Abstract. Members of the cytokine receptor family are composed of several noncovalently linked chains with sequence and structure homologies in their extracellular domain. Receptor subfamily members share at least one component: thus the receptors for interleukin (IL) 2 and ILl5 have common ~ and 3" chains, while those for IL2, 4, 7, and 9 have a common 3' chain. The intracellular pathway followed by IL2 receptors after ligand binding and endocytosis was analyzed by immunofluorescence and confocal microscopy in a human T lymphocytic cell line. Surprisingly, the a, ~, and 3' chains had different intracellular localizations after being endocytosed together. The ct chain was always in transferl-in-positive compartments (early/recycling endosomes), both at early and late internalization times, but was never detected in rab7-positive compartments (late endosomes). On the other hand, at late internalization times, the/3 and 3, chains were excluded from transferrin-positive organelles and did not colocalize with ~. Furthermore, /3 could be found in rab7-positive vesicles. These differences suggest that the ot chain recycles to the plasma membrane, while the ~ and 3' chains are sorted towards the degradation pathway. The half-lives of these three chains on the cell surface also reflect their different intracellular fates after endocytosis. The and 3' chains are very short-lived polypeptides since their half-life on the surface is only =1 h, whereas c~ is a much more stable surface protein. This shows for the first time that components of a multimeric receptor can be sorted separately along the endocytic pathway.

The commonly accepted view that enteroinvasive bacteria enter cells of the intestinal epithelial lining through the apical surface can be challenged in the case of shigellosis. This study is based on in vitro experiments that showed that the invasion of human colonic Caco-2 cells by Shigella flexneri occurred through the basolateral pole of these cells. In these experiments, the few bacteria that interacted with the apical surface either bound to microvilli of the cell dome without causing detectable alteration or bound at the level of intercellular junctions at which they demonstrated a limited capacity for paracellular invasion, which permitted subsequent entry through the lateral domain of the cells. Treatment of Caco-2 cell monolayers with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), which disrupts intercellular junctions, greatly enhanced the rate of cell infection. These observations suggest a physiopathological paradox that may have important consequences for the understanding of the process of colonic invasion in vivo during shigellosis.

Abstract. Small rab/Yptl/Sec4 GTPase family have been involved in the regulation of membrane traffic along the biosynthetic and endocytic pathways in eucaryotic cells. Polarized epithelial cells have morphologically and functionally distinct apical and basolateral surfaces separated by tight junctions. The establishment and maintenance of these structures require delivery of membrane proteins and lipids to these domains. In this work, we have isolated a cDNA clone from a human intestinal cDNA library encoding a small GTPase, rabl3, closely related to the yeast Sec4 protein. Confocal microscopy analysis on polarized Caco-2 cells shows that rabl3 protein colocalized with the tight junction marker ZO-1. Cryostat sections of tissues confirm that rabl3 localized to the junctional complex region of a variety of epithelia, including intestine, kidney, liver, and of endothelial cells. This localization requires assembly and integrity of the tight junctions. Disruption of tight junctions by incubation in low Ca 2+ media induces the redistribution of rabl3. In cells devoid of tight junctions, rabl3 was found associated with vesicles dispersed throughout the cytoplasm. Cell-cell contacts initiated by E-cadherin in transfected L cells do not recruit rabl3 to the resulting adherens-like junction complexes. The participation of rabl3 in polarized transport, in the assembly and/or the activity of tight junctions is discussed.UCARYOTIC cells contain a variety of small 21-27 kd guanine nucleotide-binding proteins, in addition to H-, K-, and N-p21ras. Although these proteins are structurally distinct from each other and from p21ras, they share significant homologies with p21ras, particularly in the domains involved in GTP/GDP binding and in GTP hydrolysis. Numerous members of the ras-related GTPase superfamily have been identified and classified according to their sequence similarities (for review see Valencia et al., 1991). Among them, the proteins of the rab/Ypt/Sec4 family (>30 members) have been proposed as key regulators in vesicular traffic (Touchot et al