A Kaleidoscope of Projects
Plenty of mornings, Loren Looger shows up at the Janelia Farm Research Campus not knowing what kind of science he’ll do that day. He devotes most of his time to the short list of projects he expects will offer the greatest potential payoff, but some days he is diverted to a new intellectual challenge. Some days, he says, “it’s just a matter of the squeakiest wheel.”
Even with the availability of new genetically encoded calcium indicators, researchers still need alternative ways to visualize neural activity. Additional layers of communication are encoded in the chemical signals other than calcium that neurons send to one another, for example. One such chemical is the neurotransmitter GABA, which, when it lands on receptors on the surface of a receiving neuron, has a quieting effect on the cell. Tracing GABA would give researchers a clearer picture of how chemical and electric signals work together to influence a cell’s behavior. Finding a way to do that, Looger says, is high on his priority list right now. For hints about what a GABA-detecting protein should look like, Looger is searching far beyond the nervous system to other cells that have evolved to tune in to the presence of GABA. The most relevant exchange appears to take place between plants and symbiotic bacteria, where GABA acts as a chemical alarm that signals the need to mount a defense against nematodes and fungi lurking in a plant’s root nodules.
As he scrutinizes the genome of such a bacterium for signs of a GABA sensor, Looger juggles progress on other sensors in development. A version of GCaMP that lights up red will send a signal that travels farther through tissue than the original green sensor, letting researchers explore deeper in the brain with noninvasive techniques. To offer a faster readout for nerve cell firing than calcium can provide, his team is helping to tweak molecules that directly monitor neurons’ electrical properties. And a separate effort focuses on developing a sensor whose signal lingers, rather than flickering and then burning out, so that researchers accustomed to probing within a microscope’s narrow field of view no longer need to worry about looking at the right place at the right time.
Looger recently designed a system with Janelia Farm chemist Luke Lavis to target chemicals to specific cells by masking them with chemical shields that can be removed only by a corresponding enzyme. The technique, which gives scientists a new way to visualize a single cell type in a tangle of neurons, built upon existing dyes that are protected by a simple chemical group called an ester. The enzymes that set them free, called esterases, are so abundant that colleagues told them they would never create an ester–esterase pair that would offer the specificity they were after: a chemical bond strong enough to resist a cell’s natural enzymes and an enzyme strong enough to cleave it.
By examining the structure and activity of a diverse range of natural esterases, Looger and Lavis succeeded and reported their technique in the Proceedings of the National Academy of Sciences in March 2012. They are now adapting their approach to small pharmaceuticals, such as drugs that block protein synthesis or alter neural activity, so that researchers can direct the drugs to specific cells instead of administering them indiscriminately, enabling more refined functional studies.
Looger has also begun applying his bag of tricks to more theoretical problems. When his mother-in-law, Duke developmental biologist Blanche Capel, mentioned over cocktails one evening that her team had traced the factors that help determine the sex of mouse embryos to a broad swath of the genome but with existing genetic tools could not determine which of the 60 genes inside that region were involved, Looger argued that the problem just needed a new perspective. By examining individual genetic variation within the regions and searching for hints of the kinds of proteins that might influence complex signaling pathways like those involved in sex determination, he zeroed in on genes encoding four proteins he suspected might be involved. “They’ve gone back to validate the proteins, and the biology seems to match,” he says.
He has since offered similar observations on genetic data shared by lupus researcher Swapan Nath from the Oklahoma Medical Research Foundation. Through large-scale statistical analyses of genetic data, Nath had found a handful of variants of a single gene that appear to increase the risk of lupus in African Americans. Looger’s analysis suggested how the sequence changes might cause the cellular dysfunctions associated with lupus by disrupting the encoded protein’s function or altering its interactions with other proteins—predictions that Nath says have matched up well with experimental data from his lab. It’s not tool building, but many of the same insights apply, and Looger says he anticipates taking on more bioinformatic challenges, where his knowledge and instinct can help biologists home in on the pieces of their data that are most likely to be biologically relevant.
“I love living outside my comfort zone,” he says. “It keeps everything so exciting.”
-- Jennifer Michalowski
HHMI Bulletin, May 2012