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R Schwimmer

Publications and source records attributed to R Schwimmer.

14 recordsLinked to original sources
PubMed2001

Integrin regulation of cell-cell adhesion during epithelial tubule formation.

The extracellular matrix plays an important role in regulation of epithelial development and organization. To determine more precisely the function of extracellular matrix in this process, the initial steps in collagen-mediated formation of epithelial tubules were studied using a model cell culture system. Previous studies have demonstrated that incubation of Madin-Darby canine kidney (MDCK) epithelial cells with a collagen gel overlay induces (beta)1 integrin-regulated epithelial remodeling accompanied by extensive cell rearrangements and formation of epithelial tubules. During epithelial remodeling there was extensive disruption of the epithelial junctional complex. Progressive opening of tight junctions was observed over 8 hours using transepithelial resistance measurements and immunofluorescence microscopy demonstrated that tight and adherens junction proteins were dispersed throughout the apical and basolateral membranes. Junction complex disruption allowed the formation of apical cell extensions and subsequent migration of selected cell sheets from the epithelial monolayer. Confocal microscopy demonstrated the presence of adherens junction (E-cadherin, (alpha)-catenin, (beta)-catenin, plakoglobin) and desmosomal (desmoplakin-1/2, plakoglobin) proteins on, and within, cell extensions demonstrating that cell junctions had undergone considerable disassembly. However, groups of cell extensions appeared to be associated by E-cadherin/catenin-mediated interactions. Association of E-cadherin/catenin complexes with the epithelial cytoskeleton was analyzed by differential detergent extraction. SDS-PAGE and immunoblot analysis demonstrated that adherens junction proteins were primarily cytoskeleton-associated in control cells. During integrin-regulated remodeling, there was a progressive reduction in the interaction of adherens junction proteins with the cytoskeleton suggesting that they play an important role in the maintenance of epithelial integrity. Since loss of transepithelial electrical resistance and disruption of junctional complexes were inhibited by an antifunctional integrin antibody, we propose that activation of integrin signaling pathways regulate junctional complex stability, cell-cell interactions and cell migration. These observations provide evidence that integrin-regulated MDCK epithelial tubule formation can serve as a model system for studying rearrangements of epithelial sheets which occur during development.

Animals↗
PubMed1999

Expression and localization of Rab3D in rat parotid gland.

Rab3 proteins (isoforms A, B, C and D) are low molecular weight GTP-binding proteins proposed to be involved in regulated exocytosis. In the present study, Rab3 protein expression and localization was examined in rat parotid gland by reverse transcription (rt) PCR, Western blotting and immunocytochemistry. An approximately 200 bp PCR product was obtained from parotid RNA by rtPCR and this fragment was cloned and sequenced. Nucleotide and deduced amino acid sequences obtained from five clones were identical to rab3D. Membrane and cytosolic fractions prepared from parotid acini were immunoblotted with antisera specific for each of the four Rab3 isoforms. A 28 kDa protein was detected with Rab3D-specific antisera in both fractions with staining being more intense in the membrane fraction. No other Rab3 isoforms were detected by immunoblotting, a result consistent with those obtained by rtPCR. Rab3D was enriched in zymogen granule membranes and Triton X-114 extraction revealed that this isoform is predominantly lipid-modified in parotid. Localization of Rab3D was done on frozen sections of parotid gland by immunofluorescence microscopy. Staining was observed primarily in the acinar cells and was adjacent to the acinar lumen. Incubation of dispersed acini with isoproterenol and substance P stimulated amylase secretion 4- and 2-fold above basal, respectively. Isoproterenol, but not substance P, induced redistribution of Rab3D from the cytosol to the membrane fraction in dispersed parotid acini. Consistent with these findings, isoproterenol injections into fasted rats also resulted in increased membrane-associated Rab3D in the parotid acini. These results indicate that Rab3D is: (1) the major Rab3 isoform expressed in rat parotid gland; (2) localized to zymogen granule membranes; and (3) involved with regulated enzyme secretion in acinar cells.

Amino Acid Sequence↗
PubMed1995

The alpha 2 beta 1 integrin regulates collagen-mediated MDCK epithelial membrane remodeling and tubule formation.

