AfCS > Project Management Plan

Our rate of accomplishment has accelerated dramatically during this initial funding period. The first year was to a large extent devoted to hiring and training of personnel, implementing procedures, developing reagents, establishing a useful web site, and deploying a laboratory information management system. Considerable progress was then made with primary cells, but we were ultimately stymied, particularly by problems related to use of RNA interference technology. The RAW 264.7 cell became our experimental model at the beginning of year four, and we believe we have convincingly displayed our worth since that time (slightly more than one year). We have assembled an efficient data-gathering machine that is poised to face the challenges of the next five-year funding cycle. The ligand screening phase of the project is nearly complete, and the efforts of the AfCS will now be focused entirely on the elucidation and quantitative modeling of the signaling networks of the RAW 264.7 cell. These efforts will follow three major avenues: perturbations to analyze connectivity in networks, development and implementation of a variety of additional assays to monitor discrete signaling modes, and establishment of quantitative models based on AfCS-generated data, supplemented with information from the literature. No new bridging projects are proposed for the new funding period.

Perturbation Analysis. Perturbation analysis, using the combined approaches of pharmacological agents, RNAi technology, and dominant negative or constitutively active mutant constructs, will involve the combined contributions of all seven AfCS experimental laboratories. Additional support will be provided by the Data Management, Analysis, & Bioinformatics group. These coordinated activities, which are currently well underway, will continue throughout the entire funding period of the project, although at varying levels of intensity. At the current throughput of four knockdown cell lines per week, it will be possible to generate and analyze the entire collection of ~200 proteins that constitutes the first focus project, the FXM. However, throughput may be limited initially because of the need to implement three necessary components of the plan. First, knockdown cell lines demonstrating interesting phenotypes in the initial screening will be subjected to more in-depth experimental analysis. This will include more detailed examination of time courses and ligand concentration-response relationships, selected utilization of additional existing assays (e.g., lipid arrays, transcript analysis, and broader assessment of protein phosphorylation.), and testing of relevant non-additive combinations of ligands. Second, to take advantage of knockdown cell lines as they are being cultured in the laboratories, analyses beyond the boundaries of the FXM will be expanded to include screening for effects on cAMP accumulation and cytokine release (see Program Summary). Third, laboratory personnel and resources will also be involved in implementing new assays, including development of novel biosensors, to increase coverage of signaling readouts (see next section). The completion of the FXM phase of the perturbation analysis is therefore projected into the second year of the funding period. Expansion of this effort, as described in the Program Summary, will continue for the duration of the funding period.

Development and Implementation of Additional Assays. Starting now, during the last year of current funding, and continuing especially for the first two years of the next proposed funding cycle, we will devote substantial effort to increase the number of assays available to measure information flow through the RAW cell signaling network. We estimate that up to 30-50% of the combined efforts of five AfCS laboratories will be devoted to this endeavor. These groups will focus on 1) adaptation or development and deployment of fluorescent biosensor assays (e.g., FRET, BRET, and translocation assays), 2) increasing both the throughput and the repertoire of protein phosphorylation sites that can be evaluated quantitatively, especially including use of Bioplex technology and new mass spectroscopic approaches – AQUA peptides and SILAC, and 3) refining and deploying assays for phosphoinositides, inositol phosphates, diacylglycerol, and other lipids and second messengers. Participating laboratories will particularly include the Cell Preparation & Analysis, Microscopy, Lipidomics, Antibody, and Protein Chemistry Labs, with critical support from the Molecular Biology Lab. These activities will be scaled back in subsequent years, and freed resources will be directed to perturbation analysis and quantitative evaluation of information flow through both wild type RAW cells and those manipulated by critically effective perturbants. With these assays and cells in hand, they will be exposed to especially interesting combinations of interacting ligands.

Data Management and Bioinformatics. The Data Management, Analysis, & Bioinformatics Lab will provide support for the three initiatives outlined above. During year one, infrastructure for the collection, display, and statistical assessment of the perturbation analysis will be completed. Handling, display, and analysis of data from each new assay platform will follow within months of deployment of that assay. Support for mathematical modeling efforts will require the creation of interfaces for the transfer of information to the Data Modeling & Network Analysis Lab, including both AfCS experimental data and AfCS Molecule Page data.

Mathematical Modeling. Starting now in the last year of the present funding period, the DMNA Lab will continue to build the initial models of the signal transduction pathways converging on Ca²⁺ dynamics. The basic model structure will be evaluated mid-year by the Macrophage Committee to ensure that the right molecules and interactions are included. These interim models will be compared to the data generated by the perturbation analysis. The results of these initial analyses – their utility in explaining the data and in generating suggestions for experiments – will be evaluated at the end of this year by the Macrophage Committee and during a signal transduction jamboree. In the first year of new funding, we will be extending the model building and model analysis tools in collaboration with the DMAB Lab. The utility of the model building extensions will be tested by disbursement of the tool to various AfCS laboratories so they can track model progress and add models/annotations of their own. Feedback will be compiled every quarter and drive the next versions of the tool. Following this there will be semiannual reviews of the software usability and the effectiveness and utility of model results. The other product of these reviews will be refocusing of what systems and questions the DMNA Lab will be addressing. Tested models, model output, and data used to validate the model will be compiled and posted for public consumption several times each year.

Evaluation of Progress. We will continue to evaluate progress as we have during the latter years of the initial funding period. AfCS laboratories submit monthly progress reports that are disseminated by email and posted (behind a firewall) on the AfCS website. Each lab presents their work once every other month at scheduled AfCS-wide (videoconference) lab meetings. Scheduled monthly meetings of the Macrophage Committee provide feedback from experts in macrophage biology and other aspects of cell signaling. Issues with data analysis and operational coordination of laboratories participating in the FXM are discussed at their respective monthly meetings. Members of the Steering Committee (the scientific subcommittee) attend all of these meetings. The scheduled monthly meetings of the Steering Committee, as well as frequent informal meeting between individuals or small groups, serve as venues to evaluate ongoing progress of individual laboratories and the overall goals of the project. Annual progress is assessed at the yearly AfCS meeting by our External Advisory Committee. This 3-day meeting is attended by AfCS personnel and staff, as well as NIH and corporate sponsors.