Calcium-induced desmosome formation in cultured kidney epithelial cells

1986 ◽  
Vol 85 (1) ◽  
pp. 95-111
Author(s):  
D.L. Mattey ◽  
D.R. Garrod
Keyword(s):  
Epithelial Cells ◽  
Mdck Cells ◽  
Fluorescent Antibody ◽  
Cell Types ◽  
Cell Periphery ◽  
Cell Type ◽  
Antibody Staining ◽  
Mdbk Cells ◽  
Triton X ◽  
Do So

Previous work has shown that cultured keratinocytes do not form desmosomes at low [Ca2+] (less than 0.1 mM) but may be induced to do so by raising [Ca2+] to physiological levels (1.8-2 mM). Here, fluorescent antibody staining with specific anti-desmosomal antibodies and electron microscopy have been used to determine whether Ca2+-induced desmosome formation also occurs in simple epithelial cells. Both Madin-Darby canine and bovine kidney cells (MDCK and MDBK) exhibit Ca2+-induced desmosome formation, but there are significant differences between them. MDCK cells resemble keratinocytes in showing rapid desmosome formation characterized by the simultaneous appearance of four desmosomal antigens at the cell periphery within 15–20 min of raising the [Ca2+]. In contrast MDBK cells take between 7 and 8 h to form desmosomes after Ca2+ switching, and this is characterized by slow appearance of two desmosomal antigens, the 175–164(X 10(3)) Mr glycoprotein and desmoplakin, at the cell periphery. Differences in the pattern of staining for desmosomal antigens between the two cell types in low and high [Ca2+] are described and discussed in relation to desmosome formation and internalization. Triton X-100 extractability of desmosomal antigen staining is also considered. While most is non-extractable, staining for the glycoproteins known as desmocollins is completely extractable from MDCK cells in low [Ca2+], but that which reaches the cell periphery after Ca2+ switching becomes non-extractable. Although neither cell type forms desmosomes in low [Ca2+], both possess zonulae adhaerentes, suggesting a difference in Ca2+ requirement for formation of these two junctions.

Splitting and internalization of the desmosomes of cultured kidney epithelial cells by reduction in calcium concentration

1986 ◽  
Vol 85 (1) ◽  
pp. 113-124 ◽  
Author(s):  
D.L. Mattey ◽  
D.R. Garrod
Keyword(s):  
Cell Surface ◽  
Calcium Concentration ◽  
Mdck Cells ◽  
Fluorescent Antibody ◽  
Antibody Staining ◽  
Mdbk Cells ◽  
Standard Culture ◽  
Lateral Fusion ◽  
First Time ◽  
Standard Culture Medium

Desmosome assembly may be induced in simple epithelial (MDBK and MDCK) cells maintained in low calcium medium (LCM: [Ca2+] less than 0.05 mM) by raising [Ca2+] to that of standard culture medium (SM: [Ca2+] = 1.8 mM). Here it is shown that if cells in SM are simply returned to LCM, their desmosomes split in the intercellular region within 15 min and the desmosomal halves are internalized within 30 min. This is the first time that desmosome splitting has been shown to occur in response to a reduction in [Ca2+] rather than Ca2+ chelation. Fluorescent antibody staining shows that the desmosomal glycoproteins as well as the plaque constituents are internalized, although a pool of the glycoproteins known as desmocollins remains at the cell surface, apparently unassociated with other desmosomal components. Desmosomal halves that have been recently internalized in response to LCM treatment do not return to the cell surface to participate in new desmosome formation. MDCK cells are able to form new desmosomes rapidly (15–30 min) while old desmosomes continue to be internalized. The desmosomes of MDBK cells remain sensitive to splitting and internalization in response to reduction in [Ca2+] for up to 14 days of culture in SM. In contrast, the desmosomes of MDCK cells become resistant to reduction in [Ca2+], as well as Ca2+ chelation by EGTA, after 4–5 days in SM. When treated with LCM or EGTA, MDCK cells with ‘stabilized’ desmosomes partially separate but remain attached to each other at some points. Regions of attachment stain brightly with anti-desmosomal antibodies and are characterized by ‘giant’ desmosomes, up to 4 micron long, roughly 20 times larger than those formed in cells in SM. These giant desmosomes may form by lateral fusion of small desmosomes.

