After he built some of the world’s first microscopes, Leeuwenhoek used them to discover an entire living world in a drop of pond water and establish the field of microbiology. His many critics, who saw only drops of water, didn’t refuse to make the same discoveries—they simply lacked the technology to see what he saw. The lesson here is that oftentimes, scientific progress occurs not only as scientists think harder about the natural world but as they develop new tools that allow them to observe and interact with it. An example of this concept is described in a recent publication in eLife from the Sullivan Lab in the Fred Hutch Human Biology Division, which reports the creation of a new tool for interrogating intracellular aspartate metabolism.
The Sullivan Lab studies the metabolic roles of the mitochondria—that kidney-bean shaped organelle which you probably know as a cellular ATP factory (powerhouse, anyone?). There’s no doubt that cells need energy, but it turns out that these diminutive organelles do a lot more for cells than just make ATP. In addition to their catabolic roles in producing cellular energy, mitochondria enable numerous anabolic pathways which together produce many of the molecular building blocks that cells are made of: DNA, RNA, proteins, and membranes, to name a few. Among these building blocks, the amino acid aspartate stands out as a crucial output—so much so that it's often aspartate, not ATP, which becomes growth-limiting in cells upon mitochondrial inhibition. Aspartate is also an endogenous metabolic limitation of tumor growth, making the study of aspartate metabolism relevant from both basic and clinical perspectives.
So, aspartate is important, and scientists in the Sullivan Lab and elsewhere would like to study aspartate metabolism, but here’s where we run into a bit of a limitation. The current state-of-the-art method for measuring intracellular aspartate, a tool called liquid chromatography-mass spectrometry (LCMS), is exquisitely sensitive but ‘single use’: cells must be killed and their metabolites extracted to be run on the instrument. This isn’t an issue if your question is ‘how much aspartate is in my cells?’ but if you’re wondering where in your cells the aspartate is, or how intracellular aspartate levels change over time in a particular experimental condition, then LCMS has notable limitations. A fundamentally different approach for measuring aspartate involves the use of a genetically encoded biosensor: essentially, an engineered protein which specifically binds aspartate and changes fluorescence upon doing so. Biosensors are being developed and used to measure a growing list of metabolites, but until recently, aspartate-ologists had no biosensor to call their own.
The Sullivan Lab’s recent study, spearheaded by graduate student Kristian Davidsen, tackles this opportunity head-on. In collaboration with Jonathan Marvin and colleagues at the Howard Hughes Medical Institute (HHMI) Janelia Research Campus, the team developed the first reported aspartate biosensor by taking an existing glutamate biosensor and mutating a few key residues in the glutamate binding pocket to switch the sensor’s specificity to aspartate (glutamate and aspartate have similar chemical structures, which makes this possible). Mechanistically, the sensor is composed of two pieces: a green fluorescent protein (GFP) which has been cleverly disabled by fusing it to a portion of a bacterial aspartate/glutamate binding protein. Aspartate binding to this protein causes a conformational change which restores GFP fluorescence—the more aspartate is around the sensor, the more binding events occur and the higher the fluorescence. After the team confirmed that their new biosensor, dubbed jAspSnFR3, specifically binds aspartate and changes fluorescence intensity in a dose-responsive manner in vitro and in cells, Davidsen used it to track intracellular aspartate levels over time in several different cell lines treated with mitochondrial inhibitors (which, as noted above, impair cell growth by limiting aspartate). While the purpose of the study was primarily to introduce and validate this new tool, the team was already able to leverage the temporal resolution it provides to produce novel biological insights—for example, to substantiate claims that two similar, commonly-used mitochondrial inhibitors (rotenone and metformin) differ markedly in their temporal effects on aspartate limitation.
Overall, the team points out that they have only scratched the surface of what’s possible using a biosensor approach to measure aspartate—future iterations of the technology could target the sensor to specific subcellular locales (like the mitochondrion) to assess compartmentalized aspartate levels, and since the sensor is genetically encoded, it could be introduced into other model systems (mouse brains or zebrafish embryos, to name a few) to study aspartate metabolism in diverse biological contexts beyond cancer. “We’re excited to use this new tool to better understand aspartate metabolism in cancer and provide it as a freely available resource for anyone in the field to use,” notes Dr. Sullivan. “On its own, it’s powerful for providing spatial and temporal resolution that is difficult using traditional LCMS approaches; however, we can also use insights gained from the biosensor to better conduct our LCMS experiments and get the best of both worlds.” A new tool for a new field; different from Leeuwenhoek, but not so different.
The spotlighted research was funded by the National Institutes of Health and the Howard Hughes Medical Institute.
Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium member Dr. Lucas Sullivan contributed to this study.
Davidsen, K., Marvin, J. S., Aggarwal, A., Brown, T. A., & Sullivan, L. B. 2023. An engineered biosensor enables dynamic aspartate measurements in living cells. eLife [Preprint]. 12:RP90024