I obtained my undergraduate degree in 1971 from Cambridge
University, UK, and then was fortunate to be accepted by Dennis
Bray for my Ph.D. in the MRC Laboratory of Molecular Biology (LMB),
also in Cambridge. In Dennis’s lab, I worked on myosins in
non-muscle cells. The existence of myosin outside of muscle had
only recently been recognized. Using biochemical techniques, I
was able to show that at least two distinct types of myosin II
exist in non-muscle cells and that some cells expressed both
types1. After
publication, this work was largely forgotten, and the existence
of two nonmuscle myosin II isoforms was rediscovered about 15
years later using molecular techniques.
Working as a student in Dennis Bray’s lab was an incredible
experience. Not only did he give me enormous freedom, but LMB
provided a very rich scientific environment. The floor on which
I worked was headed up jointly by Francis Crick and Sydney
Brenner. The Institute was directed by Max Perutz. Fred Sanger
was working on the floor above us, as was Cesar Milstein. While
I was there, Georges Kohler in Milstein’s lab developed the
first monoclonal antibodies, although I suspect that few of us
appreciated how significant this work would turn out to be. In
adjacent labs, Bob Horvitz and John Sulston were in the early
stages of their work on C. elegans, the study of which
had recently been initiated by Sydney Brenner. Up one floor,
Roger Kornberg was a postdoc working on chromatin structure
under the supervision of Aaron Klug and Francis Crick.
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“...focal adhesions will continued to be studied
because they provide an easily visualized model
for integrin-mediated adhesion.” |
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While working on my Ph.D. I decided that I would like to
change research areas for my postdoc. I was intrigued by the
work being performed on SV40 and other DNA tumor viruses and was
accepted to work in Joe Sambrook’s lab at Cold Spring Harbor.
These were the early days of recombinant DNA technology and my
plan was to study SV40 transcription. However, shortly after
arriving at Cold Spring Harbor in May 1975, my intentions to
become a virologist were unexpectedly cut short by Jim Watson,
the Director of Cold Spring Harbor. Jim suggested that it would
be better for me to continue working on the cytoskeleton. Who
was I to argue with Jim Watson?
So, after a few days at Cold Spring Harbor, I found myself in
the strange position of being a postdoc with Jim Watson. The
real reason for Jim’s desire for me to work on the cytoskeleton
only became apparent with hindsight. At Cold Spring Harbor,
there had been a small group of cell biologists who had
pioneered the use of immunofluorescence in cell biology, but
when I arrived, this group was disintegrating. By recruiting me
and a couple of others to this group, Jim kept cell biology
alive at Cold Spring Harbor.
A year previously, Elias Lazarides, a graduate student at
Cold Spring Harbor, had been able to develop an antibody against
actin (contrary to prevailing dogma). Very quickly, it was
realized that the antibody could be used to visualize actin
filaments in cells by immunofluorescence. Today, this is a
routine activity but, surprisingly, back in 1974, as a technique
for cell biology, immunofluorescence was in its infancy. The
images that Elias and the others generated were striking and
gave views of cytoskeletal organization that had not been
appreciated previously. Up until then, electron microscopy (EM)
had been the primary tool to examine the cytoskeleton, but EM
analysis is slow and usually only gives views of small regions
of cells. Suddenly, it was possible to rapidly examine the
distribution of actin or other cytoskeletal systems in thousands
of cells. There was a flurry of papers from the lab looking at
actin organization in normal and transformed cells. Antibodies
against other cytoskeletal components were being generated or
obtained from collaborators. Jim Watson was very supportive of
this work and appreciated its significance to cell biology. I
realize now that my recruitment provided a pair of hands for its
continuation.
Elias Lazarides left for his postdoc in Colorado four months
after I arrived, but as soon as I got to Cold Spring Harbor we
discovered that we had both independently generated antibodies
against the muscle protein a -actinin
and used these for immunofluorescence localization of the
protein in fibroblasts. I had done this work in Cambridge but
had not written it up before I left. Rather than writing two
separate and competing manuscripts, we pooled our results and
submitted a single paper to Cell. Compared with the
routine rejections that I get from Cell today, they
welcomed our very simple paper and even asked us to supply more
figures to pad its length2.
Our paper demonstrated that a -actinin
distributed periodically along stress fibers, the bundles of
actin filaments that are prominent in many cells growing in
tissue culture. We also noted that there was a concentration of
a -actinin in plaques at the ends of
stress fibers. These regions would several years later come to
be known as focal adhesions. a -actinin
was the first protein found to be concentrated at these sites,
which are where stress fibers attach to the cytoplasmic face of
the plasma membrane and where tension is transmitted across the
membrane to the underlying extracellular matrix (ECM) on the
outside.
