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Regulating Tissue Size
Summary: Richard Gomer is interested in understanding molecular mechanisms responsible for cell-number counting in Dictyostelium.
Our laboratory is interested in the general problem of tissue size regulation. We are trying to understand at a molecular level how embryonic cells form organs of a specific size. Such mechanisms would be centrally involved in the regulation of growth during development, wound healing, and regeneration, while defects in these mechanisms could lead to birth defects or tumors. Using the slime mold Dictyostelium discoideum as a model system, we have found a simple and elegant mechanism that can generate structures of a specific size.
Dictyostelium exists as undifferentiated single cells, called amoebae, that eat bacteria in soil and decaying leaves and proliferate by cell division. When the amoebae eventually overgrow their food supply and starve, they aggregate together in groups of about 20,000. Roughly 80 percent of these cells become spores. (A spore is a cell with a tough outer coat that forms an "escape capsule.") The remaining 20 percent of the cells form a stalk about 2 mm high that holds the spore cells off the ground. A spore, dispersed by the wind, will crack open to release an amoeba, which may luckily find itself in the midst of a new supply of bacteria. The thin stalk with a mass of spores on top of it is called a fruiting body. If a fruiting body is too small, the spores will be too close to the ground for efficient spore dispersal. If the fruiting body is too large, the whole structure will topple over or collapse. Thus, when starving
Dictyostelium cells aggregate, they carefully regulate the size of the groups that they form. The advantage of studying this organism is its simplicity: cells differentiate into just two main types, and the structures that they form are readily visible.
To learn how Dictyostelium cells form groups of a specific size, we developed a mutagenesis procedure called shotgun antisense that allows us to identify quickly which gene is affected in a given mutant. We generated a mutant called smlA– that forms very small fruiting bodies. The mutation is in a gene that regulates secretion of some proteins. We found that the smlA– cells oversecrete a signal that, when added to wild-type cells, causes these cells to form small fruiting bodies. We purified this signal and found that it is a complex of four proteins, which we named counting factor (CF). Using diffusion calculations and experimental observations, we found that as the number of cells in a group increases, the concentration of CF increases. To examine the function of CF, we created a set of mutants; each mutant in this set lacks a different component of CF. In each case, when cells lack a component of CF, the remaining CF complex is still secreted but is inactive, so the cells form huge fruiting bodies. This indicated that CF represses group size, but raises the intriguing question of why CF is a complex of proteins when a single molecule ought to be able to function as a secreted signal.
To understand how CF regulates group size, we wrote a computer simulation of aggregating cells. After exploring various scenarios and parameters, we realized that CF might be able to regulate group size if it could decrease cell-cell adhesion and increase cell motility. If a group is too big, as indicated to the cells by a high concentration of CF, a decrease in adhesion and an increase in motility would cause cells to wander away from the group.
We found that CF does indeed reduce cell-cell adhesion, and that CF decreases the levels of a known adhesion molecule. Artificially decreasing the adhesion of cells by blocking adhesion sites causes the group to become smaller. In addition, high concentrations of CF cause the cells to move about more, which helps them to break connections and dissociate. Two key molecules—actin and myosin—are involved in motility, and CF regulates both of them. Artificially decreasing motility by adding drugs that interfere with the function of actin causes cells to form larger groups. These observations showed that CF regulates both adhesion and motility and that altering adhesion and motility affects group size in a way predicted by the computer simulations.
We are now working to determine why CF has multiple components and how CF regulates adhesion and motility. Two of the protein components of CF bind to cells and have different effects on cells; a third component does not seem to bind. This suggests that some of the CF components have unique functions. We have generated a mutant that lacks three of the components of CF, with the goal of generating a mutant lacking all four of the components so that we can add components back to determine their precise function. We are using biochemical methods and a genetic screen to find the receptor protein or proteins to which these CF components bind. The biochemical assays suggest there is a receptor for one component of CF and a different receptor for a second component, and we are examining possible candidates for these receptors.
We have also found that once CF binds to cells, there is a rapid decrease in the levels of the sugar glucose in cells. High levels of glucose cause cells to increase their adhesion and decrease their motility, and we hypothesize that glucose levels are a key part of the mechanism used by CF to regulate group size. We found that CF rapidly blocks the activity of an enzyme that synthesizes glucose. To determine how glucose regulates adhesion and motility, we have characterized mutants that block the ability of cells to sense CF and have identified a novel protein that appears to regulate the ability of glucose to alter motility. Another mutant we are studying has a disruption of a metabolic enzyme and forms huge groups. Our goal is to understand at a molecular level why a secreted factor has many different components rather than being a single protein or smaller molecule, and how this factor affects cell metabolism to regulate tissue size. Because cells secrete and sense a variety of factors, and these signals can influence each other, we are (in a study supported by the Welch Foundation) examining how a secreted protein factor that allows cells to sense their local density interacts with a small chlorinated hydrocarbon factor.
An exciting offshoot from the work with CF is the identification of a factor called serum amyloid P (SAP) in human blood that blocks the ability of a subset of white blood cells to become fibrocytes, a cell type that appears to participate in wound healing. Unfortunately, some patients with diseases such as scleroderma and pulmonary fibrosis have an excess of fibrocytes. We found that some scleroderma patients have abnormally low levels of SAP, suggesting the possibility that giving these patients injections of SAP might therapeutically reduce the number of fibrocytes. In work supported by the Scleroderma Foundation, we are testing whether injections of SAP reduce fibrosis in a rat model of pulmonary fibrosis.
Last updated November 05, 2004
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