Abstract:
The fungal toxin sporidesmin causes photosensitization of livestock through hepatobiliary damage that blocks excretion of light absorbing chlorophyll and its degradation products. The effects of sporidesmin on cultured liver cells include disruption of actin microfilament stress fibers, induced filopod formation, thickening of cortical actin and cell rounding followed by the loss of cell adhesion. To investigate the dynamics of actin stress fiber disruption and its relationship to the loss of cell adhesion, we investigated change in actin filaments in HepG2 cells treated with 1μg.ml-1 sporidesmin. Confocal fluorescence microscopy of cells exposed to sporidesmin for time course showed early disruption of stress fibers. However actin microfilaments underlying the cell cortex appeared more abundant with increased exposure time to the toxin that suggested the specific disruption of stress fibers. Other early events were pronounced filopod extension and the loss of cell-cell adhesion, followed by cell rounding and detachment from the substratum.
To investigate the role of actin microfilament disruption on the loss of cell adhesion, we used the cell-permeant drug Jasplakinolide (JAS) to stabilize actin microfilaments in HepG2 cells. JAS blocked binding of phalloidin to actin and therefore indirect immunofluorescence was additionally used to detect actin microfilaments using tropomyosin as a marker.
Pretreatment with JAS blocked sporidesmin-induced loss of cell adhesion that requires the integrity of an intact actin cytoskeleton. These results suggest that sporidesmin-induced loss of cell adhesion is secondary to, and dependent upon, the disruption of stress fibers.
The pattern of sporidesmin-induced change strongly resembles the effects of modification of some proteins of the RhoA pathway. Most notably the expression of inactive LIM-Kinase are known to result in stress fiber disruption, filopod formation and thickening of cortical actin followed by cell rounding. These changes are similar to those caused by sporidesmin and suggests that the RhoA pathway should be investigated further as a target of this toxin.