Previous studies have demonstrated that incubation of MDCK cell epithelial cysts in collagen gel induced a reversal in cell surface polarity that was regulated by beta 1 integrins. Further experiments were done to identify the specific collagen binding integrin involved by applying collagen gel overlays to the apical membrane of subconfluent MDCK monolayers. Cell surface levels of the apical membrane glycoprotein gp135 were monitored by ELISA to quantitate the extent of collagen-mediated membrane remodeling. After an 8 hour incubation with collagen, there was a 35% reduction in gp135 while the cell surface levels of the alpha 2, alpha 3 and beta 1 integrin subunits were not affected. Immunofluorescence microscopy confirmed the loss of gp135 from selected regions of the apical cell surface while the alpha 2 and beta 1 integrin subunits were distributed in small clusters over the entire apical membrane in both control and collagen-treated monolayers. Collagen-mediated loss of gp135 was inhibited by monoclonal antibodies which recognize either the alpha 2 or beta 1 integrin subunits but not by a monoclonal antibody against the alpha 6 beta 1 integrin. These results demonstrated that remodeling of the apical membrane had occurred, allowing the selective retention of beta 1 integrins but not gp135. They were supported by the observation that collagen-mediated loss of apical membrane microvilli was inhibited by the monoclonal antibody against the alpha 2 integrin subunit. Incubation of confluent monolayers with collagen gel induced the formation of polarized epithelial tubules within 16 hours. Epithelial tubule biogenesis was completely inhibited by monoclonal antibodies against either the alpha 2 or beta 1 integrin subunits, providing strong evidence that the alpha 2 beta 1 integrin is essential for collagen-mediated epithelial membrane remodeling and tubule formation.

Animals↗
PubMed1994

Regulation of epithelial cell surface polarity reversal by beta 1 integrins.

The role of extracellular matrix in the regulation of epithelial cell surface polarity development was studied using MDCK cells. Previous work has demonstrated that MDCK cells cultured in suspension form epithelial cysts having polarized cell surface distributions of several membrane proteins. When MDCK suspension cysts are incubated within collagen gel, a dynamic epithelial membrane remodeling occurs that is accompanied by the reversal of cell surface polarity (Wang et al., 1990b, J. Cell Sci. 95, 153-165), suggesting that extracellular matrix is important in the modulation of epithelial polarity development. To determine if members of the integrin receptor family were involved, MDCK cyst binding studies were done utilizing antifunctional monoclonal antibodies (AIIB2 and AJ2) against the beta 1 integrin subunit. These antibodies inhibited cyst binding to type I collagen, type IV collagen and laminin, providing evidence that functional beta 1 integrin heterodimers were present on the cyst outer membrane. Integrin localization on suspension cysts demonstrated that the alpha 2, alpha 3 and alpha 6 integrin subunits had a non-polarized cell surface distribution and were localized to both the apical and basolateral membranes. Interestingly, immunofluorescence microscopy determined that the beta 1 subunit had a polarized, basolateral membrane distribution although cyst binding studies using inhibitory monoclonal antibodies suggested that functional beta 1 subunits were present on the cyst outer membrane. After incubation of suspension cysts in collagen gel for 8 hours, the beta 1 integrin subunit was detected on the outer membrane, suggesting that the formation of additional integrin alpha/beta heterodimers could be involved in epithelial remodeling. To establish the role of beta 1 integrins in polarity reversal, experiments were done on cysts incubated in collagen gel. After 6 hours in collagen gel, considerable membrane remodeling had occurred as determined by a reduction in outer membrane microvilli. However, the presence of monoclonal antibody AIIB2 inhibited membrane remodeling by preventing both microvillar loss and the endocytosis of the apical membrane glycoprotein gp135. These results provide strong evidence that members of the beta 1 integrin family are involved in the regulation of epithelial polarity reversal, and demonstrate that MDCK cysts constitute an excellent model system for studying the role of cell-extracellular matrix interactions in the regulation of epithelial plasticity and cell surface polarity development.

Animals↗
PubMed1992

Antimicrotubule drugs inhibit the polarized insertion of an intracellular glycoprotein pool into the apical membrane of Madin-Darby canine kidney (MDCK) cells.

