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.

scholarly journals Identification of desmosomal surface components (desmocollins) and inhibition of desmosome formation by specific Fab'

1984 ◽  
Vol 70 (1) ◽  
pp. 41-60
Author(s):  
P. Cowin ◽  
D. Mattey ◽  
D. Garrod
Keyword(s):  
Cell Surface ◽  
Fluorescent Antibody ◽  
Protein A ◽  
Antibody Staining ◽  
Quantitative Measurements ◽  
Mdbk Cells ◽  
Cell Surface Staining ◽  
Lateral Constraint ◽  
Gold Labelling ◽  
Fluorescent Antibody Staining

Specific antibodies against the components of desmosomes, the adhesive junctions of epithelial cells, have been used to determine which components are located on the cell surface. Three criteria have been used: fluorescent antibody staining, immuno-gold labelling and electron microscopy, and quantitative measurements of antibody binding using [125I]protein A. When these techniques were applied to living Madin-Darby bovine kidney (MDBK) cells, antibodies against only two desmosomal components, glycoproteins of approximately 115 X 10(3) Mr and 100 X 10(3) Mr, bound to the cell surface. Antibodies against all other components, the 230 and 205 X 10(3) Mr proteins (desmoplakins), the 150 X 10(3) Mr glycoprotein and the 82 and 86 X 10(3) Mr proteins reacted in fluorescent antibody staining only after cells had been fixed and made permeable. MDBK cells were cultured in the presence of univalent fragments (Fab') of anti-desmosomal antibodies for periods from 24 h to 72 h. After these times cells were fixed, made permeable, and stained with anti-desmoplakin antibody to assay for desmosome formation. Fab' derived from anti-100 X 10(3) Mr protein specifically inhibited desmosome formation, whereas Fab's from anti-desmoplakin, anti-150 X 10(3) Mr and anti-82 and 86 X 10(3) Mr proteins were without effect. We conclude that the 100 X 10(3) Mr and the immunologically related 115 X 10(3) Mr components are located on the cell surface and are directly involved in cell-cell adhesion. We have named them desmocollins to denote that they are involved in the adhesive function of desmosomes. The modulation of desmocollin distribution during monolayer formation and establishment of epithelial polarity has also been studied. Fluorescent and immuno-gold labelling using Fab' or IgG at 4 degrees C revealed that desmocollins were initially evenly dispersed over the cell surface. Staining with IgG at 37 degrees C caused the desmocollins to “patch' but not to “cap'. With the establishment of confluency, desmocollins were gradually removed from the upper surfaces of the cells (or masked and rendered inaccessible to antibody) being confined to the lateral and probably basal regions of the cells. Treatment of confluent monolayers with 3 mM-EGTA rendered the desmocollins stainable, probably by causing their release from lateral constraint. Desmocollin staining at the cell surface was not appreciably reduced during 5 h of EGTA treatment, suggesting that desmocollins, unlike desmosomal plaques, may not be internalized after junction breakdown.

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.

An investigation of the molecular components of desmosomes in epithelial cells of five vertebrates

1986 ◽  
Vol 81 (1) ◽  
pp. 223-242 ◽  
Author(s):  
A. Suhrbier ◽  
D. Garrod
Keyword(s):  
Molecular Weight ◽  
Mdck Cells ◽  
Fluorescent Antibody ◽  
Cross Reactivity ◽  
Protein Domain ◽  
Nasal Epithelium ◽  
Molecular Weights ◽  
Antibody Staining ◽  
A Cell ◽  
Molecular Components

We have shown previously, by fluorescent antibody staining, that desmosomal antigens are widely distributed in the tissues of vertebrate animals. Furthermore, we have demonstrated mutual desmosome formation between cells derived from man, cow, dog, chicken and frog. In this paper we have studied the components of desmosomes in a tissue or a cell line from each of these animals by immunoblotting with antibodies raised against the desmosomal components isolated from bovine nasal epithelium. Blotting was carried out on bovine nasal epithelial desmosomal cores, desmosome-enriched fractions derived from chicken and frog epidermis, nuclear matrix-intermediate filament scaffolds derived from Madin-Darby bovine and canine cells (MDBK and MDCK), and unextracted cultured human foreskin keratinocytes. The results show that desmosomes from all these sources contain high molecular weight proteins (desmoplakins) of similar or identical molecular weights (250 000 and 215 000). Antibodies against the two lower molecular weight desmosomal proteins (83 000 and 75 000) always recognized one or two bands in very similar molecular weight regions of the gels. The desmosomal glycoproteins were found to be much more variable than the proteins: they vary between sources in molecular weight, heterogeneity and antibody cross-reactivity. For instance, antibody specific for a group of glycoprotein bands of 175 000, 169 000 and 164 000 (Mr) in bovine nasal epithelium recognizes three bands of 245 000, 230 000 and 210 000 in MDCK cells but only a single band of 190 000 in keratinocytes. In mammals, the 175 000–164 000 glycoproteins and the desmosomal adhesion molecules, the desmocollins (Mr 130 000 and 115 000 in cow's nose), are immunologically distinct. In chicken and frog, however, there are glycoproteins that react with both anti-175 000–164 000 and anti-desmocollin antibodies, but there are also distinct desmocollin bands. The significance of these results is discussed in relation to conservation of desmosomal components and adhesion mechanisms. It is suggested that adhesion may be performed by a well-conserved protein domain and that the variation between desmosomal glycoproteins from different sources may be due to differences in their carbohydrate composition.

