Single-molecule tracker illuminates workings of cancer-related proteins

Peng lab member and study co-first-author João Shida prepares to image nanoparticles using the lab's custom-built microscope.
Photo Credit: Allison Colorado, Broad Communications

Scientific Frontline: Extended "At a Glance" Summary: Single-Molecule Tracking Using Nanoparticles

The Core Concept: Single-molecule tracking is an advanced imaging method that utilizes highly stable nanoparticle probes to tag and continuously monitor the real-time activity of individual proteins within living cells. This technique allows researchers to map the complete lifespan and movement of cellular molecules in their native environment.

Key Distinction/Mechanism: Existing contrast agents, such as fluorescent dyes, suffer from photobleaching and burn out after a few seconds of laser excitation. This new method employs upconverting nanoparticles containing rare-earth ions that remain stable and luminesce for minutes to hours, enabling uninterrupted, long-term observation of receptor signaling and pairing dynamics.

Major Frameworks/Components: 

• Upconverting Nanoparticles: Customizable, long-lasting imaging probes engineered with rare-earth ions that emit varied colors based on ion type and dose.
• EGFR Family Receptors: The specific cancer-related cell receptors (EGFR, HER2, and HER3) targeted and tagged to study cellular signaling behaviors.
• Receptor Dimerization: The biological process where cell receptors pair up to initiate signals, which can lead to uncontrolled cell growth if prolonged by mutations.

Branch of Science: Nanotechnology, Molecular Biology, Oncology, and Chemistry.

Future Application: The technology establishes a new framework for advanced drug screening by revealing exactly how potential therapeutics alter individual molecules over time. Future developments aim to engineer probes that are smaller, brighter, and capable of emitting a wider array of colors for multiplexed targeting.

Why It Matters: By providing an unprecedented look at how mutated receptors form stable pairings to drive excessive cell growth, this tool offers critical new insights into cancer biology. It enables the observation of dynamic biological processes at high spatiotemporal resolution, opening the door for highly targeted and effective cancer treatments.

Using a powerful single-molecule imaging method they developed, a Broad Institute research team has unveiled a dynamic view of how some cancer-related proteins interact in living cells. The technique relies on highly stable nanoparticle probes that brightly illuminate individual molecules for long periods of time. The researchers used their method to observe, for the first time, individual receptors as they move around the cell membrane, attaching to and then letting go of other receptors to alter signaling within the cell.

Described in 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.

“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,” said study leader Sam Peng, a Broad core institute member and assistant professor of chemistry at MIT.

Molecular Movies

Peng’s method solves a problem with 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, which means scientists can only use them to take a few snapshots of cell receptors and not follow them over the entirety of 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. The nanoparticles contain rare-earth ions that continue to luminesce for minutes, hours, and potentially years. In addition, by altering the type and doses of the ions, scientists can engineer probes emitting in many different colors, enabling the tracking of multiple targets in a single experiment.

In the current study, the researchers aimed to uncover new biology by focusing on the EGFR family of cell receptors, which have been linked to several kinds of cancer. They collaborated with EGFR experts Matthew Meyerson and Heidi Greulich of 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.

For a better and more sustained look at the receptors, the research team customized their upconverting nanoparticles to tag EGFR and the related receptors HER2 and HER3, which are linked to cancer, and used them to track the molecules in living human cells.

A microscopy video shows upconverting nanoparticles tagged to EGFR receptors (labeled pink and green), which track individual receptors as they dimerize.
Video Credit: Courtesy of the Peng lab

A New View of Protein Pairings

In this study, Peng and his 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 abnormal cell growth and cancer.

When the EGFR molecules carried cancer-related mutations, the dimers became more stable, with the more stabilizing mutations linked to more potent cancers in people. In addition, the mutated receptors could form stable dimers even without an external stimulus prompting them to dimerize. The finding helps explain how EGFR mutations can lead to uncontrolled cell growth and cancer, and it could inform efforts to target this process therapeutically.

A video created using the Peng lab's single-molecule tracking technology, which allowed them to track individual EGFR, HER2, and HER3 receptors (in green, pink, and blue, respectively) as they moved across the surface of a living cell.
Video Credit: Courtesy of the Peng lab

The team discovered several other new and surprising details about how HER2 and HER3 form stable pairings with themselves, which helps illuminate 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 over and over again.

Beyond shedding light on EGFR biology, the 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,” said Peng.

They are also planning to explore the method’s use in studying the mechanisms of drug action to reveal how potential therapeutics alter individual molecules over time. In addition, they will continue to improve their methods, such as by making the probes smaller, brighter, and able to emit more colors.

Funding: The study was supported by the Broad Institute of MIT and Harvard, the MIT Charles E. Reed Faculty Initiatives Fund, the National Institutes of Health, the Alfred P. Sloan Foundation Matter-to-Life Award, and the G. Harold and Leila Y. Mathers Charitable Foundation.

Published in journal: Cell

Title: ErbB family receptor dimerization dynamics and dysregulation via long-term single-molecule imaging

Authors: Kaibo Ma, Xiaojie Ma, João F. Shida, Zijian Niu, Yuzhu Karlie Lin, Saptarshi Mandal, Alexandra Dobbins, Lior Golomb, Michael J. Eck, Heidi Greulich, Matthew Meyerson, and Chunte Sam Peng

Source/Credit: Broad Institute | Leah Eisenstadt

Reference Number: nt050226_01

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