Polarity is a central feature of eukaryotic cells and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) has a central role in the polarization of neurons and chemotaxing cells. In polarized epithelial cells, PtdIns(3,4,5)P3 is stably localized at the basolateral plasma membrane, but excluded from the apical plasma membrane, as shown by localization of GFP fused to the PtdIns(3,4,5)P3-binding pleckstrin-homology domain of Akt (GFP-PH-Akt), a fusion protein that indicates the location of PtdIns(3,4,5)P3. Here, we ectopically inserted exogenous PtdIns(3,4,5)P3 into the apical plasma membrane of polarized Madin-Darby canine kidney (MDCK) cells. Within 5 min many cells formed protrusions that extended above the apical surface. These protrusions contained basolateral plasma membrane proteins and excluded apical proteins, indicating that their plasma membrane was transformed from apical to basolateral. Addition of PtdIns(3,4,5)P3 to the basolateral surface of MDCK cells grown as cysts caused basolateral protrusions. MDCK cells grown in the presence of a phosphatidylinositol 3-kinase inhibitor had abnormally short lateral surfaces, indicating that PtdIns(3,4,5)P3 regulates the formation of the basolateral surface.

We previously found that water transport across hepatocyte plasma membranes occurs mainly via a nonchannel mediated pathway. Recently, it has been reported that mRNA for the water channel, aquaporin-8 (AQP8), is present in hepatocytes. To further explore this issue, we studied protein expression, subcellular localization, and regulation of AQP8 in rat hepatocytes. By subcellular fractionation and immunoblot analysis, we detected an N-glycosylated band of ϳ34 kDa corresponding to AQP8 in hepatocyte plasma and intracellular microsomal membranes. Confocal immunofluorescence microscopy for AQP8 in cultured hepatocytes showed a predominant intracellular vesicular localization. Dibutyryl cAMP (Bt 2 cAMP) stimulated the redistribution of AQP8 to plasma membranes. Bt 2 cAMP also significantly increased hepatocyte membrane water permeability, an effect that was prevented by the water channel blocker dimethyl sulfoxide. The microtubule blocker colchicine but not its inactive analog lumicolchicine inhibited the Bt 2 cAMP effect on both AQP8 redistribution to cell surface and hepatocyte membrane water permeability. Our data suggest that in rat hepatocytes AQP8 is localized largely in intracellular vesicles and can be redistributed to plasma membranes via a microtubule-depending, cAMP-stimulated mechanism. These studies also suggest that aquaporins contribute to water transport in cAMP-stimulated hepatocytes, a process that could be relevant to regulated hepatocyte bile secretion.Bile is formed primarily by hepatocytes and subsequently delivered to the bile ducts where it is modified by cholangiocytes (i.e. the epithelial cells that line the bile ducts). Bile secretion by hepatocytes involves the active transport of solutes followed by the passive movement of water into the bile canaliculus in response to osmotic gradients created by these solutes (1, 2). Although a substantial amount of data have been published about the molecular identification of solute transporters and the mechanisms regulating solute transport by hepatocytes (3), little attention has been focused on the mechanistic and regulatory aspects involved in hepatocyte water transport.Water can cross cellular plasma membranes through the lipid portion of the bilayer by a diffusion mechanism or through aquaporin water channels. Aquaporins, a family of recently identified integral membrane proteins, increase cell membrane water permeability facilitating rapid movement of water in response to osmotic gradients (4, 5).We previously found based on biophysical and molecular biology studies that water transport across hepatocyte plasma membranes occurs mainly via a non-channel mediated pathway (6). As this observation seems to be in contradiction with the recent identification of transcript for the water channel aquaporin-8 (AQP8) 1 in hepatocytes (7-9), we further explored this issue by studying the protein expression, subcellular localization, and possible regulation of AQP8 water channels in isolated rat hepatocytes. MATERIALS AND METHODSIsolation and Incubation of ...