Previous experiments on MDCK cells have demonstrated that the polarized appearance of a 135 kDa glycoprotein (gp135) on the apical plasma membrane can occur through the insertion of both newly synthesized gp135 as well as a pre-existing gp135 intracellular pool. In this study, anticytoskeletal drugs were utilized to determine the role of the cytoskeleton in the polarized delivery of gp135. Colchicine and nocodazole produced a 15-20% inhibition in the apical surface accumulation of newly synthesized gp135 and inhibited the appearance of the gp135 pool by approximately 33%, while cytochalasin D had no affect on the apical accumulation of either newly synthesized gp135 or the gp135 pool. These results indicate that microtubules, but not microfilaments, are involved in the intracellular targeting of gp135. Quantitative immunogold electron microscopy of nocodazole-treated cells demonstrated that gp135 was not mistargeted to the basolateral membrane, suggesting the possibility that some vesicles containing gp135 did not fuse with the apical membrane and remained in the cells. These experiments demonstrate that microtubules are an important component of gp135 insertion into the apical membrane. They also suggest that gp135 resides within vesicles which have an apical membrane recognition signal and cannot fuse with the basolateral membrane. The possibility that these data, and those of others, could support a hypothesis for the presence of two constitutive apical transport pathways is discussed.

Animals↗
PubMed1990

Polarized insertion of an intracellular glycoprotein pool into the apical membrane of MDCK cells.

A monoclonal antibody that recognizes a 135-kDa glycoprotein (GP135) on the apical membrane of Madin-Darby canine kidney (MDCK) cells was used to identify and characterize an intracellular pool of GP135. Mild trypsin treatment at 4 degrees C removed approximately 95% of the GP135, and after warming to 37 degrees C, the reappearance of GP135 on the apical membrane was monitored by radioimmunoassay. Incorporation of GP135 into the apical cell surface after trypsin treatment consisted of two components, a rapidly inserted, cycloheximide-insensitive portion (defined as the GP135 pool), which leveled off within 1 h, followed by a slower insertion of newly synthesized GP135. Immunogold electron microscopy demonstrated that the GP135 pool was targeted in a polarized manner and was only detected on the apical membrane. Temperature shift and retrypsinization experiments provided evidence that the GP135 pool consisted of intracellular vesicles that could fuse with the plasma membrane. This was confirmed by immunofluorescence microscopy demonstrating that GP135 was localized within large cytoplasmic vesicles residing at varying distances from the apical cell surface. These data provide evidence for the presence of a regulated pathway in MDCK cells and support the possibility that the GP135 pool functions as an intracellular reserve which can exhibit polarized insertion into the plasma membranes similar to that described for other epithelial cells.

Animals↗
PubMed1989

Human plasma fibronectin as a substrate for human urokinase.

An early event in malignant transformation is the increased expression of proteases, such as plasminogen activator, which can degrade surrounding extracellular matrices, thereby conferring an advantage for tumour cell invasion and metastasis. The present studies provide evidence that plasma fibronectin (Fn), which is a component of the extracellular matrix, is a direct substrate for the plasminogen activator urokinase (UK). Human plasma Fn was incubated with human UK under plasminogen-free conditions. Fn cleavage was both time- and dose-dependent and was evident within 30 min. The proteolytic digestion was limited and complete within 12 h at an enzyme/substrate ratio of 1:20. Analysis of the final proteolytic digestion products demonstrated the disappearance of the native dimeric 440 kDa structure of Fn with the concomitant appearance of three proteolytic fragments of 210, 200 and 25 kDa. Since two large fragments of similar size to the 220 kDa monomeric chains of Fn were obtained following proteolysis, it is proposed that UK cleaves Fn at two sites, one towards the N-terminal and one close to the C-terminal, but N-terminal to its interchain disulphide bonds. These studies suggest that the local proteolytic digestion and release of Fn from the extracellular matrix by tumour cells possessing high levels of UK may involve the direct proteolytic breakdown of Fn by UK.

Fibrinolysin↗
PubMed1988

The polarized distribution of an apical cell surface glycoprotein is maintained by interactions with the cytoskeleton of Madin-Darby canine kidney cells.

