Expression of fibronectin splice variants in the postischemic rat kidney.

Using an in vivo rat model of unilateral renal ischemia, we previously showed that the expression and distribution of fibronectin (FN), a major glycoprotein of plasma and the extracellular matrix, dramatically changes in response to ischemia-reperfusion. In the distal nephron in particular, FN accumulates in tubular lumens, where it may contribute to obstruction. In the present study, we examine whether the tubular FN is the plasma or cellular form, each of which is produced by alternative splicing of a single gene transcript. We demonstrate that FN in tubular lumens does not contain the extra type III A (EIIIA) and/or the extra type III B (EIIIB) region, both of which are unique to cellular FN. It does, however, contain the V95 region, which in the rat is a component of FNs in both plasma and the extracellular matrix. Expression of FN containing EIIIA increases dramatically in the renal interstitium after ischemic injury and continues to be produced at high levels 6 wk later. V95-containing FN also increases in the interstitial space, albeit more slowly and at lower levels than FN containing EIIIA; it also persists 6 wk later. FN containing the EIIIB region is not expressed in the injured kidney. The presence of V95 but not the EIIIA or EIIIB regions of FN in tubular lumens identifies the origin of FN in this location as the plasma; tubular FN is ultimately voided in the urine. The data indicate that both plasma and cellular FNs containing the V95 and/or EIIIA regions may contribute to the pathogenesis of acute renal failure and to the repair of the injured kidney.

[Nobel prize in physiology or medicine for the discovery that proteins have intrinsic signals regulating their transport and localization within the cell]. / Nobelprijs Fysiologie of Geneeskunde 1999 voor de ontdekking dat eiwitten intrinsieke signalen hebben voor transport en lokalisatie binnen de cel.

The Nobel prize in Physiology or Medicine 1999 was awarded to Dr. Günter Blobel for the discovery that proteins contain one or more signals in their structure that act as address labels. These are recognized by one of various transport machineries that direct the protein to a specific location in the cell, or into the secretory pathway out of the cell. His work joined traditional morphology and classic biochemistry in the notion of molecular topology, and led to the insight that proteins contain domains that specifically recognize domains in other proteins, nucleic acids, lipids et cetera. Specificity of recognition is provided by information that resides in the amino acid sequence of the domain. This work has had a great impact on pathophysiology as we now understand that malfunction of proteins may be due to incorrect localization in the cell, and thus due to a defect in transport and targeting. Typical example is the mislocalization of CFTR protein in cystic fibrosis. In a broader sense, malfunctioning of proteins often results from defective interactions with other proteins. The major challenge in the post-genomic era will be the unravelling of all signals that govern interactions between the 140,000 different proteins that are encoded in our DNA, to understand functions of these macromolecular interactions in the cell, and finally to trace and cure diseases that are caused by defective signals and interactions.

Polarity, integrin, and extracellular matrix dynamics in the postischemic rat kidney.

Acute renal failure (ARF) as a consequence of ischemic injury is a common disease affecting 5% of the hospitalized population. Despite the fact that mortality from ARF is high, there has been little improvement in survival rates over the last 40 years. The pathogenesis of ARF may be related to substantial changes in cell-cell and cell-extracellular matrix interactions mediated by beta1-integrins. On the basis of in vitro and in vivo studies, reorganization of beta1-integrins from basal to apical surfaces of injured tubular epithelia has been suggested to facilitate epithelial detachment, contributing to tubular obstruction and backleak of glomerular filtrate. In this study, we examine integrin and extracellular matrix dynamics during epithelial injury and repair using an in vivo rat model of unilateral ischemia. We find that, soon after reperfusion, beta1-integrins newly appear on lateral borders in epithelial cells of the S3 segment but are not on the apical surface. At later times, as further injury and regeneration coordinately occur, epithelia adherent to the basement membrane localize beta1 predominantly to basal surfaces even while the polarity of other marker proteins is lost. At the same time, amorphous material consisting of depolarized exfoliated cells fills the luminal space. Notably, beta1-integrins are not detected on exfoliated cells. A novel finding is the presence of fibronectin, a glycoprotein of plasma and the renal interstitium, in tubular spaces of the distal nephron and to a lesser extent S3 segments. These results indicate that beta1-integrins dramatically change their distribution during ischemic injury and epithelial repair, possibly contributing to cell exfoliation initially and to epithelial regeneration at later stages. Together with the appearance of large amounts of fibronectin in tubular lumens, these alterations may play a significant role in the pathophysiology of ARF.

