The terminal step of cytokinesis in animal cells is the abscission of the midbody, a cytoplasmic bridge that connects the two prospective daughter cells. Here we show that two members of the SNARE membrane fusion machinery, syntaxin 2 and endobrevin/VAMP-8, specifically localize to the midbody during cytokinesis in mammalian cells. Inhibition of their function by overexpression of nonmembrane-anchored mutants causes failure of cytokinesis leading to the formation of binucleated cells. Time-lapse microscopy shows that only midbody abscission but not further upstream events, such as furrowing, are affected. These results indicate that successful completion of cytokinesis requires a SNARE-mediated membrane fusion event and that this requirement is distinct from exocytic events that may be involved in prior ingression of the plasma membrane.

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The apical- and basolateral-specific distribution of target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (t-SNAREs) of the syntaxin family appear to be critical for polarity in epithelial cells. To test whether differential SNARE expression and/or subcellular localization may contribute to the known diversity of trafficking phenotypes of epithelial cell types in vivo, we have investigated the distribution of syntaxins 2, 3, and 4 in epithelial cells along the renal tubule. Syntaxins 3 and 4 are restricted to the apical and basolateral domains, respectively, in all cell types, indicating that their mutually exclusive localizations are important for cell polarity. The expression level of syntaxin 3 is highly variable, depending on the cell type, suggesting that it is regulated in concert with the cellular requirement for apical exocytic pathways. While syntaxin 4 localizes all along the basal and lateral plasma membrane domains in vivo, it is restricted to the lateral membrane in Madin-Darby canine kidney (MDCK) cells in two-dimensional monolayer culture. When cultured as cysts in collagen, however, MDCK cells target syntaxin 4 correctly to the basal and lateral membranes. Unexpectedly, the polarity of syntaxin 2 is inverted between different tubule cell types, suggesting a role in establishing plasticity of targeting. The vesicle-associated (v)-SNARE endobrevin is highly expressed in intercalated cells and colocalizes with the H+-ATPase in α- but not β-intercalated cells, suggesting its involvement in H+-ATPase trafficking in the former cell type. These results suggest that epithelial membrane trafficking phenotypes in vivo are highly variable and that different cell types express or localize SNARE proteins differentially as a mechanism to achieve this variability.

In polarized Madin-Darby canine kidney epithelial cells, components of the plasma membrane fusion machinery, the t-SNAREs syntaxin 2, 3, and 4 and SNAP-23, are differentially localized at the apical and/or basolateral plasma membrane domains. Here we identify syntaxin 11 as a novel apical and basolateral plasma membrane t-SNARE. Surprisingly, all of these t-SNAREs redistribute to intracellular locations when Madin-Darby canine kidney cells lose their cellular polarity. Apical SNAREs relocalize to the previously characterized vacuolar apical compartment, whereas basolateral SNAREs redistribute to a novel organelle that appears to be the basolateral equivalent of the vacuolar apical compartment. Both intracellular plasma membrane compartments have an associated prominent actin cytoskeleton and receive membrane traffic from cognate apical or basolateral pathways, respectively. These findings demonstrate a fundamental shift in plasma membrane traffic toward intracellular compartments while protein sorting is preserved when epithelial cells lose their cell polarity. INTRODUCTIONTraffic between membranous compartments is mediated by the soluble N-ethylmaleimide-sensitive factor attachment protein (SNARE) machinery in virtually all membrane traffic pathways investigated so far (Rothman and Warren, 1994;Hanson et al., 1997;Hay and Scheller, 1997;Nichols and Pelham, 1998). During vesicle docking, membrane proteins on the vesicle membrane (v-SNAREs) and the target membrane (t-SNAREs) bind to each other to form a complex that ultimately leads to fusion of the lipid bilayers. One aspect of the SNARE hypothesis is that successful membrane fusion requires the binding of matching combinations of v-and t-SNAREs, thereby ensuring the necessary specificity of vesicle fusion. Accordingly, each membrane organelle and each class of transport vesicles should be defined by a certain set of t-and v-SNARE isoforms. Many SNAREs have been identified to date, and protein sequence analysis has shown that v-and t-SNAREs of the currently known SNARE subfamilies are evolutionarily related to each other and belong to a common superfamily (Weimbs et al., 1997b(Weimbs et al., , 1998. It is conceivable that the specificity of vesicle fusion is not directly determined by t-SNARE/v-SNARE interactions per se but rather by interactions involving larger complexes, including SNAREs and their regulatory proteins, such as those of the rab and sec1 protein families (Christoforidis et al., 1999;Gonzalez and Scheller, 1999;Pfeffer, 1999).Epithelial cells display an additional layer of complexity in that they are typically polarized and possess two distinct plasma membrane domains (Louvard et al., 1992;Simons et al., 1992;Le Gall et al., 1995;Drubin and Nelson, 1996;Yeaman et al., 1999). The apical and basolateral plasma membrane domains have different protein and lipid compositions that reflect the different functions of these domains. This plasma membrane polarity is established and maintained by protein sorting and specific vesicle trafficking routes in th...

Summary The objective of this study was to determine the effects of the level of inhaled oxygen during resuscitation on the levels of free radicals and anti-oxidative capacity in the heparinized venous blood of preterm infants. Forty four preterm infants <35 weeks of gestation with mild to moderate neonatal asphyxia were randomized into two groups. The first group of infants were resuscitated with 100% oxygen (100% O2 group), while in the other group (reduced O2 group), the oxygen concentration was titrated according to pulse oximeter readings. We measured total hydroperoxide (TH) and redox potential (RP) in the plasma within 60 min of birth. The integrated excessive oxygen (∑(FiO2-0.21) × Time(min)) was higher in the 100% O2 group than in the reduced O2 group (p<0.0001). TH was higher in the 100% O2 group than in the reduced O2 group (p<0.0001). RP was not different between the 100% O2 and reduced O2 groups (p = 0.399). RP/TH ratio was lower in the 100% O2 group than in the reduced O2 group (p<0.01). We conclude that in the resuscitation of preterm infants with mild to moderate asphyxia, oxidative stress can be reduced by lowering the inspired oxygen concentration using a pulse oximeter.