Some of the most exciting areas of research are those dynamic ones in which we are just embarking. Velia Fowler, who is a professor in the Department of Cell Biology, has spent several years studying how actin filaments work to stabilize the shape of a cell, and how actin filament organization is controlled by actin binding proteins.
"[I've been] interested in how the cell regulates actin polymerization and generates specific actin architectures in different cells," says Fowler.
Now Fowler will be pursuing a whole new direction in her researchinvestigating the dynamic properties of actin and how cells control the polymerization of actin to control cell motility, shape changing, crawling, and phagocytosis.
Muscles of Steel
Actin is a structural protein that forms filaments made up of monomers added one onto the other, and actin bundles or networks are groups of these fibers bound together with other proteins. Like the steel girders that stabilize a building beneath the glass and mortar, the different arrangements of actin filaments into bundles or networks are the supporting structures that give cells different morphologies, or shapes. These shapes go hand and hand with the functional properties of the cell, and an important part of development is the formation of the correct actin cytoskeleton.
"The form is designed for the function, and it's dependent on actin polymerization and the integration of actin filaments into higher-order structures," says Fowler.
Epithelial cells that line the intestines have long, finger-like projections into the intestine supported by bundles of actin filaments that increase the surface area of the intestinal lining. This improves the absorption of nutrients that are then transported through the blood stream.
Red blood cells, which are responsible for transporting oxygen through the bloodstream, have a two-dimensional network of short filaments of actin connected by linker proteins that give the cells their flattened, biconcave disc shape. This network is flexible and can be distorted so that a red blood cell can squeeze through capillaries one fourth their size.
Actin also plays a key role in the physiology of muscles. Heart and skeletal muscle cells have parallel bundles of actin filaments arranged end-to-end in long strings of regular repeating units called sarcomeres. In these sarcomeres, bipolar filaments of myosin, another protein fiber in muscles, pull on the opposing sets of actin filaments from each end to shorten the sarcomeres. This generates muscle contractions that powers the beating of the heart and movements of skeletal muscles. Remarkably, the lengths of the actin filaments in the muscle sarcomeres are held constant and do not vary over many cycles of contraction.
However, mechanical stress and other environmental cues influence this length.
For example, actin filament lengths in sarcomeres vary between fast and slow twitch skeletal muscles. Different muscle groups in the body have different combinations of fast and slow twitch muscles, and in athletes, the combination is largely determined by the type of training that they do. Long distance runners, for instance, have a different combination than sprinters. And if a runner were to switch from 100-yard dashes to marathons, his/her muscle groups would slowly adapt to the new demands.
"When you think of architecture, you think of something stable, fixeda defined form," says Fowler. And even though cells have apparently stable actin filaments, they are not like steel girdersformed once and fixed in their shape. Instead, there are cycles of polymerization used by the cell to create structures and change their length as opposed to making completely new ones from scratch.
"Cells need to be plastic," she says. "They have to change in time and respond to physiological conditions."
This Candle Burns at Both Ends
Fowler is interested in what controls the length of an actin filament. She uses muscle cells to study the dynamics of actin filaments, since actin filaments in muscle are about one micron long, which can easily be viewed with a light microscope. "We can study the dynamics of actin in musclesthe engine that leads to the contractionand we can study it as it happens," she says.
To do this, Fowler's research focuses on a protein that caps one end of the actin filaments and controls their length by controlling the subunit exchange of bound and free actin subunits.
Actin filaments are polarized, and the two ends are dissimilar both in terms of their appearance under a microscope and the types of other proteins that they bind to. The "barbed" end is where much of the subunit exchange takes place and where many different types of capping proteins, one of which was identified in the Fowler laboratory, exist to regulate this exchange. But the other, "pointed," end is what particularly holds Fowler's interest.
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 typesneurons, 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 timedays evenbut 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.
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.