Emily Fuller1, Craig Duckham, Edward Wood. 1. School of Biochemistry and Microbiology, Garstang Building, University of Leeds, Leeds, LS2 9JT, UK. fulleremily@yahoo.co.uk
Abstract
PURPOSE: 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. CONCLUSION: 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.
PURPOSE: 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. CONCLUSION: 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.
Authors: A F Kotzé; H L Luessen; B J de Leeuw; A G de Boer; J C Verhoef; H E Junginger Journal: J Control Release Date: 1998-01-23 Impact factor: 9.776
Authors: Yasuhiro Kimura; Marie van der Merwe; Stine B Bering; Himabindu Penmatsa; Veronica G Conoley; Per T Sangild; Anjaparavanda P Naren; Randal K Buddington Journal: Cytotechnology Date: 2013-10-29 Impact factor: 2.058