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

1995 ◽  
Vol 108 (6) ◽  
pp. 2487-2498 ◽  
Author(s):  
R. Schwimmer ◽  
G.K. Ojakian
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Cell Surface ◽  
Apical Membrane ◽  
Apical Cell ◽  
Collagen Gel ◽  
Integrin Subunit ◽  
Membrane Remodeling ◽  
Tubule Formation ◽  
Beta 1 Integrin

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.

Regulation of epithelial cell surface polarity reversal by beta 1 integrins

1994 ◽  
Vol 107 (3) ◽  
pp. 561-576 ◽  
Author(s):  
G.K. Ojakian ◽  
R. Schwimmer
Keyword(s):  
Extracellular Matrix ◽  
Cell Surface ◽  
Outer Membrane ◽  
Collagen Gel ◽  
Polarity Reversal ◽  
Integrin Subunit ◽  
Membrane Remodeling ◽  
Surface Polarity ◽  
Beta 1 Integrin

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.

Beta 1-integrin is a maternal protein that is inserted into all newly formed plasma membranes during early Xenopus embryogenesis

Development ◽  
1992 ◽  
Vol 115 (2) ◽  
pp. 595-605 ◽  
Author(s):  
V. Gawantka ◽  
H. Ellinger-Ziegelbauer ◽  
P. Hausen
Keyword(s):  
Monoclonal Antibody ◽  
Outer Surface ◽  
Unknown Function ◽  
Plasma Membranes ◽  
Integrin Subunit ◽  
Membrane Domains ◽  
Mature Form ◽  
Maternal Mrna ◽  
Beta 1 Integrin ◽  
Early Gastrula

A monoclonal antibody (mAb 8C8) that recognizes the Xenopus beta 1-integrin chain was used to study the appearance, synthesis and distribution of this integrin subunit during the early development of Xenopus. Both the precursor and the mature form of beta 1-integrin are provided maternally. They do not increase significantly in amount until early gastrula when the level of both forms begins to rise gradually. Synthesis of beta 1-integrin from maternal mRNA is observed throughout the pregastrula phase, though it seems to add only little to the total beta 1-integrin of the embryo. Until late blastula only small amounts of precursor are processed into the mature form. Starting with the formation of the first cleavage membrane, mature beta 1-integrin is inserted into the newly formed plasma membranes of all cells. The membrane domains forming the outer surface of the embryo remain devoid of the antigen. The data suggest an as yet unknown function of beta 1-integrin during the cleavage phase.

A scale associated protein of Apedinella radians (Pedinellophyceae) and its possible role in the adhesion of surface components

1993 ◽  
Vol 104 (2) ◽  
pp. 391-398
Author(s):  
A. Koutoulis ◽  
M. Ludwig ◽  
R. Wetherbee
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Cell Membrane ◽  
Cell Surface ◽  
Immunofluorescence Microscopy ◽  
Endomembrane System ◽  
Thin Sections ◽  
Specific Location ◽  
Whole Cells

Monoclonal antibodies have been generated against cell surface components of the unicellular phytoflagellate Apedinella radians (Pedinellophyceae). One monoclonal antibody, designated Arg 1E5/1B1, labels a scale associated protein (SAP) of 145 kDa. Immunofluorescence microscopy of whole cells as well as immunoelectron microscopy of whole cell mounts and thin sections using Arg 1E5/1B1 have shown that the SAP is located on the proximal surface of body scales and spine-scales. Its specific location suggests that the SAP may play a role in the adhesion of these surface components to the cell membrane and/or to one another. The potential of monoclonal antibody Arg 1E5/1B1 as a tool to study cell surface morphogenesis and the role of the endomembrane system in A. radians is discussed.

