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.

Protein trafficking in the exocytic pathway of polarized epithelial cells.

Ten years ago, we knew much about the function of polarized epithelia from the work of physiologists, but, as cell biologists, our understanding of how these cells were constructed was poor. We knew proteins were sorted and targeted to different plasma membrane domains and that, in some cells, the Golgi was the site of sorting, but we did not know the mechanisms involved. Between 1991 and the present, significant advances were made in defining sorting motifs for apical and basal-lateral proteins, describing the sorting machinery in the trans-Golgi network (TGN) and plasma membrane, and in understanding how cells specify delivery of transport vesicles to different membrane domains. The challenge now is to extend this knowledge to defining molecular mechanisms in detail in vitro and comprehending the development of complex epithelial structures in vivo.

The Sec6/8 complex in mammalian cells: characterization of mammalian Sec3, subunit interactions, and expression of subunits in polarized cells.

The yeast exocyst complex (also called Sec6/8 complex in higher eukaryotes) is a multiprotein complex essential for targeting exocytic vesicles to specific docking sites on the plasma membrane. It is composed of eight proteins (Sec3, -5, -6, -8, -10, and -15, and Exo70 and -84), with molecular weights ranging from 70 to 144 kDa. Mammalian orthologues for seven of these proteins have been described and here we report the cloning and initial characterization of the remaining subunit, Sec3. Human Sec3 (hSec3) shares 17% sequence identity with yeast Sec3p, interacts in the two-hybrid system with other subunits of the complex (Sec5 and Sec8), and is expressed in almost all tissues tested. In yeast, Sec3p has been proposed to be a spatial landmark for polarized secretion (1), and its localization depends on its interaction with Rho1p (2). We demonstrate here that hSec3 lacks the potential Rho1-binding site and GFP-fusions of hSec3 are cytosolic. Green fluorescent protein (GFP)-fusions of nearly every subunit of the mammalian Sec6/8 complex were expressed in Madin-Darby canine kidney (MDCK) cells, but they failed to assemble into a complex with endogenous proteins and localized in the cytosol. Of the subunits tested, only GFP-Exo70 localized to lateral membrane sites of cell-cell contact when expressed in MDCK cells. Cells overexpressing GFP-Exo70 fail to form a tight monolayer, suggesting the Exo70 targeting interaction is critical for normal development of polarized epithelial cells.

Performance improvement teams for better psychiatric rehabilitation.

Despite the promise of psychiatric rehabilitation, many programs fail to incorporate innovative rehabilitation practices into their day-to-day regimens. Performance improvement is an effective paradigm for helping agencies improve the quality of their programs. Four phases of performance improvement are reviewed in this article: organizing for change, preparing the environment, focusing the environment, and maintaining improvement. Implementing the four phases of performance improvement is illustrated in a case study. Methodological rigor of data generated by performance improvement teams is also discussed.

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.

The O-glycosylated stalk domain is required for apical sorting of neurotrophin receptors in polarized MDCK cells.

Delivery of newly synthesized membrane-spanning proteins to the apical plasma membrane domain of polarized MDCK epithelial cells is dependent on yet unidentified sorting signals present in the luminal domains of these proteins. In this report we show that structural information for apical sorting of transmembrane neurotrophin receptors (p75(NTR)) is localized to a juxtamembrane region of the extracellular domain that is rich in O-glycosylated serine/threonine residues. An internal deletion of 50 amino acids that removes this stalk domain from p75(NTR) causes the protein to be sorted exclusively of the basolateral plasma membrane. Basolateral sorting stalk-minus p75(NTR) does not occur by default, but requires sequences present in the cytoplasmic domain. The stalk domain is also required for apical secretion of a soluble form of p75(NTR), providing the first demonstration that the same domain can mediate apical sorting of both a membrane-anchored as well as secreted protein. However, the single N-glycan present on p75(NTR) is not required for apical sorting of either transmembrane or secreted forms.

The neural cell adhesion molecule expresses a tyrosine-independent basolateral sorting signal.

Transmembrane isoforms of the neural cell adhesion molecule, N-CAM (N-CAM-140 and N-CAM-180), are vectorially targeted from the trans-Golgi network to the basolateral domain upon expression in transfected Madin-Darby canine kidney cells (Powell, S. K., Cunningham, B. A., Edelman, G. M., and Rodriguez-Boulan, E. (1991) Nature 353, 76-77). To localize basolateral targeting information, mutant forms of N-CAM-140 were constructed and their surface distribution analyzed in Madin-Darby canine kidney cells. N-CAM-140 deleted of its cytoplasmic domain shows a non-polar steady state distribution, resulting from delivery from the trans-Golgi network to both the apical and basolateral surfaces. This result suggests that entrance into the basolateral pathway may occur without cytoplasmic signals, implying that apical targeting from the trans-Golgi network is not a default mechanism but, rather, requires positive sorting information. Subsequent construction and analysis of a nested set of C-terminal deletion mutants identified a region of 40 amino acids (amino acids 749-788) lacking tyrosine residues required for basolateral targeting. Addition of these 40 amino acids is sufficient to restore basolateral targeting to both the non-polar cytoplasmic deletion mutant of N-CAM as well as to the apically expressed cytoplasmic deletion mutant of the p75 low affinity neurotrophin receptor (p75(NTR)), indicating that this tyrosine-free sequence is capable of functioning independently as a basolateral sorting signal. Deletion of both cytoplasmic and transmembrane domains resulted in apical secretion of N-CAM, demonstrating that the ectodomain of this molecule carries recessive apical sorting information.

