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C L A S S I C E X P E R I M E N T
BRINGING CELLS TOGETHER
Nagafuchi, A., et al., 1987, Nature 329:341343
The surfaces of many animal cells are
coated with cell adhesion molecules in-
cluding integral membrane proteins
that mediate the cell-cell interactions
critical for tissue formation. In the late
1970s and early 1980s, biologists be-
gan to identify some of these mole-
cules. During this time, the Japanese
scientist Masatoshi Takeichi showed
that some molecules mediate cell-cell
interactions only in the presence of
Ca2 ions. This observation led to the
discovery of a new class of adhesion
molecules, the cadherins.
Background
In multicellular organisms, groups of
specialized cells come together to form
tissues. This grouping of cells is not ran-
dom; specific cell types must adhere to
one another. Specific interactions be-
tween cells assure that cellular composi-
tion is correct: epithelial cells are found
in epithelia, hepatocytes in the liver, and
neurons in neuronal tissues such as the
brain. During tissue formation, it is
sometimes necessary for cells of the same
type to interact with one another and
avoid interactions with other cell types.
In organs, where cells of many types
work together, the interaction between
different cell types is specific. Clearly,
there must be a mechanism to assure that
tissues and organs maintain the correct
cellular compositions and geometries.
Many researchers study the adhe-
sive interactions that occur in tissues us-
ing embryonic cells in culture. These
cells will adhere to one another via
proteinprotein interactions so tightly
that a protease must be added to break
them apart. Classic experiments per-
formed in the 1960s showed that when
different cell types are placed in the
same Petri dish, they separate from each
other like oil and water. Thus, in cell
culture, just as in the body, cells can ad-
here to cells of the same type and avoid
contact with different types of cells. But
1 9 . 1
the question remained, how are these
specific adhesive interactions achieved?
In the late 1970s, Masatoshi Takeichi
studied the interactions between cells in
cultures and attempted to find the con-
ditions under which they would and
would not adhere to one another. At the
same time, other researchers began to
identify specific molecules that mediate
cell adhesive interactions. Taken to-
gether, these two approaches would lead
to the discovery of cadherins, a group of
cell adhesive molecules critical for tissue
formation during development.
The Experiment
In the late 1970s, when Takeichi stud-
ied the adhesive properties of a lung cell
line in culture, he observed that calcium
was critical for some forms of cell adhe-
sion. Similar to other cultured cells, the
lung cells would readily dissociate in the
presence of the protease trypsin. The
dissociated cells would normally reag-
gregate when the trypsin was washed
away. However, when Takeichi at-
tempted to replicate these results in a
different laboratory, he found that the
cells remained dissociated after trypsin
treatment, and once dissociated, the
cells would never aggregate again under
the new conditions.
Puzzled by his difficulty in repeating
this seemingly basic experimental pro-
cedure, Takeichi looked at the chemical
compositions of the solutions used in
his new laboratory. He found that the
trypsin solution he used in the new lab-
oratory contained EDTA, a chemical
that sequesters divalent cations from
the solution and thus from the lung
cells. Previously, Takeichi had used a
solution that did not contain EDTA.
Perhaps a divalent cation was involved
in the adhesive interactions between
these cells? To find out, Takeichi began
investigating the effects of divalent
cations on the adhesive properties of the
lung cells. He found that the cells would
not dissociate in the presence of Ca2
and that dissociated cells would reasso-
ciate only when Ca2 was added to the
medium. These observations led him to
propose that some types of cell adhe-
sions depend on calcium.
Next, Takeichi set out to identify
the specific molecules involved in
Ca2-dependent cell adhesion. He used
antibodies raised against cell surface
proteins involved in cell-cell adhesions
to identify the specific proteins, a strat-
egy similar to that used to identify
other cell adhesion proteins. The basis
of this strategy is the observation that
when cultured cells are treated with
these antibodies, the binding sites of
adhesion molecules are blocked and
the interactions between them cannot
take place. As a result, the cells no
longer adhere in culture. Once such an
antibody is found, researchers use it to
identify a specific protein involved in
the cell adhesion. At first, Takeichi used
the same lung cell line that he used to
demonstrate that some cell adhesions
are Ca2-dependent. However, he was
unable to find an antibody that would
block cell adhesion in this cell line.
