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For many years it was believed that there was no capping at
the pointed end and that, in general, the pointed end was not
very important and certainly not very interesting. If the pointed
end did anything at all, it was simply to disassemble. Actin,
so it was believed, assembled at the barbed end, until it was
capped to stop growth, and then disassembled at the pointed
end, where it was not capped. Then Fowler found the first pointed
end capping protein and quickly became interested in the pointed
end's role in regulating actin dynamics and stability.
This protein, tropomodulin, is about 40,000 daltons and
is encoded by one of four genes in the genome. These four
are specific for various cell types—neurons, cardiac
and skeletal muscles, red blood cells, eye lens cells, and
endothelial cells.
Actin filaments in these cells grow to be a certain length
and then are maintained at that length by capping proteins
and other molecules in the cell. Actin filaments are stable
in that they exist for a long period of time—days even—but
during this time, the subunits exchange while the filaments
maintain a consistent length.
Fowler uses green fluorescent protein-tagged tropomodulin
to study this protein's interactions with actin, and what
she has found is a picture of the pointed end remarkably different
than the one scientists had previously envisioned. Rather
than a stable cap on the pointed filament end, tropomodulin
is in constant exchange, coming on and off all the time, and
actin is polymerizing and depolymerizing all the time while
the overall length of the filament is kept constant.
Filaments are stable in that they exist for several days
at one fixed length, but this length is controlled dynamically
by tropomodulin regulation of exchange of actin monomer subunits
at the pointed end. If you add more tropomodulin to a muscle
cell, you inhibit the exchange of subunits and the filaments
shrink. Conversely, if tropomodulin activity is inhibited
by microinjection of function blocking antibodies into the
muscle cell, more actin subunits add onto the end, and the
filaments grow longer.
In fact, in a recent study, Fowler's laboratory showed that
adding an excess amount of the Drosophila equivalent
of tropomodulin regulates the elongation of the actin filaments
in the indirect flight muscles in the species. By overexpressing
the tropomodulin-like protein during the formation of these
muscles while the Drosophila were developing, the filaments
stopped elongating and the adult files were unable to fly.
From Flying to Crawling
Since the capping of filaments at their pointed ends by tropomodulin
is important in maintaining stable filament lengths, Fowler
hypothesized that the protein might also be important in processes
where the length of actin filaments is not stable, as in motility.
She studies the crawling of endothelial cells, the single
layer of flattened cells that line internal body cavities,
such as veins and organs. In angiogenesis, the formation of
new blood vessels, endothelial cells have to crawl in order
to form the new blood vessels and actin polymerization is
responsible for this crawling.
In the leading edge of the crawling cells, dynamic actin
filaments polymerize, and this forces the cell wall to expand
out into a lamella and begin to move. Interestingly, these
dynamic actin filaments also have tropomodulin present. Staining
for the protein shows that it is enriched in the leading edge
of endothelial cells when those cells are sending out lamellae
and crawling.
"This was a big surprise," says Fowler, "because based on
our preconceived idea that tropomodulin was only capping very
stable filaments in cells such as muscle, it was not supposed
to be there."
Altering the level of tropomodulin in the cells alters their
crawling speed. Too much tropomodulin caps all the pointed
ends, stabilizing the actin filaments and slowing down the
crawling of cells.
Eye Grant
Fowler is also the director of one of the modules of the
new Core Center for Vision Research at TSRI. Earlier this
year, the National Eye Institute (NEI) announced multi-year
funding for the core, which will support shared resources
for several TSRI researchers who have independent programs
in vision science funded through the NEI and a few more such
researchers from the University of California, San Diego (UCSD).
The core center will operate several core modules, including
one dedicated to imaging, which will be managed by Fowler
together with Drs. David Williams and David Rapaport at UCSD.
This module will offer three types of microscopy to principal
investigators on the grant: electron microscopy, light microscopy,
and live cell imaging. For the live cell imaging, the grant
provided the funds to purchase a new microscope which will
be housed in Fowler's laboratory space.
In addition to managing this module, Fowler plans to use
the live cell imaging microscope to study how dynamic actin
polymerization is important for the form and function of eye
lens cells. Lens cells start life as a thin layer of short
cuboidal epithelial cells and with time, elongate over 100-fold
to become incredibly long and thin fiber cells, packed tightly
side-to-side in the eye lens. Proper elongation and cell-cell
packing of the lens fiber cells is critically important for
the shape and clarity of the lens; problems in this process
can lead to cataracts and impairment of vision. Fowler's interests,
of course, lie in elucidating how tropomodulin regulates actin
dynamics in the lens cells to direct actin polymerization,
cell elongation and formation of cell-cell associations in
living lens cells.
She also looks forward to collaborating with the other principal
investigators on the grant, in the department, and at TSRI.
And next year, when Fowler moves her laboratory into the new
CarrAmerica Building, she will be inhabiting a new space designed
to do just that.
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Transient overexpression of Drosophila
tropomodulin during indirect flight myofibril assembly irreversibly
arrested elongation of pre-existing thin filaments in the
myofibril core. The lengths of peripheral thin filaments assembled
after tropomodulin levels had declined were normal. Isolated
myofibrils were fixed and stained with bodipy-phallacidin
(green) and tropomodulin antibodies (Red) or anti-TM antibodies
(Red) and tropomodulin antibodies (Green). From Mardahl-Dumesnil,
M. and Fowler, V.M. "Thin filaments elongate from their pointed
ends during myofibril assembly in Drosophila
indirect flight muscle" J. Cell Biol., in press, (2001).
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