The Unit is engaged in two major research projects and several ongoing collaborative studies:
Intracellular pathways for trafficking ATP binding cassette transporters to the bile canalicular domain; development of new MDCK cell system for imaging intracellular trafficking
Previously, we discovered two pathways by which apical membrane proteins traffic from the Golgi to the bile canaliculus in mammalian hepatocytes and polarized WIFB9 cells, which are a hybrid of a rat hepatoma and human fibroblasts. Canalicular ATP binding cassette (ABC) proteins, such as BSEP (bile acid transporter), MRP2 (nonbile acid organic anion transporter), and MDR 1 (organic cation transporter), enter a large intracellular rab 11a-enriched endosomal pool, from which they cycle to the apical plasma membrane. In contrast, single transmembrane proteins, such as cCAM105 and 5´ nucleotidase, traffic from the Golgi to the basolateral plasma membrane domain, from which they undergo transcytosis to the apical membrane. We identified critical roles for tubulin, actin, GGA, hax 2, myosin vb, PI 3-kinase, and rab 11a in the direct trafficking pathway. Live-cell imaging of BSEP-YFP constructs reveals downstream docking sites in the canalicular membrane, sites that we seek to identify. Current studies focus on the role of dynamic and static microtubules in cellular polarity and trafficking of ABC transporters, mainly the bile acid transporter (ABCB11) to the canalicular domain.
The specific requirement of identified co-factors, chaperones and other molecules required for the direct pathway of ABC trafficking and recycling systems is tested using viral-RNAi constructs and pharmacologic inhibitors.
Progress in these studies has permitted further investigation of molecular mechanisms of acquired cholestasis due to selective drugs, viruses, alimentation satus and hypoxia. Our working hypothesis is that acquired cholestasis results from an “intracellular traffic jam” which we seek to define and, hopefully, discover therapies based on alternative pathways.
The role of rab 11a, myosin Vb, and other proteins in canalicular polarity
While studying mechanisms of apical targeting in WIFB9 cells, we observed that rab 11a and myosin Vb are required for canalicular formation. Expression of dominant negative constructs or RNAi prevented polarization and resulted in trafficking patterns found in nonpolarized cells. These observations prompt revision of current polarity concepts and suggest that polarization is initiated upon delivery of rab 11a-, myosin Vb-containing vesicles to the surface, which causes plasma membrane at the site of delivery to differentiate into the apical domain (bile canaliculus).
Further studies are being performed in longterm (40 day) cultures of rodent and human hepatocytes using a novel system discovered by colleagues at MIT. In contrast to other methods for primary culture of hepatoyctes, the new system of nondividing cells maintains hepatocellular structure, function and gene expression, and is suitable for live cell imaging.
Physiologic effect of in vivo expression of adenoviral rab 11a-YFP and myosin Vb-CFP dominant negative constructs in rat liverUsing adenoviral YFP and CFP constructs of rab 11a and BSEP, we expanded our in vivo cell-biologic studies in rats. The viral constructs are abundantly expressed in most hepatocytes but not in other cells. Changes in ABC transporter distribution and function were similar to those observed in cell cultures. The in vivo studies provide an exciting opportunity to explore the molecular mechanisms of bile-secretory failure (cholestasis) and the effect of various cholestatic drugs, viruses, diets, and development while suggesting possibilities for the creation of new therapies.
Other collaborative studies:
Role of rab 3D in transcytosis
Janet Larkin, PhD; in collaboration with Alan Remaley, NHLBI Transcytosis of membrane proteins in polarized cells is functionally important; however, the underlying molecules and cellular mechanisms and their regulation are poorly understood. Live-cell imaging and biochemical studies suggest that rab 3D may be critical in transcytosis. We are studying this process by using molecular knockdown methodology, expression of dominant negative constructs, and mice in which rab3D has been deleted.
Biology and pathobiology of fenestrae in hepatic endothelial cells
In collaboration with Victoria Coggin, PhD; ANZAC Institute, Sydney, Australia, and Sandra Branch, PhD; Mass. Institute of Technology, Cambridge, Mass Hepatic endothelial cells are heavily fenestrated. Our previous studies indicated that the fenestrae are formed on an actin-myosin-based cytoskeleton and that their contraction can be regulated physiologically. Given that there is no basement membrane in hepatic sinusoids, fenestrae constitute the only barrier between the circulation and the plasma membrane of hepatocytes. Using a newly described hepatic endothelial cell line with regulatable fenestrae, we are exploring the cell biology and physiology of fenestrae. In addition, scanning electron microscopy of liver from mice with deleted caveolin 1 revealed highly abnormal fenestrae with reduced number, size, and configuration. The relation of caveolin 1 to fenestra formation is under study.
Role of specific decapeptide in regulating PI 3 kinase activity and in intracellular trafficking of ABC transporters in collaboration with Paul Jamney, PhD, University Pennsylvania; Cynde Leveille-Webster,DVM, Tufts University. We continue exploring the role of a decapeptide that enhances PI 3-kinase activity in cells and in vivo by rendering the substrate phosphatidylinositol 4,5-bisphosphate (PI45P2) more susceptible to the enzyme. Our studies reveal that 3′ phosphoinositides are required for trafficking of ABC transporters and for their activity in the plasma membrane. Furthermore, we showed that the rhodamine-conjugated decapeptide is a potent choleretic agent in vivo; thus, it may be useful therapeutically.
Gene expression in cholestasis associated with hyperalimentation in collaboration with Anna Calgona, PhD, NCI.
We are exploring gene expression patterns in clinical and experimental cholestasis associated with hyperalimentation.
Molecular pathogenesis of progressive familial intrahepatic cholestasis, type 1 in collaboration with Matt Harris,PhD, University of Sydney, Australia and Benjamin Schneider, MD, University of Pittsburgh, PA.
We study the cellular and molecular pathogenesis of progressive familial intrahepatic cholestasis, type 1 (PFIC 1). FIC1 encodes a P-type ATPase, which, as we previously showed, functions as an aminophospholipid flippase in the basalateral plasma membrane of hepatocytes and small intestinal cells. FIC1 regulates FXR, a nuclear transcription factor, which, in turn, regulates the activity of BSEP and other apical ABC transporters. Using molecular and imaging techniques, we seek to elucidate how the P-ATPase and its lipid traffic regulate bile acid secretion.