Biology Reference
In-Depth Information
1970 immunofluorescence paper by Frye and Edidin
[18]
. Subsequent, improved methodol-
ogies to follow lateral diffusion include fluorescence recovery after photobleaching (FRAP)
and single particle tracking (SPT), discussed below.
The Frye-Edidin Experiment
By 1970 it was evident that cells were not rigid structures, but changed shape as the cell
moved or developed pseudopodia. The objective of the Frye-Edidin experiment was to
prove that plasma membrane (surface) proteins could move laterally in the membrane.
These investigators used two very different cell lines, one a human line, the other a mouse
line. The experiment is depicted in
Figure 9.12
. Each cell type had different surface anti-
gens and so could be readily distinguished by fluorescent-labeled antibodies. The tech-
nique is referred to as immunofluorescence. The antibody for the mouse antigen was
labeled with fluorescein and so fluoresced green, while the antibody against the human
antigen was labeled with rhodamine and appeared red. The cells were fused at 37
C
with a Sendai virus producing a heterokaryon that initially had one half of the fused cell
green and one half red. After 40 minutes the red and green colors were totally mixed.
One interpretation of this observation was that the plasma membrane proteins were free
to diffuse laterally in the plane of the membrane. However, Frye and Edidin did realize
that other explanations were possible and they tested these. Protein synthesis inhibitors
(puromycin, cycloheximide, and chloramphenicol) and the uncoupler 2,4-dinitrophenol
had no effect on the process. Therefore, intermixing of the surface antigens was not the
result of newly synthesized antigens at various locations in the plasma membrane, nor
was the intermixing due to an energy-dependent translocation. However, if the tempera-
ture was reduced from 37
Cto15
C, the process was greatly slowed. The dependence
on temperature was consistent with a change in membrane viscosity that would affect
diffusion rates. This paper concluded that a membrane 'is not a rigid structure, but is fluid
enough to allow free diffusion of surface antigens resulting in their intermingling within
minutes after the initiation of fusion'.
This seminal paper in membrane studies was one of the very first in Michael Edidin's
(
Figure 9.13
) long and distinguished career at Johns Hopkins. He is also well respected
in an area far removed from membranes. He is a world expert on the history of watch
making and repair!
Fluorescence Recovery after Photobleaching (FRAP)
While the Frye-Edidin experiment clearly established that membrane proteins can
diffuse laterally, it proved to be a difficult method to obtain accurate rates of diffusion.
The Sendai virus is not a reliable fusogen. Its fusion agent is likely a component picked
up from the host cell as the virus escapes and so varies tremendously in effectiveness
from preparation to preparation. In addition, while the time that fusion begins (T
0)
can be accurately determined, the time at which complete mixing is achieved is imprecise.
Therefore other, more reliable and accurate methods to measure lateral diffusion in
membranes were sought. Advances in laser technology led to the accelerated development
of what is now the major technique in determining lateral diffusion of lipids and proteins in
ΒΌ
