Throughout development and in the adult, cells receive specific signals that they must interpret precisely according to the signal's strength. Secreted proteins of the Wnt family represent one such signal. Wnts elicit precise changes in gene expression mediated by increased levels of the transcription factor Beta-catenin. Too much, or too little, of this signal leads to patterning defects and/or disease.
Wnts induce a Beta-catenin signal by blocking the function of the central pathway regulator, a protein complex assembled around the scaffold protein Axin. Multiple, potentially conflicting, inputs interact within the Axin complex. How these different inputs are integrated and dynamically controlled during Wnt signal transduction remains unclear. The focus of this project is to establish a dynamic model of Wnt/B-catenin signaling by identifying the location of the Axin complex and its sub-complexes during signal transduction. Genetic manipulations will then be used to determine the functional significance of the findings.
The current model of the Wnt/B-catenin pathway is based largely on studies using overexpression in tissue culture, and protein-protein interactions in vitro. The functional significance of these findings for normal signaling in vivo remains unclear. Significantly, in the proposed studies, the Wnt/B-catenin pathway will be analyzed in the model organism Drosophila, allowing its analysis in the normal cellular context and under physiological conditions. Additionally, visualization of the Axin protein complexes in real-time will establish a dynamic picture of Wnt signaling not achieved in previous studies. These studies are expected to identify novel regulatory interactions and may open the Wnt pathway to pharmacological intervention. This project will provide an opportunity for one graduate student, and two undergraduate students to engage in cutting edge research. In addition, it will allow the PI to introduce K-12 students to basic development and genetics and to promote a science-based curriculum in local schools.
Proper organismal development relies on number of signals that must be precise both in timing and signal intensity. Secreted molecules of the Wnt family provide one of the most critical signals, yet our knowledge of the intracellular signaling mechanism is incomplete. The reason lies in its unusual nature. The pathway uses components that have functions outside the Wnt/beta-catenin pathway, presumably because they were available during evolution, and were incorporated into the Wnt pathway. This fact has greatly complicated analysis, resulting in about a dozen profoundly different pathway models have been proposed. Not all are mutually exclusive and may in fact be relevant in different contexts for the precise modulation of the signal. This project established Bimolecular Fluorescence Complementation (BiFC), a ‘split-GFP’ approach, as a method allowing the PI to detect pair-wise protein-protein interactions directly in vivo at physiologically relevant conditions using transgenic Drosophila. Importantly, this method provides molecular resolution and real time analysis. For the first time in vivo and in cells the central regulator of the Wnt pathway, the destruction complex, could be detected and localized. Importantly, this approach also identifies a novel mechanistic model of how the Wnt signal blocks destruction complex activity. These major breakthroughs have allowed the PI to identify the dynamic sequence of events whereby receptor activation is followed by several steps that ultimately cause the inactivation of the destruction complex. These findings provide the framework to further refine the signaling mechanism. In particular, it will allow to test whether and how published models fit this framework. It will provide the basis to build a unified Wnt pathway model. Significantly, it will also identify instances where a published model does not describe an event in the core Wnt pathway but instead a subsequent, modulatory mechanism. Such distinctions are critical both to our understanding of Wnt signaling in development, as well as to targeting therapies in the treatment of Wnt-associated diseases. This NSF-funded project provided training opportunities for eight undergraduate students (four women), as well as two female high school students including two summer students. Students are trained in strategies for experimental design, accurate data recording and analysis. Both high school students presented their results at school and at a symposium. This type of intensive exposure to our active research projects has proven effective at fostering continued interest into careers in science and education. Additional continuing outreach included the interactions with local elementary and local high schools (by both the PI and members of the laboratory), to introduce students to basic development and genetics (using Drosophila and the moth Manduca) and to promote a strong science curriculum in local schools.