Endosomal fractions from viral K-ras-transformed MDCK cells reveal transformation specific changes on two-dimensional gel maps.

We have investigated the effects of viral Kirsten ras oncogene expression in Madin-Darby canine kidney (MDCK) II epithelial cell on the differential protein expression of organelle proteins. MDCK cells, stably transformed via infection with a helper-independent retroviral vector construct, were grown on permeable filter supports. Whereas normal cells form highly polarized monolayers, ras-transformed cells display an unpolarized phenotype, detaching from the substratum and developing multilayers (Schoenenberger, C.-A. et al., J. Cell Biol. 1991, 112, 873-889). We postulate that this breakdown of epithelial polarity reflects disturbed intracellular protein transport and sorting, namely, proteins will no longer be sorted correctly in intracellular organelles and will therefore not reach their appropriate target membrane. Here we emphasize the role of endosomes as sorting platform in epithelial cells. We found significant differences in the molecular composition of endosomes from normal vs. oncogenic transformed epithelial cells, strengthening previous evidence indicating that oncogenic transformation results in abnormal expression of normal genes (Celis, J. E., Olsen, E., Electrophoresis 1994, 15, 309-344) as well as the expression of new ones (Huber, L. A. et al., Electrophoresis 1994, 15, 468-473).

Apical beta 1 integrin in polarized MDCK cells mediates tubulocyst formation in response to type I collagen overlay.

A number of epithelia form tubulocysts in vitro when overlaid with type I collagen gel. Because collagen receptors are generally believed to be expressed on the basolateral domain, the mechanism by which collagen elicits this morphogenetic response from the apical surface is unclear. To investigate the role of beta 1 integrins, the major receptor family for collagen, in this process, we overlaid polarized monolayers of MDCK II cells grown on permeable supports with type I collagen gel and correlated integrin polarity with the polarity of other apical and basolateral membrane markers during tubulocyst formation. Polarized monolayers of one clone of MDCK II cells, referred to as Heidelberg MDCK, initially respond to collagen overlay by stratifying; within 48 hours, lumena develop between the cell layers giving rise to tubulocysts. Tight junctions remain intact during tubulocyst formation because transepithelial electrical resistance does not significantly change. Major alterations are observed, however, in the expression and localization of apical and basolateral membrane markers. beta 1 integrins are necessary for tubulocyst morphogenesis because a function-blocking antibody administered to the apical pole of the cells completely inhibits the formation of these structures. To determine how apical-cell collagen interactions elicit tubulocyst formation, we examined whether beta 1 integrins are mobilized to apical plasma membranes in response to collagen overlay. We found that in the absence of collagen, polarized monolayers of Heidelberg MDCK cells endogenously express on apical plasma membranes a small pool of the beta 1 family, including alpha 2 beta 1 and alpha 3 beta 1. Collagen overlay does not mobilize additional beta 1 integrins to apical domains. If beta 1 integrins are not already apically expressed, as in the C6 MDCK cell line (Schoenenberger et al. (1994) J. Cell Biol. 107, 527-541), beta 1 integrins are not directed apically and tubulocysts do not develop in response to collagen. Thus, interaction of beta 1 integrin pre-existing on apical plasma membranes of polarized epithelia with type I collagen gel is the mechanism by which apical application of collagen elicits the formation of tubulocysts. Depolarized integrins on apical plasma membranes of polarized epithelia may be relevant to the pathogenesis of disease and injury.

An antiglycolipid antibody inhibits Madin-Darby canine kidney cell adhesion to laminin and interferes with basolateral polarization and tight junction formation.

Epithelial cells polarize not only in response to cell-cell contacts, but also to contacts with a substratum composed of extracellular matrix molecules. To probe the role of specific matrix constituents in epithelial cell polarization, we investigated the effects of an adhesion-blocking mAb, 12B12, on initial polarization of MDCK cells. The 12B12 antibody, raised against whole MDCK cells, blocks adhesion to laminin by 65% but has no effect on adhesion of cells to collagen type I. Taking advantage of this antibody's function-blocking activity, as well as the fact that MDCK cells secrete laminin, the role of endogenous laminin in polarization was examined by plating cells on collagen-coated substrata in the presence of the antibody. Under these conditions, cell spreading was reduced 1.5h after plating, and cells were flatter and had fewer microvilli after 24 h. Even though lateral cell membranes were closely apposed, transepithelial resistance in the presence of the antibody was significantly reduced relative to controls. When the polarization of specific apical and basolateral markers was examined both biochemically and immunocytochemically in the presence of the antibody, we observed that the apical marker polarized at normal rates while basolateral markers did not. Surprisingly, the 12B12 antibody was not directed against any known cell adhesion protein but reacted specifically with Forssman antigen, a glycosphingolipid. These results suggest that glycolipids may play a significant role in cell adhesion via laminin and in epithelial cell polarization.

