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Background & Aims: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity in hepatocellular carcinoma cells is mediated by lysosomal permeabilization. Our aims were to determine which TRAIL receptor, death receptor (DR) 4 or DR5, mediates lysosomal permeabilization and assess whether receptor endocytosis followed by trafficking to lysosomes contributes in this process., Methods: TRAIL ligand internalization in Huh-7 cells was examined by confocal microscopy using Flag-tagged TRAIL, whereas DR4- and DR5-enhanced green fluorescent protein internalization was assessed by total internal reflection microscopy. Clathrin-dependent endocytosis was inhibited by expressing dominant negative dynamin., Results: Although Huh-7 cells express both TRAIL receptors, short hairpin RNA silencing of DR5 but not DR4 attenuated TRAIL-mediated lysosomal permeabilization and apoptosis. The TRAIL/DR5 complex underwent rapid cellular internalization upon ligand stimulation, whereas the TRAIL/DR4 complex was not efficiently internalized. DR5-enhanced green fluorescent protein internalization was dependent on a dileucine-based internalization motif. Endocytosis of the TRAIL/DR5 complex was dynamin dependent and was required for rapid lysosomal permeabilization and apoptosis in multiple malignant hepatocellular and cholangiocarcinoma cell lines. Upon TRAIL treatment, DR5 colocalized with lysosomes after internalization. Inhibition of DR5 trafficking to lysosomes by Rab7 small interfering RNA also reduced TRAIL-mediated lysosomal disruption and apoptosis., Conclusions: TRAIL-mediated endocytosis of DR5 with trafficking to lysosomes contributes to lysosomal protease release into the cytosol and efficient apoptosis in malignant liver cell lines.

Background & Aims: The pancreatic acinar cell is known to regulate exocytosis, total protein synthesis, and secretory protein transport in response to a secretory stimulus. Whether secretory vesicle formation also is regulated is unclear. In this study, we determined whether agonist stimulation induces morphologic alterations in the acinar cell Golgi apparatus, and we evaluated the role of the vesicle severing protein dynamin., Methods: Changes in Golgi structural integrity by examining the distribution of various Golgi and TGN lipid and protein markers in live and fixed cells on stimulation with cholecystokinin were noted in a primary pancreatic acinar cell model. Multiple dynamin reagents were used to examine the distribution and function of this molecular pinchase in resting and stimulated cells., Results: Regulated secretion in acinar cells induced (1) marked fragmentation of the trans-Golgi network (TGN) that corresponded temporally with an increase in cytoplasmic calcium whereas pre-TGN compartments of the Golgi and regions of the TGN involved in the generation of constitutively trafficking vesicles were unaffected by agonist, and (2) significant recruitment of dynamin to the acinar cell Golgi apparatus that appeared to potentiate fragmentation of the TGN., Conclusions: These results suggest that the TGN is a dynamic organelle that fragments in response to cholecystokinin stimulation, a process that may contribute to zymogen granule formation.

Background & Aims: Cryptosporidium parvum invasion of epithelia requires polymerization of host cell actin at the attachment site. We analyzed the role of host cell c-Src, a cytoskeleton-associated protein tyrosine kinase, in C. parvum invasion of biliary epithelia., Methods: In vitro models of biliary cryptosporidiosis using a human biliary epithelial cell line were used to assay the role of c-Src signaling pathway in C. parvum invasion., Results: c-Src and cortactin, an actin-binding protein and a substrate for c-Src, were recruited to the parasite-host cell interface during C. parvum invasion. Tyrosine phosphorylation of cortactin in infected cells was also detected. Inhibition of host cell c-Src significantly blocked C. parvum -induced accumulation and tyrosine phosphorylation of cortactin and actin polymerization at the attachment sites, thereby inhibiting C. parvum invasion of biliary epithelial cells. A triple mutation of tyrosine of cortactin in the epithelia also diminished C. parvum invasion. In addition, proteins originating from the parasite were detected within infected cells at the parasite-host cell interface. Antiserum against C. parvum membrane proteins blocked accumulation of c-Src and cortactin and significantly decreased C. parvum invasion. No accumulation of the endocytosis-related proteins, dynamin 2 and clathrin, was found at the parasite-host cell interface; also, inhibition of dynamin 2 did not block C. parvum invasion., Conclusions: C. parvum invasion of biliary epithelial cells requires host cell tyrosine phosphorylation of cortactin by a c-Src-mediated signaling pathway to induce actin polymerization at the attachment site, a process associated with microbial secretion but independent of host cell endocytosis.

