Effect of membrane potential depolarization on the organization of the actin cytoskeleton of eye epithelia. The role of adherens junctions.
We have previously determined that the depolarization of the plasma membrane potential of confluent bovine corneal endothelial cells in culture, provokes a characteristic reorganization of the actin cytoskeleton. The purposes of the present work are to investigate whether similar responses are exhibited by other epithelia, irrespectively of their specific functions and embryonic origin, and to test the hypothesis that the cytoskeletal reorganization induced by membrane depolarization requires of a well-organized circumferential actin disposition in order to take place. For this, we have performed studies on three different cultured epithelia of the eye: bovine corneal endothelium, bovine retinal pigment epithelium and cellular lines of murine lens epithelium. For all of these cells, we explored the effects of plasma membrane depolarization, achieved via the incorporation of gramicidin D to the bathing media or by the replacement of extracellular sodium chloride by potassium gluconate, on the cadherin and actin distribution. The membrane potential changes were monitored by fluorescence microscopy using oxonol V; fluorescent probes were also used for F-actin and cadherin. Detergent extraction and Western blot analysis were employed to reveal the relative amount of cadherin attached to the cytoskeleton. The main findings of this study are that different confluent cultured epithelia exhibiting a well-defined circumferential pattern of actin distribution respond to plasma membrane depolarization by similar modifications in the actin cystoskeleton to those reported for bovine corneal endothelial cells. On the other hand, epithelia that do not exhibit such actin pattern in confluence, as well as non-confluent monolayers, do not display noticeable actin modifications in response to the depolarizing procedures. While in the former cells, cadherin is predominantly located at the lateral membrane domain, the cells that do not respond to membrane depolarization mainly display their cadherin in the intracellular compartment. We suggest that the typical peripheral disposition of actin, associated to well-established epithelial-type adherens junctions (i.e. zonula adherens), is a pre-requisite for the cytoskeletal organizational modifications exhibited by epithelial cells in response to the depolarization of the plasma membrane potential.
Chifflet
Silvia
S
Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Gral. Flores 2125, 11800 Montevideo, Uruguay. schiffle@mednet.org.uy
Correa
Valeria
V
Nin
Verónica
V
Justet
Cristian
C
Hernández
Julio A
JA
D000199:Actins / Q000378:metabolism*; D022005:Adherens Junctions / Q000502:physiology*; D000818:Animals; D015820:Cadherins / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism*; D019573:Epithelium, Corneal / Q000378:metabolism; D005123:Eye / Q000378:metabolism*; D007908:Lens, Crystalline / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D010857:Pigment Epithelium of Eye / Q000378:metabolism
D000199:Actins; D015820:Cadherins; C502406:fat1 protein, mouse