Integrin expression and localization in normal MDCK cells and transformed MDCK cells lacking apical polarity.

Epithelial cells polarize in response to contacts with the extracellular matrix and with neighboring cells. Interactions of cells with the extracellular matrix are mediated mainly by the integrin family of receptors. To begin to understand the role of integrins in polarization, we have investigated the expression and localization of three integrin families in the polarized Madin-Darby canine kidney (MDCK) epithelial cell line and in transformed MDCK cells lacking apical polarity. We find that MDCK cells express several beta 1 integrins, including alpha 2 beta 1, alpha 3 beta 1, and an unidentified integrin designated alpha x beta 1. The beta 1 integrins are the major receptors for collagens I and IV and laminin in MDCK cells, since a blocking anti-beta 1 antibody almost totally abolishes adhesion to these proteins. They also express a vitronectin receptor tentatively identified as alpha v beta 3, and the epithelial-specific integrin alpha 6 beta 4. The latter is not a laminin receptor in MDCK cells because a function blocking anti-alpha 6 antibody has no effect on cell adhesion to laminin. All three integrin families are expressed exclusively on both the basal and lateral surfaces, as determined by immunofluorescence microscopy and surface biotinylation. Transformed MDCK cells express beta 1 integrins as well as alpha v beta 3 and alpha 6 beta 4, but show alterations in the beta 1 family. Expression of alpha x is lacking, and the relative amount of the beta 1 subunit is diminished, resulting in the accumulation of Endo-H-sensitive alpha 3. In addition, surface biotinylation and immunofluorescence indicate that significant amounts of both alpha 2 beta 1 and alpha 3 beta 1 appear on not only the basolateral but also the apical plasma membrane. These results indicate that integrins are the major receptors for the extracellular matrix in MDCK cells, and that they may affect epithelial cell polarization by mediating not only cell-substratum but also cell-cell contacts.

W(h)ither default? Sorting and polarization in epithelial cells.

The discovery of basolateral sorting signals in the past year may leave the default pathway with nowhere to go. With new results suggesting that even more GTP-binding proteins and coatamers might be involved in transport and targeting, it is clear that the age of mapmaking in polarization research is nearly over.

Identification of a 200-kD, brefeldin-sensitive protein on Golgi membranes.

A mAb AD7, raised against canine liver Golgi membranes, recognizes a novel, 200-kD protein (p200) which is found in a wide variety of cultured cell lines. Immunofluorescence staining of cultured cells with the AD7 antibody produced intense staining of p200 in the juxtanuclear Golgi complex and more diffuse staining of p200 in the cytoplasm. The p200 protein in the Golgi complex was colocalized with other Golgi proteins, including mannosidase II and beta-COP, a coatomer protein. Localization of p200 by immunoperoxidase staining at the electron microscopic level revealed concentrations of p200 at the dilated rims of Golgi cisternae. Biochemical studies showed that p200 is a peripheral membrane protein which partitions to the aqueous phase of Triton X-114 solutions and is phosphorylated. The p200 protein is located on the cytoplasmic face of membranes, since it was accessible to trypsin digestion in microsomal preparations, and is recovered in approximately equal amounts in membrane pellets and in the cytosol of homogenized cells. Immunofluorescence staining of normal rat kidney cells exposed to the toxin brefeldin A (BFA), showed that there was very rapid redistribution of p200, which was dissociated from Golgi membranes in the presence of this drug. The effect of BFA was reversible, since upon removal of the toxin, AD7 rapidly reassociated with the Golgi complex. In the BFA-resistant cell line PtK1, BFA failed to cause redistribution of p200 from Golgi membranes. Taken together, these results indicate that the p200 Golgi membrane-associated protein has many properties in common with the coatomer protein, beta-COP.

Cell polarity and epithelial oncogenesis.

Pathologists have long recognized that tumour formation in epithelia leads to disruption of normal epithelial cell polarity. Despite this, few studies have taken advantage of new information on the biogenesis of cell polarity to analyse the process of epithelial oncogenesis. Recent studies of epithelial cell lines now indicate that the pattern of breakdown of polarity during oncogenesis may reflect the way in which normal epithelial cells achieve polarity. These results suggest not only a novel way to study the development of polarity in vitro, but also new ideas for the early detection of cancer.

