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A study by scientists at the Centre for Genomic Regulation (CRG) in Barcelona has found that collagen, the protein that builds skin, bones, tendons, and organs, exists inside cells as a liquid-like droplet rather than as the long, rigid rod-like structure we might find in textbooks.

The team used techniques including high-resolution live-cell imaging to generate what they say is the first direct observation of how the most abundant protein in the human body, which accounts for around a third of total protein mass, exists naturally inside living cells.

Collagen is built inside a cellular compartment called the endoplasmic reticulum (ER). The study specifically looked at a precursor form inside cells called procollagen 1 (PC1), which matures into type 1 collagen. Type 1 collagen is the most common type of collagen, consisting of around 90% of the body’s total collagen.

“Inside a cell, collagens are not rigid molecules as one had assumed,” said ICREA research professor Vivek Malhotra, PhD, senior author of the study at the CRG. They are, in fact, very pliable, taking a liquid condensate form much like oil in a drop of water.”

The liquid-like state may serve a protective function. Collagen’s job, once outside the cell, is to assemble into the rigid fibers that hold tissues together. The same process inside the cell would be catastrophic. “This is another way by which cells ensure that collagens probably never become fibrous inside the cell,” said Malhotra. “Because if it were to become fibrous, it would kill the cell.”

The new findings have implications for how the body exports its primary structural building block from production sites inside cells. The researchers suggest cells avoid using conventional receptors or vesicles, which is the route established by work carried out in the 1980s and 1990s and recognized with a Nobel prize in 2013.

Instead, they propose a “liquid extrusion” hypothesis, whereby collagen moves from its site of synthesis to the next compartment of the secretory pathway through capillary action. The new theory has important implications for wound healing, fibrosis, and cancer.

Malhotra and colleagues describe their study and results in a paper in the Journal of Cell Biology titled “Procollagen 1 assembles into phase-separated condensates in the endoplasmic reticulum.”

“Procollagen I (PC1) is assembled into a trimer within the lumen of the endoplasmic reticulum (ER),” the authors explained. Under a microscope, purified collagen looks like long, rigid rods of up to 400 nm in length, and this conformation is presumed to represent their assembled state in vivo, the team continued. “However, there is currently no direct experimental evidence demonstrating that PC1 adopts or is maintained in such a rigid, extended conformation within the ER lumen in vivo,” they wrote. Also, the vesicles that transport proteins out from their site of synthesis to the cell’s exterior are only 60 to 90 nanometers in diameter.

Since collagen’s structure was first described more than half a century ago, the field of cell biology has asked how such large molecules can be transported out of cells. The canonical picture of the protein describes collagen only after it has left cells and assembled into the fibers that hold tissues together. The newly reported findings suggest that inside the cell, collagen is not yet assembled into that rod structure.

Using high-resolution live-cell imaging of human hepatic stellate cells—the liver cells that produce collagen and drive scarring in liver fibrosis—the team showed that collagen inside the cell gathers into small droplets that merge, split, and exchange material with their surroundings. These are all signatures of a condensate, compartments of proteins that become so concentrated they disassociate from their surroundings, like droplets of oil in water.

Most of cell biology has focused on condensates in the nucleus and on stress granules in the cytosol, said first author Soumya Bhattacharyya, PhD, a postdoctoral researcher in Malhotra’s lab. “We’re just beginning to understand condensates inside the endoplasmic reticulum.”

The findings emerged from microscopy images taken by Bhattacharyya in May 2024. Bhattacharyya was using the liver cell system as a tool to study what happens when collagen production is increased in fibrotic cells. “I had no idea what it would lead to. But when we took the samples, what struck me were these bright spherical structures you can’t miss,” recalled Bhattacharyya.

The initial reaction in the laboratory to a finding that challenged cell biology dogma was sceptical. “I thought it must be an artefact,” said Malhotra. In the months that followed, the team had to settle whether the protein clumping they observed inside the endoplasmic reticulum was junk. Cells have an elaborate system for detecting badly folded proteins and either refolding them or marking them for destruction, centered on a chaperone called BiP.

If the collagen droplets were heaps of misfolded protein, the researchers would detect high levels of BiP. The droplets contained, instead, a mixture of helper proteins, including chaperones that specifically recognize properly folded collagen.

The study also clarifies the function of TANGO1, a protein discovered by the Malhotra lab roughly two decades ago and known to be required for collagen export. When the researchers depleted TANGO1, the collagen droplets still formed but were no longer positioned at the ER exit sites (ERES) where cargo leaves the compartment. Collagen secretion dropped accordingly. “PC1 condensates were still formed after TANGO1 knockdown, indicating that TANGO1 is not required for condensate formation per se,” the investigators stated. “However, TANGO1 depletion caused a marked reduction in the association of PC1 condensates with ERES …”

The discovery suggests TANGO1 acts as a mooring point that holds the droplet at the export site rather than as a conventional cargo receptor. The authors propose that collagen then leaves the cell by a physical process called wetting, in which the liquid droplet attaches to and flows through the exit site.

Malhotra offers two possible physical mechanisms for this transfer. “Imagine you have a rubber ball with a nozzle, filled with liquid. You squeeze it, you force the liquid to come out of this little orifice. Is that the mechanism? Or is the liquid rising by capillary forces, just like nutrients flow up against gravity in plants by capillary action?”

The proposed liquid extrusion mechanism remains a model, but the next experiments to obtain direct visualization of the export mechanism are already underway. The team also plans to develop a mouse model, in collaboration with external partners, to confirm the findings in living tissue. If the model is confirmed, the work has implications for several pathological conditions in which excess collagen secretion plays a central role, including liver, lung, and skin fibrosis, as well as for targeting the dense matrix that tumors use to shield themselves from chemotherapy and the immune system.

“One of the major problems in cancer is that the cells secrete so many collagens and other proteins out into the extracellular matrix that they hide in a shell made of these components and become chemo- and immuno-refractory, meaning they are not seen by the chemical therapeutics or by the immune system,” Malhotra said. “People are trying to find ways to break this tissue cement, and our study could help inform those strategies.”

The proposed collagen secretion model suggests that either degrading TANGO1 to prevent cargo from being captured at the exit site or dissolving the condensate itself to prevent the cargo from being properly organized in the first place could be new strategies worth exploring.

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