Arginine vasopressin and forskolin regulate apical cell surface expression of epithelial Na+ channels in A6 cells

1994 ◽  
Vol 266 (3) ◽  
pp. F506-F511 ◽  
Author(s):  
T. R. Kleyman ◽  
S. A. Ernst ◽  
B. Coupaye-Gerard
Keyword(s):  
Cell Surface ◽  
Apical Membrane ◽  
Apical Cell ◽  
Brefeldin A ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Apical Plasma Membrane ◽  
Na Channels ◽  
Na Transport ◽  
A6 Cells

Both arginine vasopressin (AVP) and forskolin regulate vectorial Na+ transport across high-resistance epithelia by increasing the Na+ conductance of the apical membrane mediated by amiloride-sensitive Na+ channels. Pretreatment of A6 cells with brefeldin A partially inhibited the increase in Na+ transport in response to forskolin, suggesting recruitment of Na+ channels from an intracellular pool. The activation of Cl- secretion was not affected. Apical cell surface expression of Na+ channels was examined following activation of transepithelial Na+ transport across the epithelial cell line A6 by AVP or forskolin. Apical cell surface radioiodinated Na+ channels were immunoprecipitated to quantify the biochemical pool of Na+ channels at the apical plasma membrane and to determine whether an increment in the biochemical pool of Na+ channels expressed at the apical cell surface is a potential mechanism by which AVP and forskolin increase apical membrane Na+ conductance. The activation of Na+ transport across A6 cells by AVP was accompanied by a significant increase in the biochemical pool of Na+ channels at the apical plasma membrane within 5 min after addition of hormone, which was sustained for at least 30 min. The increase in apical cell surface expression of Na+ channels was also observed 30 min after application of forskolin. No changes in the oligomeric subunit composition of the channel were noted. Brefeldin A inhibited the forskolin-stimulated increase in apical cell surface expression of Na+ channels. These results suggest that AVP and forskolin regulate Na+ transport, in part, via rapid recruitment of Na+ channels to the cell surface, perhaps from a pool of channels in the subapical cytoplasm.

Immunotherapy of Mouse Leukemia with Monoclonal Antibodies Directed against Type-C Virus Structural Proteins

1984 ◽  
Vol 70 (1) ◽  
pp. 9-16
Author(s):  
Mauro Boiocchi ◽  
Piera Mondellini
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Cell Surface ◽  
Therapeutic Effect ◽  
Structural Proteins ◽  
Leukemia Cells ◽  
Envelope Glycoproteins ◽  
Mouse Leukemia ◽  
Type C

The monoclonal antibody A6, isolated during a study on the natural immunoresponse of BALB/c mice against leukemia cells (4), reacts with the envelope glycoproteins gp70 of the MuLV and with the cell surface of the SL2 AKR leukemia. In the present paper, we describe the in vivo immunotherapeutic effect exerted by the A6 monoclonal antibody on the growth of the transplanted leukemia SL2. The greater therapeutic effect observed when the A6 was used with exogenous complement cooperation suggests that the immunotherapeutic activity is mediated by C'-dependent cytotoxicity.

scholarly journals Anti-Mac-1 selectively inhibits the mouse and human type three complement receptor.

1982 ◽  
Vol 156 (4) ◽  
pp. 1000-1009 ◽  
Author(s):  
D I Beller ◽  
T A Springer ◽  
R D Schreiber
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Cell Surface ◽  
Polymorphonuclear Leukocytes ◽  
Specific Cell ◽  
Complement Receptor ◽  
Murine Macrophage ◽  
Differentiation Antigen ◽  
Human Macrophages ◽  
Specific Cell Surface

