“If you’re in a bar or nightclub that’s not very crowded, you might not talk to anyone or dance with anyone,” said Liam Holt, a cell biologist at New York University Langone Health. “But at midnight when the bar is jam-packed, you’re more likely to talk or dance with someone next to you. But if you see someone across the room, it’s harder to get to them.”

The new findings are confounding scientists’ expectations, raising questions about how exactly molecules can encounter their reactive partners in a teeming, crowded space — and therefore how cells can possibly function.

The Biophysical Knife’s Edge

Cells may be biological entities, but they are not exempt from the laws of physics. In his 1944 book What Is Life? The Physical Aspect of the Living Cell, the quantum physicist Erwin Schrödinger argued that living things, like their nonliving counterparts, must submit to governance by physical laws. His vision has inspired biology-minded physicists and physics-minded biologists ever since.

But studying the physics of eukaryotic cells — the kind that make up our bodies and those of other multicellular organisms — presented a challenge: How do you study an individual cell buried deep within the body of a person, a mouse, or even a simple worm?

At first, scientists got around this problem by removing those cells from humans and animals and growing them in test tubes or petri dishes. Early studies hinted that cells are subject to a Goldilocks phenomenon: They function best when their cytoplasm — everything enclosed by a cell’s membrane, including organelles, molecular structures such as ribosomes, and the gel-like cytosol and its dissolved molecules — has some level of crowding, but not too much. In the 1980s, a team of researchers found that if they diluted the cytoplasm extracted from frog eggs even a little bit, vital biochemical reactions such as mitosis and DNA replication ceased. Other studies found that overcrowding could be equally disastrous, causing the chemical machinery of life to freeze up.

Cells are constantly churning through energy to stir things up, keep the cytosol fluid, and encourage molecules to collide and react more often than they would through simple diffusion. Even so, cellular life appears to balance on a knife’s edge. If cells were any less crowded, molecules would wander aimlessly and only rarely encounter their partner (or partners) in the chemical reactions that power life — metabolism, protein synthesis, growth, division, and more. In that situation, cellular life would wither. If, on the other hand, cells were much more crowded, molecules would be stuck in place, unable to move much at all, let alone come across their reaction partners. Life would grind to a halt.

Evolution seemed to have struck a delicate balance between over- and undercrowding, with large molecules such as ribosomes typically representing between 30% and 40% of the volume of dissolved macromolecules in the cytosol, Holt said. “It seems that much of biology is tuned to have a very similar level of crowding.” But to confirm this view, researchers would need to find a way to track molecules moving through a cell. They would need a tracer of the right size.

Crowd Control

Crowding is relative. While a person might have trouble moving through Holt’s imagined club at midnight, a cat or mouse would not find it too crowded to navigate. To study crowding in cells, biologists needed a proxy molecule in the same size range — a tracer roughly as large as the large molecules involved in most cellular reactions.

In the mid-2010s, Holt introduced genetically encoded multimeric nanoparticles, or GEMs, which are naturally occurring spherical proteins about 40 nanometers in diameter — around the same size as ribosomes, the molecular machines that build proteins. Using genetic engineering, researchers can decorate the surfaces of GEMs with glowing green fluorescent tags and then track their movements through a cell’s cytoplasm under a microscope.

In 2018, this approach gave Holt and his colleagues fresh insight into how cells manage their crowdedness. They put GEMs inside yeast and human cells in culture and measured how long it took the particles to percolate through different areas of the cell. Strangely, in cells grown under different nutritional conditions, the crowding of the entire cell seemed to change. “This led me to ask what was going on,” Holt said.

He suspected that mTORC1 was involved. The main nutrient sensor in eukaryotic cells, mTORC1 is a master regulator of cell growth; based on nutrient levels, it can boost production of ribosomes to build more proteins faster. “The rate at which organisms can grow is fundamentally limited by how many ribosomes they can produce,” Holt said. Indeed, when his team chemically suppressed mTORC1, the concentration of ribosomes decreased, and GEMs flowed through the cytoplasm much more easily.