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The FXM Signaling Network PI(3,4,5)P3Ca 2+ IgG2a UTP/UDP C5a Testing Ground
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Define the system for study Plan and organize experiments Reference tool Template for future modeling ?
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We asked... What signaling molecules might be involved? Are these signaling proteins specifically expressed in RAW 264.7 cells? What is the presumed role of each signaling molecule in the network? Which signaling proteins shall be targets of perturbations and other experiments?
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We asked... What signaling molecules might be involved? Are these signaling proteins specifically expressed in RAW 264.7 cells? What is the presumed role of each signaling molecule in the network? Which signaling proteins shall be targets of perturbations and other experiments?
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We asked... What signaling molecules might be involved? Are these signaling proteins specifically expressed in RAW 264.7 cells? What is the presumed role of each signaling molecule in the network? Which signaling proteins shall be targets of perturbations and other experiments?
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We asked... What signaling molecules might be involved? Are these signaling proteins specifically expressed in RAW 264.7 cells? What is the presumed role of each signaling molecule in the network? Which signaling proteins shall be targets of perturbations and other experiments?
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Initial Goals of FXM Project Develop a parts list of specific proteins that constitute the FXM network. Develop a detailed map describing known or suspected reactions occurring within FXM pathways.
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We built an initial crude network... …to establish a basic framework
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Initial perturbation targets (test method w/ the obvious) Parts List More comprehensive map Y2H targets
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FXM Parts List Started with an inclusive list based on legacy Considered all possible isoforms
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Compiled in Excel Worksheets
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Expression in RAW 264.7 Cells Expression tracked by Affymetrix arrays and RT-PCR In some cases, antibody data and/or functional data are available Goal: trim down list
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Overview of Parts 247 proteins (including isoforms) considered 204 proteins remain on list following expression analysis ~40 mRNA confirmed present by RT-PCR ~27 proteins confirmed present by antibody ~64 expression is uncertain (i.e., Affy unclear, RT not yet done)
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Building a detailed network map Collaboration with Adam Arkin Lab Development of map using PathwayBuilder software (DARPA BioSPICE) Tested both the method for creating a signaling map and the suitability of the software
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Content of a Pathway Signaling Map NOT a connection map of all possible protein interactions A more defined map: Assign a role for each signaling molecule (protein and non-protein) within the FXM network Molecular states and complexes Create a set of hypotheses to be tested by experimental perturbation A tool and template for modelers
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Pathway Description
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Process-based diagrams Process could be... Enzymatic Reaction Regulated Reaction Binding interaction Unknown mechanism
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Nodes Each node represents a specific state of a molecule or group of molecules Nodes are unique Nodes are designated as proteins, non-proteins, or complexes ProteinNon-Protein Complex
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Sample Pathway
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Putting it all together GPCR Calcium Fc Receptor PIP3
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Overview of Map Contains 273 nodes Connected by 234 processes (reactions) Complexes constitute 123 nodes Some molecules such as Lyn, PIP3, or Ca 2+ might participate in as many as 15 distinct processes. Not yet finished!
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Parts List Pathway Map Experiments
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Credits Lily Jiang Madhu Natarajan Alex Gilman Adam Arkin Elliott Ross Al Gilman Mike Berridge Henry Bourne Rick Brown Lew Cantley Mike Gold Kelly Smith Jim Stull
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