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 have used conventional and confocal microscopy of living cells and immunohistochemistry to examine the migration and cytoskeleton of chick embryo corneal fibroblasts electroporated while cultured within collagen gels. Fibroblasts cultured in collagen (1 mg/ml) are successfully electroloaded (0.5-1.0 kVcm-1/960 microF 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 depolymerization of microtubules and/or a perinuclear collapse of vimentin filaments, seemingly caused by millimolar levels of Ca2+ ions derived from culture medium following electroporation. Fibroblasts loaded in a buffer which resembles intracellular fluid (< or = 10 microM Ca2+) maintain their cytoskeleton and continue to migrate, when returned to culture medium within 10 min. Using this novel approach, we have loaded fibroblasts migrating through extracellular matrix (ECM) with rhodamine phalloidin and monitored the behavior of the labeled actin cortex by confocal microscopy. During migration phalloidin-actin accumulates 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 tubulin cytoskeleton.
Harkin
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DG
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115-5729, USA.
Hay
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D000199:Actins / Q000378:metabolism; D000818:Animals; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D002642:Chick Embryo; D003094:Collagen / Q000502:physiology; D003315:Cornea / Q000166:cytology; D003593:Cytoplasm / Q000201:enzymology; D018274:Electroporation; D005347:Fibroblasts / Q000187:drug effects; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D008870:Microtubules / Q000378:metabolism; D011554:Pseudopodia / Q000502:physiology; D012996:Solutions / Q000494:pharmacology; D013997:Time Factors; D014404:Tubulin / Q000378:metabolism*; D014746:Vimentin / Q000378:metabolism
D000199:Actins; D012996:Solutions; D014404:Tubulin; D014746:Vimentin; D003094:Collagen