Pervanadate activation of intracellular kinases leads to tyrosine phosphorylation and shedding of syndecan-1.

Syndecan-1 is a transmembrane haparan sulphate proteoglycan that binds extracellular matrices and growth factors, making it a candidate to act between these regulatory molecules and intracellular signalling pathways. It has a highly conserved transmembrane/cytoplasmic domain that contains four conserved tyrosines. One of these is in a consensus sequence for tyrosine kinase phosphorylation. As an initial step to investigating whether or not phosphorylation of these tyrosines is part of a signal-transduction pathway, we have monitored the tyrosine phosphorylation of syndecan-1 by cytoplasmic tyrosine kinases in intact cells. Tyrosine phosphorylation of syndecan-1 is observed when NMuMG cells are treated with sodium orthovanadate or pervanadate, which have been shown to activate intracellular tyrosine kinases. Initial studies with sodium orthovanadate demonstrate a slow accumulation of phosphotyrosine on syndecan-1 over the course of several hours. Pervanadate, a more effective inhibitor of phosphatases, allows detection of phosphotyrosine on syndecan-1 within 5 min, with peak phosphorylation seen by 15 min. Concurrently, in a second process activated by pervanadate, syndecan-1 ectodomain is cleaved and released into the culture medium. Two phosphorylated fragments of syndecan-1 of apparent sizes 6 and 8 kDa remain with the cell after shedding of the ectodomain. The 8 kDa size class appears to be a highly phosphorylated form of the 6 kDa product, as it disappears if samples are dephosphorylated. These fragments contain the C-terminus of syndecan-1 and also retain at least a portion of the transmembrane domain, suggesting that they are produced by a cell surface cleavage event. Thus pervanadate treatment of cells results in two effects of syndecan-1: (i) phosphorylation of one or more of its tyrosines via the action of a cytoplasmic kinase(s) and (ii) cleavage and release of the ectodomain into the medium, producing a C-terminal fragment containing the transmembrane/cytoplasmic domain.

Polarity of TRH receptors in transfected MDCK cells is independent of endocytosis signals and G protein coupling.

Information concerning the molecular sorting of G protein-coupled receptors in polarized epithelial cells is limited. Therefore, we have expressed the receptor for thyrotropin-releasing hormone (TRH) in Madin-Darby canine kidney (MDCK) cells by adenovirus-mediated gene transfer to determine its distribution in a model cell system and to begin analyzing the molecular information responsible for its distribution. Equilibrium binding of [methyl-3H]TRH to apical and basolateral surfaces of polarized MDCK cells reveals that TRH receptors are expressed predominantly (>80%) on the basolateral cell surface. Receptors undergo rapid endocytosis following agonist binding; up to 80% are internalized in 15 min. A mutant receptor missing the last 59 residues, C335Stop, is poorly internalized (<10%) but is nevertheless basolaterally expressed (>85%). A second mutant TRH receptor, delta218-263, lacks essentially all of the third intracellular loop and is not coupled to G proteins on binding agonist. This receptor internalizes TRH approximately half as efficiently as wild-type TRH receptors but is nevertheless strongly polarized to the basolateral surface (>90%). These results indicate that molecular sequences responsible for basolateral accumulation of TRH receptors can be segregated from signals for ligand-induced receptor endocytosis and coupling to heterotrimeric G proteins.

Epithelial cell polarity: new perspectives.

All epithelial cells possess two distinct plasma membrane domains. The apical and basolateral domains differ in protein and lipid composition, and this allows the cell to perform a variety of vectorial functions. Structures involved in generating and maintaining these distinct membrane domains include the tight junction, which serves to restrict lateral diffusion within the membrane, and the cortical cytoskeleton, which can selectively bind and retain transmembrane proteins at a particular surface. A major means to generating membrane asymmetry lies in the ability of the cell to sort apical and basolateral proteins and target them to appropriate destinations. This sorting occurs predominantly at two intracellular sites: the trans-Golgi network, and the basolateral endosome. Constitutive protein traffic in epithelial cells has recently been shown to be regulated via classical signal transduction pathways involving heterotrimeric G proteins and protein kinases. The diversion of apical and basolateral proteins into specific pathways can be mediated by signals contained within these proteins. Apical sorting information is thought to be localized in the luminal domain of transmembrane proteins, and in the case of proteins anchored to the membrane via a GPI anchor, apical sorting information is provided by the lipid moiety. In contrast, basolateral signals have been identified in the cytoplasmic domain of transmembrane proteins. Shared similarities between basolateral signals and those required for endocytosis have suggested that these two sorting processes are mechanistically related.

