The rise of optogenetics — where flashes of light can manipulate brain activity and rat behavior — have excited scientists looking for more precise ways of manipulating cells and their components in the laboratory and the clinic. Two papers published this month by University of Chicago laboratories explore new methods with great scientific potential of controlling cells through light We’ll cover them in two installments.
Many important cellular functions are principally the result of factors being in the right place at the right time. The complex pathways that handle important jobs such as cell growth or migration can have hundreds of components that must arrive at the same location within the busy cellular environment. As such, biologists would like the ability to move proteins to different parts of the cell at will, giving them control over the staging of these pathways and their functions. With a new system that draws upon the work of several University of Chicago laboratories, researchers have figured out how to achieve this power with light.
Like many good ideas in science, the collaboration that created TULIPs began with a question posed at a bar. At the annual Molecular Biosciences retreat in Galena, Illinois, Devin Strickland and Tobin Sosnick challenged their colleague Michael Glotzer with a provocative question.
“They asked, ‘If you could control any protein you wanted, what would you control?’,” recalled Glotzer, Professor of Molecular Genetics & Cell Biology at the University of Chicago.
Strickland, then a graduate student in the laboratory of Sosnick, Professor and Chair of Biochemistry & Molecular Biophysics, were looking for applications of their strategy of using light to control the activity of a specific protein. Glotzer realized that the system could bring two proteins together in a cell, whenever and wherever researchers wanted. The result was an exciting new technique, called TULIPs, published this month in Nature Methods.
The acronym TULIPs stands for TUnable, Light-controlled Interacting Protein tags. Instead of the light-activated ion channels or gene transcription used by other optogenetic methods, the TULIPs use a LOV domain, a light-sensitive component from a protein plants use to detect and grow toward sunlight. By attaching different proteins to this domain and another known as an ePDZ “clamp,” the researchers built a customizable way of precisely manipulating cellular signals in both space and time.
“Basically nothing previously had been designed to control cell signaling that wasn’t engineered on a case-by-case basis,” said Strickland, now a postdoctoral researcher in Glotzer’s laboratory. “We started off wanting to be the first people to create an adaptable system.”
In the Nature Methods paper, the authors describe testing the system by attaching it to fluorescent proteins and applying it to Glotzer’s chosen target, a GTPase pathway that controls growth and genes related to mating in yeast. The experiments demonstrated both that the TULIPs worked, and that it could be easily adapted to manipulate a wide range of cellular signals.
“The idea was let’s solve this generic problem once, and then we’ll be able to reuse that solution in many different contexts,” Glotzer said. “Looking at lots of different biological phenomena, it’s pretty clear that a lot of functions correlate with changes in sub-cellular localization of specific proteins. So there’s almost no limit; you’re only limited by your imagination.”
[Light flashes on the right demonstrate the successful localization of fluorescent proteins attached to the TULIPs system. Video by Elizabeth Wagner]
The TULIP system is made up of two components that draw upon work done previously by the University of Chicago laboratories of Shohei Koide, who developed the ePDZ clamp, and Keith Moffat, who characterized the LOV domain and first suggested its use in the TULIP system.
“It’s really a nice story of how basic research can lead to things that aren’t really anticipated,” Strickland said.
To work, the light-sensitive LOV domain is attached to another domain that determines where it will be located in the cell, such as on the cell’s membrane or on the mitochondria. A second element is made up of an ePDZ clamp attached to the protein of interest. When a specific wavelength of light is shined upon the cell, the coiled Jα helix at the end of the LOV domain unfolds itself, becoming capable of binding the ePDZ clamp and recruiting the target protein to the researchers’ chosen location. By customizing the locations and target proteins, many cellular processes — such as cell polarity, migration, or division — could potentially be studied and manipulated using TULIPs.
“We can, in principle, trigger a pathway in a few seconds, so we can look at questions with a high time resolution,” Strickland said. “Both the kinetics and the reversibility are potentially very useful in answering different biological questions.”
In the yeast experiments, performed in collaboration with the laboratory of Eric Weiss at Northwestern University, the power of TULIPs was demonstrated. The researchers attached different “scaffold proteins” associated with two signaling pathways to ePDZ clamps, then engineered LOV domains that hung on the plasma membrane. When a light was shined on the cells, the scaffold proteins were attracted to the cell membrane. Depending on the ePDZ-attached protein used, the researchers were thus able to switch the pathways on or off, controlling the growth of yeast cells, the transcription of genes involved in mating, and the polarity of the cells. In addition to demonstrating the flexibility of the TULIPs, the experiments also showed just how important location can be for controlling these pathways.
“Simply putting two proteins near each other is sufficient to get activation,” Sosnick said. “There’s nothing magical…it’s just proximity and co-localization, which wasn’t obvious before.”
In the laboratory, the TULIPs system will allow researchers to test the sufficiency or necessity of other cellular signals in a similar fashion. By moving a target protein to its hypothesized functional place in the cell — or conversely, keeping it away — scientists can test whether it is an essential component of a given pathway. Farther in the future, the researchers speculated that TULIPs might have clinical potential, as a way to selectively manipulate specific cell types. The unrestrained division of tumor cells could be permanently arrested with a flash of light, or a diabetic’s insulin production could be boosted with a similar trigger. The system might even prove to be a safer and more precise way of stimulating brain or heart activity than the current electrical systems in use.
“There’s nothing easier to control than light,” Glotzer said. “It’s non-toxic, it’s specific, and it’s reversible.”
Strickland, D., Lin, Y., Wagner, E., Hope, C., Zayner, J., Antoniou, C., Sosnick, T., Weiss, E., & Glotzer, M. (2012). TULIPs: tunable, light-controlled interacting protein tags for cell biology Nature Methods, 9 (4), 379-384 DOI: 10.1038/nmeth.1904