SNARE expression and localization in renal epithelial cells suggest mechanism for variability of trafficking phenotypes

2002 ◽  
Vol 283 (5) ◽  
pp. F1111-F1122 ◽  
Author(s):  
Xin Li ◽  
Seng Hui Low ◽  
Masumi Miura ◽  
Thomas Weimbs
Keyword(s):  
Epithelial Cells ◽  
Membrane Trafficking ◽  
Mdck Cells ◽  
Cell Types ◽  
Intercalated Cells ◽  
Cell Type ◽  
Specific Distribution ◽  
Protein Receptors ◽  
Syntaxin 4

The apical- and basolateral-specific distribution of target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (t-SNAREs) of the syntaxin family appear to be critical for polarity in epithelial cells. To test whether differential SNARE expression and/or subcellular localization may contribute to the known diversity of trafficking phenotypes of epithelial cell types in vivo, we have investigated the distribution of syntaxins 2, 3, and 4 in epithelial cells along the renal tubule. Syntaxins 3 and 4 are restricted to the apical and basolateral domains, respectively, in all cell types, indicating that their mutually exclusive localizations are important for cell polarity. The expression level of syntaxin 3 is highly variable, depending on the cell type, suggesting that it is regulated in concert with the cellular requirement for apical exocytic pathways. While syntaxin 4 localizes all along the basal and lateral plasma membrane domains in vivo, it is restricted to the lateral membrane in Madin-Darby canine kidney (MDCK) cells in two-dimensional monolayer culture. When cultured as cysts in collagen, however, MDCK cells target syntaxin 4 correctly to the basal and lateral membranes. Unexpectedly, the polarity of syntaxin 2 is inverted between different tubule cell types, suggesting a role in establishing plasticity of targeting. The vesicle-associated (v)-SNARE endobrevin is highly expressed in intercalated cells and colocalizes with the H+-ATPase in α- but not β-intercalated cells, suggesting its involvement in H+-ATPase trafficking in the former cell type. These results suggest that epithelial membrane trafficking phenotypes in vivo are highly variable and that different cell types express or localize SNARE proteins differentially as a mechanism to achieve this variability.

Discordant regulatory changes in monocrotaline-induced megalocytosis of lung arterial endothelial and alveolar epithelial cells

2006 ◽  
Vol 290 (6) ◽  
pp. L1216-L1226 ◽  
Author(s):  
Somshuvra Mukhopadhyay ◽  
Pravin B. Sehgal
Keyword(s):  
Cell Cycle ◽  
Epithelial Cells ◽  
Dna Synthesis ◽  
A549 Cells ◽  
Cell Types ◽  
Alveolar Epithelial Cells ◽  
Cell Type ◽  
Alveolar Epithelial ◽  
Unfolded Protein ◽  
Cell Cycle Regulatory Proteins

Monocrotaline (MCT) causes pulmonary hypertension in the rat by a mechanism characterized by megalocytosis (enlarged cells with enlarged endoplasmic reticulum and Golgi and a cell cycle arrest) of pulmonary arterial endothelial (PAEC), arterial smooth muscle, and type II alveolar epithelial cells. In cell culture, although megalocytosis is associated with a block in entry into mitosis in both lung endothelial and epithelial cells, DNA synthesis is stimulated in endothelial but inhibited in epithelial cells. The molecular mechanism(s) for this dichotomy are unclear. While MCTP-treated PAEC and lung epithelial (A549) cells both showed an increase in the “promitogenic” transcription factor STAT3 levels and in the IL-6-induced nuclear pool of PY-STAT3, this was transcriptionally inactive in A549 but not in PAEC cells. This lack of transcriptional activity of STAT3 in A549 cells correlated with the cytoplasmic sequestration of the STAT3 coactivators CBP/p300 and SRC1/NcoA in A549 cells but not in PAEC. Both cell types displayed a Golgi trafficking block, loss of caveolin-1 rafts, and increased nuclear Ire1α, but an incomplete unfolded protein response (UPR) with little change in levels of UPR-induced chaperones including GRP78/BiP. There were discordant alterations in cell cycle regulatory proteins in the two cell types such as increase in levels of both cyclin D1 and p21 simultaneously, but with a decrease in cdc2/cdk1, a kinase required for entry into mitosis. While both cell types showed increased cytoplasmic geminin, the DNA synthesis-initiating protein Cdt1 was predominantly nuclear in PAEC but remained cytoplasmic in A549 cells, consistent with the stimulation of DNA synthesis in the former but an inhibition in the latter cell type. Thus differences in cell type-specific alterations in subcellular trafficking of critical regulatory molecules (such as CBP/p300, SRC1/NcoA, Cdt1) likely account for the dichotomy of the effects of MCTP on DNA synthesis in endothelial and epithelial cells.

scholarly journals Association of methionyl-tRNA synthetase with detergent-insoluble components of the rough endoplasmic reticulum.

