A spectroscopic method for assessing confluence of epithelial cell cultures

1991 ◽  
Vol 261 (6) ◽  
pp. C1196-C1203 ◽  
Author(s):  
B. Jovov ◽  
N. K. Wills ◽  
S. A. Lewis
Keyword(s):  
Epithelial Cell ◽  
Tight Junction ◽  
Culture Media ◽  
Spectroscopic Method ◽  
Isosbestic Point ◽  
Epithelial Cell Line ◽  
Phenol Red ◽  
Renal Epithelial Cell ◽  
Tight Junction Formation ◽  
Junction Formation

We describe a convenient nonelectrophysiological technique for assessing cell proliferation and subsequent tight junction formation for epithelial monolayers grown on permeable supports. The method involves the use of phenol red (PR), a standard pH indicator in most cell culture media. In addition, we report a systematic error in a commercially available system for measuring transepithelial electrical properties. Briefly, the flux of PR across the epithelium was measured from the serosal solution into the mucosal solution. The mucosal solution was first replaced with a PR-free solution and then collected at timed intervals. The PR concentration was measured using a spectrophotometer set at the isosbestic point for PR (479 nm). PR flux was then calculated and used as an index of the permeability of the epithelium to PR. This method was tested using the renal epithelial cell line A6. After cell seeding, PR flux decreased in two phases: an initial large decrease, associated with cell growth and monolayer confluence, and a second decrease associated with tight junction formation [assessed by measuring transepithelial conductance (Gt)]. In addition to monitoring tight junction formation, PR flux measurements were also used to estimate the net movement of solution by the epithelial cells between the mucosal and serosal compartments. For convenience, Gt was initially measured in culture dishes using a commercially available “chopstick” electrode system. However, the chopstick system yielded Gt values that were on average 51% lower than values for the same preparations when measured in standard Ussing-type chambers. The discrepancy was due to a nonuniform current field produced by the chopstick electrodes.

scholarly journals Participation of plasma membrane proteins in the formation of tight junction by cultured epithelial cells

1983 ◽  
Vol 96 (3) ◽  
pp. 693-702 ◽  
Author(s):  
EB Griepp ◽  
WJ Dolan ◽  
ES Robbins ◽  
DD Sabatini
Keyword(s):  
Protein Synthesis ◽  
Steady State ◽  
Tight Junction ◽  
Messenger Rna ◽  
Freeze Fracture ◽  
Epithelial Cell Line ◽  
Experimental Conditions ◽  
Tight Junction Formation ◽  
Junction Formation ◽  
Spinner Culture

Measurements of the transepithelial electrical resistance correlated with freeze-fracture observations have been used to study the process of tight junction formation under various experimental conditions in monolayers of the canine kidney epithelial cell line MDCK. Cells derived from previously confluent cultures and plated immediately after trypsin- EDTA dissociation develop a resistance that reaches its maximum value of several hundred ohms-cm(2) after approximately 24 h and falls to a steady-state value of 80-150 ohms- cm(2) by 48 h. The rise in resistance and the development of tight junctions can be completely and reversibly prevented by the addition of 10 μg/ml cycloheximide at the time of plating, but not when this inhibitor is added more than 10 h after planting. Thus tight junction formation consists of separable synthetic and assembly phases. These two phases can also be dissociated and the requirement for protein synthesis after plating eliminated if, following trypsinization, the cells are maintained in spinner culture for 24 h before plating. The requirement for protein synthesis is restored, however, if cells maintained in spinner culture are treated with trypsin before plating. Actinomycin D prevents development of resistance only in monolayers formed from cells derived from sparse rather than confluent cultures, but new mRNA synthesis is not required if cells obtained from sparse cultures are maintained for 24 h in spinner culture before plating. Once a steady-state resistance has been reached, its maintenance does not require either mRNA or protein synthesis; in fact, inhibition of protein synthesis causes a rise in the resistance over a 30-h period. Following treatments that disrupt the junctions in steady- state monolayers recovery of resistance also does not require protein synthesis. These observations suggest that proteins are involved in tight junction formation. Such proteins, which do not turn over rapidly under steady-state conditions, are destroyed by trypsinization and can be resynthesized in the absence of stable cell-cell or cell-substratum contact. Messenger RNA coding for proteins involved in tight junction formation is stable except when cells are sparsely plated, and can also be synthesized without intercellular contacts or cell-substratum attachment.

scholarly journals Shank2 Binds to aPKC and Controls Tight Junction Formation with Rap1 Signaling during Establishment of Epithelial Cell Polarity

Cell Reports ◽  
2020 ◽  
Vol 31 (1) ◽  
pp. 107407
Author(s):  
Kazunori Sasaki ◽  
Noriko Kojitani ◽  
Hiroko Hirose ◽  
Yohei Yoshihama ◽  
Hidefumi Suzuki ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Tight Junction ◽  
Cell Polarity ◽  
Epithelial Cell Polarity ◽  
Tight Junction Formation ◽  
Junction Formation

scholarly journals The MAGUK Protein MPP7 Binds to the Polarity Protein hDlg1 and Facilitates Epithelial Tight Junction Formation

2007 ◽  
Vol 18 (5) ◽  
pp. 1744-1755 ◽  
Author(s):  
Volker M. Stucke ◽  
Evy Timmerman ◽  
Joel Vandekerckhove ◽  
Kris Gevaert ◽  
Alan Hall
Keyword(s):  
Epithelial Cell ◽  
Tight Junction ◽  
Tight Junctions ◽  
Cell Polarity ◽  
Functional Organization ◽  
Epithelial Cell Polarity ◽  
Tight Junction Formation ◽  
Evolutionarily Conserved ◽  
Junction Formation ◽  
Epithelial Tight Junction

Three groups of evolutionarily conserved proteins have been implicated in the establishment of epithelial cell polarity: the apically-localized proteins of the Par (Par3-Par6-aPKC-Cdc42) and Crumbs groups (Crb3-PALS1-PATJ) and the basolaterally localized proteins of the Dlg group (Dlg1-Scribble-Lgl). During epithelial morphogenesis, these proteins participate in a complex network of interdependent interactions that define the position and functional organization of adherens junctions and tight junctions. However, the biochemical pathways through which they control polarity are poorly understood. In this study, we identify an interaction between endogenous hDlg1 and MPP7, a previously uncharacterized MAGUK-p55 subfamily member. We find that MPP7 targets to the lateral surface of epithelial cells via its L27N domain, through an interaction with hDlg1. Loss of either hDlg1 or MPP7 from epithelial Caco-2 cells results in a significant defect in the assembly and maintenance of functional tight junctions. We conclude that the formation of a complex between hDlg1 and MPP7 promotes epithelial cell polarity and tight junction formation.

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