HMN 2026: How Live-cell tracking reveals dynamic interaction between protein folding helpers and newly produced proteins

Proteins are the molecular machines of cells. They are produced in protein factories called ribosomes based on their blueprint—the genetic information. Here, the basic building blocks of proteins, amino acids, are assembled into long protein chains. Like the building blocks of a machine, individual proteins must have a specific three-dimensional structure to properly fulfill their functions.

To achieve this, the newly produced protein chains in human cells are folded into their stable and functional form with the help of various protein folding helper proteins, known as chaperones, such as TRiC/PFD, or HSP70/40. The protein folding helpers isolate the amino acid chains, which have different chemical properties depending on the amino acid, from the cellular environment. This prevents the newly produced protein chains from clumping together and causing disease.

F.-Ulrich Hartl, a director at the Max Planck Institute of Biochemistry, has spent decades studying the mechanisms of protein folding. Niko Dalheimer, a scientist in Hartl’s department and one of the two lead authors of a new study published in Nature, explains: “Much of what we know about protein folding has been learned from studies conducted in test tubes. However, it is virtually impossible to faithfully replicate the cellular environment in vitro.

“Unlike a test tube, a cell is a highly complex environment filled with many different macromolecules, like proteins, nucleic acids and lipids. To fully understand how chaperones work, we examined the protein folding dynamics of TRiC and PFD in its natural environment—intact human cells—using single-particle tracking on a fluorescence microscope, an approach that has only recently become feasible thanks to advances in live-cell fluorescent labeling.”

TRiC and prefoldin

A newly synthesized protein is gradually released as an amino acid chain by the ribosomes through a channel. To prevent the newly synthesized protein from clumping together, the free amino acid residues are captured and protected by prefoldin, or PFD for short, a co-chaperone of TRiC. The co-chaperone then passes the protein on to the chaperonin TRiC for folding.

TRiC is a barrel-shaped protein folding helper and is related to the bacterial GroEL/ES. Rongqin Li, scientist and co-first author of the study, states, “Although TRiC only helps 10% of the proteins in a cell to fold, many of them are particularly important for the cell, including actin and tubulin, which are building blocks of the cytoskeleton. That’s why we looked at this part of protein folding. We used actin as a test protein to understand the folding dynamics in cells.”

Single particle tracking sheds light on the unknown

In order to follow the real-time interaction of all components involved in protein folding, the researchers labeled TRiC and prefoldin, as well as the actin nascent chain as direct chaperone substrate and ribosomes and mRNAs as proxies for chaperone substrates with two colors, green and magenta, in different settings. If the two components were in close proximity, i.e., less than 500 nanometers apart, the colors overlapped and were visible under the microscope as white dots.

Dalheimer explains, “There are approximately 10 million ribosomes in a single cell. To enable us to track individual ribosomes and other components under the microscope, we stained only a small proportion of the ribosomes, rather than all of them and used the TIRF method to track the individual molecules and their interactions with chaperones. It is like a diver exploring a pitch-dark deep sea: by shining a light on just a few spots at a time, the diver can get a glimpse of the hidden dynamic life and activity around them.”

‘Seeing is believing’

The scientists observed that TRiC and PFD repeatedly probe with the newly synthesized actin protein chain emerging from the ribosome for approximately one second. PFD holds the nascent chain shortly before actin is being released from the ribosome and hands it over to the TRiC chaperone for folding completion.

Li adds, “Interestingly, the contact between TRiC and actin mutants, i.e., protein chains into which we introduce errors to disrupt its proper folding, was significantly longer. In contrast to the normal condition, the folding-defective actin undergoes multiple rounds of attempted folding by the chaperonin system and is ultimately targeted for degradation.

F.-Ulrich Hartl summarizes, “For decades, we and others have studied chaperone-mediated protein folding primarily through biochemical experiments, which have been essential for defining how this process is controlled. With live-cell single-particle tracking, we can now examine these concepts directly in living cells. In doing so, we have confirmed key findings from classical biochemical experiments, while at the same time uncovering features—such as the protected folding zone—that could not have been detected with ensemble-based assays.

“This is the first time these processes have been visualized at the single-molecule level in living cells. As I often tell my colleagues, ‘seeing is believing.'”

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