1983 ◽  
Vol 96 (4) ◽  
pp. 1138-1147 ◽  
Author(s):  
C V Dang ◽  
D C Yang ◽  
T D Pollard
Keyword(s):  
Endoplasmic Reticulum ◽  
Fluorescent Antibody ◽  
Insoluble Fraction ◽  
Trna Synthetase ◽  
Insoluble Residue ◽  
Synthetase Activity ◽  
Fluorochrome Staining ◽  
Antibody Staining ◽  
Triton X ◽  
Methionyl Trna Synthetase

Using fluorescent antibody staining, we have established the association of methionyl-tRNA synthetase with the endoplasmic reticulum in PtK2 cells. After Triton X-100 extraction, 70% of the recovered aminoacyl-tRNA synthetase activity was found in the detergent-insoluble fraction. This fraction of the enzyme remained localized with insoluble endoplasmic reticulum antigens and with ribosomes, which were stained with acridine orange. By both fluorescence microscopy and electron microscopy the organization of the detergent-insoluble residue was found to depend on the composition of the extracting solution. After extraction with a microtubule-stabilizing buffer containing EGTA, Triton X-100, and polyethylene glycol (Osburn, M., and K. Weber, 1977, Cell, 12:561-571) the ribosomes were aggregated in large clusters with remnants of membranes. After extraction with a buffer containing Triton X-100, sucrose, and CaCl2 (Fulton, A. B., K. M. Wang, and S. Penman, 1980, Cell, 20:849-857), the ribosomes were in small clusters and there were few morphologically recognizable membranes. In both cases the methionyl-tRNA synthetase and some endoplasmic reticulum antigens retained approximately their normal distribution in the cell. Double fluorochrome staining showed no morphological association of methionyl-tRNA synthetase with the microtubule, actin, or cytokeratin fiber systems of PtK2 cells. These observations demonstrate that detergent-insoluble cellular components, sometimes referred to as "cytoskeletal" preparations, contain significant amounts of nonfilamentous material including ribosomes, and membrane residue. Caution is required in speculating about intermolecular associations in such a complex cell fraction.

Immune responses to hapten conjugates in vitro

1975 ◽  
Vol 8 (4) ◽  
pp. 507-522
Author(s):  
Sirkka Kontiainen ◽  
O. Mäkelä ◽  
M. Hurme
Keyword(s):  
Tissue Culture ◽  
Immune Responses ◽  
Cell Types ◽  
Antibody Responses ◽  
Tissue Cultures ◽  
Cell Type ◽  
Animal Body ◽  
Do So

Several functions of the animal body can take place in cell or tissue cultures with almost unreduced efficiency and precision. Functions, where only one cell type is involved, often do so, but also some differentiation steps where interactions between two or more cell types are clearly needed can take place in tissue culture (Saxén et al. 1968).Most immune responses require collaboration between two or more cell types (Claman, Chaperon & Triplett, 1966; Miller & Mitchell, 1968; Feldmann & Nossal 1972c). Some of them can be easily induced in vitro but others cannot. Even when antibody responses can be induced in vitro their intensity varies a great deal. With some antigens and under some circumstances a response in vitro can be nearly as strong as one in vivo. A crude comparison can be derived from responses in vitro and in vivo to the same antigen, conjugate of hapten NIP and pneumococcal polysaccharide type III (NIP-SIll, Nakamura, Ray & Mäkelä, 1973).

Fibronectin, intercellular junctions and the sorting-out of chick embryonic tissue cells in monolayer

1982 ◽  
Vol 54 (1) ◽  
pp. 357-372
Author(s):  
A. Nicol ◽  
D.R. Garrod
Keyword(s):  
Corneal Epithelium ◽  
Fluorescent Antibody ◽  
Intercellular Junctions ◽  
Liver Parenchyma ◽  
Cell Types ◽  
Limb Bud ◽  
Antibody Staining ◽  
Intercellular Contacts ◽  
Mesenchyme Cells ◽  
Chick Embryonic Tissue