Multilayering and loss of apical polarity in MDCK cells transformed with viral K-ras.

The effects of viral Kirsten ras oncogene expression on the polarized phenotype of MDCK cells were investigated. Stable transformed MDCK cell lines expressing the v-K-ras oncogene were generated via infection with a helper-independent retroviral vector construct. When grown on plastic substrata, transformed cells formed continuous monolayers with epithelial-like morphology. However, on permeable filter supports where normal cells form highly polarized monolayers, transformed MDCK cells detached from the substratum and developed multilayers. Morphological analysis of the multilayers revealed that oncogene expression perturbed the polarized organization of MDCK cells such that the transformed cells lacked an apical--basal axis around which the cytoplasm is normally organized. Evidence for selective disruption of apical membrane polarity was provided by immunolocalization of membrane proteins; a normally apical 114-kD protein was randomly distributed on the cell surface in the transformed cell line, whereas normally basolateral proteins remained exclusively localized to areas of cell contact and did not appear on the free cell surface. The discrete distribution of the tight junction-associated ZO-1 protein as well as transepithelial resistance and flux measurements suggested that tight junctions were also assembled. These findings indicate that v-K-ras transformation alters cell-substratum and cell-cell interactions in MDCK cells. Furthermore, v-K-ras expression perturbs apical polarization but does not interfere with the development of a basolateral domain, suggesting that apical and basolateral polarity in epithelial cells may be regulated independently.

Microtubule perturbation inhibits intracellular transport of an apical membrane glycoprotein in a substrate-dependent manner in polarized Madin-Darby canine kidney epithelial cells.

The effects of microtubule perturbation on the transport of two different viral glycoproteins were examined in infected Madin-Darby canine kidney (MDCK) cells grown on both permeable and solid substrata. Quantitative biochemical analysis showed that the microtubule-depolymerizing drug nocodazole inhibited arrival of influenza hemagglutinin on the apical plasma membrane in MDCK cells grown on both substrata. In contrast, the microtubule-stabilizing drug taxol inhibited apical appearance of hemagglutinin only when MDCK cells were grown on permeable substrata. On the basis of hemagglutinin mobility on sodium dodecyl sulfate gels and its sensitivity to endo H, it was evident that nocodazole and taxol arrested hemagglutinin at different intracellular sites. Neither drug caused a significant increase in the amount of hemagglutinin detected on the basolateral plasma membrane domain. In addition, neither drug had any noticeable effect on the transport of the vesicular stomatitis virus (VSV)-G protein to the basolateral surface. These results shed light on previous conflicting reports using this model system and support the hypothesis that microtubules play a role in the delivery of membrane glycoproteins to the apical, but not the basolateral, domain of epithelial cells.

Type I collagen gel induces Madin-Darby canine kidney cells to become fusiform in shape and lose apical-basal polarity.

In the embryo, epithelia give rise to mesenchyme at specific times and places. Recently, it has been reported (Greenburg, G., and E. D. Hay. 1986. Dev. Biol. 115:363-379; Greenberg, G., and E. D. Hay. 1988. Development (Camb.). 102:605-622) that definitive epithelia can give rise to fibroblast-like cells when suspended within type I collagen gels. We wanted to know whether Madin-Darby canine kidney (MDCK) cells, an epithelial line, can form mesenchyme under similar conditions. Small explants of MDCK cells on basement membrane were suspended within or placed on top of extracellular matrix gels. MDCK cells on basement membrane gel are tall, columnar in shape, and ultrastructurally resemble epithelia transporting fluid and ions. MDCK explants cultured on type I collagen gel give rise to isolated fusiform-shaped cells that migrate over the gel surface. The fusiform cells extend pseudopodia and filopodia, lose cell membrane specializations, and develop an actin cortex around the entire cell. Unlike true mesenchymal cells, which express vimentin and type I collagen, fusiform cells produce both keratin and vimentin, continue to express laminin, and do not turn on type I collagen. Fusiform cells are not apically-basally polarized, but show mesenchymal cell polarity. Influenza hemagglutinin and virus budding localize to the front end or entire cell surface. Na,K-ATPase occurs intracellularly and also symmetrically distributes on the cell surface. Fodrin becomes diffusely distributed along the plasma membrane, ZO-1 cannot be detected, and desmoplakins distribute randomly in the cytoplasm. The loss of epithelial polarity and acquisition of mesenchymal cell polarity and shape by fusiform MDCK cells on type I collagen gel was previously unsuspected. The phenomenon may offer new opportunities for studying cytoplasmic and nuclear mechanisms regulating cell shape and polarity.