A Most Probable Number Method for the Enumeration of Legionella Bacteria in Water

1991 ◽  
Vol 24 (2) ◽  
pp. 143-147 ◽  
Author(s):  
N. A. Grabow ◽  
R. Kfir ◽  
W. O. K. Grabow
Keyword(s):  
Water Samples ◽  
Membrane Filtration ◽  
Quantitative Method ◽  
Fluorescent Antibody ◽  
Most Probable Number ◽  
Probable Number ◽  
Comparative Tests ◽  
Antibody Staining ◽  
Direct Fluorescent Antibody ◽  
Yeast Extract Agar

A new quantitative method for the enumeration of Legionella bacteria in water is described. Appropriate tenfold serial dilutions of water samples concentrated by membrane filtration are plated in triplicate on buffered charcoal yeast extract agar. After incubation for 3 days representative smears from individual plates are tested for the presence of Legionella by direct fluorescent antibody staining. The number of positive plates in each dilution is used to calculate the Legionella count by means of conventional most probable number statistics. In comparative tests on a variety of water samples this method yielded significantly higher counts than previously used procedures.

scholarly journals Remodeling the cell surface distribution of membrane proteins during the development of epithelial cell polarity.

1992 ◽  
Vol 116 (4) ◽  
pp. 889-899 ◽  
Author(s):  
D A Wollner ◽  
K A Krzeminski ◽  
W J Nelson
Keyword(s):  
Epithelial Cell ◽  
Membrane Proteins ◽  
Cell Surface ◽  
Mdck Cells ◽  
Apical Cell ◽  
Cell Contact ◽  
Surface Polarity ◽  
Lateral Membrane ◽  
E Cadherin ◽  
Cell Cell

The development of polarized epithelial cells from unpolarized precursor cells follows induction of cell-cell contacts and requires resorting of proteins into different membrane domains. We show that in MDCK cells the distributions of two membrane proteins, Dg-1 and E-cadherin, become restricted to the basal-lateral membrane domain within 8 h of cell-cell contact. During this time, however, 60-80% of newly synthesized Dg-1 and E-cadherin is delivered directly to the forming apical membrane and then rapidly removed, while the remainder is delivered to the basal-lateral membrane and has a longer residence time. Direct delivery of greater than 95% of these proteins from the Golgi complex to the basal-lateral membrane occurs greater than 48 h later. In contrast, we show that two apical proteins are efficiently delivered and restricted to the apical cell surface within 2 h after cell-cell contact. These results provide insight into mechanisms involved in the development of epithelial cell surface polarity, and the establishment of protein sorting pathways in polarized cells.

Intracellular traffic of the ecto-nucleotide pyrophosphatase/phosphodiesterase NPP3 to the apical plasma membrane of MDCK and Caco-2 cells: apical targeting occurs in the absence of N-glycosylation

2000 ◽  
Vol 113 (23) ◽  
pp. 4193-4202 ◽  
Author(s):  
N.R. Meerson ◽  
V. Bello ◽  
J.L. Delaunay ◽  
T.A. Slimane ◽  
D. Delautier ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mdck Cells ◽  
Transmembrane Proteins ◽  
Cell Types ◽  
Apical Plasma Membrane ◽  
Apical Surface ◽  
Intracellular Traffic ◽  
Transmembrane Glycoprotein ◽  
Direct Demonstration ◽  
Targeting Signal