Anti-Mac-1 (M1/70), a rat monoclonal antibody that reacts with mouse and human macrophages, polymorphonuclear leukocytes (PMNL), and natural killer cells, selectively inhibited complement receptor-mediated rosetting by murine macrophages and human PMNL. Preincubation of macrophages with anti-Mac-1 inhibited formation of rosettes with sheep erythrocytes bearing IgM antibody and murine C3 fragments. No inhibition was observed when other monoclonal antibodies that react with macrophages (such as anti-Ly5, anti-H-2, or anti-pan-leukocyte) were tested at 10-fold higher concentrations. Anti-Mac-1 did not affect macrophage Fc receptor-mediated rosetting. Erythrocytes bearing homogeneous human C3 fragments C3b (EC3b) or C3bi (EC3bi) were used to test the specificity of the murine macrophage and human PMNL complement receptor inhibited by anti-Mac-1. In both cases, anti-Mac-1 inhibited CR3-mediated rosetting of EC3bi but not CR1-dependent rosetting of EC3b. The results show that Mac-1 is either identical to CR3 or closely associated with CR3 function. This is one of the first cases in which a monoclonal antibody-defined differentiation antigen has been associated with a specific cell surface function.

Determinants of apical membrane formation and distribution in multicellular epithelial MDCK cysts

1994 ◽  
Vol 267 (2) ◽  
pp. C473-C481 ◽  
Author(s):  
A. Z. Wang ◽  
J. C. Wang ◽  
G. K. Ojakian ◽  
W. J. Nelson
Keyword(s):  
Membrane Proteins ◽  
Cell Surface ◽  
De Novo ◽  
Apical Membrane ◽  
Three Dimensional ◽  
Collagen Gel ◽  
Free Cell ◽  
Surface Polarity ◽  
Marker Proteins ◽  
Spinner Culture

Madin-Darby canine kidney epithelial cells form three-dimensional cysts in spinner culture with a defined cell surface polarity. Transfer of cysts from spinner culture to a collagen gel matrix results in rapid loss of apical membrane proteins from the outside surface of the cyst, degradation of extracellular matrix (ECM) from the cyst lumen, and de novo formation of the apical membrane at the luminal surface. Degradation of endogenous ECM was inhibited with 1,10-phenanthroline, an inhibitor of metalloproteinases, resulting in cysts in which cells are surrounded by either cell-cell or cell-substratum contacts. The consequence of the lack of a free cell surface on the formation of a new apical membrane domain in these cysts was analyzed. Changes in cell surface polarity were followed with antibodies to marker proteins of the apical or basolateral membranes. In the absence of a free cell surface, the apical membrane formed de novo by accumulation and fusion of presorted vesicles containing apical membrane proteins; the coalescence of these vesicles results in the formation of a central lumen. These results provide novel insights into the generation of membrane domains and formation of a lumen in complex, three-dimensional epithelial structures in development.

scholarly journals Inhibition of the formation of the complement membrane-attack complex by a monoclonal antibody to the complement component C8 α subunit

1989 ◽  
Vol 264 (3) ◽  
pp. 933-936 ◽  
Author(s):  
A Abraha ◽  
J P Luzio
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Cell Surface ◽  
Membrane Attack Complex ◽  
Complement Component ◽  
Alpha Subunit ◽  
Α Subunit ◽  
Complement Component C8

The effect of nine monoclonal antibodies to complement component C8 on the interaction of C9 with preformed cell-surface C5b-8 complexes and on the functional insertion of C8 into the membrane-attack complex (MAC) was investigated. None of the antibodies prevented C9 insertion into a preformed C5b-8 complex. One antibody (F1) directed to the C8 alpha subunit clearly inhibited formation of a functional MAC. It is proposed that this antibody prevents the C8 alpha subunit unfolding and distorting the bilayer to allow C9 insertion.