To overcome this problem, Take-
ichi began studying cell adhesion in a
different cell line, a teratocarcinoma
cell line, where Ca2-dependent cell
adhesion also occurs. He grew cells in
the presence of Ca2, used trypsin to
dissociate them, and then injected
them into rabbits, whose immune sys-
tem generated antibodies that recog-
nize proteins on the surface of these
cells. To purify these antibodies, he
treated the teratocarcinoma cells that
had not been exposed to calcium with
antiserum taken from the injected rab-
bits. This treatment removed all the
antibodies that bound to teratocarci-
noma cells in the absence of Ca2.
What remained were antibodies that
specifically bind proteins in the presence
of Ca2 that are on the cell surface.
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(a) (b)  Fibroblasts expressing E-cadherin adhere in

culture. Cells in (a) and (c) are from a fibroblast cell line
growing in culture. Cells in (b) and (d) are fibroblasts from
the same cell line transfected with the cDNA encoding
E-cadherin. (a) Light micrograph showing that fibroblasts
do not form adhesive interactions in culture. Notice how
the cells seem to overlap one another. (b) Light micrograph
showing fibroblasts expressing E-cadherin in culture. These
cells adhere to one another, as demonstrated by the easily
definable boundaries between cells. (c) Immunofluorescence
experiment showing that fibroblasts in culture do not
normally express E-cadherin. (d) Immunofluorescence
staining shows that fibroblasts transfected with the cDNA
encoding E-cadherin express the molecule on their cell
surfaces, suggesting that E-cadherin is in fact mediating
(c) (d) cell adhesion. (Nagafuchi, A., et al. [1987]. Nature 329:
341343.)

To find the molecule involved in
2
Ca -dependent cell adhesion, Takeichi
compared cultures grown in the pres-
ence of Ca2 with cultures of cells
grown in the presence of EDTA. Both
groups of cells were dissociated with
the protease trypsin before the experi-
ment began. Using a technique called
immunoprecipitation, he showed that
his antibody specifically interacted
with a 140 kDa protein on the surface
of the Ca2-treated cells while it did
not specifically interact with any pro-
tein on the EDTA-treated cells. He
named this protein cadherin for calcium-
dependent adhesion protein. As more
cadherins were discovered, the original
protein he discovered became known
as E-cadherin because it mediates the
adhesion of epithelial cells.
Takeichis identification of E-cadherin
demonstrated that the protein is in-
volved in Ca2-mediated cell adhesion,
but his research did not prove that
E-cadherin is primarily responsible for
this type of adhesion. To do so, Takeichi
and colleagues cloned the gene encod-
ing E-cadherin. Once the gene was
cloned, he could express E-cadherin in
a fibroblast cell line that neither ex-
pressed E-cadherin nor demonstrated
Ca2-dependent adhesion. Rather than
form cell-cell adhesions, fibroblasts
grow on top of one another in culture
(Figure a). However, fibroblasts that
express E-cadherin adhered to one an-
other in the presence of Ca2 (Figure b).
Thus, the expression of one protein, E-
cadherin, changed the adhesive proper-
ties of the fibroblast cell line. Finally,
Takeichi used immunofluorescence
microscopy to show that the E-cadherin
molecules are found at the cell surface
of the fibroblasts and thus were proba-
bly responsible for their newly acquired
adhesive property (Figures c and d).
Discussion
Over a 10-year period, Takeichi and col-
leagues used his observation about the
dependency of some adhesive interac-
tions on the divalent cation Ca2 to dis-
cover a key class of cell surface mole-
cules and to show that they are critical
for Ca2-dependent cell adhesions. The
discovery of Ca2-dependent cell adhe-
sion was prompted when an experiment
that had always worked, the reassocia-
tion of cells that had been dissociated by
trypsin, suddenly stopped working in the
conditions of the new laboratory. By
tracking down the difference between
the conditions of the laboratories, Take-
ichi made the initial observations that
led to the discovery of a critical family of
cell adhesion molecules. His work shows
that sometimes great science comes from
what looks like a failed experiment.
Today, different cadherins have been
identified on various types of tissues
from the placenta to neurons. Later ex-
periments would show that each type of
cadherin interacts specifically with the
same molecule on an adjacent cell; in
other words, an E-cadherin interacts
with another E-cadherin but not with an
N-cadherin. These homophilic interac-
tions provide some of the specificity that
allows tissues to form. The discovery of
cadherins led the way to our under-
standing of how tissues form.