The Drosophila (fruit fly) model system has been instrumental in our current understanding of human biology, development, and diseases. Here, we used a high-throughput yeast two-hybrid (Y2H)-based technology to screen 102 bait proteins from Drosophila melanogaster, most of them orthologous to human cancer-related and/or signaling proteins, against high-complexity fly cDNA libraries. More than 2300 protein-protein interactions (PPI) were identified, of which 710 are of high confidence. The computation of a reliability score for each protein-protein interaction and the systematic identification of the interacting domain combined with a prediction of structural/functional motifs allow the elaboration of known complexes and the identification of new ones.

Abstract. Plasmalemmal caveolae are a membrane specialization that mediates transcytosis across endothelial cells and the uptake of small molecules and ions by both epithelial and connective tissue cells. Recent findings suggest that caveolae may, in addition, be involved in signal transduction. To better understand the molecular composition of this membrane specialization, we have developed a biochemical method for purifying caveolae from chicken smooth muscle cells. Biochemical and morphological markers indicate that we can obtain ~1.5 mg of protein in the caveolae fraction from •100 g of chicken gizzard. Gel electrophoresis shows that there are more than 30 proteins enriched in caveolae relative to the plasma membrane. Among these proteins are: caveolin, a structural molecule of the caveolae coat; multiple, glycosylphosphatidylinositol-anchored membrane proteins; both G~ and Ga subunits of heterotrimeric GTP-binding protein; and the Ras-related GTP-binding protein, RaplA/B. The method we have developed will facilitate future studies on the structure and function of caveolae.T HF.RE is increasing evidence that plasmalemmal caveolae are a membrane specialization capable of sealing off from the extracellular environment to create a unique, membrane bound compartment at the cell surface. The dynamics of caveolae opening and closing is best observed in endothelial cells (46,47), where they appear to form plasmalemmal vesicles that move across the cell and fuse with the abluminal membrane. Each round of caveolaemediated transcytosis transports a portion of molecules from the blood to the tissue space without merging with other endocytic pathways. Although in other cell types the budding event has not been seen with the electron microscope, biochemical studies have shown (1%19) that caveolae can sequester membrane bound ligands away from the extracellular space and facilitate their delivery to the cytoplasm of the cell. This process is called potocytosis (3).What distinguishes potocytosis from other endocytic pathways is the use of glycosylphosphatidylinositol (GPI) 1-anchored membrane proteins to concentrate low molecular weight molecules and ions in closed caveolae (22,41). Morphological (54) and biochemical (5, 7) methods have

Lethal toxin (LT) fromClostridium sordellii is one of the high molecular mass clostridial cytotoxins. On cultured cells, it causes a rounding of cell bodies and a disruption of actin stress fibers. We demonstrate that LT is a glucosyltransferase that uses UDP-Glc as a cofactor to covalently modify 21-kDa proteins both in vitro and in vivo. LT glucosylates Ras, Rap, and Rac. In Ras, threonine at position 35 was identified as the target amino acid glucosylated by LT. Other related members of the Ras GTPase superfamily, including RhoA, Cdc42, and Rab6, were not modified by LT. Incubation of serumstarved Swiss 3T3 cells with LT prevents the epidermal growth factor-induced phosphorylation of mitogen-activated protein kinases ERK1 and ERK2, indicating that the toxin blocks Ras function in vivo. We also demonstrate that LT acts inside the cell and that the glucosylation reaction is required to observe its dramatic effect on cell morphology. LT is thus a powerful tool to inhibit Ras function in vivo.Several different species of the genus Clostridium produce large molecular mass (ϳ250 -300 kDa) cytotoxins that cause effects on the actin cytoskeleton, including disruption of actin stress fibers and rounding of cell bodies. This subgroup of clostridial cytotoxins includes toxins A and B from Clostridium difficile, lethal toxin (LT) 1 and hemorrhagic toxin from Clostridium sordellii, and Clostridium novyi ␣-toxin (1). Recently, toxins A and B from C. difficile, the causative agent of antibiotic-associated diarrhea (2), were shown to covalently modify the mammalian protein Rho by UDP-Glc-dependent glucosylation of threonine 37 (3, 4). Rho is a small Ras-related GTPbinding protein involved in the control of actin polymerization (5). Glucosylation of threonine 37 of Rho by C. difficile toxin A or B apparently inactivates this protein and results in a loss of actin stress fiber assembly.C. sordellii produces two toxins, LT and hemorrhagic toxin, two major virulence factors inducing gas gangrene and hemorrhagic diarrhea in humans and animals (6). These C. sordellii toxins have some similarities to toxins A and B from C. difficile in terms of amino acid sequences and immunological epitopes (7). Despite these similarities, it seems that LT and toxins A and B affect different intracellular target proteins. LT causes morphological and cytoskeletal effects different from those elicited by the C. difficile toxins. The effects consist of the rounding of cell bodies with the reorganization of F-actin structures into numerous cell-surface filopodia and a loss of actin stress fibers (8, 9). In addition, we have recently shown that overexpression of RhoA, RhoB, or RhoC cDNA in HeLa cells protects these cells from the effects of toxins A and B, but not from those of LT (9). These observations clearly pointed out that Rho small GTPbinding proteins were the main substrate for the C. difficile toxins and that the targets of LT were distinct.A mutant hamster fibroblast cell line has been described that is resistant to toxins A and B from C. dif...

At the onset of mitosis, most adherent cells undergo cell retraction characterised by the disassembly of focal adhesions and actin stress fibres. Mitosis takes place in rounded cells, and the two daughter cells spread again after cytokinesis. Because of the well-documented ability of the small GTPase Rap1 to stimulate integrin-dependent adhesion and spreading, we assessed its role during mitosis. We show that Rap1 activity is regulated during this process. Changes in Rap1 activity play an essential role in regulating cell retraction and spreading, respectively, before and after mitosis of HeLa cells. Indeed, endogenous Rap1 is inhibited at the onset of mitosis; conversely, constitutive activation of Rap1 inhibits the disassembly of premitotic focal adhesions and of the actin cytoskeleton, leading to delayed mitosis and to cytokinesis defects. Rap1 activity slowly increases after mitosis ends; inhibition of Rap1 activation by the ectopic expression of the dominant-negative Rap1[S17A] mutant prevents the rounded cells from spreading after mitosis. For the first time, we provide evidence for the direct regulation of adhesion processes during mitosis via the activity of the Rap1 GTPase.