Precise spatial and temporal control of G protein signaling in single cells using opsins.

We have developed a method based on the color opsin from the human retina that allows G protein pathways to be activated in a spatiotemporally confined manner in a single cell. Using this method we have shown that immune cell migration and neuron differentiation can be optically controlled. The method allows intracellular molecules to be quantitatively imaged simultaneously while a cell behavior is orchestrated using a beam of light. 

Opsins and their absorption maxima

Karunarathne et al, doi: 10.1073/pnas.1220697110 PNAS April 23, 2013 vol. 110 no. 17 E1565-E1574

Optical control of neuron differentiation.

Develop a library of spectrally selective opsins that activate distinct signaling pathways so that cell behavior can be controlled in opposing directions using different wavelengths of light.

Optically perturb the spatial and temporal patterns of signaling during neuron differentiation and identify how information in terms of second messengers is encoded for executing neuron differentiation.                           

Karunarathne et al, PNAS (2013); 10.1073/pnas.1220697110.

Optical control of immune cell migration

We demonstrate that the ability to use a visual opsin to recruit endogenous signaling pathways and optically orchestrate immune cell migration show an untrasensitive switch-like PIP3 response at the leading edge. At the switch stage of PIP3 response, the cell initiates migration explaining how a migratory cell filters background noise and makes the decision to migrate.
migration to a persistent signal gradient across the cell.

 

Optical control of select proteins downstream of receptor activation

The above results show that optical control of receptor activation can be used to orchestrate complex cell behaviors. However, understanding the roles of downstream signaling proteins in generating these behaviors will require new tools that provide dynamic control over their signaling activities. We developed two such tools that act on heterotrimeric G protein subunits.

The first tool, CRY2-RGS4, provides optically triggered inhibition of heterotrimeric G proteins. Using this tool, we demonstrated that a G protein signaling gradient is sufficient to trigger directed migration of macrophage cells.

O'Neill et al. Mol Biol Cell (2014); doi: 10.1091/mbc.E14-04-0870

 

The second tool, CRY2-GRK2ct, can be used to generate a gradient of G protein betagamma subunit signaling across a cell. We found that this is sufficient to induce a polarized cellular response in a macrophage cell line. The cells produce lamellipodia and accumulate PIP3 in a directionally sensitive manner (Fig below). However, the cells do not demonstrate migratory movement. This suggests that mechanisms other than a Gbetagamma gradient may govern the overall migratory response. 

Together this approach shows it can be powerful in breaking down the individual steps that together constitute cellular migration and decipher the molecular basis.
 

O'Neill et al. Mol Biol Cell (2014); doi: 10.1091/mbc.E14-04-0870