Research highlights
'Blue' lighting cell signaling research
Nature Methods 6, 10 (01 October 2009) | doi:10.1038/nmeth1009-694a
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By fusing a light-sensitive domain of an oat plant protein to Rac1, researchers created a genetically encoded protein fusion that can be reversibly activated with blue light and control cell movement—an attractive alternative to current caging tools.
Protein caging tools are a staple in the arsenal of cell biologists. Though researchers are constantly developing new means to study rapid, spatiotemporally controlled processes, the drawbacks of current tools include irreversible activation and/or cell damage when the caged molecules are introduced into cells or uncaged with UV light.
Exposure of PA-Rac1 to blue light results in unwinding of the Jα helix, which frees the previously caged Rac1 GTPase to interact with effector proteins. Adapted from Nature.
In their quest to hone in on cell signals more precisely, Klaus Hahn, with postdoc Yi Wu, at the University of North Carolina, Chapel Hill, and their colleagues took a different caging approach, creating genetically encoded photoactivatable GTPases. As the 'cage' they used the photoreactive light oxygen voltage (LOV) domain of the Avena sativa (common oat) phototropin 1 protein (Wu et al., 2009). “It's like a yo-yo,” explains Hahn: “it's a globular protein on a string, which is an alpha helix that changes length depending on the irradiation.”
In the dark, this LOV domain interacts with a C-terminal alpha helix (Jα) and, when fused to a target protein, sterically blocks effector protein binding. When it is exposed to light—458- or 473-nm blue light—photon absorption results in a conformational change and subsequent dissociation and unwinding of the Jα helix, which releases the steric block and allows the fused protein to interact with downstream signaling molecules. As controls, a light-insensitive mutant as well as a constantly 'lit' mutant of the LOV domain can be used.
Using this approach, the researchers tagged Rac1, a GTPase that regulates actin cytoskeletal dynamics. After optimizing the fusion, they could turn on this photoactivatable Rac1 (PA-Rac1) by irradiation with blue light—reversibly and repeatedly—generating cell protrusions and ruffling in the activated area of the cell. Combining this tool with a Rho protein biosensor, they found that in mouse fibroblasts Rac1 inhibits RhoA, another protein involved in cytoskeleton dynamics. “We saw that Rho was specifically blocked in particular regions of the cell, so this now allows us to look at pathway interactions spatially in different spots and see that they are in fact different in different spots,” says Hahn.
The group also tagged a similar GTPase, Cdc42, with LOV-Jα. The strategy used for PA-Rac1 resulted in a Cdc42 fusion with residual activity, but structural modeling of the interface pointed to a mutation that would stabilize the LOV-Cdc42 interaction with additional hydrogen bonds in the caged, 'dark' state, allowing the researchers to make a PA-Cdc42. This suggests that on the basis of a structural understanding of the steric block for the protein family, the interface can be optimized for each individual protein to create other caged GTPases.
Hahn hopes that the LOV domain has an ideal structure to become a general caging tool, but he notes that the tagging approach will need to be worked out for each individual protein family. “The GTPases have a similar enough structure that we could come up with a general solution,” he says. “But if you now try to do something with a different shape, you are going to have to ask how you put the yo-yo on a string,... but I hope that there will be broadly applicable solutions for each family.” His group is now working with other protein families to see how general the approach can be.
And as more such caged proteins are made, tools will be needed to keep track of them. In a companion paper (Machacek et al., 2009), Hahn's group, along with Gaudenz Danuser's team at Scripps, used a computational multiplexing approach to delineate the spatiotemporal relationships between activities of the GTPases Rac1, RhoA and Cdc42 during cell protrusion.
As for PA-Rac1, because the protein controls cell motility, according to Hahn others are using it to make cells move around in animals. So more work is yet to come on many fronts.