A monoclonal antibody made against a 135-kD glycoprotein (gp135) on the plasma membrane of Madin-Darby canine kidney (MDCK) cells was used to study the development and maintenance of epithelial cell surface polarity. Immunofluorescence microscopy and immunogold electron microscopy of confluent monolayers demonstrated that gp135 had a polarized cell surface distribution and was only localized on the apical surface. The role of membrane contacts in establishing gp135 polarity was determined by plating cells in low Ca++-medium to prevent the formation of intercellular junctions. Quantitative immunogold electron microscopy demonstrated that gp135 had a polarized distribution on cells lacking membrane contacts and was observed on the apical surface at a density 24 times that of the basal membrane contacting the substratum. The possibility that gp135 was associated with components of the apical cytoskeleton was investigated using cytoskeleton-disrupting drugs. Incubation in cytochalasin D produced a clustering of both actin and gp135, and double-label fluorescence microscopy demonstrated that these proteins were colocalized. Experiments using nocodazole had no effect, suggesting that gp135 could be interacting with actin microfilaments, but not microtubules. Treatment with Triton X-100 extracted approximately 50% of the gp135 and immunofluorescence microscopy indicated that the gp135 which remained associated with the detergent-insoluble cytoskeleton had a distribution identical to that of control cells. Experiments demonstrating that gp23, a nonpolarized glycoprotein, was preferentially extracted from the apical membrane suggested that the improperly sorted apical gp23 did not interact with the cytoskeleton. These results provided evidence that the polarized cell surface distribution of gp135 was maintained through its interaction with actin in the apical cytoskeleton.

Actins↗
PubMed1987

Limited cleavage of cellular fibronectin by plasminogen activator purified from transformed cells.

The substrate specificity and direct catalytic activity of plasminogen activator (PA) was examined under conditions where its natural substrate, plasminogen, was missing or inhibited. PA, purified from cultures of transformed chicken fibroblasts, was incubated with purified preparations of potential substrates. The adhesive glycoprotein fibronectin, isolated from normal chicken fibroblast extracellular matrix, underwent limited but specific cleavage by PA in the absence of plasminogen. Analysis of the cleavage products by polyacrylamide gels under both reducing and nonreducing conditions indicated that PA-mediated cleavage occurred near the carboxyl terminus of fibronectin but on the amino-terminal side of the interchain disulfide bridge, thus disrupting the native dimeric fibronectin molecule. Under the identical conditions, chicken ovalbumin was not cleaved while the established substrate, chicken plasminogen, was extensively converted to plasmin. A monoclonal antibody, directed against avian PA and shown to inhibit plasminogen-free, cell-mediated matrix degradation, specifically inhibited the fibronectin cleavage. A human PA, urokinase, also cleaved fibronectin under plasminogen-free conditions yielding a limited number of high molecular weight cleavage products.

Animals↗
PubMed1985

The extracellular matrix of normal chick embryo fibroblasts: its effect on transformed chick fibroblasts and its proteolytic degradation by the transformants.

Extracellular matrix (ECM), prepared from chick embryo fibroblasts, contains fibronectin as the major structural protein along with collagen and other polypeptides as less abundant protein components. When Rous sarcoma virus-transformed chick embryo fibroblasts are cultured on the ECM in the presence of the tumor promoter tetradecanoyl phorbol acetate, the transformed cells lose their characteristic rounded morphology and align on and within the ECM fibrillar network. This restrictive aspect of ECM is only temporary, however, and with time (24-72 h) the transformed cells progressively degrade the ECM fibers and resume their rounded appearance. The matrix degradation can be monitored by employing biosynthetically radiolabeled ECM. The addition of purified chicken plasminogen to the Rous sarcoma virus-transformed chick embryo fibroblast cultures enhances the rate and extent of ECM degradation, due to the elevated levels in the transformed cultures of plasminogen activator. Plasminogen-dependent and -independent degradation of ECM has been characterized with regard to sensitivity to various natural and synthetic protease inhibitors and to the requirement of cell/ECM contact. Plasminogen-dependent degradation of ECM occurs rapidly when ECM and cells are in contact or separated, whereas plasminogen-independent degradation is greatly reduced when ECM and cells are separated, which suggests that cell surface-associated proteolytic enzymes are involved. A possible role in ECM degradation has been indicated for cysteine proteases, metallo enzymes, and plasminogen activator, the latter as both a zymogen activator and a direct catalytic mediator.

Animals↗