Simon Ebbinghaus and Mailin Becker (right) have developed a new imaging technique.
Photo Credit: © Lehrstuhl für Biophysikalische Chemie
Scientific Frontline: Extended "At a Glance" Summary: Confocal Fast Relaxation Imaging (cFReI)
The Core Concept: Confocal fast relaxation imaging (cFReI) is a novel experimental technique utilized to measure the stability and unfolding behavior of proteins at specific, localized points within a single living cell.
Key Distinction/Mechanism: Unlike traditional methods, cFReI enables the direct, simultaneous comparison of protein stability within membraneless organelles (MLOs) and the surrounding cytoplasm. This clarifies whether these specific compartments actively protect the cell by sequestering misfolded proteins, or if they act as environments that promote harmful clumping.
Major Frameworks/Components:
- Membraneless Organelles (MLOs): Cellular compartments formed through a process known as "liquid-liquid phase separation," which functions similarly to oil separating in water, allowing proteins and RNA to accumulate without a physical membrane barrier.
- Protein Aggregates: Incorrectly folded, unraveled, or clumped proteins that are directly linked to the pathology of neurodegenerative conditions.
- Superoxide Dismutase 1 (SOD1): A specific protein variant known to accumulate in cellular structures known as "stress granules," which researchers analyzed to test the efficacy of the cFReI method.
Branch of Science: Cellular Biology, Biophysical Chemistry, and Molecular Biology.
Future Application: Clarifying the specific role of MLOs in protein misfolding could ultimately lead to the development of highly targeted therapies and cellular interventions for severe neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer's, and Parkinson's.
Why It Matters: By revealing whether localized cellular environments trigger or prevent harmful protein clumping, this imaging technique provides crucial insights into the fundamental mechanisms of neurodegeneration, addressing a critical knowledge gap regarding disease progression at the cellular level.
A new method makes it possible to measure the stability of proteins at various points within an individual cell, shedding more light on the development of neurodegenerative diseases.
Protein aggregates are associated with the development of diseases such as Alzheimer’s and Parkinson’s. These aggregates are found more frequently in certain cellular regions known as membraneless organelles. Do these organelles promote this protein clumping, or do they protect the cell by temporarily absorbing the aggregates? Professor Simon Ebbinghaus and Mailin Becker from Ruhr University Bochum, Germany, set out to answer this question with their newly developed method, “confocal Fast Relaxation Imaging,” or cFReI.
Proteins and RNA Accumulate
Proteins are involved in numerous processes within the cell. To ensure these processes run smoothly, the cell is divided into different compartments known as organelles. Some of these, such as the nucleus and mitochondria, are surrounded by a membrane. “However, there are also organelles that are not encased by a membrane,” Becker says. Proteins and RNA accumulate in these membraneless organelles (MLOs). “They are formed through liquid-liquid phase separation,” she explains. “This process is similar to the formation of oil droplets in water.”
Many of these MLOs contain proteins that fold incorrectly or unravel and subsequently clump together. The resulting protein aggregates are linked to neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Alzheimer’s, and Parkinson’s. However, it is not yet clear what role MLOs play in the development of these diseases. On one hand, MLOs could protect the cell by temporarily absorbing misfolded proteins; on the other hand, they could serve as sites that promote protein misfolding, thereby harming the cell. “To understand how these diseases develop, it is important to investigate the unfolding of proteins directly within the complex environment of living cells,” Ebbinghaus says.
Direct Comparison of Protein Stability Within a Cell
For this purpose, the researchers at Ruhr University Bochum and the Research Center Chemical Sciences and Sustainability developed a novel experimental method known as “confocal Fast Relaxation Imaging,” or cFReI. This technique makes it possible to directly compare the stability—and, thus, the unfolding—of proteins within MLOs and in the surrounding cytoplasm of the same cell.
Using this method, they examined a variant of the protein superoxide dismutase (SOD1) that accumulates in stress granules. Stress granules are MLOs that form in the cytoplasm in response to cellular stress and help the cell overcome it.
Observation Provides Surprising Insight
“Contrary to our expectations from in vitro experiments, we observed that SOD1 is not destabilized within the stress granules,” Becker reports. “In fact, the protein actually stabilized within the stress granules in most cells.” This, she says, indicates that MLOs could protect the cell against misfolded proteins by temporarily absorbing them instead of promoting their misfolding.
The new method is not restricted to SOD1 or stress granules—it can also be applied to other biomolecules in different organelles, thereby providing important insights into the development of neurodegenerative diseases. In the long term, it could also reveal new starting points for therapeutic strategies.
Funding: The work was supported by the German Research Foundation as part of the Focus Program SPP 2191 (project No. 402723784) and the Research Group FOR 5872 (project No. 545039200).
Published in journal: PRX Life
Authors: Mailin Becker, Nirnay Samanta, Erik Tsvetaev, Miká Vollet, Sara S. Ribeiro, and Simon Ebbinghaus
Source/Credit: Ruhr-Universität Bochum | Meike Drießen
Edited by: Scientific Frontline
Reference Number: cbio072026_01