Subcellular localization of transporters along the rat blood-brain barrier and blood-cerebral-spinal fluid barrier by in vivo biotinylation.

Nutrient transporters and ABC efflux pumps at the blood-brain barrier are major determinants of drug penetration into the brain. Immunohistochemical analysis of transporter subcellular localization is challenging due to the close apposition of the luminal and abluminal microvessel plasma membranes. We employed in vivo perfusion of biotinylation reagent through rat brain microvessels to domain-specifically label proteins exposed on the microvessel luminal surface. Using this approach, we analyzed the luminal/abluminal localization of a number of blood-brain barrier transporters identified by quantitative PCR profiling as being highly expressed and enriched in rat brain endothelial cells compared with whole brain. We also examined the apical/basal-lateral distribution of transporters in the choroid plexus, a secondary site for transport of nutrients between the blood and CNS. We detected P-glycoprotein (Pgp) (Abcb1), ATP-binding cassette (Abc) g2, multidrug resistance protein (Mrp) 4 (Abcc4), glucose transporter 1 (Glut1) (Slc2a1), Lat1 (Slc7a5), and monocarboxylate transporter-1 (Mct1) (Slc16a1) on the luminal surface of rat cerebral microvessels by both immunofluorescence staining and Western blotting of in vivo biotinylated proteins. Mrp1 (Abcc1) appeared primarily abluminal by immunofluorescence staining, and was barely detectable in the biotinylated protein fraction. Organic anion transporter (Oat) 3 (Slc22a8), organic anion transporter polypeptide (Oatp) 2b1 (Slco2b1, Oatpb), and Mrp5 (Abcc5) were not detected on the luminal surface using either method, while Oatp1a4 (Slco1a4, Oatp2) appeared to partially localize to the microvessel lumen by immunofluorescence staining, but was not detected in the biotinylated protein fraction by Western blotting. Lat1, Mrp1 and Mrp4 were detected on the basal-lateral surface of lateral ventricle choroid plexus epithelial cells. Mrp5, Oct3 and Oatp2b1 (Oatpb) were detected in the ependymal cells lining the ventricle. We did not detect Pgp expression in choroid plexus by immunofluorescence staining. In vivo biotinylation provides a method for domain-specific labeling of luminal surface proteins within the capillaries of the blood-brain barrier, allowing for biochemical analysis of protein localization and facilitating optical discrimination of the luminal and abluminal endothelial surfaces.

Sec6/8 complexes on trans-Golgi network and plasma membrane regulate late stages of exocytosis in mammalian cells.

Sec6/8 complex regulates delivery of exocytic vesicles to plasma membrane docking sites, but how it is recruited to specific sites in the exocytic pathway is poorly understood. We identified an Sec6/8 complex on trans-Golgi network (TGN) and plasma membrane in normal rat kidney (NRK) cells that formed either fibroblast- (NRK-49F) or epithelial-like (NRK-52E) intercellular junctions. At both TGN and plasma membrane, Sec6/8 complex colocalizes with exocytic cargo protein, vesicular stomatitis virus G protein (VSVG)-tsO45. Newly synthesized Sec6/8 complex is simultaneously recruited from the cytosol to both sites. However, brefeldin A treatment inhibits recruitment to the plasma membrane and other treatments that block exocytosis (e.g., expression of kinase-inactive protein kinase D and low temperature incubation) cause accumulation of Sec6/8 on the TGN, indicating that steady-state distribution of Sec6/8 complex depends on continuous exocytic vesicle trafficking. Addition of antibodies specific for TGN- or plasma membrane-bound Sec6/8 complexes to semiintact NRK cells results in cargo accumulation in a perinuclear region or near the plasma membrane, respectively. These results indicate that Sec6/8 complex is required for several steps in exocytic transport of vesicles between TGN and plasma membrane.

Cell polarity: Versatile scaffolds keep things in place.

Protein scaffolds organize transmembrane and cytoplasmic proteins and serve to integrate both structure and signaling at the apical junctional complex of polarized epithelial cells. These scaffolds are important in coordinating local and global changes in cell organization.

New perspectives on mechanisms involved in generating epithelial cell polarity.

