The molecular mechanisms underlying G protein signaling have so far been elucidated using purified proteins and lysed cells. Little information is available about the behavior of G protein pathways in a living mammalian cell. The broad aim is to visualize the functioning of a G protein signaling pathway and its cross-talk with other signaling pathways both spatially and temporally in live cells. G protein based biosensors have been designed containing signaling proteins fused to cyan and yellow fluorescent proteins (CFP & YFP). The sensors respond to receptor stimulation or inactivation with a corresponding fluorescence resonance energy transfer (FRET) signal change. Images of cells expressing the sensors acquired during signal processing provide direct information in real time on the properties of the pathway in different parts of the cell.
The specific aims will be (i) to develop methods for imaging G protein signaling activity in mammalian cells. (ii) To test models about (a) the spatial distribution of signaling machinery and (b) the spatio-temporal progression of localized G protein activation. (iii) To test the hypotheses that a G protein pathway affects the spatio-temporal dynamics of (a) another G protein signaling activity and (b) ras and rap activity. Experiments will be performed with transfected and endogenous receptors. A vascular smooth muscle cell line will be used to examine other endogenous pathways. The majority of commercially available drugs are targeted at G protein coupled receptors. The biosensors developed here can be of considerable value pharmacologically. They can be used to screen for novel drugs and identify ligands for orphan receptors in non-invasive assays. In the longterm, this approach can help observe networks of signaling pathways functioning in a cell as it senses, processes and responds to a signal.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM069027-03
Application #
7028870
Study Section
Pharmacology A Study Section (PHRA)
Program Officer
Dunsmore, Sarah
Project Start
2004-04-01
Project End
2008-03-31
Budget Start
2006-04-01
Budget End
2007-03-31
Support Year
3
Fiscal Year
2006
Total Cost
$313,749
Indirect Cost
Name
Washington University
Department
Anesthesiology
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Meshik, Xenia; O'Neill, Patrick R; Gautam, N (2018) Optogenetic Control of Cell Migration. Methods Mol Biol 1749:313-324
O'Neill, Patrick R; Castillo-Badillo, Jean A; Meshik, Xenia et al. (2018) Membrane Flow Drives an Adhesion-Independent Amoeboid Cell Migration Mode. Dev Cell 46:9-22.e4
Kim, Seungil; Barry, Devin M; Liu, Xian-Yu et al. (2016) Facilitation of TRPV4 by TRPV1 is required for itch transmission in some sensory neuron populations. Sci Signal 9:ra71
O'Neill, Patrick R; Kalyanaraman, Vani; Gautam, N (2016) Subcellular optogenetic activation of Cdc42 controls local and distal signaling to drive immune cell migration. Mol Biol Cell 27:1442-50
Karunarathne, W K Ajith; O'Neill, Patrick R; Gautam, Narasimhan (2015) Subcellular optogenetics - controlling signaling and single-cell behavior. J Cell Sci 128:15-25
O'Neill, P R; Gautam, N (2015) Optimizing optogenetic constructs for control over signaling and cell behaviours. Photochem Photobiol Sci 14:1578-85
O'Neill, Patrick R; Giri, Lopamudra; Karunarathne, W K Ajith et al. (2014) The structure of dynamic GPCR signaling networks. Wiley Interdiscip Rev Syst Biol Med 6:115-23
Zhao, Zhong-Qiu; Liu, Xian-Yu; Jeffry, Joseph et al. (2014) Descending control of itch transmission by the serotonergic system via 5-HT1A-facilitated GRP-GRPR signaling. Neuron 84:821-34
O'Neill, Patrick R; Gautam, N (2014) Subcellular optogenetic inhibition of G proteins generates signaling gradients and cell migration. Mol Biol Cell 25:2305-14
Giri, Lopamudra; Patel, Anilkumar K; Karunarathne, W K Ajith et al. (2014) A G-protein subunit translocation embedded network motif underlies GPCR regulation of calcium oscillations. Biophys J 107:242-54

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