Several Pseudomonas aeruginosa strains are internalized by epithelial cells in vitro and in vivo, but the host pathways usurped by the bacteria to enter nonphagocytic cells are not clearly understood. Here, we report that internalization of strain PAK into epithelial cells triggers and requires activation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B/Akt (Akt). Incubation of Madin-Darby canine kidney (MDCK) or HeLa cells with the PI3K inhibitors LY294002 (LY) or wortmannin abrogated PAK uptake. Addition of the PI3K product phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ] to polarized MDCK cells was sufficient to increase PAK internalization. PtdIns(3,4,5)P 3 accumulated at the site of bacterial binding in an LY-dependent manner. Akt phosphorylation correlated with PAK invasion. The specific Akt phosphorylation inhibitor SH-5 inhibited PAK uptake; internalization also was inhibited by small interfering RNA-mediated depletion of Akt phosphorylation. Expression of constitutively active Akt was sufficient to restore invasion when PI3K signaling was inhibited. Together, these results demonstrate that the PI3K signaling pathway is necessary and sufficient for the P. aeruginosa entry and provide the first example of a bacterium that requires Akt for uptake into epithelial cells. INTRODUCTIONPseudomonas aeruginosa is one of the leading causes of nosocomial infections in humans (reviewed in Engel, 2003). This Gram negative opportunistic pathogen causes acute infections of the respiratory and urinary tract, skin, and eye in the setting of preexisting epithelial tissue damage and/or host immunocompromise. P. aeruginosa is also a cause of chronic lung infections and ultimately death in patients with cystic fibrosis.Although usually considered an extracellular pathogen, ϳ50% of clinical, laboratory, and environmental P. aeruginosa isolates demonstrate measurable internalization in vivo as well as in vitro (Chi et al., 1991;Fleiszig et al., 1994Fleiszig et al., , 1995Fleiszig et al., , 1997bFleiszig et al., , 1998Hirakata et al., 1998;Grassmé et al., 2000). These two different phenotypes correlate with the differences in type III secreted effectors (reviewed in Engel, 2003). Both classes of strains are virulent in animal models of P. aeruginosa infection. The noninvasive, cytotoxic strains secrete ExoU (Hauser et al., 1998), a potent phospholipase (Sato et al., 2003), and ExoT, a bifunctional enzyme with N-terminal GAP activity toward Rho family GTPases (Krall et al., 2000;Kazmierczak and Engel, 2002) and C-terminal ADP ribosyltransferase (ADPRT) activity toward Crk (Sun and Barbieri, 2003). Both domains of ExoT contribute to its anti-internalization activity (Garrity-Ryan et al., 2000;Garrity-Ryan et al., 2004). The invasive strains are much less cytotoxic due to the loss of the ExoU gene (Allewelt et al., 2000). Interestingly, these strains secrete ExoT and a closely related protein ExoS that also possesses an N-terminal GAP domain whose substrates include Rho family GTPases (Goehring et al., 1999...

Pseudomonas aeruginosa, an important human pathogen, preferentially binds and enters injured cells from the basolateral (BL) surface. We previously demonstrated that activation of phosphatidylinositol 3-kinase (PI3K) and Akt are necessary and sufficient for P. aeruginosa entry from the apical (AP) surface and that AP addition of phosphatidylinositol 3,4,5-trisphosphate (PIP3) is sufficient to convert AP into BL membrane (Kierbel, A., A. Gassama-Diagne, K. Mostov, and J.N. Engel. 2005. Mol. Biol. Cell. 16:2577–2585; Gassama-Diagne, A., W. Yu, M. ter Beest, F. Martin-Belmonte, A. Kierbel, J. Engel, and K. Mostov. 2006. Nat. Cell Biol. 8:963–970). We now show that P. aeruginosa subverts this pathway to gain entry from the AP surface. In polarized monolayers, P. aeruginosa binds near cell–cell junctions without compromising them where it activates and recruits PI3K to the AP surface. Membrane protrusions enriched for PIP3 and actin accumulate at the AP surface at the site of bacterial binding. These protrusions lack AP membrane markers and are comprised of BL membrane constituents, which are trafficked there by transcytosis. The end result is that this bacterium transforms AP into BL membrane, creating a local microenvironment that facilitates its colonization and entry into the mucosal barrier.