Polarized epithelial cells form barriers that separate biological compartments and regulate homeostasis by controlling ion and solute transport between those compartments. Receptors, ion transporters and channels, signal transduction proteins, and cytoskeletal proteins are organized into functionally and structurally distinct domains of the cell surface, termed apical and basolateral, that face these different compartments. This review is about mechanisms involved in the establishment and maintenance of cell polarity. Previous reports and reviews have adopted a Golgi-centric view of how epithelial cell polarity is established, in which the sorting of apical and basolateral membrane proteins in the Golgi complex is a specialized process in polarized cells, and the generation of cell surface polarity is a direct consequence of this process. Here, we argue that events at the cell surface are fundamental to the generation of cell polarity. We propose that the establishment of structural asymmetry in the plasma membrane is the first, critical event, and subsequently, this asymmetry is reinforced and maintained by delivery of proteins that were constitutively sorted in the Golgi. We propose a hierarchy of stages for establishing cell polarity.

Sec6/8 complex is recruited to cell-cell contacts and specifies transport vesicle delivery to the basal-lateral membrane in epithelial cells.

In budding yeast, the Sec6/8p complex is essential for generating cell polarity by specifying vesicle delivery to the bud tip. We show that Sec6/8 homologs are components of a cytosolic, approximately 17S complex in nonpolarized MDCK epithelial cells. Upon initiation of calcium-dependent cell-cell adhesion, approximately 70% of Sec6/8 is rapidly (t(1/2) approximately 3-6 hr) recruited to sites of cell-cell contact. In streptolysin-O-permeabilized MDCK cells, Sec8 antibodies inhibit delivery of LDL receptor to the basal-lateral membrane, but not p75NTR to the apical membrane. These results indicate that lateral membrane recruitment of the Sec6/8 complex is a consequence of cell-cell adhesion and is essential for the biogenesis of epithelial cell surface polarity.

Apiconuclear organization of microtubules does not specify protein delivery from the trans-Golgi network to different membrane domains in polarized epithelial cells.

In nonpolarized epithelial cells, microtubules originate from a broad perinuclear region coincident with the distribution of the Golgi complex and extend outward to the cell periphery (perinuclear [PN] organization). During development of epithelial cell polarity, microtubules reorganize to form long cortical filaments parallel to the lateral membrane, a meshwork of randomly oriented short filaments beneath the apical membrane, and short filaments at the base of the cell; the Golgi becomes localized above the nucleus in the subapical membrane cytoplasm (apiconuclear [AN] organization). The AN-type organization of microtubules is thought to be specialized in polarized epithelial cells to facilitate vesicle trafficking between the trans-Golgi Network (TGN) and the plasma membrane. We describe two clones of MDCK cells, which have different microtubule distributions: clone II/G cells, which gradually reorganize a PN-type distribution of microtubules and the Golgi complex to an AN-type during development of polarity, and clone II/J cells which maintain a PN-type organization. Both cell clones, however, exhibit identical steady-state polarity of apical and basolateral proteins. During development of cell surface polarity, both clones rapidly establish direct targeting pathways for newly synthesized gp80 and gp135/170, and E-cadherin between the TGN and apical and basolateral membrane, respectively; this occurs before development of the AN-type microtubule/Golgi organization in clone II/G cells. Exposure of both clone II/G and II/J cells to low temperature and nocodazole disrupts >99% of microtubules, resulting in: 1) 25-50% decrease in delivery of newly synthesized gp135/170 and E-cadherin to the apical and basolateral membrane, respectively, in both clone II/G and II/J cells, but with little or no missorting to the opposite membrane domain during all stages of polarity development; 2) approximately 40% decrease in delivery of newly synthesized gp80 to the apical membrane with significant missorting to the basolateral membrane in newly established cultures of clone II/G and II/J cells; and 3) variable and nonspecific delivery of newly synthesized gp80 to both membrane domains in fully polarized cultures. These results define several classes of proteins that differ in their dependence on intact microtubules for efficient and specific targeting between the Golgi and plasma membrane domains.

Cell polarity: par for the polar course.

The nematode PAR-1 gene is required for asymmetric cell divisions during development. Recently identified mammalian Par-1 homologues are kinases that phosphorylate microtubule-associated proteins; their overexpression disrupts the microtubule cytoskeleton, and alters cellular structure and organization.

Translational regulation of Na,K-ATPase alpha1 and beta1 polypeptide expression in epithelial cells.

