EMBL Images Ribosome Assembly Inside Human Cells

EMBL researchers have visualised ribosome assembly inside intact human cells by combining cryo-electron tomography with correlative light and electron microscopy, imaging the nucleolus at sufficient resolution to capture structures of pre-ribosome intermediates along the assembly pathway. The work, published in Science, complements earlier structural insights obtained outside the native cellular context and reveals binding and maturation patterns that challenge long-standing assumptions. EMBL News report.

Combining cryo-ET with correlative microscopy

Ribosomes are complex molecular machines, and much of what is known about how they are put together came from studying their components outside the native cellular context. To observe assembly where it actually happens, the collaborating EMBL Heidelberg groups turned to cryo-electron tomography combined with correlative light and electron microscopy, a pairing that lets researchers locate a target and then image it at high resolution.

Whole human cells are too thick for direct electron microscopy, so they are flash-frozen and cut into thin slices that can be observed under the microscope, producing tomograms. Because the nucleolus is a small, dense, low-contrast compartment within the much larger cell, fluorescence labelling was used to locate the components of interest before high-resolution tomograms were collected at that exact spot.

Related coverage: Related research.

Lipid droplets as shared landmarks

Aligning fluorescence images with electron tomograms requires landmarks visible in both kinds of microscopy. Lipid droplets, small fat-storing organelles, served that role: they appear in electron microscopy and in fluorescence microscopy, allowing the images to be matched precisely on top of one another.

The thin slices initially contained too few lipid droplets for reliable registration. The team addressed this by feeding the cells oleic acid, which induced enough lipid droplets to support image alignment. The resulting correlated in-cell maps were built from more than 300 tomograms.

Related coverage: Sector coverage.

An assembly factor binds earlier than expected

The in-cell snapshots confirmed mechanisms of human ribosomal subunit assembly previously identified outside cells and revealed additional interaction partners, including in-cell structures of two key subunit precursors, the SSU processome and the pre-60S. The researchers describe the resolution as short of atomic but close enough to reveal secondary structure elements of the proteins and RNAs involved.

One notable finding concerned a protein factor long thought to bind only late in assembly, when it helps export an almost-completed subunit from the nucleus. The team found that it binds at a much earlier stage, offering insight into the spatial and temporal order of events in the assembly pathway.

Related coverage: Further reporting.

Maturity does not follow nucleolar position

To probe regulation, the scientists blocked RNA polymerase I, the molecular machine that makes ribosomal RNA at the start of the process. The composition of the assembling particles changed markedly, and one subunit precursor disappeared from the nucleolus; the team suggests one subunit may have exited quickly while the other became trapped. Small-subunit assembly also appeared faster than large-subunit assembly, possibly pointing to different regulatory mechanisms.

The data also tested a long-standing assumption that ribosomes mature steadily as they travel from the centre of the nucleolus to its edge. Instead, maturity did not map cleanly onto location. One possible explanation is that assembly proceeds from several centres, with gradients from different sites overlapping and mixing together.

Related coverage: More developments.

What This Means for Practitioners

OUR recommendations: treat this work as a structural-characterisation advance, not a therapeutic or commercial result. Laboratories studying large nuclear complexes should consider correlative cryo-ET workflows and plan landmark strategies, such as lipid-droplet labelling, for image registration in frozen slices. Note the limitations: the earliest and smallest assembly intermediates remain unresolved, and size is a general constraint of in-cell cryo-ET. Future work is proposed rather than demonstrated, including transport of particles out of the nucleolus and applying the approach to other nuclear complexes.