Using a powerful single-molecule imaging method developed by a research team from the Broad Institute of MIT and Harvard, a dynamic view of how some cancer-related proteins interact in living cells has been unveiled. The technique relies on highly stable nanoparticle probes that brightly illuminate individual molecules for extended periods. For the first time, researchers observed individual receptors moving around the cell membrane, attaching to, and releasing other receptors, thereby altering signaling within the cell. Published in the journal Cell, the work demonstrates the method’s potential for investigating other receptors and molecules, and for improved drug screening to better understand the effects of therapeutics on living cells. Study leader Sam Peng stated, "With our photostable probes, we can map out the entire lifespan of these molecules in their native environment and see things that have never been observable before."
Molecular Movies
Peng's method addresses a limitation of existing contrast agents used in single-molecule tracking, such as dyes. Under the laser light used to excite these dyes, they burn out after a few seconds in a phenomenon known as photobleaching, meaning scientists could only take a few snapshots of cell receptors and could not follow them throughout the signaling process. For a longer and richer view, Peng's lab developed long-lasting probes, known as upconverting nanoparticles, which emit signals that remain stable under laser excitation. These nanoparticles contain rare-earth ions that continue to luminesce for minutes, hours, and potentially years. Moreover, by altering the type and doses of the ions, scientists can engineer probes emitting in many different colors, enabling tracking of multiple targets in a single experiment.
In the current study, researchers aimed to uncover new biology by focusing on the EGFR family of cell receptors, which have been linked to several types of cancer. They collaborated with EGFR experts Matthew Meyerson and Heidi Greulich from the Broad’s Cancer Program. They knew that EGFR receptors need to pair up, or “dimerize,” to initiate signaling within the cell, but they wanted to learn more about the dynamics of these pairings—what the receptors partner with, how long they stay together, and how they find new partners.
A New View of Protein Pairings
To better observe the receptors, the research team customized their upconverting nanoparticles to tag EGFR and related receptors HER2 and HER3, which are linked to cancer, and used them to track the molecules in living human cells. The team observed that, when activated with a stimulating molecule, EGFR receptors can pair up and stay dimerized for several minutes, something not observable using traditional dyes. Excessive and prolonged dimerization can lead to too much cell growth and cancer. A microscopy video shows upconverting nanoparticles tagged to EGFR receptors (labeled pink and green), tracking individual receptors as they dimerize.
When EGFR molecules carried cancer-related mutations, the dimers became more stable, with more stabilizing mutations linked to more potent cancers in people. Additionally, the mutated receptors could form stable dimers even without an external stimulus prompting them to dimerize. This finding helps explain how EGFR mutations can lead to uncontrolled cell growth and cancer, potentially informing efforts to target this process therapeutically. The team also discovered several other new and surprising details about how HER2 and HER3 form stable pairings with themselves, illuminating the role of these molecules in related cancers. When the research team tagged all three receptor types in one experiment, they observed a vibrant scene with receptors navigating the cell surface, finding partners, unpairing, and then finding new partners repeatedly.
Beyond shedding light on EGFR biology, scientists hope that collaborators in other fields will apply their method to ask new scientific questions about other proteins of interest. "We think this technique could be transformative for studying molecular biology, because it enables dynamic biological processes to be observed with high spatiotemporal resolution over unprecedented timescales," says Peng. They are also planning to explore the method’s use in studying the mechanism of drug action, to reveal how potential therapeutics alter individual molecules over time. Additionally, they will continue to improve their methods, such as making the probes smaller, brighter, and able to emit more colors.
Blogger's Review: This research not only showcases a breakthrough in single-molecule tracking technology but also provides a fresh perspective on the dynamic interactions of cancer-related proteins. With deeper exploration, we anticipate these techniques to play a significant role in drug development and personalized therapies, advancing the field of precision medicine.