Glycosylation was considered the major signal candidate for apical targeting of transmembrane proteins in polarized epithelial cells. However, direct demonstration of the role of glycosylation has proved difficult because non-glycosylated apical transmembrane proteins usually do not reach the cell surface. Here we were able to follow the targeting of the apical transmembrane glycoprotein NPP3 both when glycosylated and non-glycosylated. Transfected in polarized MDCK and Caco-2 cells, NPP3 was exclusively expressed at the apical membrane. The transport kinetics of the protein to the cell surface were studied after metabolic (35)S-labeling and surface immunoprecipitation. The newly synthesized protein was mainly targeted directly to the apical surface in MDCK cells, whereas 50% transited through the basolateral surface in Caco-2 cells. In both cell types, the basolaterally targeted pool was effectively transcytosed to the apical surface. In the presence of tunicamycin, NPP3 was not N-glycosylated. The non-glycosylated protein was partially retained intracellularly but the fraction that reached the cell surface was nevertheless predominantly targeted apically. However, transcytosis of the non-glycosylated protein was partially impaired in MDCK cells. These results provide direct evidence that glycosylation cannot be considered an apical targeting signal for NPP3, although glycosylation is necessary for correct trafficking of the protein to the cell surface.

Ingestion and survival of Y. pseudotuberculosis in HeLa cells

1975 ◽  
Vol 21 (12) ◽  
pp. 1997-2007 ◽  
Author(s):  
Åke Bovallius ◽  
Gustaf Nilsson
Keyword(s):  
Cell Culture ◽  
Hela Cells ◽  
Yersinia Pseudotuberculosis ◽  
Fluorescent Antibody ◽  
Cell System ◽  
Viable Count ◽  
Linear Increase ◽  
Intracellular Bacteria ◽  
Antibody Staining ◽  
Upper Limit

HeLa cells were infected with Yersinia pseudotuberculosis for 0.5–3 h. Intracellular bacteria could then be demonstrated by three different techniques: viable count, fluorescent-antibody staining, and electron microscopy. Most of the bacteria seemed to be viable, since there was a good positive correlation (0.94) between viable and fluorescent bacteria. The bacterial uptake seemed to be mediated by a phagocytic-like procedure. The intracellular bacteria seemed to reside in vacuoles some of which increased in size as a function of time. The kinetics of infection was studied after addition of 107 or 109 bacteria per cell culture (2 × 106 cells). After a lag period of about 30 min there was a linear increase of intracellular bacteria, and this uptake proceeded for 1–2 h until most of the bacteria were ingested or an upper limit of ingested bacteria was reached. The upper limit was calculated to be a mean of 60 per infected cell in the cell culture. More than 90% of the cells could be infected and a reasonable number of the bacteria survive in the cells for at least 3 days, as demonstrated by the viable-count technique.The bacteria–cell system may be used to study, for example, the effect of antibiotics or antibodies on intracellular bacteria and pathogenicity of intracellular diseases.

scholarly journals The effect of Malaysian stingless bee, Trigona spp. honey in promoting proliferation of the undifferentiated stem cell

2019 ◽  
pp. 10-19 ◽  
Author(s):  
Mohd Amin Marwan Mohamad ◽  
Muhammad Alif Mazlan ◽  
Muhammad Ibrahim ◽  
Afzan Mat Yusof ◽  
Shamsul Azlin Ahmad Shamsuddin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Culture Media ◽  
Proliferative Effect ◽  
Natural Medicine ◽  
Ability To Regenerate ◽  
Potential Applications ◽  
Standard Culture ◽  
The Stability ◽  
Dose Dependent ◽  
Standard Culture Medium

Stem cells provide various potential applications in regenerative medicine through its ability of self-renewal and differentiation. Among the various stem cells, dental pulp stem cells (DPSCs) have shown encouraging results in their ability to regenerate. Honey has been used in traditional culture as a natural medicine in supporting wound healing. Yet, very few studies on honey were conducted for its potential as a proliferative agent for stem cells. The aim of this study is to evaluate the stability of two Trigona spp. honeys (1 and 2) added in culture media and its proliferative effect on DPSCs. Both honeys were diluted with standard culture medium through dilution process to prepare the concentrations of 0.01%, 0.04%, 0.10% and 0.25%. DPSCs were treated with the diluted honeys for 24 hours. The proliferative activity was determined through the images taken using an inverted microscope for every six hours. In addition, the MTT assay was conducted to determine the cell viability of DPSCs when treated with both honey 1 and 2 at various concentrations. The results showed a stable culture media added with honey for three days and a dose-dependent proliferative effect of both Trigona spp. honey samples on DPSCs. Optimum proliferative effects were observed at 24 hours for both Trigona spp. honey 1 and 2 on DPSCs. The optimum concentration of Trigona spp. honey 1 was from 0.04% to 0.10% and Trigona spp. honey 2 was below 0.01%. It is concluded that Trigona spp. honey has a promising proliferative effect on DPSCs.

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