A karst fruitfly is easy to spot—provided you know what you're looking for. Under the microscope, the bulbous red eyes are rough instead of smooth. The see-through wings are curled, like dying leaves. A dark substance called hemolymph, the equivalent of blood, leaks from the shunt-like trachea.
Zoom in on those red eyes and you'll notice a sort of scarring across the many-faceted surface, like burrs or imperfections in glass. To Graham Thomas, Penn State assistant professor of biochemistry and molecular biology, this roughness—at even higher magnification—looked like the karst knolls of Bohol Island in the Philippines, land forms whose photograph he had seen in an atlas. ("My father is a geomorphologist," he explains.) Since Thomas isolated this particular mutation while a postdoctoral fellow at Harvard, his was the privilege of naming it.
Karst flies are missing a protein called β Heavy-spectrin, or βH. Large and rod-shaped, βH is one of a family of cytoskeletal proteins which bind with other tiny filaments to give shape to epithelial cells—the type of cells that make up skin, and line the human gut. Thomas's study of what happens when it's absent, however, suggests that βH may have other important jobs beyond structural support.
Most karst flies die as larvae, notes Daniela Zarnescu, a graduate student in Thomas's lab. Those few that make it to adulthood exhibit the above-mentioned defects in eyes and wings—both of which originate as epithelial tissue. "In order to understand the karst phenotype," she says, "we have to look at how the eye develops."
Epithelial cells are tightly bound to one another. Once joined, they exhibit polarity—they have a well-defined top and bottom. This polarity is crucial to communication: the cell-to-cell signaling that is necessary for both development and mature function.
Under the microscope, using fine-point tweezers and a tiny scalpel, Zarnescu dissects the third-stage larva of an ordinary fruit-fly, Drosophila melanogaster. With exquisite care, she removes the eye imaginal disc—the sheet of epithelial tissue that would have become the adult eye. Taking the disc to a more powerful microscope, she stains the tissue with antibodies that will indicate where βH is found in the cells.
According to the stain, βH "localizes" precisely at the place where cells join—the so-called adherens junction. This placement suggests an adhesion or signaling function.
Next, Zarnescu looks at the eye disc of a mutant fly. In fly development, she explains, the third-stage larva is where "the magic happens": the epithelial sheet begins to differentiate into its mature form. Obeying some elemental signal, a wave of change called the morphogenetic furrow sweeps across the tissue, leaving a new sort of order in its wake. "The cells are getting information, gathering in clusters," Zarnescu explains. Some of these clusters will become neurons, the photoreceptors of the adult eye. The furrow advances in orderly fashion, transforming a new row of cells every two hours. By fixing tissue at the right times, Zarnescu can capture a whole series of developmental stages.
In normal cells, as the images on her computer screen show, the epithelial sheet is all symmetry: Neat rows of cells, the future neurons forming as flower-shaped clusters.
In the mutant disc, by contrast, the rows of cells are jumbled like cars stuck in traffic. The clusters are more like random piles. Most striking to the untrained eye, the junctions between cells are no longer clearly drawn. The boundary lines are spidery and uneven, frequently broken as though partially erased.
"It seems clear that β H is important to the normal development of the eye," Zarnescu says. To pinpoint its role will mean first shifting from genetics to biochemistry. "We can isolate the complex of proteins located at the junction, and identify exactly what proteins are there by a process called co-immunoprecipitation," she says. If βH shows up along with known junctional proteins, as expected, "this would be a good confirmation of our staining result. And we may find some proteins we haven't yet seen."
Zarnescu and her lab mates are also employing the tools of molecular biology. "βH is a large protein," she explains. "We think of it as a scaffold on which many different proteins can interact." By selectively cloning pieces of this framework and watching for the effects of over-expression, they hope to determine the function of each interaction.
"We are looking at the fundamental processes of epithelial cells," Zarnescu explains. "The fly eye is different from the human eye, but in terms of what happens in epithelia, how cells connect with each other and signal each other, the mechanisms are very similar."
The junctions between these cells are important, she adds, for reasons beyond structural support. Without good connections, the cell-to-cell signaling necessary for differentiation is lost. Understanding how this communication breaks down could help solve other, higher profile mysteries. Signaling problems and changes in adhesion, for instance, are two of the peculiarities associated with cancer cells. "As they become invasive," Zarnescu notes, "they lose their adherence and start wandering around on their own."
Daniela Zarnescu is a Ph.D. student in biochemistry and molecular biology in the Eberly College of Science, 107 Althouse Laboratory, University Park, PA 16802; 814-863-0718; dcz102@psu.edu. Her adviser is Graham H. Thomas, Ph.D., assistant professor of biology and biochemistry and molecular biology, 208 Mueller Laboratory, 863-0716; gxt5@psu.edu. Visit the Thomas lab on the World Wide Web at http://www.bmb.psu.edu/thomas/default.htm. The research reported above is funded by the National Institutes of Health.