Membrane potential and endocytic activity control disintegration of cell-cell adhesion and cell fusion in vinculin-injected MDBK cells.
Cell fusion occurs during fertilization and in the formation of organs such as muscles, placenta, and bones. We have developed an experimental model for epithelial cell fusion which permits analysis of the processes during junction disintegration and formation of polykaryons (Palovuori and Eskelinen [2000] Eur. J. Cell. Biol. 79: 961-974). In the present work, we analyzed the process in detail. Cell fusion was achieved by microinjecting into the cytoplasm of kidney epithelial Madin-Darby bovine kidney (MDBK) cells TAMRA-tagged vinculin, which incorporated into lateral membranes, focal adhesions and nucleus, and, prior fusion, induced internalization of actin, cadherin and plakoglobin to small clusters in cytoplasm. Injected vinculin was still visible at lateral membranes after removal of junctional proteins indicating that it was tightly associated and perturbed the cell-cell contact sites resulting in membrane fragmentation. Injection of active Rac together with vinculin induced accumulation of cadherin to the membranes, but did not affect vinculin-membrane association. However, it hampered cell fusion probably by supporting adherens junctions. In order to stop endocytosis, we lowered intracellular pH of vinculin-injected cells to 5.5 with the aid of nigericin in KCl buffer. In acidified cells, injected vinculin delineated lateral membranes as thick layers, cadherin remained in situ, and cell fusion was completely inhibited. Since this treatment also leads to cell depolarization, we checked the vinculin incorporation in a KCl solution containing nigericin at neutral pH. In these circumstances, both endogenous and injected vinculin delineated lateral membranes as very thin discontinuous layers, but still fusion was hampered most likely due to perturbation in the initial vinculin-membrane association. We suggest that vinculin might function as a sensor of the environment triggering cell fusion during development in circumstances where membrane potential and local and transient pH gradients play a role.
Palovuori
Riitta
R
Biocenter Oulu and the Department of Pathology, University of Oulu, University of Oulu, Finland.
Myrsky
Essi
E
Eskelinen
Sinikka
S
D022005:Adherens Junctions / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D002417:Cattle; D002448:Cell Adhesion; D002450:Cell Communication; D002459:Cell Fusion; D002460:Cell Line; D004705:Endocytosis; D004847:Epithelial Cells / Q000378:metabolism / Q000502:physiology; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D007668:Kidney / Q000166:cytology; D008564:Membrane Potentials; D018613:Microscopy, Confocal; D013997:Time Factors; D016596:Vinculin / Q000378:metabolism / Q000502:physiology*; D020744:rac GTP-Binding Proteins / Q000378:metabolism
D005456:Fluorescent Dyes; D016596:Vinculin; D020744:rac GTP-Binding Proteins; D016650:Fluorescein-5-isothiocyanate