila melanogaster metamorphosis, based on data from in situ hybridization. The transcripts of this gene, thought to encode a basement membrane type IV collagen, are mainly accumulated during ecdysis in wandering haemocytes. Our results demonstrate that haemocytes contribute to extracellular matrix deposition and seem to perform a fibroblastic function during Drosophila development.

Based on data from developmental RNA profiles and in situ hybridization, we report a direct examination of the expression of one collagen gene (Dcg1) during drosophila melanogaster life cycle. These studies show, for the first time, that the expression of a collagen gene is both differential and tissue-specific during the course of development. Moreover, they demonstrate that the connective tissues in Drosophila do contain a collagen type synthesized by mesodermal tissues. Indeed the accumulation of Dcg1 transcripts was located mainly within the second instar fat bodies, the third instar lymph glands, and over adepithelial cells associated with third instar imaginal discs. In addition, these results seem to confirm the interpretation that wandering hemocytes released by the lymph glands could contribute in extracellular matrix composition in some tissues in the larva.