To investigate the regulation of the Na,K-ATPase, we have studied the expression of the Na,K-ATPase polypeptides in several mammalian cell lines using the vaccinia virus/T7 RNA polymerase expression system. Infection of several fibroblast-like cell lines with viral recombinants containing the Na,K-ATPase alpha and beta isoforms, the glucose transporters, GLUT 1 and GLUT 4, or the capsid protein of the Sindbis virus all result in the production of the appropriate protein products. However, all epithelial cell lines tested fail to synthesize the Na,K-ATPase viral recombinants, yet they efficiently express the other virally directed polypeptides. While Madin-Darby canine kidney (MDCK) epithelial cells infected with the Na,K-ATPase alpha1 or beta1 recombinant viruses produce both mRNAs, the messages are inefficiently translated. Furthermore, the RNA from infected MDCK cells does not direct the in vitro synthesis of the beta1 polypeptide, whereas the message from infected fibroblast-like BSC 40 cells is efficiently translated both in vivo and in vitro. Moreover, the synthesis of the H,K-ATPase alpha subunit is also limited in MDCK cells, although the H,K-ATPase beta subunit is efficiently expressed. Expression of chimeras constructed between the Na+ pump beta1 isoform and the H,K-ATPase beta subunit indicates that sequences in the 5' coding region of the beta1 message have an inhibitory effect; however, the stringent translational regulation of the beta1 isoform in MDCK cells requires the 5' and 3' regions of the coding sequence. The ability of the polarized cell lines to limit the synthesis of the Na+ pump polypeptides while expressing other vaccinia recombinants at high levels suggests that the polarized cells possess a stringent mechanism for the specific translational regulation of a select set of messages.

Effect of Gibberellin and Heat Shock on the Lipid Composition of Endoplasmic Reticulum in Barley Aleurone Layers.

The heat-shock responses of barley (Hordeum vulgare L. cv Hi- malaya) aleurone layers incubated with or without gibberellic acid (GA3) were compared. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that heat shock blocked the synthesis and secretion of secretory proteins from GA3-treated layers but not untreated layers. This suppression of secretory protein synthesis has been correlated with changes in endoplasmic reticulum (ER) membranes (F.C. Belanger, M. R. Brodl, T.-h.D. Ho [1986] Proc Natl Acad Sci USA 83: 1354-1358; L. Sticher, A.K. Biswas, D.S. Bush, R.L. Jones [1990] Plant Physiol 92: 506-513). Our secretion data suggested that the ER membranes of aleurone layers incubated without GA3 may be more heat shock tolerant. To investigate this, the lipid profiles of membrane extracts in aleurone layers labeled with [14C]glycerol were examined. Heat shock markedly increased [14C]glycerol incorporation into phosphatidylcholine (PC), and gas chromatography revealed an increase in the amount of saturated fatty acids associated with thin layer chromatography-purified PC in GA3-treated layers. In contrast, aleurone layers incubated without GA3 at normal temperature contained PC-associated fatty acids with a greater degree of saturation than GA3-treated layers. Heat shock modestly increased the degree of fatty acid saturation in untreated aleurone layers. This same trend was noted in fatty acids isolated from ER membranes purified by continuous sucrose density centrifugation. We propose that increased fatty acid saturation may help sustain ER membrane function in heat-shocked aleurone layers incubated in the absence of GA3.

Characterization of Na,K-ATPase isoform expression and activity in MDCK and Caco-2 epithelial cells.

The asymmetric distribution of the Na,K-ATPase in the plasma membrane of epithelial cells is essential to the establishment of the transepithelial Na+ gradient that supports the vectorial transport of ions and solutes. To investigate the changes that occur during the development of polarity, we have characterized Na,K-ATPase expression and activity in two epithelial cell culture lines, Madin Darby canine kidney (MDCK) and Caco-2 cells. RNA and immunoblot analysis of both cell lines demonstrate that only the alpha 1 and beta 1 isoforms are expressed in nonpolarized and polarized cultures. Interestingly, alpha 1 and beta 1 message increases in MDCK cells with the development of polarity, yet there is little change in the amount of protein for either subunit. In contrast, alpha 1 and beta 1 polypeptide expression increases in Caco-2 cells with the development of polarity, even though the amount of both transcripts decreases. The lack of correlation between the changes that occur at the level of the message and protein suggest that appropriate expression is mediated in part by a combination of transcriptional and translational events. Furthermore, while there was a slight decrease in activity in polarized MDCK cells, there was a 1.9 fold increase in Na,K-ATPase activity in the polarized Caco-2 cells as compared to nonpolarized cells. These results demonstrate that the regulation of Na,K-ATPase alpha 1 and beta 1 isoform expression is mediated by a combination of transcriptional and translational events during the development of polarity in both cell lines.