Apical membrane targeting and trafficking of the human proton-coupled transporter in polarized epithelia

2008 ◽  
Vol 294 (1) ◽  
pp. C233-C240 ◽  
Author(s):  
Veedamali S. Subramanian ◽  
Jonathan S. Marchant ◽  
Hamid M. Said
Keyword(s):  
Cell Surface ◽  
Structure Function ◽  
Fluorescent Protein ◽  
Apical Membrane ◽  
Yellow Fluorescent Protein ◽  
Apical Cell ◽  
Membrane Targeting ◽  
Acid Uptake ◽  
Conserved Sequence ◽  
Polarized Cells

The human proton-coupled folate transporter (hPCFT) is a recently discovered intestinal transporter involved in folate uptake in epithelia (and possibly other cells). Little is currently known about the structure-function relationship of the different domains of this transporter, particularly which regions are important for substrate transport as well as targeting of the transporter to the apical cell surface of polarized cells. Here we have investigated the role of the COOH-terminal domain and a well-conserved sequence separating transmembrane (TM) domains TM2 and TM3 (DXXGRR; amino acids 109–114) speculated by others to be important for transport function. Using live cell imaging approaches, we show that 1) an hPCFT-yellow fluorescent protein construct is functionally expressed at the apical membrane domain and is localized differentially to the human reduced folate carrier; 2) the predicted cytoplasmic COOH-terminal region of hPCFT is not essential for apical targeting or transporter functionality; 3) mutations that ablate a consensus β-turn sequence separating predicted TM2 and TM3 abolished apical [3H]folic acid uptake as a consequence of endoplasmic reticulum retention of mutant, likely misfolded, transporters; and 4) cell surface delivery of hPCFT is disrupted by microtubule depolymerization or by overexpression of the dynactin complex dynamitin (p50). For the first time, our data present information regarding structure-function and membrane targeting of the hPCFT polypeptide, as well as the mechanisms that control its steady-state expression in polarized cells.

scholarly journals Altered glycosylation and cell surface expression of beta 1 integrin receptors during keratinocyte activation

1992 ◽  
Vol 103 (3) ◽  
pp. 743-753 ◽  
Author(s):  
L.T. Kim ◽  
S. Ishihara ◽  
C.C. Lee ◽  
S.K. Akiyama ◽  
K.M. Yamada ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Surface ◽  
Fold Increase ◽  
Cell Surface Expression ◽  
Activation Process ◽  
Integrin Subunit ◽  
Integrin Receptors ◽  
Serum Factors ◽  
Keratinocyte Proliferation ◽  
Beta 1 Integrin

We studied the mechanism by which cell adhesiveness becomes activated when keratinocytes are removed from skin and placed into cell culture. Our results suggest that activation involves altered beta 1 integrin subunit glycosylation accompanied by an increase in cell surface beta 1 integrin receptors. Activated keratinocytes contained two forms of the beta 1 integrin subunit, approximately 93 kDa and approximately 113 kDa. As shown by pulse-chase experiments, the smaller represented the cytoplasmic precursor of the larger, and only the 113 kDa mature form was detected in integrin receptors expressed at the cell surface. Pre-activated keratinocytes contained beta 1 integrin subunits ranging from approximately 97 to 110 kDa. These beta 1 subunits had been processed through the Golgi, based on resistance to endoglycosidase-H treatment, and were not converted to 113 kDa subunits during subsequent cell culture. Experiments with endoglycosidase-F showed that differences in the apparent sizes of beta 1 integrin subunits observed in pre-activated and activated keratinocytes could be attributed to differences in subunit glycosylation. Smaller beta 1 subunits found in pre-activated keratinocytes, like the precursor beta 1 subunits of activated cells, appeared to be less efficient in reaching the cell surface. Overall, a approximately 10-fold increase in the level of cell surface integrin receptors occurred concomitant with the increased proportion of 113 kDa beta 1 subunits found in activated cells. Endoglycosidase-F experiments also indicated that there were changes in keratinocyte alpha subunits associated with beta 1. In related experiments, keratinocytes cultured in low Ca2+, serum-free MCDB medium for 4 days proliferated but their adhesiveness did not become activated. Therefore, keratinocyte proliferation and activation of adhesion are regulated separately. Finally, substantial activation of keratinocytes was observed when serum was added to cells cultured in MCDB with serum, indicating a role for serum factors in the activation process.

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