G protein coupled receptor (GPCR) initiated signaling regulates important cell behaviors such as migration, differentiation, secretion and contraction. Considerable knowledge exists about the properties of individual molecules that constitute GPCR signaling networks. However, there is much less information about how these molecules act dynamically in a signaling network to orchestrate complex cell behaviors such as cell migration. It has not been possible to answer this question so far because of an absence of methods to exert spatiotemporal control over GPCR activity in a single living cell, evoke responses that faithfully reflect native cell behavior and quantitatively image cellular and molecular response dynamics. We have now developed an approach with all of these characteristics using opsins. Using this approach we have been able to direct cell migration using a light beam. The optically directed cell migration faithfully recapitulates molecular and cellular properties of chemoattractant induced cell migration. High resolution imaging has facilitated quantitation of signaling activity within a single cell and helped develop a computational model of migration. Here we propose to develop the optical tools further and use our optical approach to uncover molecular mechanisms at the basis of cell migration. We will use novel optical approaches to both orchestrate and perturb signaling systems that regulate migration. This will be combined with live cell imaging, biochemical methods and mathematical modeling.
Aim 1. We will develop (i) red opsins that can be used together with the blue light activated CRY2 system and (ii) opsins that can be activated for extended periods of time without deactivation.
Aim 2. We will test the model that SHIP1 acts antagonistically to PI3K to control cell migration.
Aim 3. We will identify molecular mechanisms at the basis of adaptation in cell migration. Identifying dynamic mechanisms that control cell migration can provide a better understanding of cancer metastasis, inflammatory diseases and can afford control over morphogenetic movement. Further development of the optical methods here can be of value in redirecting cell migration in the whole animal.

Public Health Relevance

The ability to control GPCR mediated single cell behaviors with different wavelengths of light can be used to interrogate their molecular basis experimentally and direct their function for therapeutic purposes.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular and Integrative Signal Transduction Study Section (MIST)
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Dunsmore, Sarah
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Washington University
Schools of Medicine
Saint Louis
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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
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