Isolated plasma membranes induce the loss of oriented detyrosinated microtubules and other contact inhibition-like responses in migrating NRK cells.
We have previously shown that detyrosinated microtubules (Glu MTs), which are oriented toward the direction of locomotion in motile fibroblasts, disappear from the area adjacent to cell-cell contact soon after a cell-cell collision. To identify cell surface molecules that trigger this phenomenon, we have established a system in which this and other cellular reactions to cell-cell contact can be reproduced by the addition of isolated plasma membranes. Experimental wounds were made in confluent monolayers of NRK cells, and cells at the wound margin were allowed to develop oriented Glu MTs. Test samples were added to these cells and after a 1 hour incubation the distributions of Glu MTs, tyrosinated MTs (Tyr MTs) and microfilaments were determined by immunofluorescence. When plasma membranes isolated from NRK cells were added, oriented Glu MTs disappeared from the leading lamella of target cells and instead a small number of Glu MTs were found clustered around the nucleus. As observed for cell-cell contact, plasma membranes did not significantly affect the distribution of Tyr MTs. We also found that both cell-cell contact and membrane treatment caused the collapse of lamellipodia and loss of associated staining with antiactin antibody. Time-lapse recordings of directed locomotion of NRK cells showed that membranes suppressed the forward movement of cells. The loss of Glu MTs from the leading lamella was the most amenable response for quantification and we used it to examine the biochemical properties of the membrane activity. The ability of membranes to induce the loss of oriented Glu MTs was observed at as low as 4 micrograms/ml of membrane protein and was detectable 10 minutes after membrane addition. The loss of oriented Glu MTs was reversible upon removal of membranes, demonstrating that the membranes were not toxic to the cells. The oriented Glu MT reducing activity could be solubilized from the membranes by detergent, was enriched in a plasma membrane fraction, and was labile to heat and acid treatment. In summary, we have successfully reconstituted a number of responses of contact inhibition using solubilized preparations of membranes. Our preliminary results suggest that there is a specific factor in plasma membranes that is capable of triggering contact inhibition. With the assay we have developed, it should now be possible to dissect contact inhibition of motility at the molecular level.