Fusion of cultured dog kidney (MDCK) cells: I. Technique, fate of plasma membranes and of cell nuclei

Abstract

The evaluation of the intracellular signal train and its regulatory function in controlling transepithelial transport with electrophysiological methods often requires intracellular measurements with microelectrodes. However, multiple impalements in epithelial cells are hampered by the small size of the cells. In an attempt to avoid these problems we fused cells of an established cell line. Madin Darby canine kidney cells, originally derived from dog kidney, to "giant" cells by applying a modified polyethylene glycol method. During trypsin-induced detachment from the ground of the petri dish, individual cells grown in a monolayer incorporate volume and mainly lose basolateral plasma membrane by extrusion. By isovolumetric cell-to-cell fusion, spherical "giant" cells are formed within 2 hr. During this process a major part of the individual cell plasma membranes is internalized. Over three weeks following cell plasma membrane fusion degradation of single cell nuclei and cell nuclear fusion occurs. We conclude that this experimental approach opens the possibility to investigate ion transport of epithelia in culture by somatic cell genetic techniques.

Patchy accumulation of apical Na+ transporters allows cross talk between extracellular space and cell nucleus.

“…In short, cells were grown in culture and harvested by trypsin/EDTA treatment. Then cells were fused to giant cells by a modified polyethylene glycol method (7). The multikarya were maintained in culture for at least 3 days until most nuclei were either fused or decomposed.…”

Fusion of cultured dog kidney (MDCK) cells: II. Relationship between cell pH and K+ conductance in response to aldosterone

“…The preceding paper describes culture and fusion techniques in detail [13]. In short MDCK cells were grown in plastic culture dishes until subconfluency was approached.…”

Acute Aldosterone Action in Renal Target Cells

“…This technique was applied to intact am phibian renal tubules [50], to cells isolated from amphibian kidney [25] and to cultured MDCK cells [9,53], In these preparations, aldosterone action was elucidated at the cel lular level for the first time. One of the unique features of a giant cell fused from the renal epithelium is its large size that enables the cell to survive multiple impalements with microelectrodes.…”