Disruption of epithelial tight junctions by yeast enhances the paracellular delivery of a model protein.
OBJECTIVE
The aim of this study was to investigate the effect of heat-killed yeast cells on the integrity of epithelial tight junctions in vitro.


METHODS
Changes in barrier potential of Caco-2 cell monolayers were assessed by transepithelial electrical resistance (TEER) measurements and by an increasing permeability to a marker protein, horse-radish peroxidase (HRP). Visualisation of tight junction disruption was carried out directly through electron microscopy and indirectly through fluorescence confocal microscopy and immunoblotting of the tight junction-associated proteins zonula occludens ZO-1, occludin and actin.


RESULTS
Yeast cells opened tight junctions in a reversible dose- and time-dependent manner, as shown by a decrease in TEER and an increase in HRP permeability. These changes to barrier potential were shown not to be due to cytotoxic effects but due to modulation of the tight junctions. ZO-1, actin and occludin proteins were demonstrated to be involved in yeast-induced tight junction opening through the use of confocal microscopy and western blotting. Electron microscopy confirmed a direct opening of tight junctions after application of yeast.


CONCLUSIONS
Yeast modulated epithelial tight junctions in a reversible manner by contraction of the actin cytoskeleton and shift of ZO-1 and occludin tight junction proteins from the membrane to cytoskeletal areas of the cell.
Fuller
Emily
E
School of Biochemistry and Microbiology, Garstang Building, University of Leeds, Leeds, LS2 9JT, UK. fulleremily@yahoo.co.uk
Duckham
Craig
C
Wood
Edward
E
D000199:Actins / Q000378:metabolism; D018938:Caco-2 Cells; D002463:Cell Membrane Permeability; D002470:Cell Survival; D003599:Cytoskeleton / Q000378:metabolism; D016503:Drug Delivery Systems; D017097:Electric Impedance; D004594:Electrophysiology; D004847:Epithelial Cells / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D006735:Horseradish Peroxidase / Q000008:administration & dosage* / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D062793:Occludin; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D013347:Subcellular Fractions / Q000378:metabolism; D019108:Tight Junctions / Q000378:metabolism / Q000502:physiology*
D000199:Actins; D005456:Fluorescent Dyes; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D006735:Horseradish Peroxidase