Background & Aims: The molecular mechanisms that contribute to the cholestatic condition in hepatocytes are poorly defined. It has been postulated that a disruption of normal vesicle-based protein trafficking may lead to alterations in hepatocyte polarity., Methods: To determine if vesicle motility is reduced by cholestasis, hepatocytes cultured from livers of bile duct ligation (BDL)- or ethinyl estradiol (EE)-injected rats, were viewed and recorded by high-resolution video microscopy. Cholestatic hepatocytes were analyzed by phalloidin staining and electron microscopy. Functional analysis was done by the sodium fluorescein sequestration assay., Results: In cholestatic hepatocytes, there was a significant decrease in the number of motile cytoplasmic vesicles observed compared with control cells. Further examination of cells from BDL- or EE-treated livers revealed the presence of numerous large intracellular lumina. More than 24% of cells in BDL-treated livers and 19% of cells in EE-treated livers displayed these structures, compared with 1.1% found in control hepatocytes. Phalloidin staining of hepatocytes showed a prominent sheath of actin surrounding the lumina, reminiscent of those seen about bile canaliculi. Electron microscopy revealed that these structures were lined by actin-filled microvilli. Further, these pseudocanaliculi perform many of the functions exhibited by bona fide canaliculi, such as sequestering sodium fluorescein., Conclusions: Both mechanically and chemically induced cholestasis have substantial effects on vesicle-based transport, leading to marked disruption of hepatocellular polarity.

Background & Aims: Ethanol is known to alter vesicle-mediated protein trafficking in hepatocytes by undefined mechanisms. In this study, the effects of long- and short-term ethanol exposure on vesicle movements were measured in isolated hepatocytes, and alterations in the function of the microtubule-associated motor enzymes dynamin, kinesin, and dynein, which are believed to support the transport and/or budding of vesicles along microtubules, were tested., Methods: Vesicular movements in isolated hepatocytes exposed to short- and long-term ethanol treatment were measured. Motor adenosine triphosphatase activities and their association with specific membrane organelles were assessed in response to long-term administration of ethanol in vivo or acetaldehyde in vitro., Results: Hepatocytes exposed to short- or long-term ethanol treatment showed a significant reduction in the number of motile vesicles. No alterations in the levels of motor messenger RNA, protein, or enzymatic activity were observed. Interestingly, ethanol had no effect on the association of dynein and kinesin with membranes, whereas there was a significant increase in the amount of dynamin associated specifically with Golgi membranes., Conclusions: Long- and short-term ethanol exposure markedly reduces hepatocellular vesicle transport by a mechanism apparently independent of any alteration in the enzymatic activity of molecular motors, possibly involving a change in the function of dynamin.

Background/aims: Vesicular transport is supported by microtubule-based, force-transducing adenosine triphosphatases (ATPases), such as kinesin, a ubiquitous motor enzyme that has been well studied in neuronal tissues. Although vesicular transport is important for hepatocellular secretory and clearance activities, the role of kinesin in liver function is poorly understood. Furthermore, the effects of bile acids on kinesin are unknown., Methods: Kinesin was purified from rat liver cytosol by conventional chromatography and microtubule affinity binding and was characterized by immunoblotting with domain-specific kinesin antibodies and amino acid sequencing of tryptic fragments. Kinesin activity was measured with and without bile acids using an in vitro motility assay and ATPase assays., Results: Immunoblot analysis and partial amino acid sequencing of purified kinesin showed that the sequence at the heavy chain of hepatic kinesin is nearly identical to that of brain kinesin. Purified kinesin transported microtubules in vitro with a velocity of approximately 0.5 microns/s; this activity was significantly inhibited by 0.5-1 mmol/L taurochenodeoxycholate but not by tauroursodeoxycholate. At a dose of 1 mmol/L, chenodeoxycholate conjugates, but not ursodeoxycholate or cholate conjugates, directly inhibited the ATPase activities of kinesin and another microtubule motor, cytoplasmic dynein., Conclusions: Cholestatic concentrations of chenodeoxycholate conjugates directly inhibit the activity of microtubule motors, suggesting a possible mechanism for impairment of vesicular transport in cholestasis.