EFFECTS OF ELECTROPORATION ON THE TUBULIN CYTOSKELETON AND DIRECTED MIGRATION OF CORNEAL FIBROBLASTS CULTURED WITHIN COLLAGEN MATRICES

Electroporation provides a useful method for loading fibroblasts with fluorescent probes for the cytoskeleton, but the possible deleterious effects of this loading technique on cell motility are unknown. We hav e used conventional and confocal microscopy of living cells and immuno histochemistry to examine the migration and cytoskeleton of chick embr yo corneal fibroblasts electroporated while cultured within collagen g els. Fibroblasts cultured in collagen (1 mg/ml) are successfully elect roloaded (0.5-1.0 kV cm(-1)/960 mu F in DMEM/F12/20 mM Hepes, pH 7.2) with dextran (4-150 kDa) and immunoglobulin, but subsequently display uncoordinated pseudopodia and hence are unable to migrate effectively in any one direction. The lack of directed movement is due to depolyme rization of microtubules and/or a perinuclear collapse of vimentin fil aments, seemingly caused by millimolar levels of Ca2+ ions derived fro m culture medium following electroporation. Fibroblasts loaded in a bu ffer which resembles intracellular fluid (less than or equal to 10 mu M Ca2+) maintain their cytoskeleton and continue to migrate, when retu rned to culture medium within 10 min. Using this novel approach, we ha ve loaded fibroblasts migrating through extracellular matrix (ECM) wit h rhodamine phalloidin and monitored the behavior of the labeled actin cortex by confocal microscopy. During migration phalloidin-actin accu mulates near the base of pseudopodia and at the rear of the cell where it is subsequently left behind. We conclude that electroporation is a valuable technique for loading fibroblasts to study migration within ECM, provided that the conditions used support stability of the tubuli n cytoskeleton. (C) 1996 Wiley-Liss, Inc.