While researchers accumulated support for the idea that proteins were embedded in the lipid bilayer and projecting out beyond the inner and outer surfaces, a classic experiment by Frye & Edidin (1970) was adding to our knowledge of what a membrane was like. Citing the ability of animal cell membranes to move (as with the formation of pseudopods by the amoebas that you might remember from junior high biology class), Frye & Edidin hypothesized that cell membranes must be fluid – the components of the membrane must have some freedom of movement relative to other components.
To test this, they fused mouse and human cells together to form cell hybrids and heterokaryons. Each type of cell has distinct molecules on its surface (called surface antigens) that would identify it as being either a mouse cell or a human cell. Scientists can make antibodies to bind to specific antigens. Frye & Edidin attached fluorescent molecules to the antibodies so that they could track where the antibodies were (one color for antibodies that bound to mouse antigens, and another color for antibodies that bound to human antigens). Since the antibodies would bind to the antigens, knowing where the antibodies were also told them where the antigens were. Right after fusion of the two cells, the antigens were found only on half the cell.
To help visualize this, imagine the blue circle below is a human cell. If scientists attached antibodies to antigens on the cell's surface and attached a blue molecule to the antibodies, then the human cell would look blue. The presence of the blue color indicates that human antigens are also present. The same would be true for a mouse cell, except the scientists attach a red molecule to the antibodies bound to mouse antigens. Right after fusing the human cell and the mouse cell, you'd have something that looked like the image on the right: all the human antigens in the membrane that came from the human cell and all the mouse antigens in the membrane that came from the mouse cell.
But over time (it took about 40 minutes at 37 °C) the antigens from the mouse half would spread out across the whole cell and the human antigens did the same. Frye & Edidin ended up with a mosaic of mouse and human antigens over the surface of the heterokaryon (shown on the far right, below). One likely explanation is that the antigens in the membrane moved, producing the mosaic pattern that they observed.
One of the things that makes this a classic experiment is that instead of simply concluding, “Yep, our initial hypothesis was right,” Frye & Edidin considered other explanations for how these mosaics had formed. They offered four possibilities: (1) the surface antigens of the hybrid cell were being rapidly removed from the membrane and then replaced by newly-made copies of the antigen molecule, (2) new copies of the antigens were added to the membrane from a pre-existing pool of antigen molecules stored in the cell, (3) the antigen molecules moved (by diffusion) from place to place within the membrane [this was their favored hypothesis], and (4) antigen molecules were taken out of the membrane, into the center of the cell, and then put back into the membrane somewhere else.
They excluded the other possibilities by altering the conditions of the experiments and seeing what happened. For example, if explanation (1) is correct, then new antigen molecules must be built inside the fused cell. Frye & Edidin added a chemical to a batch of the fused cells that would prevent the synthesis of new antigen molecules. This should have prevented the mosaic pattern from forming, but it formed anyway. This suggests that explanation (1) is incorrect. By eliminating possibilities, you become more confident in the possibilities that remain. This is a hallmark of good science: test multiple, competing hypotheses.
At the end of the day, what we learned from Frye & Edidin's experiment is that the cell membrane is fluid - the parts move around, they're not fixed in place. In a future post, I'll describe how all of the results described so far were pulled together to produce one coherent model of what cell membranes are like.
Frye, L.D. and Edidin, M. (1970) “The rapid intermixing of cell surface antigens after formation of mouse-human heterokaryons.” Journal of Cell Science 7: 319-335