Researchers have devised a way to control cell movement using flashes of blue light and have used the technique to uncover the function of a protein crucial to cell motility, they report online in Nature today (August 19th).

"This is going to promote studies of cellular behavior and even of organismal behavior," Keith Moffat, a University of Chicago biophysicist who was not involved with the study, told The Scientist. "I think it's really cool, frankly."

Activation of the protein Rac in the red circle (left) causes localized cell protrusion and movement of another protein PAK (shown in red at right) to the cell edge Image: Y.Wu, UNC-Chapel Hill

Other methods exist to control protein activity with light, but Klaus Hahn, a cell biologist and chemist at the University of North Carolina who led the research team, said that the method he and his colleagues developed is a big improvement. Previously, researchers have attached small, light-sensitive organic molecules to peptides or inhibitors, and injected these large constructs through the membrane and into a cell. A light flash would then break off the organic molecule, exposing biochemically active parts of the peptide or inhibitor.

That approach, though, required disrupting the cell membrane, and the wavelength of light needed to irreversibly break the covalent bond between the photoactive organic molecule and the peptide of interest was toxic to the cell. Finding the exact spot to link the photosensitive molecule so that it covered active sites on large, complex proteins was also problematic. "That was cumbersome, and it was also very much an engineering problem," Hahn said.

Hahn and his team instead turned to the photoreactive light oxygen voltage (LOV) domain of the phototropin protein, which mediates light sensitive behavior in plants and bacteria and responds to blue light. The researchers created a genetic construct combining LOV with their enzyme of interest, Rac1 (ras-related C3 botulinum toxin substrate 1), which regulates actin cytoskeletal dynamics in moving cells. When they expressed the construct in mouse and human cells, a targeted burst of non-toxic blue light separated LOV from Rac1, uncoiling a helix connecting the two molecules and freeing the protein to participate in biochemical pathways.

When the light was switched off, the helix sprang back into shape, again rendering Rac1 inactive. "The LOV domain is built kind of like a yo-yo," Hahn noted. "The beauty of it is that when you turn the light off, the thing winds up again." Switching the light on and off in specific spots in the cell resulted in protrusions and ruffles on the cell surface as Rac1 triggered actin polymerization.

Because Hahn's photoactive Rac1 is genetically encoded, the technique doesn't compromise the cell membrane. Also, the reaction is reversible, unlike previous techniques in which photosensitive proteins could only be activated once. "[Hahn's] approach is far superior to the previously used chemical approaches," said Moffat.

Also, Hahn explained, knocking out or silencing protein encoding genes may provide cells sufficient time to adapt to those changes, potentially muddling the results of protein function studies using such methods. "Those techniques give the cell an opportunity to compensate before you study it." Using light to control protein behavior allows unparalleled spatial and temporal specificity, and "Cells don't know what hit them until the light comes on," Hahn said.

Using the technique, Hahn and his collaborators tracked the interaction of Rac1 with another protein that functions in cell motility. By attaching a biosensor to RhoA (Ras homolog gene family, member A) while activating Rac1, the researchers were able to show definitively, for the first time, that Rac1 inhibits RhoA in mouse embryonic fibroblasts.

Recently, Moffat used LOV domains to manipulate the behavior of DNA binding proteins and a family of histodine kinases, but this is the first time the technique has been applied to a protein that plays a crucial role in cell motility. "Light is an extremely versatile way of controlling systems in biology," Moffat said. "This application is really neat."

Stanford University bioengineer Karl Deisseroth, who was not involved with the study, told The Scientist that the paper presents an interesting experimental construct that should be applied in living organisms. "[Hahn and colleagues] can regulate these aspects of motility, and an interesting avenue for further work will be to see if this will work in vivo," he said.

In fact, Hahn said that he has been doing just that. With collaborators at the University of Wisconsin and Johns Hopkins University, Hahn has begun using the technique in intact embryos to study the effects of manipulating the movement of dividing cells on development.


Related stories:

LOV story
[May 2009]

Sean Crosson: Bacteria in LOV
[December 2007]

Bacteria see the light
[23rd August 2007]