Differential extractability of influenza virus hemagglutinin during intracellular transport in polarized epithelial cells and nonpolar fibroblasts.

Biochemical changes in the influenza virus hemagglutinin during intracellular transport to the apical plasma membrane of epithelial cells were investigated in Madin-Darby canine kidney (MDCK) cells and in LLC-PK1 cells stably transfected with a hemagglutinin gene. After pulse-labeling a substantial fraction of hemagglutinin was observed to become insoluble in isotonic solutions of Triton X-100. Insolubility of hemagglutinin was detected late in the transport pathway after addition of complex sugars in the Golgi complex but before insertion of the protein in the plasma membrane. Insolubility was not dependent on oligosaccharide modification since deoxymannojirimycin (dMM), which inhibits mannose trimming, failed to prevent its onset. Insolubility was not due to assembly of virus particles at the plasma membrane because insoluble hemagglutinin was also observed in transfected cells. Hemagglutinin insolubility was also seen in MDCK cells cultured in suspension and in chick embryo fibroblasts, indicating that insolubility and plasma membrane polarity are not simply correlated. In addition to insolubility, an apparent transport-dependent reduction of the disulfide bond linking HA1 and HA2 in hemagglutinin was detected. Because of the timing of both insolubility and the loss of the disulfide bond, these modifications may be important in the delivery of the hemagglutinin to the cell surface.

Reduced extracellular pH reversibly inhibits oligomerization, intracellular transport, and processing of the influenza hemagglutinin in infected Madin-Darby canine kidney cells.

Incubation of Madin-Darby canine kidney cells infected with influenza virus in medium of pH 5.8-6.0 blocks transport of newly synthesized hemagglutinin and processing of the hemagglutinin oligosaccharides to a form resistant to endo H digestion. Upon restoration of the culture medium to pH 7.4, arrested hemagglutinin is processed and then appears on the cell surface, indicating that exclusively transport and not oligosaccharide processing is inhibited. Based upon kinetic data and localization of blocked hemagglutinin by immunofluorescence, the point of inhibition appears to be a discrete step in transport located in a pre-Golgi compartment. This conclusion is supported by the observation that trimerization of hemagglutinin, which is believed to occur in the endoplasmic reticulum, is also inhibited by acidic medium.

Ammonium chloride slows transport of the influenza virus hemagglutinin but does not cause mis-sorting in a polarized epithelial cell line.

The effects of the weak base ammonium chloride on the intracellular transport and sorting of the influenza hemagglutinin to the apical plasma membrane of polarized epithelial cells were examined in infected Madin-Darby canine kidney cells. Ammonium chloride was found to significantly retard cell surface appearance of the hemagglutinin but to have no effect on either the initial sorting or steady-state levels of hemagglutinin on the apical domain. Based on the rate of acquisition of resistance to endo H, the timed addition of ammonium chloride, and dissociation by reduced temperature incubation of cell surface appearance of the hemagglutinin from early stages of transport and processing, it was determined that the likely site of ammonium chloride action was the trans Golgi.

Sorting of an apical plasma membrane glycoprotein occurs before it reaches the cell surface in cultured epithelial cells.

In Madin-Darby canine kidney (MDCK) cells (a polarized epithelial cell line) infected with influenza virus, the hemagglutinin behaves as an apical plasma membrane glycoprotein. To determine biochemically the domain on the plasma membrane, apical or basolateral, where newly synthesized hemagglutinin first appears, cells were cultured on Millipore filters to make both cell surface domains independently accessible. Hemagglutinin in virus-infected cells was pulse-labeled, chased, and detected on the plasma membrane with a sensitive trypsin assay. Under all conditions tested, newly made hemagglutinin appeared simultaneously on both domains, with the bulk found in the apical membrane. When trypsin was continuously present on the basolateral surface during the chase, little hemagglutinin was cleaved relative to the amount transported apically. In addition, specific antibodies against the hemagglutinin placed basolaterally had no effect on transport to the apical domain. These observations suggested that most newly synthesized hemagglutinin does not transiently appear on the basolateral surface but rather is delivered directly to the apical surface in amounts that account for its final polarized distribution.