Nonspecific depolarization of the plasma membrane potential induces cytoskeletal modifications of bovine corneal endothelial cells in culture.
Modifications in the cell membrane potential have been suggested to affect signaling mechanisms participating in diverse cellular processes, many of which involve structural cellular alterations. In order to contribute some evidence in this respect, we explored the effects of several depolarizing procedures on the structure and monolayer organization of bovine corneal endothelial cells in culture. Visually confluent cell monolayers were incubated with or without the depolarizing agent, either in a saline solution or in culture medium for up to 30 min. Membrane potential was monitored by fluorescence microscopy using oxonol V. Fluorescent probes were employed for F-actin, microtubules, and vinculin. Depolarization of the plasma membrane, achieved via the incorporation of gramicidin D into confluent endothelial cells or by modifications of the extracellular saline composition, provoked an increment of oxonol fluorescence and changes in cell morphology, consisting mainly of modifications in the cytoskeletal organization. In some areas, noticeable intercellular spaces appear. The cytoskeleton modifications mainly consist of a marked redistribution of F-actin and microtubules, with accompanying changes in vinculin localization. The results suggest that the depolarization of the plasma membrane potential may participate in mechanisms involved in cytoskeleton organization and monolayer continuity in corneal endothelial cells in culture.
Chifflet
Silvia
S
Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Gral Flores 2125, 11800 Montevideo, Uruguay. schiffle@mednet.org.uy
Hernández
Julio A
JA
Grasso
Silvina
S
Cirillo
Angela
A
D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D000818:Animals; D002417:Cattle; D002450:Cell Communication / Q000187:drug effects / Q000502:physiology*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D002478:Cells, Cultured; D002794:Choline / Q000494:pharmacology; D003315:Cornea / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D005942:Gluconates / Q000494:pharmacology; D006096:Gramicidin / Q000494:pharmacology; D007150:Immunohistochemistry; D007555:Isoxazoles; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D012965:Sodium Chloride / Q000378:metabolism; D016596:Vinculin / Q000187:drug effects / Q000378:metabolism
D005942:Gluconates; D007555:Isoxazoles; D016596:Vinculin; D006096:Gramicidin; D012965:Sodium Chloride; C013633:oxonol V; D002794:Choline; C030691:gluconic acid