A hierarchy of relative cohesiveness in monolayer of four different embryonic chick tissues was determined in a previous study. The hierarchy is: corneal epithelium congruent to liver parenchyma greater than pigmented epithelium greater than limb bud mesenchyme. The purpose of this paper is to describe the correlation between these adhesive relationships and, firstly, the amount of the adhesive glycoprotein, fibronectin, associated with the cells and, secondly, the morphology of their intercellular contacts. Fluorescent antibody staining of the cells with anti-fibronectin antibody showed that limb bud mesenchyme cells, the most weakly cohesive, had much more fibronectin than the other cell types. Thus there was a negative correlation between the amount of fibronectin and cellular cohesiveness. Analysis of intercellular contacts by electron microscopy showed that the most strongly cohesive cell types, corneal epithelium and liver parenchyma, were also those that possessed desmosomes.

scholarly journals Autocrine signals enable chondrocytes to survive in culture.

1994 ◽  
Vol 126 (4) ◽  
pp. 1069-1077 ◽  
Author(s):  
Y Ishizaki ◽  
J F Burne ◽  
M C Raff
Keyword(s):  
Epithelial Cells ◽  
Cell Density ◽  
Mammalian Cells ◽  
Cell Types ◽  
High Density ◽  
Lens Epithelial Cells ◽  
Low Density ◽  
Cell Type ◽  
Adult Rats ◽  
And Cartilage

We recently proposed that most mammalian cells other than blastomeres may be programmed to kill themselves unless continuously signaled by other cells not to. Many observations indicate that some mammalian cells are programmed in this way, but is it the case for most mammalian cells? As it is impractical to test all of the hundreds of types of mammalian cells, we have focused on two tissues--lens and cartilage--which each contain only a single cell type: if there are cells that do not require signals from other cells to avoid programmed cell death (PCD), lens epithelial cells and cartilage cells (chondrocytes) might be expected to be among them. We have previously shown that rat lens epithelial cells can survive in serum-free culture without signals from other cell types but seem to require signals from other lens epithelial cells to survive: without such signals they undergo PCD. We show here that the same is true for rat (and chick) chondrocytes. They can survive for weeks in culture at high cell density in the absence of other cell types, serum, or exogenous proteins or signaling molecules, but they die with the morphological features of apoptosis in these conditions at low cell density. Medium from high density cultures, FCS, or a combination of known growth factors, all support prolonged chondrocyte survival in low density cultures, as long as antioxidants are also present. Moreover, medium from high density chondrocyte cultures promotes the survival of lens epithelial cells in low density cultures and vice versa. Chondrocytes isolated from adult rats behave similarly to those isolated from developing rats. These findings support the hypothesis that most mammalian cells require signals from other cells to avoid PCD, although the signals can sometimes be provided by cells of the same type, at least in tissues that contain only one cell type.

scholarly journals Control of lens epithelial cell survival.

1993 ◽  
Vol 121 (4) ◽  
pp. 899-908 ◽  
Author(s):  
Y Ishizaki ◽  
J T Voyvodic ◽  
J F Burne ◽  
M C Raff
Keyword(s):  
Epithelial Cells ◽  
Cell Types ◽  
Lens Epithelial Cell ◽  
High Density ◽  
Lens Epithelial Cells ◽  
Cell Contact ◽  
Low Density ◽  
First Time ◽  
Cells Cultured ◽  
Do So

We have studied the survival requirements of developing lens epithelial cells to test the hypothesis that most cells are programmed to kill themselves unless they are continuously signaled by other cells not to do so. The lens cells survived for weeks in both explant cultures and high-density dissociated cell cultures in the absence of other cells or added serum or protein, suggesting that they do not require signals from other cell types to survive. When cultured at low density, however, they died by apoptosis, suggesting that they depend on other lens epithelial cells for their survival. Lens epithelial cells cultured at high density in agarose gels also survived for weeks, even though they were not in direct contact with one another, suggesting that they can promote one another's survival in the absence of cell-cell contact. Conditioned medium from high density cultures promoted the survival of cells cultured at low density, suggesting that lens epithelial cells support one another's survival by secreting survival factors. We show for the first time that normal cell death occurs within the anterior epithelium in the mature lens, but this death is strictly confined to the region of the anterior suture.

scholarly journals The cytoplasmic tail of CD44 is required for basolateral localization in epithelial MDCK cells but does not mediate association with the detergent-insoluble cytoskeleton of fibroblasts.

