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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).