Basolateral but not apical application of protease results in a rapid rise of transepithelial electrical resistance and formation of aberrant tight junction strands in MDCK cells.
In the presence of Ca2+, application of trypsin to the basolateral surface of confluent MDCK cell monolayers with formed tight junctions (TJ), induces the formation of basolaterally oriented aberrant TJ strands. Induction of aberrant TJ strands is accompanied by an increase in transepithelial electrical resistance (TER), up to 90%, which upon addition of trypsin inhibitor is maintained for up to 1 h. Thereafter TER returns slowly to baseline values. Under similar conditions, application of trypsin to the apical surface has little or no effect on either TER or the number of aberrant TJ strands. Confocal microscopy of monolayers, immunostained for ZO-1, revealed that this TJ associated cytoplasmic protein, extended below the TJ along the basolateral surface following brief exposure to trypsin. Removing Ca2+ after treatment of the monolayer with basolaterally applied trypsin resulted, after 20 min, in the increased partitioning of TJ particles onto the E fracture face, of both normal and aberrant TJ strands. Like the TJ strands themselves, therefore, aberrant strands may be linked to cytoskeletal elements. Aberrant TJ strands do not form when monolayers, maintained in low Ca2+ medium, are exposed to trypsin, suggesting that under these conditions TJ precursors, and/or trypsin-sensitive proteins regulating TJ strand assembly, are sequestered in a vesicular compartment that is inaccessible to exogenous trypsin. Prolonged exposure of the apical surface of an established, polarized epithelium with intact TJ to trypsin, had little effect on TJ integrity and did not induce aberrant strands.
Lynch
R D
RD
Department of Biological Sciences, University of Massachusetts at Lowell, USA.
Tkachuk-Ross
L J
LJ
McCormack
J M
JM
McCarthy
K M
KM
Rogers
R A
RA
Schneeberger
E E
EE
D000818:Animals; D002118:Calcium / Q000378:metabolism; D016764:Cell Polarity / Q000502:physiology*; D002478:Cells, Cultured; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004553:Electric Conductivity; D004847:Epithelial Cells; D004848:Epithelium / Q000187:drug effects / Q000502:physiology; D005614:Freeze Fracturing; D007365:Intercellular Junctions / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D007668:Kidney / Q000166:cytology / Q000502:physiology; D008565:Membrane Proteins / Q000302:isolation & purification; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D010750:Phosphoproteins / Q000302:isolation & purification; D014357:Trypsin / Q000494:pharmacology*; D062826:Zonula Occludens-1 Protein
D008565:Membrane Proteins; D010750:Phosphoproteins; D062826:Zonula Occludens-1 Protein; D014357:Trypsin; D002118:Calcium