Although focal adhesions have been the center of my research
for nearly 30 years, it was not until Jim Feramisco arrived as a
postdoc at Cold Spring Harbor three years later that I returned
to working on a -actinin. At the
time, there was no easy way to purify the protein, and we set
out to establish a rapid purification from smooth muscle. While
developing this procedure, we noticed that we had inadvertently
purified another protein of unknown function. We were curious
about this protein and rather slowly began to study it and again
to make antibodies against it for localization studies. Unknown
to us, Benny Geiger, then a postdoc with John Singer at UCSD,
had similarly stumbled into this protein while purifying
a -actinin. Unlike us, he rapidly
generated antibodies and used these to show that the protein was
concentrated in focal adhesions. Benny published his discovery
of this protein, subsequently named by him vinculin, in the fall
of 19793, and Jim and I
published our work a few months later in early 19804.
More than any other work, Benny Geiger’s discovery of vinculin
established the field of focal adhesion research.
I left Cold Spring Harbor in 1981 for a faculty position at
the University of North Carolina at Chapel Hill, where I
continued to work on focal adhesions. Together with a talented
technician, Laurie Connell, I discovered talin5
as another focal adhesion protein and then in collaboration with
Rick Horwitz’s lab showed that talin bound to the cytoplasmic
domains of integrins6,
the receptors for many ECM proteins. Integrins are clustered at
focal adhesions and provide the primary link between the ECM on
the outside and the cytoskeleton on the inside.
Whereas focal adhesions were originally studied in the
context of being structural links between the force generating
cytoskeleton within cells and the ECM to which cells are
adhering, it became apparent in the early 1990s that focal
adhesions are also major sites of signal transduction. Many
signaling components are concentrated in focal adhesions,
including tyrosine kinases, such as the focal adhesion kinase (FAK)
and members of the Src family. The last 15 years of research in
this area have been dominated by efforts to understand the
signaling pathways that emanate from focal adhesions. It is now
generally acknowledged that focal adhesions are sites of
mechanotransduction, where cells monitor the physical and
chemical properties of the ECM to which they adhere. ECM
adhesion affects the behavior of cells in many ways, influencing
not only their morphology and migratory properties, but also
their growth, survival, and differentiation. Consequently,
understanding the signals that are transmitted at focal
adhesions is relevant to understanding how cells respond to the
ECM and how these may change under different conditions of
health and disease.
A seminal paper in the field came in 1992 when Ridley and
Hall demonstrated that focal adhesions and their associated
stress fibers were formed in response to active Rho, a GTP-binding
protein of the Ras superfamily7.
Following this work, Magdalena Chrzanowska-Wodnicka, a graduate
student in the lab, and I showed that Rho induces the assembly
of focal adhesions by stimulating myosin-based contractility. We
demonstrated that the resulting tension leads to the clustering
of integrins8, thereby
forming the focal adhesion scaffold upon which other structural
and signaling proteins assemble.
For all the work that many of us have directed to the study
of focal adhesions, ironically, they are something of a tissue
culture artifact. Although there are examples of cells
developing focal adhesions in tissues, they are rare compared
with the prominence of focal adhesions in tissue culture. In
part, this appears to be due to the artificial situation of
growing cells on rigid substrates that promote the development
of isometric tension. Additionally, cells in culture are
typically grown in the presence of serum, which contains several
factors that activate Rho, thereby promoting contraction and the
development of both stress fibers and focal adhesions.
Nevertheless, the study of focal adhesions has facilitated the
identification of structural and signaling components that are
concentrated at sites where cells engage and transmit tension to
the ECM.
Undoubtedly, focal adhesions will continue to be studied
because they provide an easily visualized model for integrin-mediated
adhesion. In the future, however, I predict that attention will
be increasingly turned to studying adhesions that form in
three-dimensional cultures. These are more difficult to
visualize than focal adhesions but they resemble more closely
the adhesions made between cells and the ECM in tissues within
the body.
Keith Burridge, Ph.D.
Department of Cell and
Developmental Biology
University of North
Carolina School of Medicine
Chapel Hill, NC, USA
cites
:
n
the essay below, Professor Keith Burridge writes about his
highly cited work on focal adhesions. This work has garnered
him a ranking in the top 1% in the field of Molecular
Biology & Genetics in
