Tiny traps, big trouble: Small regions within cells aggregate proteins linked to ALS, dementia

(Press-News.org) ANN ARBOR—Inside the cell reside many tiny assembly factories and warehouses that gather together all of the proteins and RNAs—which carry out instructions from our DNA—that a living being needs. 

These assemblies, called biomolecular condensates, help regulate how cells divide and respond to stress by sequestering and processing RNA and proteins. However, they don't have a membrane that separates them from the rest of the nucleus. Instead, their machinery condenses together, similar to how water vapor fleetingly condenses to form clouds in the sky, says University of Michigan professor of chemistry Nils Walter. 

But scientists aren't sure how the processes work that control the fate of these biomolecular droplets. Imaging these processes has proven difficult: Everything within a cell moves and is hard to pinpoint, and biomolecular condensates have a tendency to roll around when placed on a microscope slide for examination.


Now, U-M researchers led by Walter have developed a method to examine the interior of the droplets, focusing on a protein called fused in sarcoma, or FUS, which often condenses in patients with the neurodegenerative disease amyotrophic lateral sclerosis, or ALS. They found that the movements of RNA and protein molecules within these biomolecular condensates are slowed down within distinct, infinitesimal areas that the researchers call nanodomains. Over time—as the condensates age—the nanodomains move to the droplet's surface.

The researchers also applied drugs used to treat ALS and similar diseases to the biomolecular condensates, and saw that the drugs may act, in part, by speeding up the nanodomain movement to the droplet surface, accelerating the formation of fibrils there. These fibrils are thought to protect neurons from degrading since they soak up smaller toxic aggregates during the progression of ALS. 

The findings, which were supported by the National Institutes of Health, U.S. National Science Foundation and Chan Zuckerberg Initiative, are reported in Nature Nanotechnology.

"There is a lot of hope that by manipulating these condensates, we can use them for medical purposes, such as slowing neurodegenerative disease, making them a repository for drugs that can be released slowly over time, or sequestering unwanted proteins such as those that are cancer- or virus-related by inducing them to form condensates," said Walter, director of the Center for RNA Biomedicine at U-M. "Understanding how they form—and what develops inside of them as they age—is essential for finding ways to influence the process beneficially."

FUS is a central regulator of cellular RNA metabolism and condenses when cells undergo a stress conduction called hyposmotic phase separation. Under this condition, the cell experiences a higher than normal concentration of salt, which causes the cell to adapt by shedding water and shrinking by up to 50%. After a while, when the stress is not released, cells start gene expression programs to inflate themselves again. FUS condensation may play a role in facilitating the correct programs. 

Certain genetic mutations of FUS lead to cancers and neurodegenerative diseases like ALS. Upon mutation, FUS accumulates and then condenses in the cell's cytoplasm. The clumping of FUS over time is linked to ALS and frontotemporal dementia. But capturing the progression of this pathological change is difficult, the researchers say, as is capturing images of how FUS condenses and the condensates age.

To study how FUS condenses, the researchers purified a full-length form of the protein. They first added a type of sugar to the protein that prevents it from condensing, and which they could remove at will, triggering the protein's condensation. The researchers tagged an RNA probe and the protein with two different colors of fluorescent dyes. This allowed them to use fluorescence microscopy to follow the diffusion of single RNA and protein molecules in the condensed droplets.

But they had one more hurdle to overcome: the biomolecular condensate droplets are difficult to hold in place in order for a microscope to examine them, Walter says.

"If you make a condensate and put it on a microscope slide, it can roll over the surface or wiggle back and forth. If that happens, then the particle tracking gets messed up," Walter said. "So you have to immobilize the condensates on the surface, but you have to do that in a very judicial way. For example, if you have too many anchors on the surface of the condensate, it just flattens out. It becomes a pancake."

The research team found a happy medium of just enough anchors to keep the droplet still, and using a type of microscopy called HILO microscopy, the researchers could track the movements of individual molecules within the droplet. This allowed them to see where particles were congregating within the droplet.

The technique also allowed the researchers to watch fibrils form around these FUS condensates. The researchers then applied small molecule drugs used to treat ALS to the biocondensates to see how they affected the FUS proteins. They found that the drugs caused the nanodomain clusters of FUS to move more quickly to the surface of the condensate, from where the fibers grew. 

"But what our findings mean overall is that, for the first time, we see these nanodomains as potential seeds to these fibers," Walter said. "Maybe the drugs we used, edaravone and especially riluzole, have another effect beyond those known, by helping the condensates to fibralize faster and protect the neuron."

Walter says understanding the mechanisms within biomolecular condensates is an important topic for researchers right now.

"The field of phase condensation has exploded. There are many of these phase condensates in cells that either accelerate reactions or sequester things away so they cannot wreak havoc," he said. "There's a lot of biology being learned in a fast-moving area of biology."

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