386065 Analyzing Negative Feedback and Shuttling Using a Synthetic Gene Network
Analyzing Negative Feedback and Shuttling Using a Synthetic Gene Network
15D02 Intracellular Processes I
Ashley A. Jermusyk and Gregory T. Reeves
In multicellular organisms, cellular signaling events are crucial for patterning tissues, as well as for maintaining healthy adult tissues, while improper signaling can lead to disease states, such as cancer. Therefore, cellular signaling processes must be tightly regulated. Complex systems of gene regulatory circuits control these signaling processes and act to buffer these systems against noise, thereby minimizing mistakes in gene expression and preventing patterning defects or disease states. Despite their importance to patterning and development, hypotheses regarding these gene regulatory circuits have been difficult to test experimentally due to their complexity and high connectivity. Therefore, to better understand the fundamental processes involved, we created a synthetic gene network in the fruit fly Drosophila melanogaster embryo. This approach has the advantages that (1) the gene network is orthogonal to native Drosophila biology, and (2) the network is designed. These two aspects imply the connectivity of the network is understood, and thus hypotheses regarding the designed network motif can be experimentally tested with this system. We are examining expression at the syncytial blastoderm stage when the Drosophila embryo is one cell with many nuclei. By studying a single-cell system we are able to simplify what can be a very complex process (patterning) as well as gain insight into processes at varying stages of development.
Developing tissues are patterned using signaling proteins that determine expression of downstream genes based on concentration thresholds. These signaling proteins form gradients due to localized protein production and subsequent diffusion and degradation. A second protein can bind to the signaling protein to inhibit signaling (shrink the spatial gradient - negative feedback) or to facilitate transport (broaden the morphogen gradient – known as “shuttling”). Both negative feedback and shuttling have been proposed as mechanisms to increase the robustness of gene expression (Eldar et al., 2003; Haskel-Ittah et al., 2012), but this hypothesis is difficult to test experimentally. Therefore, we have created a synthetic network to test the effects of these two systems (shuttling and negative feedback) utilizing genes from yeast and E. coli, namely, gal4, gal80, and lacZ. We expressed gal4 in a graded fashion along the anterior-posterior axis of the embryo, mimicking the intracellular diffusion of Bicoid, an endogenous transcription factor and signaling molecule. As seen below in Figure 1, the Gal4 protein activates expression of UAS-linked gal80 and lacZ. Gal80 binds to Gal4, preventing Gal4 from binding to UAS and activating expression of gal80 and lacZ; this sequestration creates a negative feedback loop in our system. However, due to the spatial dynamics of the system, a shuttling mechanism can also be observed when the level and spatial expression domain of Gal80 is altered. The effects of this shuttling mechanism on the network can be determined by looking at lacZ expression, specifically changes in expression due to the addition of Gal80 to the network. These genes were chosen since they are not endogenous to Drosophila, so all interactions in this network are fully understood. Our goal is to measure the effect of Gal80-mediated Gal4 expression on the robustness of the location of the lacZ domain at varying levels of Gal80 (namely in both the negative feedback and shuttling regime). This system provides a direct experimental test of the effects of these control mechanisms in cellular signaling events, specifically whether this shuttling and negative feedback can lead to increases in robustness of the system. It is this robustness that is important for combating diseases and defects in development and maintenance of expression in the organism.
Figure 1. Network Diagram
Representation of the interactions in the studied network where arrows represent activation and flat arrowheads denote repression by binding of Gal80 to Gal4.
Eldar A, Rosin D, Shilo B, Barkai N. 2003. Self-enhanced ligand degradation underlies robustness of morphogen gradients. Dev Cell 5(4):635-46.
Haskel-Ittah M, Ben-Zvi D, Branski-Arieli M, Schejter ED, Shilo B, Barkai N. 2012. Self-organized shuttling: Generating sharp dorsoventral polarity in the early drosophila embryo. Cell 150(5):1016-28.
The focus of this session is the quantitative analysis of intracellular processes at the molecular level utilizing experimental and/or modeling techniques. Topics of interest include, but are not limited to, signal transduction, intracellular trafficking, cytoskeletal dynamics, metabolic and transcriptional networks, and subcellular imaging.