1993 ◽  
Vol 121 (6) ◽  
pp. 1299-1310 ◽  
Author(s):  
S J Neame ◽  
C M Isacke
Keyword(s):  
Epithelial Cells ◽  
Mdck Cells ◽  
Constitutive Expression ◽  
Cytoplasmic Tail ◽  
Apical Surface ◽  
Wild Type ◽  
Coated Pits ◽  
Triton X ◽  
Cortical Cytoskeleton ◽  
Human Specific

A number of recent reports on the trafficking of receptor proteins in MDCK epithelial cells have provided evidence that delivery to the basolateral domain requires a specific targeting sequence and that deletion of this sequence results in constitutive expression on the apical surface. To date, these studies have concentrated on receptors which are competent for internalization via the clathrin coated pits. We have examined the localization of a resident plasma membrane protein by transfecting human CD44 into MDCK cells. Using human specific and cross-species reactive antibodies, we show that in MDCK cells both the endogenous and transfected wild-type CD44 are found on the basolateral surface where they are restricted to the lateral domain. Deletion of the CD44 cytoplasmic tail reduces the half life of this mutant protein and causes it to be expressed both on the apical surface and to a significant extent within the cell. We have also used biochemical and morphological analysis to investigate the interaction of CD44 with the cytoskeleton in detergent extracted cells. Strikingly different extraction results were obtained between epithelial and fibroblast cells. However, there is no difference in the Triton X-100 solubility of the transfected wild-type and tail-less CD44 in fibroblasts and both forms of the protein remain associated with the cortical cytoskeleton after Triton X-100 extraction. These results demonstrate that the sequence present in the cytoplasmic domain of CD44 responsible for its distribution in epithelial cells is functionally and spatially separate from the ability of this protein to associate with the cytoskeleton.

Lixazinone Stimulates Mitogenesis of Madin-Darby Canine Kidney Cells

2006 ◽  
Vol 231 (3) ◽  
pp. 288-295 ◽  
Author(s):  
Jingfei Cheng ◽  
Michael A. Thompson ◽  
Henry J. Walker ◽  
Catherine E. Gray ◽  
Gina M. Warner ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Mdck Cells ◽  
Cell Types ◽  
Total Activity ◽  
Kinase Assay ◽  
Intracellular Camp ◽  
Tubular Epithelial Cells ◽  
Renal Tubular Epithelial Cells ◽  
Renal Tubular

Polycystic kidney diseases (PKD) are characterized by excessive proliferation of renal tubular epithelial cells, development of fluid-filled cysts, and progressive renal insufficiency. cAMP inhibits proliferation of normal renal tubular epithelial cells but stimulates proliferation of renal tubular epithelial cells derived from patients with PKD. Madin-Darby canine kidney (MDCK) epithelial cells, which are widely used as an in vitro model of cystogenesis, also proliferate in response to cAMP. Intracellular cAMP levels are tightly regulated by phosphodiesterases (PDE). Isoform-specific PDE inhibitors have been developed as therapeutic agents to regulate signaling pathways directed by cAMP. In other renal cell types, we have previously demonstrated that cAMP is hydrolyzed by PDE3 and PDE4, but only PDE3 inhibitors suppress proliferation by inhibiting Raf-1 activity (Cheng J, Thompson MA, Walker HJ, Gray CE, Diaz Encarnacion MM, Warner GM, Grande JP. Am J Physiol Renal Physiol 287:F940-F953, 2004.) A potential role for PDE isoform(s) in cAMP-mediated proliferation of MDCK cells has not previously been established. Similar to what we have previously found in several other renal cell types, cAMP hydrolysis in MDCK cells is directed primarily by PDE4 (85% of total activity) and PDE3 (15% of total activity). PDE4 inhibitors are more effective than PDE3 inhibitors in increasing intracellular cAMP levels in MDCK cells. However, only PDE3 inhibitors, and not PDE4 inhibitors, stimulate mitogenesis of MDCK cells. PDE3 but not PDE4 inhibitors activate B-Raf but not Raf-1, as assessed by an in vitro kinase assay. PDE3 but not PDE4 inhibitors activate the ERK pathway and activate cyclins D and E, as assessed by histone H1 kinase assay. We conclude that mitogenesis of MDCK cells is regulated by a functionally compartmentalized intracellular cAMP pool directed by PDE3. Pharmacologic agents that stimulate PDE3 activity may provide the basis for new therapies directed toward reducing cystogenesis in patients with PKD.

Sign in / Sign up

Export Citation Format

Share Document