Membrane-anchored proteoglycans of mouse macrophages: P388D1 cells express a syndecan-4-like heparan sulfate proteoglycan and a distinct chondroitin sulfate form.

Proteoglycan accumulation by thioglycollate-elicited mouse peritoneal macrophages and a panel of murine monocyte-macrophage cell lines has been examined to determine whether these cells express plasma membrane-anchored heparan sulfate proteoglycans. Initially, cells were screened for heparan sulfate and chondroitin sulfate glycosaminoglycans after metabolic labeling with radiosulfate. Chondroitin sulfate is secreted to a variable extent by every cell type examined. In contrast, heparan sulfate is all but absent from immature pre-monocytes and is associated predominantly with the cell layer of mature macrophage-like cells. In the P388D1 cell line, the cell-associated chondroitin sulfate is largely present as a plasma membrane-anchored proteoglycan containing a 55 kD core protein moiety, which appears to be unique. In contrast, the cell-associated heparan sulfate is composed of a proteoglycan fraction and protein-free glycosaminoglycan chains, which accumulate intracellularly. A fraction of the heparan sulfate proteoglycan contains a lipophilic domain and can be released from cells following mild treatment with trypsin, suggesting that it is anchored in the plasma membrane. Isolation of this proteoglycan indicates that it is likely syndecan-4: it is expressed as a heparan sulfate proteoglycan at the cell surface, it is cleaved from the plasma membrane by low concentrations of trypsin, and it consists of a single 37 kD core protein moiety that co-migrates with syndecan-4 isolated from NMuMG mouse mammary epithelial cells. Northern analysis reveals that a panel of macrophage-like cell lines accumulate similar amounts of syndecan-4 mRNA, demonstrating that this proteoglycan is expressed by a variety of mature macrophage-like cells. Syndecan-1 mRNA is present only in a subset of these cells, suggesting that the expression of this heparan sulfate proteoglycan may be more highly regulated by these cells.

Post-transcriptional regulation of syndecan-1 expression by cAMP in peritoneal macrophages.

Syndecan-1 is a cell surface heparan sulfate proteoglycan that is proposed to serve in cell-cell adhesion, cell-matrix anchorage, and growth factor signaling. Its expression is temporally and spatially regulated during epithelial-mesenchymal interactions in many developing tissues. In some cases, this regulation appears to be achieved at the level of transcription. However, induction of syndecan-1 expression in the embryonic kidney mesenchyme is suggested to occur at the level of mRNA translation (Vainio, S., M. Jalkanen, M. Bernfield, and L. Saxén. 1992. Dev. Biol. 152:221-232). To identify a system in which the regulatory mechanisms controlling syndecan-1 expression can be studied, cells of the monocyte-macrophage lineage, which regulate the expression of many cell surface receptors, were screened for syndecan-1 expression. The syndecan-1 gene is active in blood monocytes as well as resident and thioglycollate-elicited mouse peritoneal macrophages, but expression of the proteoglycan is regulated at two levels. First, elicited macrophages accumulate nine-fold more syndecan-1 mRNA than do resident macrophages or circulating blood monocytes. Another member of the syndecan family of proteoglycans, syndecan-4, shows a distinct pattern of expression, suggesting that this regulation is specific for syndecan-1. Second, utilization of the mRNA for syndecan-1 production encounters a post-transcriptional block in the elicited macrophages that can be overcome by triggering agents such as E-type prostaglandins or dibutyryl cAMP, which raise intracellular cAMP levels. Dibutyryl cAMP does not induce syndecan-1 expression in resident peritoneal macrophages, which lack a pool of stored mRNA. This suggests that this agent promotes the post-transcriptional utilization of stored syndecan-1 mRNA. The induced proteoglycan appears at the cell surface as a integral of 100-kD heparan sulfate-rich isoform of syndecan-1. This suggests that a cAMP-dependent post-transcriptional control mechanism may be present in a variety of tissues when syndecan-1 expression is regulated.

A quantitative solid-phase assay for identifying radiolabeled glycosaminoglycans in crude cell extracts.

Extraction of radiosulfate-labeled cell layers in denaturing urea and nonionic detergent allows the quantitative binding of GAG-containing materials from up to 96 discrete samples to a single cationic nylon blot. Free sulfate and/or sulfated lipids fail to bind. Washing the blot with differential salt concentrations discriminates between native proteoglycans and free glycosaminoglycan chains or fragments. In addition, chondroitin sulfates and heparan sulfate are identified either by prior digestion with chondroitin ABC or AC lyase, as generated disaccharides fail to bind to the blot, or by treatment of the entire blot with nitrous acid following binding. Similarly, heparan sulfate can be identified on chromatograms or Western transfers from polyacrylamide gel electrophoresis by autoradiography before and after treatment of the blot with nitrous acid.